Measuring Local Viscosities near Plasma Membranes of Living Cells with Photonic Force Microscopy

PubMed Central

Jünger, Felix; Kohler, Felix; Meinel, Andreas; Meyer, Tim; Nitschke, Roland; Erhard, Birgit; Rohrbach, Alexander

2015-01-01

The molecular processes of particle binding and endocytosis are influenced by the locally changing mobility of the particle nearby the plasma membrane of a living cell. However, it is unclear how the particle’s hydrodynamic drag and momentum vary locally and how they are mechanically transferred to the cell. We have measured the thermal fluctuations of a 1 μm-sized polystyrene sphere, which was placed in defined distances to plasma membranes of various cell types by using an optical trap and fast three-dimensional (3D) interferometric particle tracking. From the particle position fluctuations on a 30 μs timescale, we determined the distance-dependent change of the viscous drag in directions perpendicular and parallel to the cell membrane. Measurements on macrophages, adenocarcinoma cells, and epithelial cells revealed a significantly longer hydrodynamic coupling length of the particle to the membrane than those measured at giant unilamellar vesicles (GUVs) or a plane glass interface. In contrast to GUVs, there is also a strong increase in friction and in mean first passage time normal to the cell membrane. This hydrodynamic coupling transfers a different amount of momentum to the interior of living cells and might serve as an ultra-soft stimulus triggering further reactions. PMID:26331245

Immunocytochemical localization of muscarinic, adrenergic and AT1 receptors.

PubMed

Schulze, W; Fu, M L

1996-01-01

By indirect immunofluorescence and post-embedding EM gold technique, the localization of alpha 1-adrenergic, M2-muscarinic and angiotensin II receptor-I (AT1) were determinated. With antipeptide antibodies directed against the second extracellular loops of all three receptors, these receptors were found to be localized at the sarcolemma of adult rat cardiomyocytes and at the surface membranes of cultivated neonatal heart cells. Additionally, M2 receptors were localized along T-tubule membranes of both rat and human adult cardiomyocytes. alpha 1-Adrenergic receptors were found intracellular near the surface of atrial granules (ANF-granules). By using M2 and alpha 1-adrenergic receptor antibodies the strongest fluorescence was found in the right atrium of the rat. Besides the localization in cardiomyocytes, AT1 receptors were also localized in outer plasma membranes and the endoplasmic reticulum of fibroblasts, and the surface of smooth muscle cells of the major arteries and veins. Likewise, the muscarinic M2 receptors were found along the outer membranes of endothelial cells from capillaries and endocardium.

Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

PubMed Central

Lotti, L V; Lanfrancone, L; Migliaccio, E; Zompetta, C; Pelicci, G; Salcini, A E; Falini, B; Pelicci, P G; Torrisi, M R

1996-01-01

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein. PMID:8628261

Cardiolipin Synthesis and Outer Membrane Localization Are Required for Shigella flexneri Virulence.

PubMed

Rossi, Rachael M; Yum, Lauren; Agaisse, Hervé; Payne, Shelley M

2017-08-29

Cardiolipin, an anionic phospholipid that resides at the poles of the inner and outer membranes, is synthesized primarily by the putative cardiolipin synthase ClsA in Shigella flexneri An S. flexneri clsA mutant had no cardiolipin detected within its membrane, grew normally in vitro , and invaded cultured epithelial cells, but it failed to form plaques in epithelial cell monolayers, indicating that cardiolipin is required for virulence. The clsA mutant was initially motile within the host cell cytoplasm but formed filaments and lost motility during replication and failed to spread efficiently to neighboring cells. Mutation of pbgA , which encodes the transporter for cardiolipin from the inner membrane to the outer membrane, also resulted in loss of plaque formation. The S. flexneri pbgA mutant had normal levels of cardiolipin in the inner membrane, but no cardiolipin was detected in the outer membrane. The pbgA mutant invaded and replicated normally within cultured epithelial cells but failed to localize the actin polymerization protein IcsA properly on the bacterial surface and was unable to spread to neighboring cells. The clsA mutant, but not the pbgA mutant, had increased phosphatidylglycerol in the outer membrane. This appeared to compensate partially for the loss of cardiolipin in the outer membrane, allowing some IcsA localization in the outer membrane of the clsA mutant. We propose a dual function for cardiolipin in S. flexneri pathogenesis. In the inner membrane, cardiolipin is essential for proper cell division during intracellular growth. In the outer membrane, cardiolipin facilitates proper presentation of IcsA on the bacterial surface. IMPORTANCE The human pathogen Shigella flexneri causes bacterial dysentery by invading colonic epithelial cells, rapidly multiplying within their cytoplasm, and then spreading intercellularly to neighboring cells. Worldwide, Shigella spp. infect hundreds of millions of people annually, with fatality rates up to 15%. Antibiotic treatment of Shigella infections is compromised by increasing antibiotic resistance, and there is no approved vaccine to prevent future infections. This has created a growing need to understand Shigella pathogenesis and identify new targets for antimicrobial therapeutics. Here we show a previously unknown role of phospholipids in S. flexneri pathogenesis. We demonstrate that cardiolipin is required in the outer membrane for proper surface localization of IcsA and in the inner membrane for cell division during growth in the host cell cytoplasm. Copyright © 2017 Rossi et al.

The RxLR effector Avh241 from Phytophthora sojae requires plasma membrane localization to induce plant cell death.

PubMed

Yu, Xiaoli; Tang, Junli; Wang, Qunqing; Ye, Wenwu; Tao, Kai; Duan, Shuyi; Lu, Chenchen; Yang, Xinyu; Dong, Suomeng; Zheng, Xiaobo; Wang, Yuanchao

2012-10-01

• The Phytophthora sojae genome encodes hundreds of RxLR effectors predicted to manipulate various plant defense responses, but the molecular mechanisms involved are largely unknown. Here we have characterized in detail the P. sojae RxLR effector Avh241. • To determine the function and localization of Avh241, we transiently expressed it on different plants. Silencing of Avh241 in P. sojae, we determined its virulence during infection. Through the assay of promoting infection by Phytophthora capsici to Nicotiana benthamiana, we further confirmed this virulence role. • Avh241 induced cell death in several different plants and localized to the plant plasma membrane. An N-terminal motif within Avh241 was important for membrane localization and cell death-inducing activity. Two mitogen-activated protein kinases, NbMEK2 and NbWIPK, were required for the cell death triggered by Avh241 in N. benthamiana. Avh241 was important for the pathogen's full virulence on soybean. Avh241 could also promote infection by P. capsici and the membrane localization motif was not required to promote infection. • This work suggests that Avh241 interacts with the plant immune system via at least two different mechanisms, one recognized by plants dependent on subcellular localization and one promoting infection independent on membrane localization. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

The C-terminal domain of TRPV4 is essential for plasma membrane localization.

PubMed

Becker, Daniel; Müller, Margarethe; Leuner, Kristina; Jendrach, Marina

2008-02-01

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.

Reparable Cell Sonoporation in Suspension: Theranostic Potential of Microbubble.

PubMed

Nejad, S Moosavi; Hosseini, Hamid; Akiyama, Hidenori; Tachibana, Katsuro

2016-01-01

The conjunction of low intensity ultrasound and encapsulated microbubbles can alter the permeability of cell membrane, offering a promising theranostic technique for non-invasive gene/drug delivery. Despite its great potential, the biophysical mechanisms of the delivery at the cellular level remains poorly understood. Here, the first direct high-speed micro-photographic images of human lymphoma cell and microbubble interaction dynamics are provided in a completely free suspension environment without any boundary parameter defect. Our real-time images and theoretical analyses prove that the negative divergence side of the microbubble's dipole microstreaming locally pulls the cell membrane, causing transient local protrusion of 2.5 µm in the cell membrane. The linear oscillation of microbubble caused microstreaming well below the inertial cavitation threshold, and imposed 35.3 Pa shear stress on the membrane, promoting an area strain of 0.12%, less than the membrane critical areal strain to cause cell rupture. Positive transfected cells with pEGFP-N1 confirm that the interaction causes membrane poration without cell disruption. The results show that the overstretched cell membrane causes reparable submicron pore formation, providing primary evidence of low amplitude (0.12 MPa at 0.834 MHz) ultrasound sonoporation mechanism.

Bacillus subtilis actin-like protein MreB influences the positioning of the replication machinery and requires membrane proteins MreC/D and other actin-like proteins for proper localization.

PubMed

Defeu Soufo, Hervé Joël; Graumann, Peter L

2005-03-03

Bacterial actin-like proteins have been shown to perform essential functions in several aspects of cellular physiology. They affect cell growth, cell shape, chromosome segregation and polar localization of proteins, and localize as helical filaments underneath the cell membrane. Bacillus subtilis MreB and Mbl have been shown to perform dynamic motor like movements within cells, extending along helical tracks in a time scale of few seconds. In this work, we show that Bacillus subtilis MreB has a dual role, both in the formation of rod cell shape, and in chromosome segregation, however, its function in cell shape is distinct from that of MreC. Additionally, MreB is important for the localization of the replication machinery to the cell centre, which becomes aberrant soon after depletion of MreB. 3D image reconstructions suggest that frequently, MreB filaments consist of several discontinuous helical filaments with varying length. The localization of MreB was abnormal in cells with decondensed chromosomes, as well as during depletion of Mbl, MreBH and of the MreC/MreD proteins, which we show localize to the cell membrane. Thus, proper positioning of MreB filaments depends on and is affected by a variety of factors in the cell. Our data provide genetic and cytological links between MreB and the membrane, as well as with other actin like proteins, and further supports the connection of MreB with the chromosome. The functional dependence on MreB of the localization of the replication machinery suggests that the replisome is not anchored at the cell centre, but is positioned in a dynamic manner.

WAVE2 forms a complex with PKA and is involved in PKA enhancement of membrane protrusions.

PubMed

Yamashita, Hiroshi; Ueda, Kazumitsu; Kioka, Noriyuki

2011-02-04

PKA contributes to many physiological processes, including glucose homeostasis and cell migration. The substrate specificity of PKA is low compared with other kinases; thus, complex formation with A-kinase-anchoring proteins is important for the localization of PKA in specific subcellular regions and the phosphorylation of specific substrates. Here, we show that PKA forms a complex with WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) in MDA-MB-231 breast cancer cells and mouse brain extracts. Two separate regions of WAVE2 are involved in WAVE2-PKA complex formation. This complex localizes to the leading edge of MDA-MB-231 cells. PKA activation results in enlargement of the membrane protrusion. WAVE2 depletion impairs PKA localization at membrane protrusions and the enlargement of membrane protrusion induced by PKA activation. Together, these results suggest that WAVE2 works as an A-kinase-anchoring protein that recruits PKA at membrane protrusions and plays a role in the enlargement of membrane protrusions induced by PKA activation.

Macrophage NADPH oxidase flavocytochrome B localizes to the plasma membrane and Rab11-positive recycling endosomes.

PubMed

Casbon, Amy-Jo; Allen, Lee-Ann H; Dunn, Kenneth W; Dinauer, Mary C

2009-02-15

Flavocytochrome b(558), the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91(phox) (NOX2) and a smaller subunit, p22(phox). Although in neutrophils flavocytochrome b has been shown to localize to the plasma membrane and specific granules, little is known about its distribution in macrophages. Using immunofluorescent staining and live cell imaging of fluorescently tagged gp91(phox) and p22(phox), we demonstrate in a Chinese hamster ovary cell model system and in RAW 264.7 and primary murine bone marrow-derived macrophages that flavocytochrome b is found in the Rab11-positive recycling endocytic compartment, as well as in Rab5-positive early endosomes and plasma membrane. Additionally, we show that unassembled p22(phox) and gp91(phox) subunits localize to the endoplasmic reticulum, which redistribute to the cell surface and endosomal compartments following heterodimer formation. These studies show for the first time that flavocytochrome b localizes to intracellular compartments in macrophages that recycle to the plasma membrane, which may act as a reservoir to deliver flavocytochrome b to the cell surface and phagosome membranes.

Quantitative Microscopic Analysis of Plasma Membrane Receptor Dynamics in Living Plant Cells.

PubMed

Luo, Yu; Russinova, Eugenia

2017-01-01

Plasma membrane-localized receptors are essential for cellular communication and signal transduction. In Arabidopsis thaliana, BRASSINOSTEROID INSENSITIVE1 (BRI1) is one of the receptors that is activated by binding to its ligand, the brassinosteroid (BR) hormone, at the cell surface to regulate diverse plant developmental processes. The availability of BRI1 in the plasma membrane is related to its signaling output and is known to be controlled by the dynamic endomembrane trafficking. Advances in fluorescence labeling and confocal microscopy techniques enabled us to gain a better understanding of plasma membrane receptor dynamics in living cells. Here we describe different quantitative microscopy methods to monitor the relative steady-state levels of the BRI1 protein in the plasma membrane of root epidermal cells and its relative exocytosis and recycling rates. The methods can be applied also to analyze similar dynamics of other plasma membrane-localized receptors.

Septal membrane localization by C-terminal amphipathic α-helices of MinD in Bacillus subtilis mutant cells lacking MinJ or DivIVA.

PubMed

Ishikawa, Kazuki; Matsuoka, Satoshi; Hara, Hiroshi; Matsumoto, Kouji

2017-10-18

The Min system, which inhibits assembly of the cytokinetic protein FtsZ, is largely responsible for positioning the division site in rod-shaped bacteria. It has been reported that MinJ, which bridges DivIVA and MinD, is targeted to the cell poles by an interaction with DivIVA, and that MinJ in turn recruits MinCD to the cell poles. MinC, however, is located primarily at active division sites at mid-cell when expressed from its native promoter. Surprisingly, we found that Bacillus subtilis MinD is located at nascent septal membranes and at an asymmetric site on lateral membranes between nascent septal membranes in filamentous cells lacking MinJ or DivIVA. Bacillus subtilis MinD has two amphipathic α-helices rich in basic amino acid residues at its C-terminus; one of these, named MTS1 here, is the counterpart of the membrane targeting sequence (MTS) in Escherichia coli MinD while the other, named MTS-like sequence (MTSL), is the nearest helix to MTS1. These amphipathic helices were located independently at nascent septal membranes in cells lacking MinJ or DivIVA, whereas elimination of the helices from the wild type protein reduced its localization considerably. MinD variants with altered MTS1 and MTSL, in which basic amino acid residues were replaced with proline or acidic residues, were not located at nascent septal membranes, indicating that the binding to the nascent septal membranes requires basic residues and a helical structure. The septal localization of MTSL, but not of MTS1, was dependent on host cell MinD. These results suggest that MinD is targeted to nascent septal membranes via its C-terminal amphipathic α-helices in B. subtilis cells lacking MinJ or DivIVA. Moreover, the diffuse distribution of MinD lacking both MTSs suggests that only a small fraction of MinD depends on MinJ for its localization to nascent septal membranes.

The Role of Membrane Curvature in Nanoscale Topography-Induced Intracellular Signaling.

PubMed

Lou, Hsin-Ya; Zhao, Wenting; Zeng, Yongpeng; Cui, Bianxiao

2018-05-15

Over the past decade, there has been growing interest in developing biosensors and devices with nanoscale and vertical topography. Vertical nanostructures induce spontaneous cell engulfment, which enhances the cell-probe coupling efficiency and the sensitivity of biosensors. Although local membranes in contact with the nanostructures are found to be fully fluidic for lipid and membrane protein diffusions, cells appear to actively sense and respond to the surface topography presented by vertical nanostructures. For future development of biodevices, it is important to understand how cells interact with these nanostructures and how their presence modulates cellular function and activities. How cells recognize nanoscale surface topography has been an area of active research for two decades before the recent biosensor works. Extensive studies show that surface topographies in the range of tens to hundreds of nanometers can significantly affect cell functions, behaviors, and ultimately the cell fate. For example, titanium implants having rough surfaces are better for osteoblast attachment and host-implant integration than those with smooth surfaces. At the cellular level, nanoscale surface topography has been shown by a large number of studies to modulate cell attachment, activity, and differentiation. However, a mechanistic understanding of how cells interact and respond to nanoscale topographic features is still lacking. In this Account, we focus on some recent studies that support a new mechanism that local membrane curvature induced by nanoscale topography directly acts as a biochemical signal to induce intracellular signaling, which we refer to as the curvature hypothesis. The curvature hypothesis proposes that some intracellular proteins can recognize membrane curvatures of a certain range at the cell-to-material interface. These proteins then recruit and activate downstream components to modulate cell signaling and behavior. We discuss current technologies allowing the visualization of membrane deformation at the cell membrane-to-substrate interface with nanometer precision and demonstrate that vertical nanostructures induce local curvatures on the plasma membrane. These local curvatures enhance the process of clathrin-mediated endocytosis and affect actin dynamics. We also present evidence that vertical nanostructures can induce significant deformation of the nuclear membrane, which can affect chromatin distribution and gene expression. Finally, we provide a brief perspective on the curvature hypothesis and the challenges and opportunities for the design of nanotopography for manipulating cell behavior.

A Novel Phosphatidylinositol 4,5-Bisphosphate Binding Domain Mediates Plasma Membrane Localization of ExoU and Other Patatin-like Phospholipases*

PubMed Central

Tyson, Gregory H.; Halavaty, Andrei S.; Kim, Hyunjin; Geissler, Brett; Agard, Mallory; Satchell, Karla J.; Cho, Wonhwa; Anderson, Wayne F.; Hauser, Alan R.

2015-01-01

Bacterial toxins require localization to specific intracellular compartments following injection into host cells. In this study, we examined the membrane targeting of a broad family of bacterial proteins, the patatin-like phospholipases. The best characterized member of this family is ExoU, an effector of the Pseudomonas aeruginosa type III secretion system. Upon injection into host cells, ExoU localizes to the plasma membrane, where it uses its phospholipase A2 activity to lyse infected cells. The targeting mechanism of ExoU is poorly characterized, but it was recently found to bind to the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a marker for the plasma membrane of eukaryotic cells. We confirmed that the membrane localization domain (MLD) of ExoU had a direct affinity for PI(4,5)P2, and we determined that this binding was required for ExoU localization. Previously uncharacterized ExoU homologs from Pseudomonas fluorescens and Photorhabdus asymbiotica also localized to the plasma membrane and required PI(4,5)P2 for this localization. A conserved arginine within the MLD was critical for interaction of each protein with PI(4,5)P2 and for localization. Furthermore, we determined the crystal structure of the full-length P. fluorescens ExoU and found that it was similar to that of P. aeruginosa ExoU. Each MLD contains a four-helical bundle, with the conserved arginine exposed at its cap to allow for interaction with the negatively charged PI(4,5)P2. Overall, these findings provide a structural explanation for the targeting of patatin-like phospholipases to the plasma membrane and define the MLD of ExoU as a member of a new class of PI(4,5)P2 binding domains. PMID:25505182

A PI4P-driven electrostatic field controls cell membrane identity and signaling in plants

PubMed Central

Simon, Mathilde Laetitia Audrey; Platre, Matthieu Pierre; Marquès-Bueno, Maria Mar; Armengot, Laia; Stanislas, Thomas; Bayle, Vincent; Caillaud, Marie-Cécile; Jaillais, Yvon

2016-01-01

Many signaling proteins permanently or transiently localize to specific organelles for function. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. Here, we found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PI4P). Our results further reveal that, contrarily to other eukaryotes, PI4P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID, as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATORs (MAKRs) family, which are involved in brassinosteroid and receptor-like kinase signaling. We anticipate that this PI4P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition. PMID:27322096

A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants.

PubMed

Simon, Mathilde Laetitia Audrey; Platre, Matthieu Pierre; Marquès-Bueno, Maria Mar; Armengot, Laia; Stanislas, Thomas; Bayle, Vincent; Caillaud, Marie-Cécile; Jaillais, Yvon

2016-06-20

Many signalling proteins permanently or transiently localize to specific organelles. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. Here, we found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PtdIns(4)P). Our results further reveal that, contrarily to other eukaryotes, PtdIns(4)P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATOR (MAKR) family, which are involved in brassinosteroid and receptor-like kinase signalling. We anticipate that this PtdIns(4)P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition.

Motor Protein Myo1c Is a Podocyte Protein That Facilitates the Transport of Slit Diaphragm Protein Neph1 to the Podocyte Membrane ▿

PubMed Central

Arif, E.; Wagner, M. C.; Johnstone, D. B.; Wong, H. N.; George, B.; Pruthi, P. A.; Lazzara, M. J.; Nihalani, D.

2011-01-01

The podocyte proteins Neph1 and nephrin organize a signaling complex at the podocyte cell membrane that forms the structural framework for a functional glomerular filtration barrier. Mechanisms regulating the movement of these proteins to and from the membrane are currently unknown. This study identifies a novel interaction between Neph1 and the motor protein Myo1c, where Myo1c plays an active role in targeting Neph1 to the podocyte cell membrane. Using in vivo and in vitro experiments, we provide data supporting a direct interaction between Neph1 and Myo1c which is dynamic and actin dependent. Unlike wild-type Myo1c, the membrane localization of Neph1 was significantly reduced in podocytes expressing dominant negative Myo1c. In addition, Neph1 failed to localize at the podocyte cell membrane and cell junctions in Myo1c-depleted podocytes. We further demonstrate that similarly to Neph1, Myo1c also binds nephrin and reduces its localization at the podocyte cell membrane. A functional analysis of Myo1c knockdown cells showed defects in cell migration, as determined by a wound assay. In addition, the ability to form tight junctions was impaired in Myo1c knockdown cells, as determined by transepithelial electric resistance (TER) and bovine serum albumin (BSA) permeability assays. These results identify a novel Myo1c-dependent molecular mechanism that mediates the dynamic organization of Neph1 and nephrin at the slit diaphragm and is critical for podocyte function. PMID:21402783

Influence of cell shape, inhomogeneities and diffusion barriers in cell polarization models

NASA Astrophysics Data System (ADS)

Giese, Wolfgang; Eigel, Martin; Westerheide, Sebastian; Engwer, Christian; Klipp, Edda

2015-12-01

In silico experiments bear the potential for further understanding of biological transport processes by allowing a systematic modification of any spatial property and providing immediate simulation results. Cell polarization and spatial reorganization of membrane proteins are fundamental for cell division, chemotaxis and morphogenesis. We chose the yeast Saccharomyces cerevisiae as an exemplary model system which entails the shuttling of small Rho GTPases such as Cdc42 and Rho, between an active membrane-bound form and an inactive cytosolic form. We used partial differential equations to describe the membrane-cytosol shuttling of proteins. In this study, a consistent extension of a class of 1D reaction-diffusion systems into higher space dimensions is suggested. The membrane is modeled as a thin layer to allow for lateral diffusion and the cytosol is modeled as an enclosed volume. Two well-known polarization mechanisms were considered. One shows the classical Turing-instability patterns, the other exhibits wave-pinning dynamics. For both models, we investigated how cell shape and diffusion barriers like septin structures or bud scars influence the formation of signaling molecule clusters and subsequent polarization. An extensive set of in silico experiments with different modeling hypotheses illustrated the dependence of cell polarization models on local membrane curvature, cell size and inhomogeneities on the membrane and in the cytosol. In particular, the results of our computer simulations suggested that for both mechanisms, local diffusion barriers on the membrane facilitate Rho GTPase aggregation, while diffusion barriers in the cytosol and cell protrusions limit spontaneous molecule aggregations of active Rho GTPase locally.

Dynamic organization of myristoylated Src in the live cell plasma membrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Adam W.; Huang, Hector H.; Endres, Nicholas F.

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell–cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescencemore » cross-correlation spectroscopy (PIE–FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10–80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Altogether, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.« less

Dynamic organization of myristoylated Src in the live cell plasma membrane

DOE PAGES

Smith, Adam W.; Huang, Hector H.; Endres, Nicholas F.; ...

2016-01-15

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell–cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescencemore » cross-correlation spectroscopy (PIE–FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10–80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Altogether, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.« less

Impact of heat and water management on proton exchange membrane fuel cells degradation in automotive application

NASA Astrophysics Data System (ADS)

Nandjou, F.; Poirot-Crouvezier, J.-P.; Chandesris, M.; Blachot, J.-F.; Bonnaud, C.; Bultel, Y.

2016-09-01

In Proton Exchange Membrane Fuel Cells, local temperature is a driving force for many degradation mechanisms such as hygrothermal deformation and creep of the membrane, platinum dissolution and bipolar plates corrosion. In order to investigate and quantify those effects in automotive application, durability testing is conducted in this work. During the ageing tests, the local performance and temperature are investigated using in situ measurements of a printed circuit board. At the end of life, post-mortem analyses of the aged components are conducted. The experimental results are compared with the simulated temperature and humidity in the cell obtained from a pseudo-3D multiphysics model in order to correlate the observed degradations to the local conditions inside the stack. The primary cause of failure in automotive cycling is pinhole/crack formation in the membrane, induced by high variations of its water content over time. It is also observed that water condensation largely increases the probability of the bipolar plates corrosion while evaporation phenomena induce local deposits in the cell.

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells.

PubMed

Thomas, Audrey; Mariani-Floderer, Charlotte; López-Huertas, Maria Rosa; Gros, Nathalie; Hamard-Péron, Elise; Favard, Cyril; Ohlmann, Theophile; Alcamí, José; Muriaux, Delphine

2015-08-01

During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

PubMed Central

Thomas, Audrey; Mariani-Floderer, Charlotte; López-Huertas, Maria Rosa; Gros, Nathalie; Hamard-Péron, Elise; Favard, Cyril; Ohlmann, Theophile; Alcamí, José

2015-01-01

ABSTRACT During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. IMPORTANCE During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes. PMID:26018170

Cell membrane causes the lipid bilayers to behave as variable capacitors: A resonance with self-induction of helical proteins.

PubMed

Monajjemi, Majid

2015-12-01

Cell membrane has a unique feature of storing biological energies in a physiologically relevant environment. This study illustrates a capacitor model of biological cell membrane including DPPC structures. The electron density profile models, electron localization function (ELF) and local information entropy have been applied to study the interaction of proteins with lipid bilayers in the cell membrane. The quantum and coulomb blockade effects of different thicknesses in the membrane have also been specifically investigated. It has been exhibited the quantum effects can appear in a small region of the free space within the membrane thickness due to the number and type of phospholipid layers. In addition, from the viewpoint of quantum effects by Heisenberg rule, it is shown the quantum tunneling is allowed in some micro positions while it is forbidden in other forms of membrane capacitor systems. Due to the dynamical behavior of the cell membrane, its capacitance is not fixed which results a variable capacitor. In presence of the external fields through protein trance membrane or ions, charges exert forces that can influence the state of the cell membrane. This causes to appear the charge capacitive susceptibility that can resonate with self-induction of helical coils; the resonance of which is the main reason for various biological pulses. Copyright © 2015 Elsevier B.V. All rights reserved.

Simultaneous AFM topography and recognition imaging at the plasma membrane of mammalian cells.

PubMed

Chtcheglova, Lilia A; Hinterdorfer, Peter

2018-01-01

Elucidation the nano-organization of membrane proteins at/within the plasma membrane is probably the most demanding and still challenging task in cell biology since requires experimental approaches with nanoscale resolution. During last decade, atomic force microscopy (AFM)-based simultaneous topography and recognition imaging (TREC) has become a powerful tool to quickly obtain local receptor nano-maps on complex heterogeneous biosurfaces such as cells and membranes. Here we emphasize the TREC technique and explain how to unravel the nano-landscape of mammalian cells. We describe the procedures for all steps of the experiment including tip functionalization with ligand molecules, sample preparation, and localization of key molecules on the cell surface. We also discuss the current limitations and future perspectives of this technique. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Hyaluronan, CD44, and Emmprin Regulate Lactate Efflux and Membrane Localization of Monocarboxylate Transporters in Human Breast Carcinoma Cells

PubMed Central

Slomiany, Mark G.; Grass, G. Daniel; Robertson, Angela D.; Yang, Xiao Y.; Maria, Bernard L.; Beeson, Craig; Toole, Bryan P.

2013-01-01

Interactions of hyaluronan with CD44 in tumor cells play important cooperative roles in various aspects of malignancy and drug resistance. Emmprin (CD147; basigin)is a cell surface glycoprotein of the immunoglobulin superfamily that is highly up-regulated in malignant cancer cells and stimulates hyaluronan production, as well as several downstream signaling pathways. Emmprin also interacts with various monocarboxylate transporters (MCT). Malignant cancer cells use the glycolytic pathway and require MCTs to efflux lactate that results from glycolysis. Glycolysis and lactate secretion contribute to malignant cell behaviors and drug resistance in tumor cells. In the present study, we find that perturbation of endogenous hyaluronan, using small hyaluronan oligosaccharides, rapidly inhibits lactate efflux from breast carcinoma cells; down-regulation of emmprin, using emmprin small interfering RNA, also results in decreased efflux. In addition, we find that CD44 coimmunoprecipitates with MCT1, MCT4, and emmprin and colocalizes with these proteins at the plasma membrane. Moreover, after treatment of the cells with hyaluronan oligosaccharides, CD44, MCT1, and MCT4 become localized intracellularly whereas emmprin remains at the cell membrane. Together, these data indicate that constitutive interactions among hyaluronan, CD44, and emmprin contribute to regulation of MCT localization and function in the plasma membrane of breast carcinoma cells. PMID:19176383

WAVE2 Forms a Complex with PKA and Is Involved in PKA Enhancement of Membrane Protrusions*

PubMed Central

Yamashita, Hiroshi; Ueda, Kazumitsu; Kioka, Noriyuki

2011-01-01

PKA contributes to many physiological processes, including glucose homeostasis and cell migration. The substrate specificity of PKA is low compared with other kinases; thus, complex formation with A-kinase-anchoring proteins is important for the localization of PKA in specific subcellular regions and the phosphorylation of specific substrates. Here, we show that PKA forms a complex with WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) in MDA-MB-231 breast cancer cells and mouse brain extracts. Two separate regions of WAVE2 are involved in WAVE2-PKA complex formation. This complex localizes to the leading edge of MDA-MB-231 cells. PKA activation results in enlargement of the membrane protrusion. WAVE2 depletion impairs PKA localization at membrane protrusions and the enlargement of membrane protrusion induced by PKA activation. Together, these results suggest that WAVE2 works as an A-kinase-anchoring protein that recruits PKA at membrane protrusions and plays a role in the enlargement of membrane protrusions induced by PKA activation. PMID:21119216

Pinpoint Delivery of Molecules by Using Electron Beam Addressing Virtual Cathode Display.

PubMed

Hoshino, Takayuki; Yoshioka, Moto; Wagatsuma, Akira; Miyazako, Hiroki; Mabuchi, Kunihiko

2018-03-01

Electroporation, a physical transfection method to introduce genomic molecules in selective living cells, could be implemented by microelectrode devices. A local electric field generated by a finer electrode can induces cytomembrane poration in the electrode vicinity. To employ fine, high-speed scanning electrodes, we developed a fine virtual cathode pattern, which was generated on a cell adhesive surface of 100-nm-thick SiN membrane by inverted-electron beam lithography. The SiN membrane works as both a vacuum barrier and the display screen of the virtual cathode. The kinetic energy of the incident primary electrons to the SiN membrane was completely blocked, whereas negative charges and leaking electric current appeared on the surface of the dielectric SiN membrane within a region of 100 nm. Locally controlled transmembrane molecular delivery was demonstrated on adhered C2C12 myoblast cells in a culturing medium with fluorescent dye propidium iodide (PI). Increasing fluorescence of pre-diluted PI indicated local poration and transmembrane inflow at the virtual cathode position, as well as intracellular diffusion. The transmembrane inflows depended on beam duration time and acceleration voltage. At the post-molecular delivery, a slight decrease in intracellular PI fluorescence intensity indicates membrane recovery from the poration. Cell viability was confirmed by time-lapse cell imaging of post-exposure cell migration.

Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein.

PubMed

Oda, Yoshihisa; Iida, Yuki; Kondo, Yuki; Fukuda, Hiroo

2010-07-13

Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern. Copyright 2010 Elsevier Ltd. All rights reserved.

Transmembrane protein sorting driven by membrane curvature

NASA Astrophysics Data System (ADS)

Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

2015-11-01

The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

PubMed Central

Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

2000-01-01

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria. PMID:10816421

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

PubMed

Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

2000-06-01

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.

p27Kip1 localizes to detergent-insoluble microdomains within lymphocyte membranes.

PubMed Central

Yaroslavskiy, B. B.; Stolz, D. B.; Watkins, S. C.; Alber, S. M.; Bradbury, N. A.; Steinman, R. A.

2001-01-01

BACKGROUND: Low levels of the cyclin-dependent kinase inhibitor p27Kip1 are associated with poor prognosis in cancer. It is unclear whether this is related strictly to p27Kip1-mediated cell cycle inhibition or to other, possibly extranuclear, roles of this protein. In this study, we examined p27Kip1 expression in quiescent and activated lymphocytes. T-cell membranes have been shown to possess sphingolipid and cholesterol-rich microdomains that are insoluble in non-ionic detergents. These "rafts" provide a scaffold for signaling proteins. Signal transduction coincides with coalescence of these microdomains into larger complexes. METHODS: Localization of p27Kip1 was studied by electron and confocal microscopy. Association of p27Kip1 with membrane microdomains in unstimulated and stimulated lymphocytes was determined using Western blots analysis of isolated membranes variably treated with detergents. RESULTS: We demonstrated that p27Kip1 was present in clusters associated with the plasma membrane in normal lymphocytes. The solubility profile of p27Kip1 in isolated membranes indicated that it was localized to raft structures. When lymphocytes were stimulated, however, p27Kip1 was excluded from aggregated raft complexes. CONCLUSIONS: This study identifies, for the first time, the localization of p27 within a membrane microdomain associated with signaling. Because some cell surface signaling complexes lose p27Kip1 upon cellular activation, p27Kip1 may play a functional role in modulating membrane signaling. PMID:11474127

Differential Expression and Immunolocalization of Antioxidant Enzymes in Entamoeba histolytica Isolates during Metronidazole Stress

PubMed Central

Iyer, Lakshmi Rani; Singh, Nishant; Verma, Anil Kumar; Paul, Jaishree

2014-01-01

Entamoeba histolytica infections are endemic in the Indian subcontinent. Five to eight percent of urban population residing under poor sanitary conditions suffers from Entamoeba infections. Metronidazole is the most widely prescribed drug used for amoebiasis. In order to understand the impact of metronidazole stress on the parasite, we evaluated the expression of two antioxidant enzymes, peroxiredoxin and FeSOD, in Entamoeba histolytica isolates during metronidazole stress. The results reveal that, under metronidazole stress, the mRNA expression levels of these enzymes did not undergo any significant change. Interestingly, immunolocalization studies with antibodies targeting peroxiredoxin indicate differential localization of the protein in the cell during metronidazole stress. In normal conditions, all the Entamoeba isolates exhibit presence of peroxiredoxin in the nucleus as well as in the membrane; however with metronidazole stress the protein localized mostly to the membrane. The change in the localization pattern was more pronounced when the cells were subjected to short term metronidazole stress compared to cells adapted to metronidazole. The protein localization to the cell membrane could be the stress response mechanism in these isolates. Colocalization pattern of peroxiredoxin with CaBp1, a cytosolic protein, revealed that the membrane and nuclear localization was specific to peroxiredoxin during metronidazole stress. PMID:25013795

PTK6 activation at the membrane regulates epithelial-mesenchymal transition in prostate cancer.

PubMed

Zheng, Yu; Wang, Zebin; Bie, Wenjun; Brauer, Patrick M; Perez White, Bethany E; Li, Jing; Nogueira, Veronique; Raychaudhuri, Pradip; Hay, Nissim; Tonetti, Debra A; Macias, Virgilia; Kajdacsy-Balla, André; Tyner, Angela L

2013-09-01

The intracellular tyrosine kinase protein tyrosine kinase 6 (PTK6) lacks a membrane-targeting SH4 domain and localizes to the nuclei of normal prostate epithelial cells. However, PTK6 translocates from the nucleus to the cytoplasm in human prostate tumor cells. Here, we show that while PTK6 is located primarily within the cytoplasm, the pool of active PTK6 in prostate cancer cells localizes to membranes. Ectopic expression of membrane-targeted active PTK6 promoted epithelial-mesenchymal transition in part by enhancing activation of AKT, thereby stimulating cancer cell migration and metastases in xenograft models of prostate cancer. Conversely, siRNA-mediated silencing of endogenous PTK6 promoted an epithelial phenotype and impaired tumor xenograft growth. In mice, PTEN deficiency caused endogenous active PTK6 to localize at membranes in association with decreased E-cadherin expression. Active PTK6 was detected at membranes in some high-grade human prostate tumors, and PTK6 and E-cadherin expression levels were inversely correlated in human prostate cancers. In addition, high levels of PTK6 expression predicted poor prognosis in patients with prostate cancer. Our findings reveal novel functions for PTK6 in the pathophysiology of prostate cancer, and they define this kinase as a candidate therapeutic target. Cancer Res; 73(17); 5426-37. ©2013 AACR.

The Role of Cargo Proteins in GGA Recruitment

PubMed Central

Hirst, Jennifer; Seaman, Matthew N J; Buschow, Sonja I; Robinson, Margaret S

2007-01-01

Coat proteins are recruited onto membranes to form vesicles that transport cargo from one compartment to another, but the extent to which the cargo helps to recruit the coat proteins is still unclear. Here we have examined the role of cargo in the recruitment of Golgi-localized, γ-ear-containing, ADP ribosylation factor (ARF)-binding proteins (GGAs) onto membranes in HeLa cells. Moderate overexpression of CD8 chimeras with cytoplasmic tails containing DXXLL-sorting signals, which bind to GGAs, increased the localization of all three GGAs to perinuclear membranes, as observed by immunofluorescence. GGA2 was also expressed at approximately twofold higher levels in these cells because it was degraded more slowly. However, this difference only partially accounted for the increase in membrane localization because there was a approximately fivefold increase in GGA2 associated with crude membranes and a ∼12-fold increase in GGA2 associated with clathrin-coated vesicles (CCVs) in cells expressing CD8-DXXLL chimeras. The effect of cargo proteins on GGA recruitment was reconstituted in vitro using permeabilized control and CD8-DXXLL-expressing cells incubated with cytosol containing recombinant GGA2 constructs. Together, these results demonstrate that cargo proteins contribute to the recruitment of GGAs onto membranes and to the formation of GGA-positive CCVs. PMID:17451558

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

PubMed Central

Tholanikunnel, Baby G.; Joseph, Kusumam; Kandasamy, Karthikeyan; Baldys, Aleksander; Raymond, John R.; Luttrell, Louis M.; McDermott, Paul J.; Fernandes, Daniel J.

2010-01-01

β2-Adrenergic receptors (β2-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β2-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β2-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β2-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β2-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β2-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals. PMID:20739277

Myosin light chain kinase and Src control membrane dynamics in volume recovery from cell swelling

PubMed Central

Barfod, Elisabeth T.; Moore, Ann L.; Van de Graaf, Benjamin G.; Lidofsky, Steven D.

2011-01-01

 The expansion of the plasma membrane, which occurs during osmotic swelling of epithelia, must be retrieved for volume recovery, but the mechanisms are unknown. Here we have identified myosin light chain kinase (MLCK) as a regulator of membrane internalization in response to osmotic swelling in a model liver cell line. On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain. At the sides of the cell, MLCK and myosin II localized to swelling-induced membrane blebs with actin just before retraction, and MLCK inhibition led to persistent blebbing and attenuated cell volume recovery. At the base of the cell, MLCK also localized to dynamic actin-coated rings and patches upon swelling, which were associated with uptake of the membrane marker FM4-64X, consistent with sites of membrane internalization. Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures. Inhibition of either Src or MLCK led to altered patch and ring lifetimes, consistent with the concept that Src and MLCK form a swelling-induced protein complex that regulates volume recovery through membrane turnover and compensatory endocytosis under osmotic stress. PMID:21209319

Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells.

PubMed

Beckmann, Anja; Grissmer, Alexander; Krause, Elmar; Tschernig, Thomas; Meier, Carola

2016-03-01

Pannexins (Panx) are proteins with a similar membrane topology to connexins, the integral membrane protein of gap junctions. Panx1 channels are generally of major importance in a large number of system and cellular processes and their function has been thoroughly characterized. In contrast, little is known about channel structure and subcellular distribution. We therefore determine the subcellular localization of Panx1 channels in cultured cells and aim at the identification of channel morphology in vitro. Using freeze-fracture replica immunolabeling on EYFP-Panx1-overexpressing HEK 293 cells, large particles were identified in plasma membranes, which were immunogold-labeled using either GFP or Panx1 antibodies. There was no labeling or particles in the nuclear membranes of these cells, pointing to plasma membrane localization of Panx1-EYFP channels. The assembly of particles was irregular, this being in contrast to the regular pattern of gap junctions. The fact that no counterparts were identified on apposing cells, which would have been indicative of intercellular signaling, supported the idea of Panx1 channels within one membrane. Control cells (transfected with EYFP only, non-transfected) were devoid of both particles and immunogold labeling. Altogether, this study provides the first demonstration of Panx1 channel morphology and assembly in intact cells. The identification of Panx1 channels as large particles within the plasma membrane provides the knowledge required to enable recognition of Panx1 channels in tissues in future studies. Thus, these results open up new avenues for the detailed analysis of the subcellular localization of Panx1 and of its nearest neighbors such as purinergic receptors in vivo.

The basolateral vesicle sorting machinery and basolateral proteins are recruited to the site of enteropathogenic E. coli microcolony growth at the apical membrane.

PubMed

Pedersen, Gitte A; Jensen, Helene H; Schelde, Anne-Sofie B; Toft, Charlotte; Pedersen, Hans N; Ulrichsen, Maj; Login, Frédéric H; Amieva, Manuel R; Nejsum, Lene N

2017-01-01

Foodborne Enteropathogenic Escherichia coli (EPEC) infections of the small intestine cause diarrhea especially in children and are a major cause of childhood death in developing countries. EPEC infects the apical membrane of the epithelium of the small intestine by attaching, effacing the microvilli under the bacteria and then forming microcolonies on the cell surface. We first asked the question where on epithelial cells EPEC attaches and grows. Using models of polarized epithelial monolayers, we evaluated the sites of initial EPEC attachment to the apical membrane and found that EPEC preferentially attached over the cell-cell junctions and formed microcolonies preferentially where three cells come together at tricellular tight junctions. The ability of EPEC to adhere increased when host cell polarity was compromised yielding EPEC access to basolateral proteins. EPEC pedestals contain basolateral cytoskeletal proteins. Thus, we asked if attached EPEC causes reorganization the protein composition of the host cell plasma membrane at sites of microcolony formation. We found that EPEC microcolony growth at the apical membrane resulted in a local accumulation of basolateral plasma membrane proteins surrounding the microcolony. Basolateral marker protein aquaporin-3 localized to forming EPEC microcolonies. Components of the basolateral vesicle targeting machinery were re-routed. The Exocyst (Exo70) was recruited to individual EPEC as was the basolateral vesicle SNARE VAMP-3. Moreover, several Rab variants were also recruited to the infection site, and their dominant-negative equivalents were not. To quantitatively study the recruitment of basolateral proteins, we created a pulse of the temperature sensitive basolateral VSVG, VSVG3-SP-GFP, from the trans-Golgi Network. We found that after release from the TGN, significantly more VSVG3-SP-GFP accumulated at the site of microcolony growth than on equivalent membrane regions of uninfected cells. This suggests that trafficking of vesicles destined for the basolateral membrane are redirected to the apical site of microcolony growth. Thus, in addition to disrupting host cell fence function, local host cell plasma membrane protein composition is changed by altered protein trafficking and recruitment of basolateral proteins to the apical microcolony. This may aid EPEC attachment and subsequent microcolony growth.

The basolateral vesicle sorting machinery and basolateral proteins are recruited to the site of enteropathogenic E. coli microcolony growth at the apical membrane

PubMed Central

Pedersen, Gitte A.; Jensen, Helene H.; Schelde, Anne-Sofie B.; Toft, Charlotte; Pedersen, Hans N.; Ulrichsen, Maj; Login, Frédéric H.; Amieva, Manuel R.

2017-01-01

Foodborne Enteropathogenic Escherichia coli (EPEC) infections of the small intestine cause diarrhea especially in children and are a major cause of childhood death in developing countries. EPEC infects the apical membrane of the epithelium of the small intestine by attaching, effacing the microvilli under the bacteria and then forming microcolonies on the cell surface. We first asked the question where on epithelial cells EPEC attaches and grows. Using models of polarized epithelial monolayers, we evaluated the sites of initial EPEC attachment to the apical membrane and found that EPEC preferentially attached over the cell-cell junctions and formed microcolonies preferentially where three cells come together at tricellular tight junctions. The ability of EPEC to adhere increased when host cell polarity was compromised yielding EPEC access to basolateral proteins. EPEC pedestals contain basolateral cytoskeletal proteins. Thus, we asked if attached EPEC causes reorganization the protein composition of the host cell plasma membrane at sites of microcolony formation. We found that EPEC microcolony growth at the apical membrane resulted in a local accumulation of basolateral plasma membrane proteins surrounding the microcolony. Basolateral marker protein aquaporin-3 localized to forming EPEC microcolonies. Components of the basolateral vesicle targeting machinery were re-routed. The Exocyst (Exo70) was recruited to individual EPEC as was the basolateral vesicle SNARE VAMP-3. Moreover, several Rab variants were also recruited to the infection site, and their dominant-negative equivalents were not. To quantitatively study the recruitment of basolateral proteins, we created a pulse of the temperature sensitive basolateral VSVG, VSVG3-SP-GFP, from the trans-Golgi Network. We found that after release from the TGN, significantly more VSVG3-SP-GFP accumulated at the site of microcolony growth than on equivalent membrane regions of uninfected cells. This suggests that trafficking of vesicles destined for the basolateral membrane are redirected to the apical site of microcolony growth. Thus, in addition to disrupting host cell fence function, local host cell plasma membrane protein composition is changed by altered protein trafficking and recruitment of basolateral proteins to the apical microcolony. This may aid EPEC attachment and subsequent microcolony growth. PMID:28636623

Mitochondrial and Plasma Membrane Pools of Stomatin-Like Protein 2 Coalesce at the Immunological Synapse during T Cell Activation

PubMed Central

Christie, Darah A.; Kirchhof, Mark G.; Vardhana, Santosh; Dustin, Michael L.; Madrenas, Joaquín

2012-01-01

Stomatin-like protein 2 (SLP-2) is a member of the stomatin – prohibitin – flotillin – HflC/K (SPFH) superfamily. Recent evidence indicates that SLP-2 is involved in the organization of cardiolipin-enriched microdomains in mitochondrial membranes and the regulation of mitochondrial biogenesis and function. In T cells, this role translates into enhanced T cell activation. Although the major pool of SLP-2 is associated with mitochondria, we show here that there is an additional pool of SLP-2 associated with the plasma membrane of T cells. Both plasma membrane-associated and mitochondria-associated pools of SLP-2 coalesce at the immunological synapse (IS) upon T cell activation. SLP-2 is not required for formation of IS nor for the re-localization of mitochondria to the IS because SLP-2-deficient T cells showed normal re-localization of these organelles in response to T cell activation. Interestingly, upon T cell activation, we found the surface pool of SLP-2 mostly excluded from the central supramolecular activation complex, and enriched in the peripheral area of the IS where signalling TCR microclusters are located. Based on these results, we propose that SLP-2 facilitates the compartmentalization not only of mitochondrial membranes but also of the plasma membrane into functional microdomains. In this latter location, SLP-2 may facilitate the optimal assembly of TCR signalosome components. Our data also suggest that there may be a net exchange of membrane material between mitochondria and plasma membrane, explaining the presence of some mitochondrial proteins in the plasma membrane. PMID:22623988

At the border: the plasma membrane-cell wall continuum.

PubMed

Liu, Zengyu; Persson, Staffan; Sánchez-Rodríguez, Clara

2015-03-01

Plant cells rely on their cell walls for directed growth and environmental adaptation. Synthesis and remodelling of the cell walls are membrane-related processes. During cell growth and exposure to external stimuli, there is a constant exchange of lipids, proteins, and other cell wall components between the cytosol and the plasma membrane/apoplast. This exchange of material and the localization of cell wall proteins at certain spots in the plasma membrane seem to rely on a particular membrane composition. In addition, sensors at the plasma membrane detect changes in the cell wall architecture, and activate cytoplasmic signalling schemes and ultimately cell wall remodelling. The apoplastic polysaccharide matrix is, on the other hand, crucial for preventing proteins diffusing uncontrollably in the membrane. Therefore, the cell wall-plasma membrane link is essential for plant development and responses to external stimuli. This review focuses on the relationship between the cell wall and plasma membrane, and its importance for plant tissue organization. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The absence of p53 during Human Cytomegalovirus infection leads to decreased UL53 expression, disrupting UL50 localization to the inner nuclear membrane, and thereby inhibiting capsid nuclear egress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuan, Man I; O’Dowd, John M.; Fortunato, Elizabeth

Our electron microscopy study (Kuan et al., 2016) found HCMV nuclear capsid egress was significantly reduced in p53 knockout cells (p53KOs), correlating with inhibited formation of infoldings of the inner nuclear membrane (IINMs). Molecular examination of these phenomena has found p53KOs expressed UL97 and phosphorylated lamins, however the lamina failed to remodel. The nuclear egress complex (NEC) protein UL50 was expressed in almost all cells. UL50 re-localized to the inner nuclear membrane (INM) in ~90% of wt cells, but only ~35% of p53KOs. UL53 expression was significantly reduced in p53KOs, and cells lacking UL50 nuclear staining, expressed no UL53. Re-introductionmore » of p53 into p53KOs largely recovered UL53 positivity and UL50 nuclear re-localization. Nuclear rim located UL50/53 puncta, which co-localized with the major capsid protein, were largely absent in p53KOs. We believe these puncta were IINMs. In the absence of p53, UL53 expression was inhibited, disrupting formation of the NEC/IINMs, and reducing functional virion secretion. -- Highlights: •Phosphorylated nuclear lamins were inefficiently remodeled in p53KO cells. •p53KO cells expressed UL50, but it was not efficiently targeted to the nuclear rim. •UL53 was not expressed in the large majority of p53KO cells. •Cells failing to express UL53 did not localize UL50 to the nucleus. •NEC puncta/infoldings of the inner nuclear membrane were scarce in p53KO cells.« less

High-Resolution pH Imaging of Living Bacterial Cells To Detect Local pH Differences

PubMed Central

Morimoto, Yusuke V.; Kami-ike, Nobunori; Miyata, Tomoko; Kawamoto, Akihiro; Kato, Takayuki

2016-01-01

ABSTRACT Protons are utilized for various biological activities such as energy transduction and cell signaling. For construction of the bacterial flagellum, a type III export apparatus utilizes ATP and proton motive force to drive flagellar protein export, but the energy transduction mechanism remains unclear. Here, we have developed a high-resolution pH imaging system to measure local pH differences within living Salmonella enterica cells, especially in close proximity to the cytoplasmic membrane and the export apparatus. The local pH near the membrane was ca. 0.2 pH unit higher than the bulk cytoplasmic pH. However, the local pH near the export apparatus was ca. 0.1 pH unit lower than that near the membrane. This drop of local pH depended on the activities of both transmembrane export components and FliI ATPase. We propose that the export apparatus acts as an H+/protein antiporter to couple ATP hydrolysis with H+ flow to drive protein export. PMID:27923921

The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin

PubMed Central

Dyson, Jennifer M.; O'Malley, Cindy J.; Becanovic, Jelena; Munday, Adam D.; Berndt, Michael C.; Coghill, Imogen D.; Nandurkar, Harshal H.; Ooms, Lisa M.; Mitchell, Christina A.

2001-01-01

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline–rich domain. To identify proteins that bind to the SHIP-2 proline–rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton. PMID:11739414

Diffusion mediated localization on membrane surfaces

NASA Technical Reports Server (NTRS)

Weaver, D. L.

1982-01-01

Using the model of a cell membrane of a spherical surface in which membrane components may diffuse, the rate of localization due to trapping under diffusion control has been estimated by computing an analytical expression for the mean trapping time including the possibilities of a trapping probability less than one and/or the establishment of an equilibrium at the trap boundary.

The Sur7 Protein Regulates Plasma Membrane Organization and Prevents Intracellular Cell Wall Growth in Candida albicans

PubMed Central

Alvarez, Francisco J.; Douglas, Lois M.; Rosebrock, Adam

2008-01-01

The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Δ mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Δ mutant are similar to the effects of inhibiting β-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains. PMID:18799621

An ESIPT fluorescent probe sensitive to protein α-helix structures.

PubMed

Jiang, Nan; Yang, Chanli; Dong, Xiongwei; Sun, Xianglang; Zhang, Dan; Liu, Changlin

2014-07-28

A large majority of membrane proteins have one or more transmembrane regions consisting of α-helices. Membrane protein levels differ from one type of cell to another, and the expression of membrane proteins also changes from normal to diseased cells. For example, prostate cancer cells have been reported to have downregulated expression of membrane proteins, including zinc transporters, compared with normal prostate cells. These reports inspired us to design a fluorescence probe sensitive to protein α-helical structures to discriminate individual prostate cancer cells from normal ones. A benzazole derivative ( in this study) was observed to emit strong fluorescence resulting from an excited-state intramolecular proton transfer (ESIPT) in protein α-helical environments. The intensity of ESIPT fluorescence of was observed to be positively correlated with the α-helix content of proteins. The molecular docking simulation suggested that it had low energy for the binding of to proteins when the binding sites were localized within the α-helical regions of protein via H-bonds. Furthermore, was found to be localized in cell membranes through binding to transmembrane α-helical regions of membrane proteins, and was capable of probing differences in the α-helix contents of membrane proteins between normal and cancerous prostate cells through changes in the ESIPT emission intensity. These results indicated that could distinguish individual prostate cancer cells from normal ones, as the changes in the ESIPT fluorescence intensity of could reflect the regulation in expression of the membrane proteins including zinc transporters. This recognition strategy of individual prostate cancer cells might contribute to early diagnosis techniques for prostate cancer.

Cytotopographical specialization of enzymatically isolated rabbit retinal Müller (glial) cells: K+ conductivity of the cell membrane.

PubMed

Reichenbach, A; Eberhardt, W

1988-01-01

Müller (radial glial) cells were isolated from rabbit retinae by means of papaine and mechanical dissociation. Regional membrane properties of these cells were studied by intracellular microelectrode recordings of potential responses to local application of high K+ solutions. When different parts of the cell membrane were exposed to high K+, the amplitude of the depolarizing responses varied greatly, indicating a strong regional specialization of the membrane properties. Using morphometrical data of isolated rabbit Müller cells, and a simple circuit model, we calculated the endfoot membrane to constitute more than 80% of the total K+ conductance of the cell; the specific resistivity of the endfoot membrane was about 400 omega cm2, i.e., more than 40 times less than that of the membrane of the vitread process, which is immediately adjacent. This kind of regional membrane specialization seems to be optimized in respect to the Müller cells' ability to carry spatial buffering K+ currents.

Tula hantavirus L protein is a 250 kDa perinuclear membrane-associated protein.

PubMed

Kukkonen, Sami K J; Vaheri, Antti; Plyusnin, Alexander

2004-05-01

The complete open reading frame of Tula hantavirus (TULV) L RNA was cloned in three parts. The middle third (nt 2191-4344) could be expressed in E. coli and was used to immunize rabbits. The resultant antiserum was then used to immunoblot concentrated TULV and infected Vero E6 cells. The L protein of a hantavirus was detected, for the first time, in infected cells and was found to be expressed as a single protein with an apparent molecular mass of 250 kDa in both virions and infected cells. Using the antiserum, the expression level of the L protein was followed and image analysis of immunoblots indicated that there were 10(4) copies per cell at the peak level of expression. The antiserum was also used to detect the L protein in cell fractionation studies. In cells infected with TULV and cells expressing recombinant L, the protein pelleted with the microsomal membrane fraction. The membrane association was confirmed with membrane flotation assays. To visualize L protein localization in cells, a fusion protein of L and enhanced green fluorescent protein, L-EGFP, was expressed in Vero E6 cells with a plasmid-driven T7 expression system. L-EGFP localized in the perinuclear region where it had partial co-localization with the Golgi matrix protein GM130 and the TULV nucleocapsid protein.

THE LOCALIZATION OF ENZYME ACTIVITIES IN THE RAT BRAIN

PubMed Central

Becker, Norwin H.; Goldfischer, Sidney; Shin, Woo-Yung; Novikoff, Alex B.

1960-01-01

Studies with rat brain illustrate the usefulness of formol-calcium-fixed tissue for studying both enzymatic "chemoarchitectonics" and intracellular organelles. Unembedded frozen sections and polyvinyl alcohol-embedded sections may be used to demonstrate the activities of DPNH-tetrazolium reductase localized in mitochondria and ergastoplasm, TPNH-tetrazolium reductase localized in mitochondria, ATPase (and/or apyrase or ADPase) in cell membranes, and acid phosphatase in lysosomes.1 Among the observations recorded are: (1) the presence of lysosomes in all cells of the brain; (2) the presence of numerous large lysosomes near the nuclei of capillary endothelial cells; (3) a polarized arrangement of large lysosomes in epithelial cells of the ependyma and choroid plexus; (4) the presence of ATPase activity in the cell membranes of some neurons; (5) the presence of either an apyrase or combination of ATPase and ADPase in the cell membranes of neuroglia and capillaries; (6) the presence of both DPNH- and TPNH-tetrazolium reductase activities in neuroglia; (7) the presence of DPNH- and TPNH-tetrazolium reductase activities in mitochondria and of DPNH-tetrazolium reductase activity in Nissl substance. The possible functional significance of these localizations is briefly discussed, as is their relation to "quantitative histochemistry" data available in the literature. PMID:13688468

The localization of enzyme activities in the rat brain.

PubMed

BECKER, N H; GOLDFISCHER, S; SHIN, W Y; NOVIKOFF, A B

1960-12-01

Studies with rat brain illustrate the usefulness of formol-calcium-fixed tissue for studying both enzymatic "chemoarchitectonics" and intracellular organelles. Unembedded frozen sections and polyvinyl alcohol-embedded sections may be used to demonstrate the activities of DPNH-tetrazolium reductase localized in mitochondria and ergastoplasm, TPNH-tetrazolium reductase localized in mitochondria, ATPase (and/or apyrase or ADPase) in cell membranes, and acid phosphatase in lysosomes.(1) Among the observations recorded are: (1) the presence of lysosomes in all cells of the brain; (2) the presence of numerous large lysosomes near the nuclei of capillary endothelial cells; (3) a polarized arrangement of large lysosomes in epithelial cells of the ependyma and choroid plexus; (4) the presence of ATPase activity in the cell membranes of some neurons; (5) the presence of either an apyrase or combination of ATPase and ADPase in the cell membranes of neuroglia and capillaries; (6) the presence of both DPNH- and TPNH-tetrazolium reductase activities in neuroglia; (7) the presence of DPNH- and TPNH-tetrazolium reductase activities in mitochondria and of DPNH-tetrazolium reductase activity in Nissl substance. The possible functional significance of these localizations is briefly discussed, as is their relation to "quantitative histochemistry" data available in the literature.

Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane.

PubMed

Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

2018-02-28

Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca 2+ -dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca 2+ -binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae , is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly. IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ∼ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of MV within the cell. Further studies demonstrated that annexin A2 interacts with the MV matrix (M) protein and mediates the localization of the M protein at the plasma membrane where MV particles are formed. The M protein lines the inner surface of the MV envelope membrane and plays a role in MV particle formation. Our results provide useful information for the understanding of the MV replication process and potential development of anti-viral agents. Copyright © 2018 American Society for Microbiology.

Immunohistochemical localization of cannabinoid receptor 1 (CB1) in the submandibular gland of mice under normal conditions and when stimulated by isoproterenol or carbachol.

PubMed

Thoungseabyoun, Wipawee; Tachow, Apussara; Pakkarato, Sawetree; Rawangwong, Atsara; Krongyut, Suthankamon; Sakaew, Waraporn; Kondo, Hisatake; Hipkaeo, Wiphawi

2017-09-01

We wished to investigate the subcellular localization of CB1, a receptor for the endocannabinoids in mouse submandibular glands (SMGs) under normal conditions and when stimulated by adrenergic or cholinergic agonists. SMGs of both male and female adult mice were utilized for immunoblotting and immuno-light and -electron microscopic analyses. Isoproterenol and carbachol were used as adrenergic and cholinergic stimulants, respectively. SMGs were examined at 15, 30, 60 and 120min after intraperitoneal injection of these agents. Selective localization of intense immunoreactivity for CB1 in the granular convoluted ductal cells was confirmed by immunoblotting and the antigen absorption test. In SMGs of control male mice, CB1-immunoreactivity was evident on the basolateral plasma membranes, including the basal infoldings, but was absent on the apical membranes in the ductal cells. Localization and intensity of CB1-immunoreactivity were essentially the same in SMGs of female mice. The immunoreactivity was transiently localized in the apical plasmalemma of some acinar and granular ductal cells of male SMGs shortly after stimulation by isoproterenol, but not by carbachol. The present finding suggests that CB1 functions primarily in the basolateral membranes of the granular convoluted ductal cells of SMGs under normal conditions, and that the CB1 can function additionally in the apical membrane of acinar and granular ductal cells for modulation of the saliva secretory condition via adrenoceptors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Prm3p is a pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion.

PubMed

Shen, Shu; Tobery, Cynthia E; Rose, Mark D

2009-05-01

Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.

Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.

PubMed Central

Teissié, J; Ramos, C

1998-01-01

Electric field pulses have been reported to induce long-lived permeabilization and fusogenicity on cell membranes. The two membrane property alterations are under the control of the field strength, the pulse duration, and the number of pulses. Experiments on mammalian cells pulsed by square wave form pulses and then brought into contact randomly through centrifugation revealed an even stronger analogy between the two processes. Permeabilization was known to affect well-defined regions of the cell surface. Fusion can be obtained only when permeabilized surfaces on the two partners were brought into contact. Permeabilization was under the control of the pulse duration and of the number of pulses. A similar relationship was observed as far as fusion is concerned. But a critical level of local permeabilization must be present for fusion to take place when contacts are created. The same conclusions are obtained from previous experiments on ghosts subjected to exponentially decaying field pulses and then brought into contact by dielectrophoresis. These observations are in agreement with a model of membrane fusion in which the merging of local random defects occurs when the two membranes are brought into contact. The local defects are considered part of the structural membrane reorganization induced by the external field. Their density is dependent on the pulse duration and number of pulses. They support the long-lived permeabilization. Their number must be very large to support the occurrence of membrane fusion. PMID:9545050

Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.

PubMed

Teissié, J; Ramos, C

1998-04-01

Electric field pulses have been reported to induce long-lived permeabilization and fusogenicity on cell membranes. The two membrane property alterations are under the control of the field strength, the pulse duration, and the number of pulses. Experiments on mammalian cells pulsed by square wave form pulses and then brought into contact randomly through centrifugation revealed an even stronger analogy between the two processes. Permeabilization was known to affect well-defined regions of the cell surface. Fusion can be obtained only when permeabilized surfaces on the two partners were brought into contact. Permeabilization was under the control of the pulse duration and of the number of pulses. A similar relationship was observed as far as fusion is concerned. But a critical level of local permeabilization must be present for fusion to take place when contacts are created. The same conclusions are obtained from previous experiments on ghosts subjected to exponentially decaying field pulses and then brought into contact by dielectrophoresis. These observations are in agreement with a model of membrane fusion in which the merging of local random defects occurs when the two membranes are brought into contact. The local defects are considered part of the structural membrane reorganization induced by the external field. Their density is dependent on the pulse duration and number of pulses. They support the long-lived permeabilization. Their number must be very large to support the occurrence of membrane fusion.

Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase.

PubMed

Haruta, Miyoshi; Tan, Li Xuan; Bushey, Daniel B; Swanson, Sarah J; Sussman, Michael R

2018-01-01

A P-type H + -ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis ( Arabidopsis thaliana ) plant expressing H + -ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H + secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H + -ATPase. © 2018 American Society of Plant Biologists. All Rights Reserved.

Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase1[OPEN

PubMed Central

Tan, Li Xuan; Bushey, Daniel B.; Swanson, Sarah J.

2018-01-01

A P-type H+-ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis (Arabidopsis thaliana) plant expressing H+-ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H+ secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H+-ATPase. PMID:29042459

Morphological study on permeating efficiency and localization of FCLA and HpD through membrane of lung cancer cell

NASA Astrophysics Data System (ADS)

Wu, Yunxia; Xing, Da; Tang, Yonghong

2004-07-01

It is reported that apoptosis of cancer cells in photodynamic therapy (PDT) is caused by 1O2 generated in photosensitization. In order to study the mechanism of this kind of 1O2-induced apoptosis, it is necessary to establish a special technique to dynamically detect intracellular production and localization of 1O2. FCLA, as a chemiluminescence probe to detect singlet oxygen (1O2) and superoxide (O2-.), has been used successfully in photodynamic and sonodynamic diagnosis in tissue level, recently. This paper reported a preliminary result of morphological study on permeating efficiency and localization of FCLA and hematoporphyrin derivative (HpD) through cellular membrane. Human lung cancer cell line (ASTC-a-1) was used in the experiment. The result of this research showed that both HpD and FCLA could permeate through cellular membrane and localize to prinuclear area, when HpD or FCLA was incubated with cells. Although the molecular weight of HpD is close to FCLA's, the permeating efficiency of HpD through membrane was different from that of FCLA. Intracellular FCLA concentration reached a peak after incubation for only 30 - 45 minutes, but amount of HpD in cells approached the equilibrium after incubation for near 22 h. In the experiment, we did not observe the evidence of FCLA or HpD penetrating into nucleolus. This study suggests that it is possibly to use a specific chemiluminescence probe to dynamcially detect the production and localization of 1O2 or 02-. in cell.

Association of p60c-src with endosomal membranes in mammalian fibroblasts

PubMed Central

1992-01-01

We have examined the subcellular localization of p60c-src in mammalian fibroblasts. Analysis of indirect immunofluorescence by three- dimensional optical sectioning microscopy revealed a granular cytoplasmic staining that co-localized with the microtubule organizing center. Immunofluorescence experiments with antibodies against a number of membrane markers demonstrated a striking co-localization between p60c-src and the cation-dependent mannose-6-phosphate receptor (CI- MPR), a marker that identifies endosomes. Both p60c-src and the CI-MPR were found to cluster at the spindle poles throughout mitosis. In addition, treatment of interphase and mitotic cells with brefeldin A resulted in a clustering of p60c-src and CI-MPR at a peri-centriolar position. Biochemical fractionation of cellular membranes showed that a major proportion of p60c-src co-enriched with endocytic membranes. Treatment of membranes containing HRP to alter their apparent density also altered the density of p60c-src-containing membranes. Similar density shift experiments with total cellular membranes revealed that the majority of membrane-associated p60c-src in the cell is associated with endosomes, while very little is associated with plasma membranes. These results support a role for p60c-src in the regulation of endosomal membranes and protein trafficking. PMID:1378446

Damage of Escherichia coli membrane by bactericidal agent polyhexamethylene guanidine hydrochloride: micrographic evidences.

PubMed

Zhou, Z X; Wei, D F; Guan, Y; Zheng, A N; Zhong, J J

2010-03-01

The purpose of this study was to provide micrographic evidences for the damaged membrane structure and intracellular structure change of Escherichia coli strain 8099, induced by polyhexamethylene guanidine hydrochloride (PHMG). The bactericidal effect of PHMG on E. coli was investigated based on beta-galactosidase activity assay, fluorescein-5-isothiocyanate confocal laser scanning microscopy, field emission scanning electron microscopy and transmission electron microscopy. The results revealed that a low dose (13 microg ml(-1)) of PHMG slightly damaged the outer membrane structure of the treated bacteria and increased the permeability of the cytoplasmic membrane, while no significant damage was observed to the morphological structure of the cells. A high dose (23 microg ml(-1)) of PHMG collapsed the outer membrane structure, led to the formation of a local membrane pore across the membrane and badly damaged the internal structure of the cells. Subsequently, intracellular components were leaked followed by cell inactivation. Dose-dependent membrane disruption was the main bactericidal mechanism of PHMG. The formation of the local membrane pores was probable after exposure to a high dose (23 microg ml(-1)) of PHMG. Micrographic evidences were provided about the damaged membrane structure and intracellular structure change of E. coli. The presented information helps understand the bactericidal mechanism of PHMG by membrane damage.

Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

PubMed Central

van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K.; Roos, Dirk; Otto, Cees

2008-01-01

We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91phox are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91phox. By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91phox are ∼1.38 and ∼1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane. PMID:18223002

The anti-cancer peptide, PNC-27, induces tumor cell necrosis of a poorly differentiated non-solid tissue human leukemia cell line that depends on expression of HDM-2 in the plasma membrane of these cells.

PubMed

Davitt, Katlin; Babcock, Blake D; Fenelus, Maly; Poon, Chi Kong; Sarkar, Abhishek; Trivigno, Vincent; Zolkind, Paul A; Matthew, Sheena M; Grin'kina, Natalia; Orynbayeva, Zulfiya; Shaikh, Mohammad F; Adler, Victor; Michl, Josef; Sarafraz-Yazdi, Ehsan; Pincus, Matthew R; Bowne, Wilbur B

2014-01-01

We have developed the anti-cancer peptide, PNC-27, which is a membrane-active peptide that binds to the HDM-2 protein expressed in the cancer cell membranes of solid tissue tumor cells and induces transmembrane pore formation in cancer, but not in normal cells, resulting in tumor cell necrosis that is independent of p53 activity in these cells. We now extend our study to non-solid tissue tumor cells, in this case, a primitive, possible stem cell human leukemia cell line (K562) that is also p53-homozygously deleted. Our purpose was twofold: to investigate if these cells likewise express HDM-2 in their plasma membranes and to determine if our anti-cancer peptide induces tumor cell necrosis in these non-solid tissue tumor cells in a manner that depends on the interaction between the peptide and membrane-bound HDM-2. The anti-cancer activity and mechanism of PNC-27, which carries a p53 aa12-26-leader sequence connected on its carboxyl terminal end to a trans-membrane-penetrating sequence or membrane residency peptide (MRP), was studied against p53-null K562 leukemia cells. Murine leukocytes were used as a non-cancer cell control. Necrosis was determined by measuring the lactate dehydrogenase (LDH) release and apoptosis was determined by the detection of Caspases 3 and 7. Membrane colocalization of PNC-27 with HDM-2 was analyzed microscopically using fluorescently labeled antibodies against HDM-2 and PNC-27 peptides. We found that K562 cells strongly express HDM-2 protein in their membranes and that PNC-27 co-localizes with this protein in the membranes of these cells. PNC-27, but not the negative control peptide PNC-29, is selectively cytotoxic to K562 cells, inducing nearly 100 percent cell killing with LDH release. In contrast, this peptide had no effect on the lymphocyte control cells. The results suggest that HDM-2 is expressed in the membranes of non-solid tissue tumor cells in addition to the membranes of solid tissue tumor cells. Since K-562 cells appear to be in the stem cell family, the results suggest that early developing tumor cells also express HDM-2 protein in their membranes. Since PNC-27 induces necrosis of K-562 leukemia cells and co-localizes with HDM-2 in the tumor cell membrane as an early event, we conclude that the association of PNC-27 with HDM-2 in the cancer cell membrane results in trans-membrane pore formation which results in cancer cell death, as previously discovered in a number of different solid tissue tumor cells. Since K562 cells lack p53 expression, these effects of PNC-27 on this leukemia cell line occur by a p53-independent pathway. © 2014 by the Association of Clinical Scientists, Inc.

Cyclophilin A is a new M cell marker of bovine intestinal epithelium.

PubMed

Hondo, Tetsuya; Someya, Shunsuke; Nagasawa, Yuya; Terada, Shunsuke; Watanabe, Hitoshi; Chen, Xiangning; Watanabe, Kouichi; Ohwada, Shyuichi; Kitazawa, Haruki; Rose, Michael T; Nochi, Tomonori; Aso, Hisashi

2016-06-01

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches contribute to the mucosal immune response by the transcytosis of microorganisms. The mechanism by which M cells take up microorganisms, and the functional proteins by which they do this, are not clear. In order to explore one such protein, we developed a 2H5-F3 monoclonal antibody (2H5-F3 mAb) through its binding to bovine M cells, and identified the antibody reactive molecule as cyclophilin A (Cyp-A). The localization patterns of Cyp-A were very similar to the localization pattern of cytokeratin (CK) 18-positive M cells. Cyp-A was identified at the luminal surface of CK18-positive M cells in bovine jejunal and ileal FAE. The membranous localization of Cyp-A in the bovine intestinal cell line (BIE cells) increased as cells differentiated toward M cells, as determined by flow cytometry analysis. Additionally, BIE cells released Cyp-A to the extracellular space and the differentiation of BIE cells to M cells increased the secretion of Cyp-A, as determined by western blotting. Accordingly, Cyp-A may be localized in M cells in the small intestinal epithelium of cattle. The rise of the membranous localization and secretion of Cyp-A by differentiation toward M cells indicates that Cyp-A has an important role in the function of M cells. While Cyp-A of the M cell membrane may contribute to the uptake of viruses with peptidyl-prolyl cis-trans isomerase activity, in the extracellular space Cyp-A may work as a chemokine and contribute to the distribution of immuno-competent cells.

Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using /sup 125/I-alpha scorpion toxin. I. Preferential labeling of glial cells on the presynaptic side

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boudier, J.L.; Jover, E.; Cau, P.

1988-05-01

Alpha-scorpion toxins bind specifically to the voltage-sensitive sodium channel in excitable membranes, and binding is potential-dependent. The radioiodinated toxin II from the scorpion Androctonus australis Hector (alpha ScTx) was used to localize voltage-sensitive sodium channels on the presynaptic side of mouse neuromuscular junctions (NMJ) by autoradiography using both light and electron microscopy. Silver grain localization was analyzed by the cross-fire method. At the light-microscopic level, grain density over NMJ appeared 6-8x higher than over nonjunctional muscle membrane. The specificity of labeling was verified by competition/displacement with an excess of native alpha ScTx. Labeling was also inhibited by incubation in depolarizingmore » conditions, showing its potential-dependence. At the electron-microscopic level, analysis showed that voltage-sensitive sodium channels labeled with alpha ScTx were almost exclusively localized on membranes, as expected. Due to washout after incubation, appreciable numbers of binding sites were not found on the postsynaptic membranes. However, on the presynaptic side, alpha ScTx-labeled voltage-sensitive sodium channels were localized on the membrane of non-myelin-forming Schwann cells covering NMJ. The axonal presynaptic membrane was not labeled. These results show that voltage-sensitive sodium channels are present on glial cells in vivo, as already demonstrated in vitro. It is proposed that these glial channels could be indirectly involved in the ionic homeostasis of the axonal environment.« less

Front-to-rear membrane tension gradient in rapidly moving cells.

PubMed

Lieber, Arnon D; Schweitzer, Yonatan; Kozlov, Michael M; Keren, Kinneret

2015-04-07

Membrane tension is becoming recognized as an important mechanical regulator of motile cell behavior. Although membrane-tension measurements have been performed in various cell types, the tension distribution along the plasma membrane of motile cells has been largely unexplored. Here, we present an experimental study of the distribution of tension in the plasma membrane of rapidly moving fish epithelial keratocytes. We find that during steady movement the apparent membrane tension is ∼30% higher at the leading edge than at the trailing edge. Similar tension differences between the front and the rear of the cell are found in keratocyte fragments that lack a cell body. This front-to-rear tension variation likely reflects a tension gradient developed in the plasma membrane along the direction of movement due to viscous friction between the membrane and the cytoskeleton-attached protein anchors embedded in the membrane matrix. Theoretical modeling allows us to estimate the area density of these membrane anchors. Overall, our results indicate that even though membrane tension equilibrates rapidly and mechanically couples local boundary dynamics over cellular scales, steady-state variations in tension can exist in the plasma membranes of moving cells. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Local Area Water Removal Analysis of a Proton Exchange Membrane Fuel Cell under Gas Purge Conditions

PubMed Central

Lee, Chi-Yuan; Lee, Yu-Ming; Lee, Shuo-Jen

2012-01-01

In this study, local area water content distribution under various gas purging conditions are experimentally analyzed for the first time. The local high frequency resistance (HFR) is measured using novel micro sensors. The results reveal that the liquid water removal rate in a membrane electrode assembly (MEA) is non-uniform. In the under-the-channel area, the removal of liquid water is governed by both convective and diffusive flux of the through-plane drying. Thus, almost all of the liquid water is removed within 30 s of purging with gas. However, liquid water that is stored in the under-the-rib area is not easy to remove during 1 min of gas purging. Therefore, the re-hydration of the membrane by internal diffusive flux is faster than that in the under-the-channel area. Consequently, local fuel starvation and membrane degradation can degrade the performance of a fuel cell that is started from cold. PMID:22368495

Local area water removal analysis of a proton exchange membrane fuel cell under gas purge conditions.

PubMed

Lee, Chi-Yuan; Lee, Yu-Ming; Lee, Shuo-Jen

2012-01-01

In this study, local area water content distribution under various gas purging conditions are experimentally analyzed for the first time. The local high frequency resistance (HFR) is measured using novel micro sensors. The results reveal that the liquid water removal rate in a membrane electrode assembly (MEA) is non-uniform. In the under-the-channel area, the removal of liquid water is governed by both convective and diffusive flux of the through-plane drying. Thus, almost all of the liquid water is removed within 30 s of purging with gas. However, liquid water that is stored in the under-the-rib area is not easy to remove during 1 min of gas purging. Therefore, the re-hydration of the membrane by internal diffusive flux is faster than that in the under-the-channel area. Consequently, local fuel starvation and membrane degradation can degrade the performance of a fuel cell that is started from cold.

Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling

PubMed Central

Jenny, Andreas; Darken, Rachel S.; Wilson, Paul A.; Mlodzik, Marek

2003-01-01

Frizzled (Fz) signaling regulates the establishment of planar cell polarity (PCP). The PCP genes prickle (pk) and strabismus (stbm) are thought to antagonize Fz signaling. We show that they act in the same cell, R4, adjacent to that in which the Fz/PCP pathway is required in the Drosophila eye. We demonstrate that Stbm and Pk interact physically and that Stbm recruits Pk to the cell membrane. Through this interaction, Pk affects Stbm membrane localization and can cause clustering of Stbm. Pk is also known to interact with Dsh and is thought to antagonize Dsh by affecting its membrane localization. Thus our data suggest that the Stbm/Pk complex modulates Fz/Dsh activity, resulting in a symmetry-breaking step during polarity signaling. PMID:12941693

Interactions between lipids and proteins are critical for organization of plasma membrane-ordered domains in tobacco BY-2 cells.

PubMed

Grosjean, Kevin; Der, Christophe; Robert, Franck; Thomas, Dominique; Mongrand, Sébastien; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

2018-06-27

The laterally heterogeneous plant plasma membrane (PM) is organized into finely controlled specialized areas that include membrane-ordered domains. Recently, the spatial distribution of such domains within the PM has been identified as playing a key role in cell responses to environmental challenges. To examine membrane order at a local level, BY-2 tobacco suspension cell PMs were labelled with an environment-sensitive probe (di-4-ANEPPDHQ). Four experimental models were compared to identify mechanisms and cell components involved in short-term (1 h) maintenance of the ordered domain organization in steady-state cell PMs: modulation of the cytoskeleton or the cell wall integrity of tobacco BY-2 cells; and formation of giant vesicles using either a lipid mixture of tobacco BY-2 cell PMs or the original lipid and protein combinations of the tobacco BY-2 cell PM. Whilst inhibiting phosphorylation or disrupting either the cytoskeleton or the cell wall had no observable effects, we found that lipids and proteins significantly modified both the abundance and spatial distribution of ordered domains. This indicates the involvement of intrinsic membrane components in the local physical state of the plant PM. Our findings support a major role for the 'lipid raft' model, defined as the sterol-dependent ordered assemblies of specific lipids and proteins in plant PM organization.

A New Membrane Protein Sbg1 Links the Contractile Ring Apparatus and Septum Synthesis Machinery in Fission Yeast

PubMed Central

Sethi, Kriti; Palani, Saravanan; Cortés, Juan C. G.; Sato, Mamiko; Sevugan, Mayalagu; Ramos, Mariona; Vijaykumar, Shruthi; Osumi, Masako; Naqvi, Naweed I.; Ribas, Juan Carlos; Balasubramanian, Mohan

2016-01-01

Cytokinesis in many organisms requires a plasma membrane anchored actomyosin ring, whose contraction facilitates cell division. In yeast and fungi, actomyosin ring constriction is also coordinated with division septum assembly. How the actomyosin ring interacts with the plasma membrane and the plasma membrane-localized septum synthesizing machinery remains poorly understood. In Schizosaccharomyces pombe, an attractive model organism to study cytokinesis, the β-1,3-glucan synthase Cps1p / Bgs1p, an integral membrane protein, localizes to the plasma membrane overlying the actomyosin ring and is required for primary septum synthesis. Through a high-dosage suppressor screen we identified an essential gene, sbg1+ (suppressor of beta glucan synthase 1), which suppressed the colony formation defect of Bgs1-defective cps1-191 mutant at higher temperatures. Sbg1p, an integral membrane protein, localizes to the cell ends and to the division site. Sbg1p and Bgs1p physically interact and are dependent on each other to localize to the division site. Loss of Sbg1p results in an unstable actomyosin ring that unravels and slides, leading to an inability to deposit a single contiguous division septum and an important reduction of the β-1,3-glucan proportion in the cell wall, coincident with that observed in the cps1-191 mutant. Sbg1p shows genetic and / or physical interaction with Rga7p, Imp2p, Cdc15p, and Pxl1p, proteins known to be required for actomyosin ring integrity and efficient septum synthesis. This study establishes Sbg1p as a key member of a group of proteins that link the plasma membrane, the actomyosin ring, and the division septum assembly machinery in fission yeast. PMID:27749909

A New Membrane Protein Sbg1 Links the Contractile Ring Apparatus and Septum Synthesis Machinery in Fission Yeast.

PubMed

Sethi, Kriti; Palani, Saravanan; Cortés, Juan C G; Sato, Mamiko; Sevugan, Mayalagu; Ramos, Mariona; Vijaykumar, Shruthi; Osumi, Masako; Naqvi, Naweed I; Ribas, Juan Carlos; Balasubramanian, Mohan

2016-10-01

Cytokinesis in many organisms requires a plasma membrane anchored actomyosin ring, whose contraction facilitates cell division. In yeast and fungi, actomyosin ring constriction is also coordinated with division septum assembly. How the actomyosin ring interacts with the plasma membrane and the plasma membrane-localized septum synthesizing machinery remains poorly understood. In Schizosaccharomyces pombe, an attractive model organism to study cytokinesis, the β-1,3-glucan synthase Cps1p / Bgs1p, an integral membrane protein, localizes to the plasma membrane overlying the actomyosin ring and is required for primary septum synthesis. Through a high-dosage suppressor screen we identified an essential gene, sbg1+ (suppressor of beta glucan synthase 1), which suppressed the colony formation defect of Bgs1-defective cps1-191 mutant at higher temperatures. Sbg1p, an integral membrane protein, localizes to the cell ends and to the division site. Sbg1p and Bgs1p physically interact and are dependent on each other to localize to the division site. Loss of Sbg1p results in an unstable actomyosin ring that unravels and slides, leading to an inability to deposit a single contiguous division septum and an important reduction of the β-1,3-glucan proportion in the cell wall, coincident with that observed in the cps1-191 mutant. Sbg1p shows genetic and / or physical interaction with Rga7p, Imp2p, Cdc15p, and Pxl1p, proteins known to be required for actomyosin ring integrity and efficient septum synthesis. This study establishes Sbg1p as a key member of a group of proteins that link the plasma membrane, the actomyosin ring, and the division septum assembly machinery in fission yeast.

Photothermal nanoblade for patterned cell membrane cutting

PubMed Central

Wu, Ting-Hsiang; Teslaa, Tara; Teitell, Michael A.; Chiou, Pei-Yu

2010-01-01

We report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized and specifically shaped explosive vapor bubble. Rapid bubble expansion and collapse punctures a lightly-contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The membrane cutting pattern is controlled by the metallic nanostructure configuration, laser pulse polarization, and energy. Highly controllable, sub-micron sized circular hole pairs to half moon-like, or cat-door shaped, membrane cuts were realized in glutaraldehyde treated HeLa cells. PMID:21164656

Shape matters in protein mobility within membranes

PubMed Central

Quemeneur, François; Sigurdsson, Jon K.; Renner, Marianne; Atzberger, Paul J.; Bassereau, Patricia; Lacoste, David

2014-01-01

The lateral mobility of proteins within cell membranes is usually thought to be dependent on their size and modulated by local heterogeneities of the membrane. Experiments using single-particle tracking on reconstituted membranes demonstrate that protein diffusion is significantly influenced by the interplay of membrane curvature, membrane tension, and protein shape. We find that the curvature-coupled voltage-gated potassium channel (KvAP) undergoes a significant increase in protein mobility under tension, whereas the mobility of the curvature-neutral water channel aquaporin 0 (AQP0) is insensitive to it. Such observations are well explained in terms of an effective friction coefficient of the protein induced by the local membrane deformation. PMID:24706877

Cutting edge: rescue of pre-TCR but not mature TCR signaling in mice expressing membrane-targeted SLP-76.

PubMed

Bezman, Natalie A; Baker, Rebecca G; Lenox, Laurie E; Jordan, Martha S; Koretzky, Gary A

2009-05-01

SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) organizes signaling from immunoreceptors, including the platelet collagen receptor, the pre-TCR, and the TCR, and is required for T cell development. In this study we examine a mouse in which wild-type SLP-76 is replaced with a mutant constitutively targeted to the cell membrane. Membrane-targeted SLP-76 (MTS) supports ITAM signaling in platelets and from the pre-TCR. Signaling from the mature TCR, however, is defective in MTS thymocytes, resulting in failed T cell differentiation. Defective thymic selection by MTS is not rescued by a SLP-76 mutant whose localization is restricted to the cytosol. Thus, fixed localization of SLP-76 reveals differential requirements for the subcellular localization of signaling complexes downstream of the pre-TCR vs mature TCR.

NEU3 Sialidase Protein Interactors in the Plasma Membrane and in the Endosomes*

PubMed Central

Cirillo, Federica; Ghiroldi, Andrea; Fania, Chiara; Piccoli, Marco; Torretta, Enrica; Tettamanti, Guido; Gelfi, Cecilia; Anastasia, Luigi

2016-01-01

NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking. PMID:26987901

NEU3 Sialidase Protein Interactors in the Plasma Membrane and in the Endosomes.

PubMed

Cirillo, Federica; Ghiroldi, Andrea; Fania, Chiara; Piccoli, Marco; Torretta, Enrica; Tettamanti, Guido; Gelfi, Cecilia; Anastasia, Luigi

2016-05-13

NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential insertion of GPI-anchored GFPs into lipid rafts of live cells.

PubMed

Legler, Daniel F; Doucey, Marie-Agnès; Schneider, Pascal; Chapatte, Laurence; Bender, Florent C; Bron, Claude

2005-01-01

Partitioning of proteins in cholesterol and sphingolipid enriched plasma membrane microdomains, called lipid rafts, is critical for many signal transduction and protein sorting events. Although raft partitioning of many signaling molecules remains to be determined, glycosylphosphatidyl-inositol (GPI)-anchored proteins possess high affinity for lipid rafts and are currently exploited as markers to investigate fundamental mechanisms in protein sorting and signal transduction events. In this study, we demonstrate that two recombinant GPI-anchored green fluorescent proteins (GFP-GPIs) that differ in their GPI signal sequence confer distinct localization in plasma membrane microdomains. GFP fused to the GPI signal of the decay accelerating factor GFP-GPI(DAF) partitioned exclusively in lipid rafts, whereas GFP fused to the GPI signal of TRAIL-R3, GFP-GPI(TRAIL-R3), associated only minimally with microdomains. In addition, we investigated the unique ability of purified GFP-GPIs to insert into membrane microdomains of primary lymphocytes. This cell surface painting allows rapid, stable, and functional association of the GPI-anchored proteins with the target cell plasma membrane. The distinct membrane localization of the two GFP-GPIs was observed irrespective of whether the GPI-anchored molecules were painted or transfected. Furthermore, we show that painted GFP-GPI(DAF) was totally dependent on the GPI anchor and that the membrane insertion was increased by the addition of raft-associated lipids such as cholesterol, sphingomyelin, and dipalmitoyl-phosphatidylethanolamine. Thus, this study provides evidence that different GPI signal sequences lead to distinct membrane microdomain localization and that painted GFP-GPI(DAF) serves as an excellent fluorescent marker for lipid rafts in live cells.

Pepper pathogenesis-related protein 4c is a plasma membrane-localized cysteine protease inhibitor that is required for plant cell death and defense signaling.

PubMed

Kim, Nak Hyun; Hwang, Byung Kook

2015-01-01

Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis-related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT-triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease-inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H2 O2 and significant induction of some defense-response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT-triggered resistance responses, and enabled Xcv proliferation in infected leaves. H2 O2 accumulation, cell-death induction, and defense-response gene expression were distinctly reduced in CaPR4c-silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Selective cell-surface labeling of the molecular motor protein prestin

PubMed Central

McGuire, Ryan M.; Silberg, Jonathan J.; Pereira, Fred A.; Raphael, Robert M.

2011-01-01

Prestin, a multipass transmembrane protein whose N- an C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity. PMID:21651892

Fluorescent Labeling of the Nuclear Envelope by Localizing Green Fluorescent Protein on the Inner Nuclear Membrane.

PubMed

Taniyama, Toshiyuki; Tsuda, Natsumi; Sueda, Shinji

2018-06-15

The nuclear envelope (NE) is a double membrane that segregates nuclear components from the cytoplasm in eukaryotic cells. It is well-known that the NE undergoes a breakdown and reformation during mitosis in animal cells. However, the detailed mechanisms of the NE dynamics are not yet fully understood. Here, we propose a method for the fluorescent labeling of the NE in living cells, which enables the tracing of the NE dynamics during cell division under physiological conditions. In our method, labeling of the NE is accomplished by fixing green fluorescent protein carrying the nuclear localization signal on the inner nuclear membrane based on a unique biotinylation reaction from the archaeon Sulfolobus tokodaii. With this method, we observed HeLa cells during mitosis by confocal laser scanning microscopy and succeeded in clearly visualizing the difference in the timing of the formation of the NE and the nuclear lamina.

Cell Protrusion and Retraction Driven by Fluctuations in Actin Polymerization: A Two-Dimensional Model

PubMed Central

Ryan, Gillian L.; Holz, Danielle; Yamashiro, Sawako; Taniguchi, Daisuke; Watanabe, Naoki; Vavylonis, Dimitrios

2017-01-01

Animal cells that spread onto a surface often rely on actin-rich lamellipodial extensions to execute protrusion. Many cell types recently adhered on a two-dimensional substrate exhibit protrusion and retraction of their lamellipodia, even though the cell is not translating. Traveling waves of protrusion have also been observed, similar to those observed in crawling cells. These regular patterns of protrusion and retraction allow quantitative analysis for comparison to mathematical models. The periodic fluctuations in leading edge position of XTC cells have been linked to excitable actin dynamics using a one-dimensional model of actin dynamics, as a function of arc-length along the cell. In this work we extend this earlier model of actin dynamics into two dimensions (along the arc-length and radial directions of the cell) and include a model membrane that protrudes and retracts in response to the changing number of free barbed ends of actin filaments near the membrane. We show that if the polymerization rate at the barbed ends changes in response to changes in their local concentration at the leading edge and/or the opposing force from the cell membrane, the model can reproduce the patterns of membrane protrusion and retraction seen in experiment. We investigate both Brownian ratchet and switch-like force-velocity relationships between the membrane load forces and actin polymerization rate. The switch-like polymerization dynamics recover the observed patterns of protrusion and retraction as well as the fluctuations in F-actin concentration profiles. The model generates predictions for the behavior of cells after local membrane tension perturbations. PMID:28752950

The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains.

PubMed

Corcoran, Jennifer A; Salsman, Jayme; de Antueno, Roberto; Touhami, Ahmed; Jericho, Manfred H; Clancy, Eileen K; Duncan, Roy

2006-10-20

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.

Single-Molecule Imaging of Wnt3A Protein Diffusion on Living Cell Membranes.

PubMed

Lippert, Anna; Janeczek, Agnieszka A; Fürstenberg, Alexandre; Ponjavic, Aleks; Moerner, W E; Nusse, Roel; Helms, Jill A; Evans, Nicholas D; Lee, Steven F

2017-12-19

Wnt proteins are secreted, hydrophobic, lipidated proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the protein with its receptor, Frizzled, but may also regulate the transport of Wnt protein and its localization at the cell membrane. Here, by employing single-molecule fluorescence techniques, we show that Wnt proteins associate with and diffuse on the plasma membranes of living cells in the absence of any receptor binding. We find that labeled Wnt3A transiently and dynamically associates with the membranes of Drosophila Schneider 2 cells, diffuses with Brownian kinetics on flattened membranes and on cellular protrusions, and does not transfer between cells in close contact. In S2 receptor-plus (S2R+) cells, which express Frizzled receptors, membrane diffusion rate is reduced and membrane residency time is increased. These results provide direct evidence of Wnt3A interaction with living cell membranes, and represent, to our knowledge, a new system for investigating the dynamics of Wnt transport. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Nuclear localization of Klotho in brain: an anti-aging protein

PubMed Central

German, Dwight C.; Khobahy, Ida; Pastor, Johanne; Kuro-o, Makoto; Liu, Xinran

2011-01-01

Klotho is a putative age-suppressing gene whose over-expression in mice results in extension of life span. The klotho gene encodes a single-pass transmembrane protein whose extracellular domain is shed and released into blood, urine, and cerebrospinal fluid, potentially functioning as a humoral factor. The extracellular domain of Klotho has an activity that increases the expression of anti-oxidant enzymes and confers resistance to oxidative stress in cultured cells and in whole animals. The transmembrane form of the Klotho protein directly binds to multiple fibroblast growth factor receptors and modifies their ligand affinity and specificity. The purpose of the present study was to determine the precise cellular localization of Klotho in the mouse brain. Using light microscopic immunohistochemical methods, we found the highest levels of Klotho immunoreactivity in two brain regions: the choroid plexus, and cerebellar Purkinje cells. In the choroid plexus cells, Klotho was found not only on the plasma membrane but also in large amounts near the nuclear membrane. Likewise, in the Purkinje cell Klotho was found throughout the cell including dendrites, axon and soma with large amounts near the nuclear membrane. Using immunoelectron microscopy, we found Klotho in the cell membrane, but the highest concentration was localized in the peripheral portion of the nucleus and the nucleolus in both cell types. This new finding suggests that in addition to Klotho being secreted from cells in brain, it also has a nuclear function. PMID:22245317

Human cytotoxic T-lymphocyte membrane-camouflaged nanoparticles combined with low-dose irradiation: a new approach to enhance drug targeting in gastric cancer.

PubMed

Zhang, Lianru; Li, Rutian; Chen, Hong; Wei, Jia; Qian, Hanqing; Su, Shu; Shao, Jie; Wang, Lifeng; Qian, Xiaoping; Liu, Baorui

2017-01-01

Cell membrane-derived nanoparticles are becoming more attractive because of their ability to mimic many features of their source cells. This study reports on a biomimetic delivery platform based on human cytotoxic T-lymphocyte membranes. In this system, the surface of poly-lactic- co -glycolic acid nanoparticles was camouflaged using T-lymphocyte membranes, and local low-dose irradiation (LDI) was used as a chemoattractant for nanoparticle targeting. The T-lymphocyte membrane coating was verified using dynamic light scattering, transmission electron microscopy, and confocal laser scanning microscopy. This new platform reduced nanoparticle phagocytosis by macrophages to 23.99% ( P =0.002). Systemic administration of paclitaxel-loaded T-lymphocyte membrane-coated nanoparticles inhibited the growth of human gastric cancer by 56.68% in Balb/c nude mice. Application of LDI at the tumor site significantly increased the tumor growth inhibition rate to 88.50%, and two mice achieved complete remission. Furthermore, LDI could upregulate the expression of adhesion molecules in tumor vessels, which is important in the process of leukocyte adhesion and might contribute to the localization of T-lymphocyte membrane-encapsulated nanoparticles in tumors. Therefore, this new drug-delivery platform retained both the long circulation time and tumor site accumulation ability of human cytotoxic T lymphocytes, while local LDI could significantly enhance tumor localization.

The bacterial Sec system is required for the organization and function of the MreB cytoskeleton

PubMed Central

2017-01-01

The Sec system is responsible for protein insertion, translocation and secretion across membranes in all cells. The bacterial actin homolog MreB controls various processes, including cell wall synthesis, membrane organization and polarity establishment. Here we show that the two systems genetically interact and that components of the Sec system, especially the SecA motor protein, are essential for spatiotemporal organization of MreB in E. coli, as evidenced by the accumulation of MreB at irregular sites in Sec-impaired cells. MreB mislocalization in SecA-defective cells significantly affects MreB-coordinated processes, such as cell wall synthesis, and induce formation of membrane invaginations enriched in high fluidity domains. Additionally, MreB is not recruited to the FtsZ ring in secA mutant cells, contributing to division arrest and cell filamentation. Our results show that all these faults are due to improper targeting of MreB to the membrane in the absence of SecA. Thus, when we reroute RodZ, MreB membrane-anchor, by fusing it to a SecA-independent integral membrane protein and overproducing it, MreB localization is restored and the defect in cell division is corrected. Notably, the RodZ moiety is not properly inserted into the membrane, strongly suggesting that it only serves as a bait for placing MreB around the cell circumference. Finally, we show that MreB localization depends on SecA also in C. crescentus, suggesting that regulation of MreB by the Sec system is conserved in bacteria. Taken together, our data reveal that the secretion system plays an important role in determining the organization and functioning of the cytoskeletal system in bacteria. PMID:28945742

The bacterial Sec system is required for the organization and function of the MreB cytoskeleton.

PubMed

Govindarajan, Sutharsan; Amster-Choder, Orna

2017-09-01

The Sec system is responsible for protein insertion, translocation and secretion across membranes in all cells. The bacterial actin homolog MreB controls various processes, including cell wall synthesis, membrane organization and polarity establishment. Here we show that the two systems genetically interact and that components of the Sec system, especially the SecA motor protein, are essential for spatiotemporal organization of MreB in E. coli, as evidenced by the accumulation of MreB at irregular sites in Sec-impaired cells. MreB mislocalization in SecA-defective cells significantly affects MreB-coordinated processes, such as cell wall synthesis, and induce formation of membrane invaginations enriched in high fluidity domains. Additionally, MreB is not recruited to the FtsZ ring in secA mutant cells, contributing to division arrest and cell filamentation. Our results show that all these faults are due to improper targeting of MreB to the membrane in the absence of SecA. Thus, when we reroute RodZ, MreB membrane-anchor, by fusing it to a SecA-independent integral membrane protein and overproducing it, MreB localization is restored and the defect in cell division is corrected. Notably, the RodZ moiety is not properly inserted into the membrane, strongly suggesting that it only serves as a bait for placing MreB around the cell circumference. Finally, we show that MreB localization depends on SecA also in C. crescentus, suggesting that regulation of MreB by the Sec system is conserved in bacteria. Taken together, our data reveal that the secretion system plays an important role in determining the organization and functioning of the cytoskeletal system in bacteria.

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation

PubMed Central

Naegeli, Kaleb M.; Chi, Qiuyi; Ziel, Joshua W.; Hagedorn, Elliott J.; Park, Jieun E.; Jayadev, Ranjay; Sherwood, David R.

2016-01-01

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue. PMID:26765257

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation.

PubMed

Lohmer, Lauren L; Clay, Matthew R; Naegeli, Kaleb M; Chi, Qiuyi; Ziel, Joshua W; Hagedorn, Elliott J; Park, Jieun E; Jayadev, Ranjay; Sherwood, David R

2016-01-01

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.

Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes

NASA Astrophysics Data System (ADS)

Weinberger, Andreas; Walter, Vivien; MacEwan, Sarah R.; Schmatko, Tatiana; Muller, Pierre; Schroder, André P.; Chilkoti, Ashutosh; Marques, Carlos M.

2017-03-01

Although cationic cell-penetrating peptides (CPPs) are able to bind to cell membranes, thus promoting cell internalization by active pathways, attachment of cargo molecules to CPPs invariably reduces their cellular uptake. We show here that CPP binding to lipid bilayers, a simple model of the cell membrane, can be recovered by designing cargo molecules that self-assemble into spherical micelles and increase the local interfacial density of CPP on the surface of the cargo. Experiments performed on model giant unilamellar vesicles under a confocal laser scanning microscope show that a family of thermally responsive elastin-like polypeptides that exhibit temperature-triggered micellization can promote temperature triggered attachment of the micelles to membranes, thus rescuing by self-assembly the cargo-induced loss of the CPP affinity to bio-membranes.

Feedback Interactions of Polymerized Actin with the Cell Membrane: Waves, Pulses, and Oscillations

NASA Astrophysics Data System (ADS)

Carlsson, Anders

Polymerized filaments of the protein actin have crucial functions in cell migration, and in bending the cell membrane to drive endocytosis or the formation of protrusions. The nucleation and polymerization of actin filaments are controlled by upstream agents in the cell membrane, including nucleation-promoting factors (NPFs) that activate the Arp2/3 complex to form new branches on pre-existing filaments. But polymerized actin (F-actin) also feeds back on the assembly of NPFs. We explore the effects of the resulting feedback loop of F-actin and NPFs on two phenomena: actin pulses that drive endocytosis in yeast, and actin waves traveling along the membrane of several cell types. In our model of endocytosis in yeast, the actin network is grown explicitly in three dimensions, exerts a negative feedback interaction on localized patch of NPFs in the membrane, and bends the membrane by exerting a distribution of forces. This model explains observed actin and NPF pulse dynamics, and the effects of several interventions including i) NPF mutations, ii) inhibition of actin polymerization, and iii) deletion of a protein that allows F-actin to bend the cell membrane. The model predicts that mutation of the active region of an NPF will enhance the accumulation of that NPF, and we confirm this prediction by quantitative fluorescence microscopy. For actin waves, we treat a similar model, with NPFs distributed over a larger region of the cell membrane. This model naturally generates actin waves, and predicts a transition from wave behavior to spatially localized oscillations when NPFs are confined to a small region. We also predict a transition from waves to static polarization as the negative-feedback coupling between F-actin and the NPFs is reduced. Supported by NIGMS Grant R01 GM107667.

Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

PubMed

Kress, Alla; Wang, Xiao; Ranchon, Hubert; Savatier, Julien; Rigneault, Hervé; Ferrand, Patrick; Brasselet, Sophie

2013-07-02

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) alters the membrane raft location of the TSH receptor stably expressed in Chinese hamster ovary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Gregorio, Francesca; Pellegrino, Mario; Picchietti, Simona

2011-06-01

DDT is a highly lipophilic molecule known to deplete membrane rafts of their phosphoglycolipid and cholesterol contents. However, we have recently shown that DDT can also alter the thyroid homeostasis by inhibiting TSH receptor (TSHr) internalization. The present study was undertaken to verify whether DDT goitrogenic effects are due to the insecticide acting directly on TSHr or via alteration of the membrane rafts hosting the receptor itself. Our results demonstrate that, in CHO-TSHr transfected cells, TSHr is activated in the presence of TSH, while it is inhibited following DDT exposure. DDT can also reduce the endocytic vesicular traffic, alter themore » extension of multi-branched microvilli along their plasma membranes and induce TSHr shedding in vesicular forms. To verify whether TSHr displacement might depend on DDT altering the raft constitution of CHO-TSHr cell membranes the extent of TSHr and lipid raft co-localization was examined by confocal microscopy. Evidence shows that receptor/raft co-localization increased significantly upon exposure to TSH, while receptors and lipid rafts become dislodged on opposite cell poles in DDT-exposed CHO-TSHr cells. As a control, under similar culturing conditions, diphenylethylene, which is known to be a lipophilic substance that is structurally related to DDT, did not affect the extent of TSHr and lipid raft co-localization in CHO-TSHr cells treated with TSH. These findings corroborate and extend our view that, in CHO cells, the DDT disrupting action on TSHr is primarily due to the insecticide acting on membranes to deplete their raft cholesterol content, and that the resulting inhibition on TSHr internalization is due to receptor dislodgement from altered raft microdomains of the plasma membrane. - Highlights: >DDT is a pesticide with a severe environmental impact >Epidemiologic correlation exists between exposition to DDT and thyroid dysfunction >DDT is a lipophilic molecule that has been shown to inhibit TSH receptor function >DDT depletes membrane raft cholesterol content and by this way inhibits TSH receptor« less

Nance-Horan syndrome protein, NHS, associates with epithelial cell junctions.

PubMed

Sharma, Shiwani; Ang, Sharyn L; Shaw, Marie; Mackey, David A; Gécz, Jozef; McAvoy, John W; Craig, Jamie E

2006-06-15

Nance-Horan syndrome, characterized by congenital cataracts, craniofacial, dental abnormalities and mental disturbances, is an X-linked disorder with significant phenotypic heterogeneity. Affected individuals have mutations in the NHS (Nance-Horan syndrome) gene typically resulting in premature truncation of the protein. This report underlines the complexity of the regulation of the NHS gene that transcribes several isoforms. We demonstrate the differential expression of the two NHS isoforms, NHS-A and NHS-1A, and differences in the subcellular localization of the proteins encoded by these isoforms. This may in part explain the pleiotropic features of the syndrome. We show that the endogenous and exogenous NHS-A isoform localizes to the cell membrane of mammalian cells in a cell-type-dependent manner and that it co-localizes with the tight junction (TJ) protein ZO-1 in the apical aspect of cell membrane in epithelial cells. We also show that the NHS-1A isoform is a cytoplasmic protein. In the developing mammalian lens, we found continuous expression of NHS that became restricted to the lens epithelium in pre- and postnatal lens. Consistent with the in vitro findings, the NHS-A isoform associates with the apical cell membrane in the lens epithelium. This study suggests that disturbances in intercellular contacts underlie cataractogenesis in the Nance-Horan syndrome. NHS is the first gene localized at TJs that has been implicated in congenital cataracts.

The actin homologue MreB organizes the bacterial cell membrane

PubMed Central

Strahl, Henrik; Bürmann, Frank; Hamoen, Leendert W.

2014-01-01

The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate bacterial cell wall synthesis. We noticed that the MreB cytoskeleton influences fluorescent staining of the cytoplasmic membrane. Detailed analyses combining an array of mutants, using specific lipid staining techniques and spectroscopic methods, revealed that MreB filaments create specific membrane regions with increased fluidity (RIFs). Interference with these fluid lipid domains (RIFs) perturbs overall lipid homeostasis and affects membrane protein localization. The influence of MreB on membrane organization and fluidity may explain why the active movement of MreB stimulates membrane protein diffusion. These novel MreB activities add additional complexity to bacterial cell membrane organization and have implications for many membrane-associated processes. PMID:24603761

The actin homologue MreB organizes the bacterial cell membrane.

PubMed

Strahl, Henrik; Bürmann, Frank; Hamoen, Leendert W

2014-03-07

The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate bacterial cell wall synthesis. We noticed that the MreB cytoskeleton influences fluorescent staining of the cytoplasmic membrane. Detailed analyses combining an array of mutants, using specific lipid staining techniques and spectroscopic methods, revealed that MreB filaments create specific membrane regions with increased fluidity (RIFs). Interference with these fluid lipid domains (RIFs) perturbs overall lipid homeostasis and affects membrane protein localization. The influence of MreB on membrane organization and fluidity may explain why the active movement of MreB stimulates membrane protein diffusion. These novel MreB activities add additional complexity to bacterial cell membrane organization and have implications for many membrane-associated processes.

Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duangtum, Natapol; Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700; Junking, Mutita

Highlights: {yields} Impaired trafficking of kAE1 causes distal renal tubular acidosis (dRTA). {yields} The interaction between kAE1 and kinesin family member 3B (KIF3B) is reported. {yields} The co-localization between kAE and KIF3B was detected in human kidney tissues. {yields} A marked reduction of kAE1 on the cell membrane was observed when KIF3B was knockdown. {yields} KFI3B plays an important role in trafficking of kAE1 to the plasma membrane. -- Abstract: Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of {alpha}-intercalated cells of the kidney collecting duct leads to the defect of the Cl{sup -}/HCO{sub 3}{supmore » -} exchange and the failure of proton (H{sup +}) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney {alpha}-intercalated cells.« less

Interface Symbiotic Membrane Formation in Root Nodules of Medicago truncatula: the Role of Synaptotagmins MtSyt1, MtSyt2 and MtSyt3

PubMed Central

Gavrin, Aleksandr; Kulikova, Olga; Bisseling, Ton; Fedorova, Elena E.

2017-01-01

Symbiotic bacteria (rhizobia) are maintained and conditioned to fix atmospheric nitrogen in infected cells of legume root nodules. Rhizobia are confined to the asymmetrical protrusions of plasma membrane (PM): infection threads (IT), cell wall-free unwalled droplets and symbiosomes. These compartments rapidly increase in surface and volume due to the microsymbiont expansion, and remarkably, the membrane resources of the host cells are targeted to interface membrane quite precisely. We hypothesized that the change in the membrane tension around the expanding microsymbionts creates a vector for membrane traffic toward the symbiotic interface. To test this hypothesis, we selected calcium sensors from the group of synaptotagmins: MtSyt1, Medicago truncatula homolog of AtSYT1 from Arabidopsis thaliana known to be involved in membrane repair, and two other homologs expressed in root nodules: MtSyt2 and MtSyt3. Here we show that MtSyt1, MtSyt2, and MtSyt3 are expressed in the expanding cells of the meristem, zone of infection and proximal cell layers of zone of nitrogen fixation (MtSyt1, MtSyt3). All three GFP-tagged proteins delineate the interface membrane of IT and unwalled droplets and create a subcompartments of PM surrounding these structures. The localization of MtSyt1 by EM immunogold labeling has shown the signal on symbiosome membrane and endoplasmic reticulum (ER). To specify the role of synaptotagmins in interface membrane formation, we compared the localization of MtSyt1, MtSyt3 and exocyst subunit EXO70i, involved in the tethering of post-Golgi secretory vesicles and operational in tip growth. The localization of EXO70i in root nodules and arbusculated roots was strictly associated with the tips of IT and the tips of arbuscular fine branches, but the distribution of synaptotagmins on membrane subcompartments was broader and includes lateral parts of IT, the membrane of unwalled droplets as well as the symbiosomes. The double silencing of synaptotagmins caused a delay in rhizobia release and blocks symbiosome maturation confirming the functional role of synaptotagmins. In conclusion: synaptotagmin-dependent membrane fusion along with tip-targeted exocytosis is operational in the formation of symbiotic interface. PMID:28265280

Aquaporin-1 Facilitates Angiogenic Invasion in the Pathologic Neovasculature that Accompanies Cirrhosis

PubMed Central

Huebert, Robert C.; Vasdev, Meher M.; Shergill, Uday; Das, Amitava; Huang, Bing Q.; Charlton MR, Michael R.; LaRusso, Nicholas F.; Shah, Vijay H.

2010-01-01

Increasing evidence suggests that hepatic fibrosis and pathologic angiogenesis are inter-dependent processes that occur in parallel. Endothelial cell invasion is requisite for angiogenesis and thus studies of the mechanisms governing liver endothelial cell (LEC) invasion during cirrhosis are of great importance. Emerging research implicates amoeboid-type motility and membrane blebbing as features that may facilitate invasion through matrix-rich microenvironments. Aquaporins (AQPs) are integral membrane water channels, recognized for their importance in epithelial secretion and absorption. However, recent studies also suggest links between water transport and cell motility / invasion. Therefore, the purpose of this study was to test the hypothesis that AQP-1 is involved in amoeboid motility and angiogenic invasion during cirrhosis. AQP-1 expression and localization was examined in normal and cirrhotic liver tissues derived from human and mouse. AQP-1 levels were modulated in LEC using retroviral overexpression or siRNA knockdown and functional effects on invasion, membrane blebbing dynamics, and osmotic water permeability were assayed. Results demonstrate that AQP-1 is up-regulated in the small, angiogenic, neo-vasculature within the fibrotic septa of cirrhotic liver. AQP-1 overexpression promotes FGF-induced dynamic membrane blebbing in LEC which is sufficient to augment invasion through extracellular matrix. Additionally, AQP-1 localizes to plasma membrane blebs where it increases osmotic water permeability and locally facilitates the rapid, trans-membrane flux of water. CONCLUSION AQP-1 enhances osmotic water permeability and FGF-induced dynamic membrane blebbing in LEC and thereby drives invasion and pathologic angiogenesis during cirrhosis PMID:20578142

Fendiline Inhibits K-Ras Plasma Membrane Localization and Blocks K-Ras Signal Transmission

PubMed Central

van der Hoeven, Dharini; Cho, Kwang-jin; Ma, Xiaoping; Chigurupati, Sravanthi; Parton, Robert G.

2013-01-01

Ras proteins regulate signaling pathways important for cell growth, differentiation, and survival. Oncogenic mutant Ras proteins are commonly expressed in human tumors, with mutations of the K-Ras isoform being most prevalent. To be active, K-Ras must undergo posttranslational processing and associate with the plasma membrane. We therefore devised a high-content screening assay to search for inhibitors of K-Ras plasma membrane association. Using this assay, we identified fendiline, an L-type calcium channel blocker, as a specific inhibitor of K-Ras plasma membrane targeting with no detectable effect on the localization of H- and N-Ras. Other classes of L-type calcium channel blockers did not mislocalize K-Ras, suggesting a mechanism that is unrelated to calcium channel blockade. Fendiline did not inhibit K-Ras posttranslational processing but significantly reduced nanoclustering of K-Ras and redistributed K-Ras from the plasma membrane to the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and cytosol. Fendiline significantly inhibited signaling downstream of constitutively active K-Ras and endogenous K-Ras signaling in cells transformed by oncogenic H-Ras. Consistent with these effects, fendiline blocked the proliferation of pancreatic, colon, lung, and endometrial cancer cell lines expressing oncogenic mutant K-Ras. Taken together, these results suggest that inhibitors of K-Ras plasma membrane localization may have utility as novel K-Ras-specific anticancer therapeutics. PMID:23129805

[Changes of lastids in virus-infected cells of the attraction-zone from Sarracenia purpurea L].

PubMed

Barckhaus, R H; Weinert, H

1975-01-01

Viruslike particles 300-350 nm long and 70 nm in diameter were found in ultrathin sections of attraction-zone from Sarracenia purpurea. Epidermal- and mesophyll cells contained the bacilliform particles. The membrane-bound particles-most virions occured within ER-like membranes-consisted of an outer coat 70-90 A thick, an inner membrane and an axial core. The plastids of infected cells in which virus particles were localized show morphologicals changes of the organells.

Listeria membrane protrusion collapse: Requirement of Cyclophilin A for Listeria cell-to-cell spreading.

PubMed

Dhanda, Aaron S; Lulic, Katarina T; Vogl, A Wayne; Mc Gee, Margaret M; Chiu, Robert H; Guttman, Julian A

2018-05-04

Listeria generate actin-rich tubular protrusions at the plasma membrane that propel the bacteria into neighbouring cells. The precise molecular mechanisms governing the formation of these protrusions remain poorly defined. Here we demonstrate that the PPIase Cyclophilin A (CypA) is hijacked by Listeria at membrane protrusions used for cell-to-cell spreading. CypA localizes within the F-actin of these structures and is crucial for their proper formation, as in cells depleted of CypA, these extended actin-rich structures are mis-shaped and collapsed due to changes within the F-actin network. The lack of structural integrity within the Listeria membrane protrusions hampers the microbes from spreading from CypA null cells. Our results demonstrate a crucial role for CypA during Listeria infections.

Giga-ohm seals on intracellular membranes: a technique for studying intracellular ion channels in intact cells.

PubMed

Jonas, E A; Knox, R J; Kaczmarek, L K

1997-07-01

A method is outlined for obtaining giga-ohm seals on intracellular membranes in intact cells. The technique employs a variant of the patch-clamp technique: a concentric electrode arrangement protects an inner patch pipette during penetration of the plasma membrane, after which a seal can be formed on an internal organelle membrane. Using this technique, successful recordings can be obtained with the same frequency as with conventional patch clamping. To localize the position of the pipette within cells, lipophilic fluorescent dyes are included in the pipette solution. These dyes stain the membrane of internal organelles during seal formation and can then be visualized by video-enhanced or confocal imaging. The method can detect channels activated by inositol trisphosphate, as well as other types of intracellular membrane ion channel activity, and should facilitate studies of internal membranes in intact neurons and other cell types.

Polycystin-1 Surface Localization Is Stimulated by Polycystin-2 and Cleavage at the G Protein-coupled Receptor Proteolytic Site

PubMed Central

Chapin, Hannah C.; Rajendran, Vanathy

2010-01-01

Polycystin (PC)1 and PC2 are membrane proteins implicated in autosomal dominant polycystic kidney disease. A physiologically relevant cleavage at PC1's G protein-coupled receptor proteolytic site (GPS) occurs early in the secretory pathway. Our results suggest that PC2 increases both PC1 GPS cleavage and PC1's appearance at the plasma membrane. Mutations that prevent PC1's GPS cleavage prevent its plasma membrane localization. PC2 is a member of the trp family of cation channels and is an important PC1 binding partner. The effect of PC2 on PC1 localization is independent of PC2 channel activity, as tested using channel-inhibiting PC2 mutations. PC1 and PC2 can interact through their C-terminal tails, but removing the C-terminal tail of either protein has no effect on PC1 surface localization in human embryonic kidney 293 cells. Experiments in polarized LLC-PK cells show that apical and ciliary PC1 localization requires PC2 and that this delivery is sensitive to PC2 truncation. In sum, our work shows that PC2 expression is required for the movement of PC1 to the plasma and ciliary membranes. In fibroblast cells this localization effect is independent of PC2's channel activity or PC1 binding ability but involves a stimulation of PC1's GPS cleavage before the PC1 protein's surface delivery. PMID:20980620

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clarke, J.T.; Cook, H.W.; Spence, M.W.

1985-03-01

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22 h in the presence of D-(1-/sup 3/H)galactose or (/sup 3/H)GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipid-sialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellularmore » membrane fractions studied was recovered from plasma membrane and only 10-15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous (/sup 3/H)GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.« less

SChloro: directing Viridiplantae proteins to six chloroplastic sub-compartments.

PubMed

Savojardo, Castrense; Martelli, Pier Luigi; Fariselli, Piero; Casadio, Rita

2017-02-01

Chloroplasts are organelles found in plants and involved in several important cell processes. Similarly to other compartments in the cell, chloroplasts have an internal structure comprising several sub-compartments, where different proteins are targeted to perform their functions. Given the relation between protein function and localization, the availability of effective computational tools to predict protein sub-organelle localizations is crucial for large-scale functional studies. In this paper we present SChloro, a novel machine-learning approach to predict protein sub-chloroplastic localization, based on targeting signal detection and membrane protein information. The proposed approach performs multi-label predictions discriminating six chloroplastic sub-compartments that include inner membrane, outer membrane, stroma, thylakoid lumen, plastoglobule and thylakoid membrane. In comparative benchmarks, the proposed method outperforms current state-of-the-art methods in both single- and multi-compartment predictions, with an overall multi-label accuracy of 74%. The results demonstrate the relevance of the approach that is eligible as a good candidate for integration into more general large-scale annotation pipelines of protein subcellular localization. The method is available as web server at http://schloro.biocomp.unibo.it gigi@biocomp.unibo.it.

Coupled Segmentation of Nuclear and Membrane-bound Macromolecules through Voting and Multiphase Level Set

PubMed Central

Wen, Quan

2014-01-01

Membrane-bound macromolecules play an important role in tissue architecture and cell-cell communication, and is regulated by almost one-third of the genome. At the optical scale, one group of membrane proteins expresses themselves as linear structures along the cell surface boundaries, while others are sequestered; and this paper targets the former group. Segmentation of these membrane proteins on a cell-by-cell basis enables the quantitative assessment of localization for comparative analysis. However, such membrane proteins typically lack continuity, and their intensity distributions are often very heterogeneous; moreover, nuclei can form large clump, which further impedes the quantification of membrane signals on a cell-by-cell basis. To tackle these problems, we introduce a three-step process to (i) regularize the membrane signal through iterative tangential voting, (ii) constrain the location of surface proteins by nuclear features, where clumps of nuclei are segmented through a delaunay triangulation approach, and (iii) assign membrane-bound macromolecules to individual cells through an application of multi-phase geodesic level-set. We have validated our method using both synthetic data and a dataset of 200 images, and are able to demonstrate the efficacy of our approach with superior performance. PMID:25530633

Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium

PubMed Central

Ewald, Andrew J.; Huebner, Robert J.; Palsdottir, Hildur; Lee, Jessie K.; Perez, Melissa J.; Jorgens, Danielle M.; Tauscher, Andrew N.; Cheung, Kevin J.; Werb, Zena; Auer, Manfred

2012-01-01

Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in three-dimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane localization of E-cadherin and β-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes re-formed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program. PMID:22344263

Dynamic regulation of mechanosensitive channels: capacitance used to monitor patch tension in real time

NASA Astrophysics Data System (ADS)

Suchyna, Thomas M.; Besch, Steven R.; Sachs, Frederick

2004-03-01

All cells, from bacteria to human, are mechanically sensitive. The most rapid of these membrane protein transducers are mechanosensitive ion channels, ionic pores in the membrane that open and close in response to membrane tension. In specific sensory organs, these channels serve the senses of touch and hearing, and inform the central nervous system about the filling of hollow organs such as the bladder. Non-specialized cells use these channels to report on changes in cell volume and local strain. To preserve dynamic sensitivity, sensory receptors adapt to steady-state stimuli. Here we show that in rat astrocytes, the most abundant cells in the brain, this apparent adaptation to the stimulus is actually an inactivation. We have been able to track the time course of local strain by measuring attofarad changes in membrane capacitance and show that it is not correlated with loss of channel activity. The reduction in current with time is caused by an increased occupancy of low conductance states, and a reduction in the probability of opening, not a relaxation of local stress. The occupancy of these substates depends on the integrity of the cell's cytoplasm. However, while disruption of the cytoskeleton leads to a loss of inactivation, it leaves activation unaffected. The activation process is voltage-insensitive, closely correlated with changes in capacitance, and seems to arise solely from stress in the bilayer. The inactivation rate decreases with depolarization, and kinetic analysis suggests that the process involves multiple cytoplasmic ligands. Surprisingly, multivalent ions such as Gd+3 and Ca+2 that bind to the lipids and affect channel gating, do not affect the strain-induced increase in membrane capacitance; contrary to expectations, membrane elasticity is unchanged.

ATP7B mediates vesicular sequestration of copper: insight into biliary copper excretion.

PubMed

Cater, Michael A; La Fontaine, Sharon; Shield, Kristy; Deal, Yolanda; Mercer, Julian F B

2006-02-01

The Wilson protein (ATP7B) regulates levels of systemic copper by excreting excess copper into bile. It is not clear whether ATP7B translocates excess intrahepatic copper directly across the canalicular membrane or sequesters this copper into exocytic vesicles, which subsequently fuse with canalicular membrane to expel their contents into bile. The aim of this study was to clarify the mechanism underlying ATP7B-mediated copper detoxification by investigating endogenous ATP7B localization in the HepG2 hepatoma cell line and its ability to mediate vesicular sequestration of excess intracellular copper. Immunofluorescence microscopy was used to investigate the effect of copper concentration on the localization of endogenous ATP7B in HepG2 cells. Copper accumulation studies to determine whether ATP7B can mediate vesicular sequestration of excess intracellular copper were performed using Chinese hamster ovary cells that exogenously expressed wild-type and mutant ATP7B proteins. In HepG2 cells, elevated copper levels stimulated trafficking of ATP7B to pericanalicular vesicles and not to the canalicular membrane as previously reported. Mutation of an endocytic retrieval signal in ATP7B caused the protein to constitutively localize to vesicles and not to the plasma membrane, suggesting that a vesicular compartment(s) is the final trafficking destination for ATP7B. Expression of wild-type and mutant ATP7B caused Chinese hamster ovary cells to accumulate copper in vesicles, which subsequently undergo exocytosis, releasing copper across the plasma membrane. This report provides compelling evidence that the primary mechanism of biliary copper excretion involves ATP7B-mediated vesicular sequestration of copper rather than direct copper translocation across the canalicular membrane.

Chemical synthesis, characterisation, and biocompatibility of nanometre scale porous anodic aluminium oxide membranes for use as a cell culture substrate for the vero cell line: a preliminary study.

PubMed

Poinern, Gérrard Eddy Jai; Le, Xuan Thi; O'Dea, Mark; Becker, Thomas; Fawcett, Derek

2014-01-01

In this preliminary study we investigate for the first time the biomedical potential of using porous anodic aluminium oxide (AAO) membranes as a cell substrate for culturing the Cercopithecus aethiops (African green monkey) Kidney (Vero) epithelial cell line. One advantage of using the inorganic AAO membrane is the presence of nanometre scale pore channels that allow the exchange of molecules and nutrients across the membrane. The size of the pore channels can be preselected by adjusting the controlling parameters of a temperature controlled two-step anodization process. The cellular interaction and response of the Vero cell line with an in-house synthesised AAO membrane, a commercially available membrane, and a glass control were assessed by investigating cell adhesion, morphology, and proliferation over a 72 h period. The number of viable cells proliferating over the respective membrane surfaces revealed that the locally produced in-house AAO membrane had cells numbers similar to the glass control. The study revealed evidence of focal adhesion sites over the surface of the nanoporous membranes and the penetration of cellular extensions into the pore structure as well. The outcome of the study has revealed that nanometre scale porous AAO membranes have the potential to become practical cell culture scaffold substrates with the capability to enhance adhesion and proliferation of Vero cells.

Chemical Synthesis, Characterisation, and Biocompatibility of Nanometre Scale Porous Anodic Aluminium Oxide Membranes for Use as a Cell Culture Substrate for the Vero Cell Line: A Preliminary Study

PubMed Central

Poinern, Gérrard Eddy Jai; Le, Xuan Thi; Becker, Thomas; Fawcett, Derek

2014-01-01

In this preliminary study we investigate for the first time the biomedical potential of using porous anodic aluminium oxide (AAO) membranes as a cell substrate for culturing the Cercopithecus aethiops (African green monkey) Kidney (Vero) epithelial cell line. One advantage of using the inorganic AAO membrane is the presence of nanometre scale pore channels that allow the exchange of molecules and nutrients across the membrane. The size of the pore channels can be preselected by adjusting the controlling parameters of a temperature controlled two-step anodization process. The cellular interaction and response of the Vero cell line with an in-house synthesised AAO membrane, a commercially available membrane, and a glass control were assessed by investigating cell adhesion, morphology, and proliferation over a 72 h period. The number of viable cells proliferating over the respective membrane surfaces revealed that the locally produced in-house AAO membrane had cells numbers similar to the glass control. The study revealed evidence of focal adhesion sites over the surface of the nanoporous membranes and the penetration of cellular extensions into the pore structure as well. The outcome of the study has revealed that nanometre scale porous AAO membranes have the potential to become practical cell culture scaffold substrates with the capability to enhance adhesion and proliferation of Vero cells. PMID:24579077

Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18.

PubMed

Etter, E F; Minta, A; Poenie, M; Fay, F S

1996-05-28

(Ca2+)-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] measured during stimulation in intact cells. This paradox might be explained if the local [Ca2+] at the cell membrane is very different from that in the rest of the cell. Soluble Ca2+ indicators, which indicate spatially averaged cytoplasmic [Ca2+], cannot resolve these localized, near-membrane [Ca2+] signals. FFP18, the newest Ca2+ indicator designed to selectively monitor near-membrane [Ca2+], has a lower Ca2+ affinity and is more water soluble than previously used membrane-associating Ca2+ indicators. Images of the intracellular distribution of FFP18 show that >65% is located on or near the plasma membrane. [Ca2+] transients recorded using FFP18 during membrane depolarization-induced Ca2+ influx show that near-membrane [Ca2+] rises faster and reaches micromolar levels at early times when the cytoplasmic [Ca2+], recorded using fura-2, has risen to only a few hundred nanomolar. High-speed series of digital images of [Ca2+] show that near-membrane [Ca2+], reported by FFP18, rises within 20 msec, peaks at 50-100 msec, and then declines. [Ca2+] reported by fura-2 rose slowly and continuously throughout the time images were acquired. The existence of these large, rapid increases in [Ca2+] directly beneath the surface membrane may explain how numerous (Ca2+)-sensitive membrane processes are activated at times when bulk cytoplasmic [Ca2+] changes are too small to activate them.

Visualization and functional dissection of coaxial paired SpoIIIE channels across the sporulation septum

DOE PAGES

Shin, Jae Yen; Lopez-Garrido, Javier; Lee, Sang-Hyuk; ...

2015-05-07

SpoIIIE is a membrane-anchored DNA translocase that localizes to the septal midpoint to mediate chromosome translocation and membrane fission during Bacillus subtilis sporulation. Here we use cell-specific protein degradation and quantitative photoactivated localization microscopy in strains with a thick sporulation septum to investigate the architecture and function of the SpoIIIE DNA translocation complex in vivo. We were able to visualize SpoIIIE complexes with approximately equal numbers of molecules in the mother cell and the forespore. Cell-specific protein degradation showed that only the mother cell complex is required to translocate DNA into the forespore, whereas degradation in either cell reverses membranemore » fission. Our data suggest that SpoIIIE assembles a coaxially paired channel for each chromosome arm comprised of one hexamer in each cell to maintain membrane fission during DNA translocation. We show that SpoIIIE can operate, in principle, as a bi-directional motor that exports DNA.« less

Visualization and functional dissection of coaxial paired SpoIIIE channels across the sporulation septum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, Jae Yen; Lopez-Garrido, Javier; Lee, Sang-Hyuk

SpoIIIE is a membrane-anchored DNA translocase that localizes to the septal midpoint to mediate chromosome translocation and membrane fission during Bacillus subtilis sporulation. Here we use cell-specific protein degradation and quantitative photoactivated localization microscopy in strains with a thick sporulation septum to investigate the architecture and function of the SpoIIIE DNA translocation complex in vivo. We were able to visualize SpoIIIE complexes with approximately equal numbers of molecules in the mother cell and the forespore. Cell-specific protein degradation showed that only the mother cell complex is required to translocate DNA into the forespore, whereas degradation in either cell reverses membranemore » fission. Our data suggest that SpoIIIE assembles a coaxially paired channel for each chromosome arm comprised of one hexamer in each cell to maintain membrane fission during DNA translocation. We show that SpoIIIE can operate, in principle, as a bi-directional motor that exports DNA.« less

Early and late HIV-1 membrane fusion events are impaired by sphinganine lipidated peptides that target the fusion site.

PubMed

Klug, Yoel A; Ashkenazi, Avraham; Viard, Mathias; Porat, Ziv; Blumenthal, Robert; Shai, Yechiel

2014-07-15

Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus-cell and cell-cell contact sites. Overall, the findings of the present study may suggest lipid-protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.

Noncontact microsurgery of cell membranes using femtosecond laser pulses for optoinjection of specified substances into cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Il'ina, I V; Ovchinnikov, A V; Chefonov, O V

IR femtosecond laser pulses were used for microsurgery of a cell membrane aimed at local and short-duration change in its permeability and injection of specified extracellular substances into the cells. The possibility of noncontact laser delivery of the propidium iodide fluorescent dye and the pEGFP plasmid, encoding the green fluorescent protein, into the cells with preservation of the cell viability was demonstrated. (extreme light fields and their applications)

5-Aminolevulinic acid-based photochemical internalization of the immunotoxin MOC31-gelonin generates synergistic cytotoxic effects in vitro.

PubMed

Selbo, P K; Kaalhus, O; Sivam, G; Berg, K

2001-08-01

Photochemical internalization (PCI) is a novel method for the endosomal or lysosomal release of membrane-impermeable molecules into the cytosol of target cells. This novel technology is based on the photodynamically induced rupture of endocytic vesicles preloaded with molecules of therapeutic interest. PCI of the ribosome-inactivating plant toxin gelonin and the immunotoxin monoclonal antibody 31 (MOC31) gelonin has been performed previously by the use of the endocytic vesicle-localizing photosensitizers TPPS2a and AIPcS2a and light, demonstrating synergistic toxicity against the more than 20 different cell lines tested, most of them of neoplastic origin. In this study we demonstrate that 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) is also capable of inducing PCI of MOC31-gelonin in the human colon adenocarcinoma cell line WiDr. The cells were incubated with 1 mM 5-ALA for up to 8 h in serum-free medium and from 24 to 96 h in serum-containing medium. Fluorescence microscopical studies indicate a partial plasma membrane localization of PpIX when 5-ALA was applied under serum-free conditions. This plasma membrane localization was not seen when 5-ALA was given in the presence of serum. There was a granular component of the PpIX localization in addition to a diffuse cytoplasmic localization. The granular component resembled the localization of the fluorescent dye conjugate Alexa-gelonin and the lysosomal localizing dye acridine orange. Our present results provide evidence for an endocytic vesicle-associated fraction of PpIX after 5-ALA incubation of the WiDr cells. We demonstrate that PCI, by combining 5-ALA, MOC31-gelonin and light, induces a synergistic cytotoxic effect against the WiDr cells.

TMPRSS2, a novel membrane-anchored mediator in cancer pain

PubMed Central

Lam, David K.; Dang, Dongmin; Flynn, Andrea N.; Hardt, Markus; Schmidt, Brian L.

2016-01-01

More than half of all cancer patients will suffer significant pain during the course of their disease. The strategic localization of TMPRSS2, a membrane-bound serine protease, on the cancer cell surface may allow it to mediate signal transduction between the cancer cell and its extracellular environment. Here we show TMPRSS2 expression is not only dramatically increased in the primary cancers of patients but TMPRSS2-immunopositivity is also directly correlated with cancer pain severity in these patients. TMPRSS2 induced proteolytic activity, activated trigeminal neurons, and produced marked mechanical hyperalgesia when administered into the hindpaw of wild-type mice but not in PAR2-deficient mice. Co-culture of human cancer cells with murine trigeminal neurons demonstrated co-localization of TMPRSS2 with PAR2. These results point to a novel role for a cell membrane-anchored mediator in cancer pain, as well as pain in general. PMID:25734995

Enhanced transcription and translation in clay hydrogel and implications for early life evolution

PubMed Central

Yang, Dayong; Peng, Songming; Hartman, Mark R.; Gupton-Campolongo, Tiffany; Rice, Edward J.; Chang, Anna Kathryn; Gu, Zi; Lu, G. Q. (Max); Luo, Dan

2013-01-01

In most contemporary life forms, the confinement of cell membranes provides localized concentration and protection for biomolecules, leading to efficient biochemical reactions. Similarly, confinement may have also played an important role for prebiotic compartmentalization in early life evolution when the cell membrane had not yet formed. It remains an open question how biochemical reactions developed without the confinement of cell membranes. Here we mimic the confinement function of cells by creating a hydrogel made from geological clay minerals, which provides an efficient confinement environment for biomolecules. We also show that nucleic acids were concentrated in the clay hydrogel and were protected against nuclease, and that transcription and translation reactions were consistently enhanced. Taken together, our results support the importance of localized concentration and protection of biomolecules in early life evolution, and also implicate a clay hydrogel environment for biochemical reactions during early life evolution. PMID:24196527

Membrane organization determines barrier properties of endothelial cells and short-chain sphingolipid-facilitated doxorubicin influx.

PubMed

van Hell, A J; Klymchenko, A; Gueth, D M; van Blitterswijk, W J; Koning, G A; Verheij, M

2014-09-01

The endothelial lining and its outer lipid membrane are the first major barriers drug molecules encounter upon intravenous administration. Our previous work identified lipid analogs that counteract plasma membrane barrier function for a series of amphiphilic drugs. For example, short-chain sphingolipids (SCS), like N-octanoyl-glucosylceramide, effectively elevated doxorubicin accumulation in tumor cells, both in vitro and in vivo, and in endothelial cells, whereas other (normal) cells remained unaffected. We hypothesize here that local membrane lipid composition and the degree of lipid ordering define SCS efficacy in individual cells. To this end, we study the differential effect of SCS on bovine aortic endothelial cells (BAEC) in its confluent versus proliferative state, as a model system. While their (plasma membrane) lipidome stays remarkably unaltered when BAECs reach confluency, their lipids segregate to form apical and basolateral domains. Using probe NR12S, we reveal that lipids in the apical membrane are more condensed/liquid-ordered. SCS preferentially attenuate the barrier posed by these condensed membranes and facilitate doxorubicin influx in these particular membrane regions. We confirm these findings in MDCK cells and artificial membranes. In conclusion, SCS-facilitated drug traversal acts on condensed membrane domains, elicited by confluency in resting endothelium. Copyright © 2014 Elsevier B.V. All rights reserved.

Rice hypersensitive induced reaction protein 1 (OsHIR1) associates with plasma membrane and triggers hypersensitive cell death.

PubMed

Zhou, Liang; Cheung, Ming-Yan; Li, Man-Wah; Fu, Yaping; Sun, Zongxiu; Sun, Sai-Ming; Lam, Hon-Ming

2010-12-30

In plants, HIR (Hypersensitive Induced Reaction) proteins, members of the PID (Proliferation, Ion and Death) superfamily, have been shown to play a part in the development of spontaneous hypersensitive response lesions in leaves, in reaction to pathogen attacks. The levels of HIR proteins were shown to correlate with localized host cell deaths and defense responses in maize and barley. However, not much was known about the HIR proteins in rice. Since rice is an important cereal crop consumed by more than 50% of the populations in Asia and Africa, it is crucial to understand the mechanisms of disease responses in this plant. We previously identified the rice HIR1 (OsHIR1) as an interacting partner of the OsLRR1 (rice Leucine-Rich Repeat protein 1). Here we show that OsHIR1 triggers hypersensitive cell death and its localization to the plasma membrane is enhanced by OsLRR1. Through electron microscopy studies using wild type rice plants, OsHIR1 was found to mainly localize to the plasma membrane, with a minor portion localized to the tonoplast. Moreover, the plasma membrane localization of OsHIR1 was enhanced in transgenic rice plants overexpressing its interacting protein partner, OsLRR1. Co-localization of OsHIR1 and OsLRR1 to the plasma membrane was confirmed by double-labeling electron microscopy. Pathogen inoculation studies using transgenic Arabidopsis thaliana expressing either OsHIR1 or OsLRR1 showed that both transgenic lines exhibited increased resistance toward the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. However, OsHIR1 transgenic plants produced more extensive spontaneous hypersensitive response lesions and contained lower titers of the invading pathogen, when compared to OsLRR1 transgenic plants. The OsHIR1 protein is mainly localized to the plasma membrane, and its subcellular localization in that compartment is enhanced by OsLRR1. The expression of OsHIR1 may sensitize the plant so that it is more prone to HR and hence can react more promptly to limit the invading pathogens' spread from the infection sites.

Patterning and lifetime of plasma membrane-localized cellulose synthase is dependent on actin organization in Arabidopsis interphase cells.

PubMed

Sampathkumar, Arun; Gutierrez, Ryan; McFarlane, Heather E; Bringmann, Martin; Lindeboom, Jelmer; Emons, Anne-Mie; Samuels, Lacey; Ketelaar, Tijs; Ehrhardt, David W; Persson, Staffan

2013-06-01

The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In higher plants, cortical microtubules help to organize cell wall construction by positioning the delivery of cellulose synthase (CesA) complexes and guiding their trajectories to orient newly synthesized cellulose microfibrils. The actin cytoskeleton is required for normal distribution of CesAs to the plasma membrane, but more specific roles for actin in cell wall assembly and organization remain largely elusive. We show that the actin cytoskeleton functions to regulate the CesA delivery rate to, and lifetime of CesAs at, the plasma membrane, which affects cellulose production. Furthermore, quantitative image analyses revealed that actin organization affects CesA tracking behavior at the plasma membrane and that small CesA compartments were associated with the actin cytoskeleton. By contrast, localized insertion of CesAs adjacent to cortical microtubules was not affected by the actin organization. Hence, both actin and microtubule cytoskeletons play important roles in regulating CesA trafficking, cellulose deposition, and organization of cell wall biogenesis.

Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.

PubMed

de Jong, Arjan S; Wessels, Els; Dijkman, Henri B P M; Galama, Jochem M D; Melchers, Willem J G; Willems, Peter H G M; van Kuppeveld, Frank J M

2003-01-10

The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

Effectors of animal and plant pathogens use a common domain to bind host phosphoinositides.

PubMed

Salomon, Dor; Guo, Yirui; Kinch, Lisa N; Grishin, Nick V; Gardner, Kevin H; Orth, Kim

2013-01-01

Bacterial Type III Secretion Systems deliver effectors into host cells to manipulate cellular processes to the advantage of the pathogen. Many host targets of these effectors are found on membranes. Therefore, to identify their targets, effectors often use specialized membrane-localization domains to localize to appropriate host membranes. However, the molecular mechanisms used by many domains are unknown. Here we identify a conserved bacterial phosphoinositide-binding domain (BPD) that is found in functionally diverse Type III effectors of both plant and animal pathogens. We show that members of the BPD family functionally bind phosphoinositides and mediate localization to host membranes. Moreover, NMR studies reveal that the BPD of the newly identified Vibrio parahaemolyticus Type III effector VopR is unfolded in solution, but folds into a specific structure upon binding its ligand phosphatidylinositol-(4,5)-bisphosphate. Thus, our findings suggest a possible mechanism for promoting refolding of Type III effectors after delivery into host cells.

Novel Tonoplast Transporters Identified Using a Proteomic Approach with Vacuoles Isolated from Cauliflower Buds1[W][OA

PubMed Central

Schmidt, Ulrike G.; Endler, Anne; Schelbert, Silvia; Brunner, Arco; Schnell, Magali; Neuhaus, H. Ekkehard; Marty-Mazars, Daniéle; Marty, Francis; Baginsky, Sacha; Martinoia, Enrico

2007-01-01

Young meristematic plant cells contain a large number of small vacuoles, while the largest part of the vacuome in mature cells is composed by a large central vacuole, occupying 80% to 90% of the cell volume. Thus far, only a limited number of vacuolar membrane proteins have been identified and characterized. The proteomic approach is a powerful tool to identify new vacuolar membrane proteins. To analyze vacuoles from growing tissues we isolated vacuoles from cauliflower (Brassica oleracea) buds, which are constituted by a large amount of small cells but also contain cells in expansion as well as fully expanded cells. Here we show that using purified cauliflower vacuoles and different extraction procedures such as saline, NaOH, acetone, and chloroform/methanol and analyzing the data against the Arabidopsis (Arabidopsis thaliana) database 102 cauliflower integral proteins and 214 peripheral proteins could be identified. The vacuolar pyrophosphatase was the most prominent protein. From the 102 identified proteins 45 proteins were already described. Nine of these, corresponding to 46% of peptides detected, are known vacuolar proteins. We identified 57 proteins (55.9%) containing at least one membrane spanning domain with unknown subcellular localization. A comparison of the newly identified proteins with expression profiles from in silico data revealed that most of them are highly expressed in young, developing tissues. To verify whether the newly identified proteins were indeed localized in the vacuole we constructed and expressed green fluorescence protein fusion proteins for five putative vacuolar membrane proteins exhibiting three to 11 transmembrane domains. Four of them, a putative organic cation transporter, a nodulin N21 family protein, a membrane protein of unknown function, and a senescence related membrane protein were localized in the vacuolar membrane, while a white-brown ATP-binding cassette transporter homolog was shown to reside in the plasma membrane. These results demonstrate that proteomic analysis of highly purified vacuoles from specific tissues allows the identification of new vacuolar proteins and provides an additional view of tonoplastic proteins. PMID:17660356

Enhancement of Cell Membrane Invaginations, Vesiculation and Uptake of Macromolecules by Protonation of the Cell Surface

PubMed Central

Ben-Dov, Nadav; Korenstein, Rafi

2012-01-01

The different pathways of endocytosis share an initial step involving local inward curvature of the cell’s lipid bilayer. It has been shown that to generate membrane curvature, proteins or lipids enforce transversal asymmetry of the plasma membrane. Thus it emerges as a general phenomenon that transversal membrane asymmetry is the common required element for the formation of membrane curvature. The present study demonstrates that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesiculation accompanied by efficient uptake of macromolecules (Dextran-FITC, 70 kD), relative to the constitutive one. The insensitivity of proton induced uptake to inhibiting treatments and agents of the known endocytic pathways suggests the entry of macromolecules to proceeds via a yet undefined route. This is in line with the fact that neither ATP depletion, nor the lowering of temperature, abolishes the uptake process. In addition, fusion mechanism such as associated with low pH uptake of toxins and viral proteins can be disregarded by employing the polysaccharide dextran as the uptake molecule. The proton induced uptake increases linearly in the extracellular pH range of 6.5 to 4.5, and possesses a steep increase at the range of 4> pH>3, reaching a plateau at pH≤3. The kinetics of the uptake implies that the induced vesicles release their content to the cytosol and undergo rapid recycling to the plasma membrane. We suggest that protonation of the cell’s surface induces local charge asymmetries across the cell membrane bilayer, inducing inward curvature of the cell membrane and consequent vesiculation and uptake. PMID:22558127

EphA2 transmembrane domain is uniquely required for keratinocyte migration by regulating ephrin-A1 levels.

PubMed

Ventrella, Rosa; Kaplan, Nihal; Hoover, Paul; Perez White, Bethany E; Lavker, Robert M; Getsios, Spiro

2018-04-26

EphA2 receptor tyrosine kinase (RTK) is activated by ephrin-A1 ligand, which harbors a GPI-anchor that enhances lipid raft localization. While EphA2 and ephrin-A1 modulate keratinocyte migration and differentiation, the ability of this cell-cell communication complex to localize to different membrane regions in keratinocytes remains unknown. Using a combination of biochemical and imaging approaches, we provide evidence that ephrin-A1 and a ligand-activated form of EphA2 partition outside of lipid raft domains in response to calcium-mediated cell-cell contact stabilization in normal human epidermal keratinocytes (NHEKs). EphA2 transmembrane domain (TMD) swapping with a shorter and molecularly distinct TMD of EphA1 resulted in decreased localization of this RTK at cell-cell junctions and increased expression of ephrin-A1, which is a negative regulator of keratinocyte migration. Accordingly, altered EphA2 membrane distribution at cell-cell contacts limited the ability of keratinocytes to seal linear scratch wounds in vitro in an ephrin-A1-dependent manner. Collectively, these studies highlight a key role for the EphA2 TMD in receptor-ligand membrane distribution at cell-cell contacts that modulates ephrin-A1 levels to allow for efficient keratinocyte migration with relevance for cutaneous wound healing. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Region-specific spike frequency acceleration in Layer 5 pyramidal neurons mediated by Kv1 subunits

PubMed Central

Miller, Mark N; Okaty, Benjamin W; Nelson, Sacha B

2009-01-01

Separation of the cortical sheet into functionally distinct regions is a hallmark of neocortical organization. Cortical circuit function emerges from afferent and efferent connectivity, local connectivity within the cortical microcircuit, and the intrinsic membrane properties of neurons that comprise the circuit. While localization of functions to particular cortical areas can be partially accounted for by regional differences in both long range and local connectivity, it is unknown whether the intrinsic membrane properties of cortical cell-types differ between cortical regions. Here we report the first example of a region-specific firing type in layer 5 pyramidal neurons, and show that the intrinsic membrane and integrative properties of a discrete subtype of layer 5 pyramidal neurons differ between primary motor and somatosensory cortices due to region and cell-type-specific Kv1 subunit expression. PMID:19091962

Initial targets and cellular responses to PDT

NASA Astrophysics Data System (ADS)

Rodriguez, Myriam E.; Azizuddin, Kashif; Chiu, Song-mao; Delos Santos, Grace; Joseph, Sheeba; Xue, Liang-yan; Oleinick, Nancy L.

2007-02-01

Pc 4, a photosensitizer first synthesized at Case Western Reserve University and now in clinical trial at University Hospitals of Cleveland, has been shown to bind preferentially and with high affinity to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components are photodamaged. In most cancer cells, apoptosis is triggered by the initial photodamage; however, in cells deficient in one of the critical intermediates of apoptosis, this process does not occur, although the cells remain as sensitive to the lethal effects of Pc 4-PDT as the apoptosis-competent cells, when cell death is determined by colony formation. Here we report that an alternative death process, autophagy, is induced in all cells tested and becomes the dominant pathway for elimination of lethally damaged cells when apoptosis is compromised. The anti-apoptotic protein Bcl-2, when overexpressed, protects only apoptosis-competent cells against loss of clonogenicity, while the autophagy inhibitor 3-methyladenine provides a markedly greater protection to apoptosis-deficient cells. The results suggest that the primary determinant of cell death is not the final pathway for elimination of the cells but the initial photodamage to critical membrane targets. In attempts to identify those targets, we have studied the role of different membrane phospholipids in the localization of Pc 4. Cardiolipin (CL) is a phospholipid found exclusively in the mitochondrial inner membrane and at the contact sites between the inner and outer membranes. Previous fluorescence resonance energy transfer studies revealed colocalization of Pc 4 and CL, which points to CL as a possible binding site and target for Pc 4. Unilamellar liposomes with different lipid compositions were used as membrane models to test the affinity of Pc 4. As revealed by the binding constants, Pc 4 does not display preferential binding to CL in these systems. Moreover, binding affinities appear to be independent of lipid composition. Localization of Pc 4 in mitochondrial membranes is likely determined by proteins or other factors not replicated in the liposomes. Studies in cells with modified CL content could report modified binding affinities.

Selective cell-surface labeling of the molecular motor protein prestin.

PubMed

McGuire, Ryan M; Silberg, Jonathan J; Pereira, Fred A; Raphael, Robert M

2011-06-24

Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity. Copyright © 2011 Elsevier Inc. All rights reserved.

Functional mapping of cell surface proteins with localized stimulation of single cells

NASA Astrophysics Data System (ADS)

Sun, Bingyun; Chiu, Daniel T.

2003-11-01

This paper describes the development of using individual micro and nano meter-sized vesicles as delivery vessels to functionally map the distribution of cell surface proteins at the level of single cells. The formation of different sizes of vesicles from tens of nanometers to a few micrometers in diameter that contain the desired molecules is addressed. An optical trap is used to manipulate the loaded vesicle to specific cell morphology of interest, and a pulsed UV laser is used to photo-release the stimuli onto the cell membrane. Carbachol activated cellular calcium flux, upon binding to muscarinic acetylcholine receptors, is studied by this method, and the potential of using this method for the functional mapping of localized proteins on the cell surface membrane is discussed.

Multiple-Localization and Hub Proteins

PubMed Central

Ota, Motonori; Gonja, Hideki; Koike, Ryotaro; Fukuchi, Satoshi

2016-01-01

Protein-protein interactions are fundamental for all biological phenomena, and protein-protein interaction networks provide a global view of the interactions. The hub proteins, with many interaction partners, play vital roles in the networks. We investigated the subcellular localizations of proteins in the human network, and found that the ones localized in multiple subcellular compartments, especially the nucleus/cytoplasm proteins (NCP), the cytoplasm/cell membrane proteins (CMP), and the nucleus/cytoplasm/cell membrane proteins (NCMP), tend to be hubs. Examinations of keywords suggested that among NCP, those related to post-translational modifications and transcription functions are the major contributors to the large number of interactions. These types of proteins are characterized by a multi-domain architecture and intrinsic disorder. A survey of the typical hub proteins with prominent numbers of interaction partners in the type revealed that most are either transcription factors or co-regulators involved in signaling pathways. They translocate from the cytoplasm to the nucleus, triggered by the phosphorylation and/or ubiquitination of intrinsically disordered regions. Among CMP and NCMP, the contributors to the numerous interactions are related to either kinase or ubiquitin ligase activity. Many of them reside on the cytoplasmic side of the cell membrane, and act as the upstream regulators of signaling pathways. Overall, these hub proteins function to transfer external signals to the nucleus, through the cell membrane and the cytoplasm. Our analysis suggests that multiple-localization is a crucial concept to characterize groups of hub proteins and their biological functions in cellular information processing. PMID:27285823

Variable-angle epifluorescence microscopy characterizes protein dynamics in the vicinity of plasma membrane in plant cells.

PubMed

Chen, Tong; Ji, Dongchao; Tian, Shiping

2018-03-14

The assembly of protein complexes and compositional lipid patterning act together to endow cells with the plasticity required to maintain compositional heterogeneity with respect to individual proteins. Hence, the applications for imaging protein localization and dynamics require high accuracy, particularly at high spatio-temporal level. We provided experimental data for the applications of Variable-Angle Epifluorescence Microscopy (VAEM) in dissecting protein dynamics in plant cells. The VAEM-based co-localization analysis took penetration depth and incident angle into consideration. Besides direct overlap of dual-color fluorescence signals, the co-localization analysis was carried out quantitatively in combination with the methodology for calculating puncta distance and protein proximity index. Besides, simultaneous VAEM tracking of cytoskeletal dynamics provided more insights into coordinated responses of actin filaments and microtubules. Moreover, lateral motility of membrane proteins was analyzed by calculating diffusion coefficients and kymograph analysis, which represented an alternative method for examining protein motility. The present study presented experimental evidence on illustrating the use of VAEM in tracking and dissecting protein dynamics, dissecting endosomal dynamics, cell structure assembly along with membrane microdomain and protein motility in intact plant cells.

Bovine adenovirus 3 core protein precursor pVII localizes to mitochondria, and modulates ATP synthesis, mitochondrial Ca2+ and mitochondrial membrane potential.

PubMed

Anand, Sanjeev K; Gaba, Amit; Singh, Jaswant; Tikoo, Suresh K

2014-02-01

Viruses modulate the functions of mitochondria by translocating viral proteins to the mitochondria. Subcellular fractionation and sensitivity to proteinase K/Triton X-100 treatment of mitochondrial fractions of bovine adenovirus (BAdV)-3-infected/transfected cells suggested that core protein pVII localizes to the mitochondria and contains a functional mitochondrial localization signal. Moreover, mitochondrial localization of BAdV-3 pVII appears to help in the retention of mitochondrial Ca(2+), inducing a significant increase in the levels of ATP and maintaining the mitochondrial membrane potential (MMP) in transfected cells. In contrast, mitochondrial localization of BAdV-3 pVII has no significant effect on the levels of cytoplasmic Ca(2+) and reactive oxygen species production in the transfected cells. Consistent with these results, expression of pVII in transfected cells treated with staurosporine decreased significantly the activation of caspase-3. Our results suggested that BAdV-3 pVII localizes to mitochondria, and interferes with apoptosis by inhibiting loss of the MMP and by increasing mitochondrial Ca(2+) and ATP production.

A Novel Plasma Membrane-Anchored Protein Regulates Xylem Cell-Wall Deposition through Microtubule-Dependent Lateral Inhibition of Rho GTPase Domains.

PubMed

Sugiyama, Yuki; Wakazaki, Mayumi; Toyooka, Kiminori; Fukuda, Hiroo; Oda, Yoshihisa

2017-08-21

Spatial control of cell-wall deposition is essential for determining plant cell shape [1]. Rho-type GTPases, together with the cortical cytoskeleton, play central roles in regulating cell-wall patterning [2]. In metaxylem vessel cells, which are the major components of xylem tissues, active ROP11 Rho GTPases form oval plasma membrane domains that locally disrupt cortical microtubules, thereby directing the formation of oval pits in secondary cell walls [3-5]. However, the regulatory mechanism that determines the planar shape of active Rho of Plants (ROP) domains is still unknown. Here we show that IQD13 associates with cortical microtubules and the plasma membrane to laterally restrict the localization of ROP GTPase domains, thereby directing the formation of oval secondary cell-wall pits. Loss and overexpression of IQD13 led to the formation of abnormally round and narrow secondary cell-wall pits, respectively. Ectopically expressed IQD13 increased the presence of parallel cortical microtubules by promoting microtubule rescue. A reconstructive approach revealed that IQD13 confines the area of active ROP domains within the lattice of the cortical microtubules, causing narrow ROP domains to form. This activity required the interaction of IQD13 with the plasma membrane. These findings suggest that IQD13 positively regulates microtubule dynamics as well as their linkage to the plasma membrane, which synergistically confines the area of active ROP domains, leading to the formation of oval secondary cell-wall pits. This finding sheds light on the role of microtubule-plasma membrane linkage as a lateral fence that determines the planar shape of Rho GTPase domains. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sphingolipid topology and the dynamic organization and function of membrane proteins.

PubMed

van Meer, Gerrit; Hoetzl, Sandra

2010-05-03

When acquiring internal membranes and vesicular transport, eukaryotic cells started to synthesize sphingolipids and sterols. The physical differences between these and the glycerophospholipids must have enabled the cells to segregate lipids in the membrane plane. Localizing this event to the Golgi then allowed them to create membranes of different lipid composition, notably a thin, flexible ER membrane, consisting of glycerolipids, and a sturdy plasma membrane containing at least 50% sphingolipids and sterols. Besides sorting membrane proteins, in the course of evolution the simple sphingolipids obtained key positions in cellular physiology by developing specific interactions with (membrane) proteins involved in the execution and control of signaling. The few signaling sphingolipids in mammals must provide basic transmission principles that evolution has built upon for organizing the specific regulatory pathways tuned to the needs of the different cell types in the body. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

LE COMPLEXE MEMBRANAIRE SUPERFICIEL ET SON EVOLUTION LORS DE L'ELABORATION DES INDIVIDUS-FILS CHEZ TOXOPLASMA GONDII

PubMed Central

Vivier, Emile; Petitprez, André

1969-01-01

The parasitic protozoan Toxoplasma gondii has been examined with the electron microscope in order to study the fine structure and the formation of the membranes surrounding the cell. The study of the ultrastructure of the membranes covering the parasite shows the existence of a three-membraned complex. Only the outer membrane is considered to be the plasma membrane; the two membranes below it form an inseparable whole of changeable molecular architecture (modifications in appearance depending on the methods of fixation, local differentiation). During reproduction, which takes place by fission or more often by endogeny, the membranes of the daughter individuals are formed from the membranes of the parent. At first the middle and inner membranes of the parent extend, separating the cytoplasm of the daughter cells from that of the parent. The three-membrane complex of the endozoites is completed at the time of their liberation; the external membrane of the parent covers the leaving endozoites; thus, the plasma membrane of the daughter cells derives also from that of the parent. These findings on the origin and role of limiting membranes during reproduction differ entirely from those described so far for other cells. PMID:5344151

Relative quantification of membrane-associated calcium in red spruce mesophyll cells

Treesearch

Catherine H. Borer; Paul Schaberg; Jonathan R. Cumming

1997-01-01

We describe a method for localizing and comparing relative amounts of plasma membrane-associated calcium ions (mCa) in complex tissues and verify the procedure for mesophyll cells of red spruce (Picea rubens Sarg.) needles. This technique incorporates epifluorescence microscopy using the fluorescent probe chlorotetracycline (CTC) with computer image...

Rac1-mediated membrane raft localization of PI3K/p110β is required for its activation by GPCRs or PTEN loss

PubMed Central

Cizmecioglu, Onur; Ni, Jing; Xie, Shaozhen; Zhao, Jean J; Roberts, Thomas M

2016-01-01

We aimed to understand how spatial compartmentalization in the plasma membrane might contribute to the functions of the ubiquitous class IA phosphoinositide 3-kinase (PI3K) isoforms, p110α and p110β. We found that p110β localizes to membrane rafts in a Rac1-dependent manner. This localization potentiates Akt activation by G-protein-coupled receptors (GPCRs). Thus genetic targeting of a Rac1 binding-deficient allele of p110β to rafts alleviated the requirement for p110β-Rac1 association for GPCR signaling, cell growth and migration. In contrast, p110α, which does not play a physiological role in GPCR signaling, is found to reside in nonraft regions of the plasma membrane. Raft targeting of p110α allowed its EGFR-mediated activation by GPCRs. Notably, p110β dependent, PTEN null tumor cells critically rely upon raft-associated PI3K activity. Collectively, our findings provide a mechanistic account of how membrane raft localization regulates differential activation of distinct PI3K isoforms and offer insight into why PTEN-deficient cancers depend on p110β. DOI: http://dx.doi.org/10.7554/eLife.17635.001 PMID:27700986

Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stall, Richard; Ramos, Joseph; Kent Fulcher, F.

Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated thatmore » MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin.« less

Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement

PubMed Central

Sasaki, Atsuo T.; Chun, Cheryl; Takeda, Kosuke; Firtel, Richard A.

2004-01-01

During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P3, the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P3 asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal. PMID:15534002

MHC Class II and CD9 in human eosinophils localize to detergent-resistant membrane microdomains.

PubMed

Akuthota, Praveen; Melo, Rossana C N; Spencer, Lisa A; Weller, Peter F

2012-02-01

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor-stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-β-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR-containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4(+) T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils.

MHC Class II and CD9 in Human Eosinophils Localize to Detergent-Resistant Membrane Microdomains

PubMed Central

Akuthota, Praveen; Melo, Rossana C. N.; Spencer, Lisa A.

2012-01-01

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor–stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-β-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR–containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4+ T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils. PMID:21885678

Localization of aPKC lambda/iota and its interacting protein, Lgl2, is significantly associated with lung adenocarcinoma progression.

PubMed

Imamura, Naoko; Horikoshi, Yosuke; Matsuzaki, Tomohiko; Toriumi, Kentaro; Kitatani, Kanae; Ogura, Go; Masuda, Ryota; Nakamura, Naoya; Takekoshi, Susumu; Iwazaki, Masayuki

2013-12-20

Atypical protein kinase C lambda/iota (aPKC λ/ι) is expressed in several human cancers; however, the correlation between aPKC λ/ι localization and cancer progression in human lung adenocarcinoma (LAC) remains to be clarified. We found that patients with a high level of aPKC λ/ι expression in LAC had significantly shorter overall survival than those with a low level of aPKC λ/ι expression. In addition, localization of aPKC λ/ι in the apical membrane or at the cell-cell contact was associated with both lymphatic invasion and metastasis. The intercellular adhesion molecule, E-cadherin, was decreased in LACs with highly expressed aPKC λ/ι at the invasion site of tumor cells. This result suggested that the expression levels of aPKC λ/ι and E-cadherin reflect the progression of LAC. On double-immunohistochemical analysis, aPKC λ/ι and Lgl2, a protein that interacts with aPKC λ/ι, were co-localized within LACs. Furthermore, we found that Lgl2 bound the aPKC λ/ι-Par6 complex in tumor tissue by immune-cosedimentation analysis. Apical membrane localization of Lgl2 was correlated with lymphatic invasion and lymph node metastasis. These results thus indicate that aPKC λ/ι expression is altered upon the progression of LAC. This is also the first evidence to show aPKC λ/ι overexpression in LAC and demonstrates that aPKC λ/ι localization at the apical membrane or cell-cell contact is associated with lymphatic invasion and metastasis of the tumor.

Arf and Rho GAP adapter protein ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane.

PubMed

Símová, Sárka; Klíma, Martin; Cermak, Lukas; Sourková, Vladimíra; Andera, Ladislav

2008-03-01

TRAIL, a ligand of the TNFalpha family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis.

Vascularization and Cellular Isolation Potential of a Novel Electrospun Cell Delivery Vehicle

PubMed Central

Krishnan, Laxminarayanan; Touroo, Jeremy; Reed, Robert; Boland, Eugene; Hoying, James B.; Williams, Stuart K.

2014-01-01

A clinical need exists for a cell delivery device that supports long term cell viability, cell retention within the device and retrieval of delivered cells if necessary. Previously, cell isolation devices have been based on hollow fiber membranes, porous polymer scaffolds, alginate systems, or micro-machined membranes. We present the development and characterization of a novel dual porosity electrospun membrane based device, which supports cellular infiltration and vascularization of its outer porous layer and maintains cellular isolation within a lumen bounded by an inner low porosity layer. Electrospinning conditions were initially established to support electrospun fiber deposition onto nonconductive silicone surfaces. With these parameters established, devices for in vivo evaluations were produced using nylon as a nonconductive scaffold for deposition of dual porosity electrospun fibers. The outer porous layer supported the development of a penetrating microcirculation and the membrane supported the transfer of insulin from encapsulated sustained release pellets for four weeks. Viable cells implanted within the device could be identified after two weeks of implantation. Through the successful demonstration of survival and cellular isolation of human epithelial cells within the implanted devices and the ability to use the device to deliver insulin, we have established the utility of this device toward localized cell transplantation. The Cell Delivery Device establishes a platform to test the feasibility of approaches to cell dose control and cell localization at the site of implantation in the clinical use of modified autologous or allogeneic cells. PMID:23913805

Developmental Localization of Nephrin in Zebrafish and Medaka Pronephric Glomerulus

PubMed Central

Ichimura, Koichiro; Fukuyo, Yayoi; Nakamura, Tomomi; Powell, Rebecca; Sakai, Tatsuo; Janknecht, Ralf

2013-01-01

Slit diaphragm (SD) is a highly specialized intercellular junction between podocyte foot processes and plays a crucial role in the formation of the filtration barrier. In this study, we examined the developmental localization of Nephrin, an essential component of SD, in the pronephric glomerulus of zebrafish and medaka. In the mature glomerulus of both fish, Nephrin is localized along the glomerular basement membrane as seen in mammals, indicating that Nephrin is localized at the SD. Interestingly, Nephrin was detected already in immature podocytes before the SD and foot processes started to form in both fish. Nephrin was localized along the cell surface of immature podocytes but as different localization patterns. In zebrafish, Nephrin signal bordered the lateral membrane of podocytes, which were columnar in shape, as in rat immature podocytes. However, in medaka immature podocytes, Nephrin was localized in a punctate pattern among podocyte cell bodies. These findings suggest that Nephrin needs to be integrated to the membrane before the formation of the SD and then moves to the proper site to form the SD. Furthermore, a podocyte-specific marker, such as Nephrin, should be a useful tool for the future analysis of pronephric glomerular development in fish mutants and morphants. PMID:23324868

G protein-coupled estrogen receptor 1/G protein-coupled receptor 30 localizes in the plasma membrane and traffics intracellularly on cytokeratin intermediate filaments.

PubMed

Sandén, Caroline; Broselid, Stefan; Cornmark, Louise; Andersson, Krister; Daszkiewicz-Nilsson, Joanna; Mårtensson, Ulrika E A; Olde, Björn; Leeb-Lundberg, L M Fredrik

2011-03-01

G protein-coupled receptor 30 [G protein-coupled estrogen receptor 1 (GPER1)], has been introduced as a membrane estrogen receptor and a candidate cancer biomarker and therapeutic target. However, several questions surround the subcellular localization and signaling of this receptor. In native cells, including mouse myoblast C(2)C(12) cells, Madin-Darby canine kidney epithelial cells, and human ductal breast epithelial tumor T47-D cells, G-1, a GPER1 agonist, and 17β-estradiol stimulated GPER1-dependent cAMP production, a defined plasma membrane (PM) event, and recruitment of β-arrestin2 to the PM. Staining of fixed and live cells showed that GPER1 was localized both in the PM and on intracellular structures. One such intracellular structure was identified as cytokeratin (CK) intermediate filaments, including those composed of CK7 and CK8, but apparently not endoplasmic reticulum, Golgi, or microtubules. Reciprocal coimmunoprecipitation of GPER1 and CKs confirmed an association of these proteins. Live staining also showed that the PM receptors constitutively internalize apparently to reach CK filaments. Receptor localization was supported using FLAG- and hemagglutinin-tagged GPER1. We conclude that GPER1-mediated stimulation of cAMP production and β-arrestin2 recruitment occur in the PM. Furthermore, the PM receptors constitutively internalize and localize intracellularly on CK. This is the first observation that a G protein-coupled receptor is capable of associating with intermediate filaments, which may be important for GPER1 regulation in epithelial cells and the relationship of this receptor to cancer.

Exclusive photorelease of signalling lipids at the plasma membrane.

PubMed

Nadler, André; Yushchenko, Dmytro A; Müller, Rainer; Stein, Frank; Feng, Suihan; Mulle, Christophe; Carta, Mario; Schultz, Carsten

2015-12-21

Photoactivation of caged biomolecules has become a powerful approach to study cellular signalling events. Here we report a method for anchoring and uncaging biomolecules exclusively at the outer leaflet of the plasma membrane by employing a photocleavable, sulfonated coumarin derivative. The novel caging group allows quantifying the reaction progress and efficiency of uncaging reactions in a live-cell microscopy setup, thereby greatly improving the control of uncaging experiments. We synthesized arachidonic acid derivatives bearing the new negatively charged or a neutral, membrane-permeant coumarin caging group to locally induce signalling either at the plasma membrane or on internal membranes in β-cells and brain slices derived from C57B1/6 mice. Uncaging at the plasma membrane triggers a strong enhancement of calcium oscillations in β-cells and a pronounced potentiation of synaptic transmission while uncaging inside cells blocks calcium oscillations in β-cells and causes a more transient effect on neuronal transmission, respectively. The precise subcellular site of arachidonic acid release is therefore crucial for signalling outcome in two independent systems.

Chronic shear induces caveolae formation and alters ERK and Akt responses in endothelial cells

NASA Technical Reports Server (NTRS)

Boyd, Nolan L.; Park, Heonyong; Yi, Hong; Boo, Yong Chool; Sorescu, George P.; Sykes, Michelle; Jo, Hanjoong

2003-01-01

Caveolae are plasmalemmal domains enriched with cholesterol, caveolins, and signaling molecules. Endothelial cells in vivo are continuously exposed to shear conditions, and their caveolae density and location may be different from that of static cultured cells. Here, we show that chronic shear exposure regulates formation and localization of caveolae and caveolin-1 in bovine aortic endothelial cells (BAEC). Chronic exposure (1 or 3 days) of BAEC to laminar shear increased the total number of caveolae by 45-48% above static control. This increase was due to a rise in the luminal caveolae density without changing abluminal caveolae numbers or increasing caveolin-1 mRNA and protein levels. Whereas some caveolin-1 was found in the plasma membrane in static-cultured cells, it was predominantly localized in the Golgi. In contrast, chronic shear-exposed cells showed intense caveolin-1 staining in the luminal plasma membrane with minimum Golgi association. The preferential luminal localization of caveolae may play an important role in endothelial mechanosensing. Indeed, we found that chronic shear exposure (preconditioning) altered activation patterns of two well-known shear-sensitive signaling molecules (ERK and Akt) in response to a step increase in shear stress. ERK activation was blunted in shear preconditioned cells, whereas the Akt response was accelerated. These results suggest that chronic shear stimulates caveolae formation by translocating caveolin-1 from the Golgi to the luminal plasma membrane and alters cell signaling responses.

Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

PubMed

Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

2016-05-01

Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. © FASEB.

Membrane anchoring of aminoacyl-tRNA synthetases by convergent acquisition of a novel protein domain.

PubMed

Olmedo-Verd, Elvira; Santamaría-Gómez, Javier; Ochoa de Alda, Jesús A G; Ribas de Pouplana, Lluis; Luque, Ignacio

2011-11-25

Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl-, and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process also dependent on the CAAD domain.

A Novel Type III Endosome Transmembrane Protein, TEMP

PubMed Central

Aturaliya, Rajith N.; Kerr, Markus C.; Teasdale, Rohan D.

2012-01-01

As part of a high-throughput subcellular localisation project, the protein encoded by the RIKEN mouse cDNA 2610528J11 was expressed and identified to be associated with both endosomes and the plasma membrane. Based on this, we have assigned the name TEMP for Type III Endosome Membrane Protein. TEMP encodes a short protein of 111 amino acids with a single, alpha-helical transmembrane domain. Experimental analysis of its membrane topology demonstrated it is a Type III membrane protein with the amino-terminus in the lumenal, or extracellular region, and the carboxy-terminus in the cytoplasm. In addition to the plasma membrane TEMP was localized to Rab5 positive early endosomes, Rab5/Rab11 positive recycling endosomes but not Rab7 positive late endosomes. Video microscopy in living cells confirmed TEMP’s plasma membrane localization and identified the intracellular endosome compartments to be tubulovesicular. Overexpression of TEMP resulted in the early/recycling endosomes clustering at the cell periphery that was dependent on the presence of intact microtubules. The cellular function of TEMP cannot be inferred based on bioinformatics comparison, but its cellular distribution between early/recycling endosomes and the plasma membrane suggests a role in membrane transport. PMID:24710541

Ultrastructure of Bacterial Cells Infected with Bacteriophage PM2, a Lipid-containing Bacterial Virus

PubMed Central

Cota-Robles, Eugene; Espejo, Romilio Torres; Haywood, Patricia Williams

1968-01-01

The cytological pattern of infection of a host pseudomonad with PM2, a lipid-containing bacterial virus, was investigated by electron microscopy. Normal and infected cells frequently contain a myelin figure, which is found in the nucleoid region or at the periphery of the cell. The most striking finding in this investigation was that completed virions are found in the cell adjacent to or in association with the cytoplasmic membrane. This localization is precise; virions are not found elsewhere in infected cells. The completed virions occasionally appear to be attached to the cytoplasmic membrane. The virus contains a darkly staining core surrounded by a tripartite envelope of a thickness of approximately 70 A, which is identical to the thickness of the cytoplasmic membrane. Lysing cells appear to undergo extensive damage of the cytoplasmic membrane prior to rupture of the L layer of the cell wall. Images PMID:5742028

Calcium signaling and cell proliferation.

PubMed

Pinto, Mauro Cunha Xavier; Kihara, Alexandre Hiroaki; Goulart, Vânia A M; Tonelli, Fernanda M P; Gomes, Katia N; Ulrich, Henning; Resende, Rodrigo R

2015-11-01

Cell proliferation is orchestrated through diverse proteins related to calcium (Ca(2+)) signaling inside the cell. Cellular Ca(2+) influx that occurs first by various mechanisms at the plasma membrane, is then followed by absorption of Ca(2+) ions by mitochondria and endoplasmic reticulum, and, finally, there is a connection of calcium stores to the nucleus. Experimental evidence indicates that the fluctuation of Ca(2+) from the endoplasmic reticulum provides a pivotal and physiological role for cell proliferation. Ca(2+) depletion in the endoplasmatic reticulum triggers Ca(2+) influx across the plasma membrane in an phenomenon called store-operated calcium entries (SOCEs). SOCE is activated through a complex interplay between a Ca(2+) sensor, denominated STIM, localized in the endoplasmic reticulum and a Ca(2+) channel at the cell membrane, denominated Orai. The interplay between STIM and Orai proteins with cell membrane receptors and their role in cell proliferation is discussed in this review. Copyright © 2015 Elsevier Inc. All rights reserved.

Targeting of GLUT1-GLUT5 chimeric proteins in the polarized cell line Caco-2.

PubMed

Inukai, K; Takata, K; Asano, T; Katagiri, H; Ishihara, H; Nakazaki, M; Fukushima, Y; Yazaki, Y; Kikuchi, M; Oka, Y

1997-04-01

Caco-2, a human differentiated intestinal epithelial cell line, is a promising model for investigating the mechanism of polarized targeting of apical and basolateral membrane proteins. We stably transfected rat GLUT5 cDNA and rabbit GLUT1 cDNA into Caco-2 cells with an expression vector. Immunohistochemical study revealed that the GLUT5 protein expressed was localized at apical membranes and that the GLUT1 expressed was present primarily in the basolateral membranes of cells grown on permeable support. Next, to investigate the domain responsible for determining apical vs. basolateral sorting in glucose transporters, we prepared several GLUT1-GLUT5 chimeric cDNAs and transfected them into Caco-2 cells. A GLUT1 [N terminus approximately sixth transmembrane domain (TM6)]-GLUT5 [intracellular loop (IL) approximately C terminus] chimera was observed exclusively at the apical membrane, while GLUT1 (N terminus approximately IL)-GLUT5 (TM7 approximately C terminus) and GLUT1 (N terminus approximately TM12)-GLUT5 (C-terminal domain) chimeras were observed mainly at the basolateral membrane, a localization similar to that of GLUT1. Moreover, using a recombinant adenovirus expression system, we expressed a GLUT5 (N terminus approximately TM6)-GLUT1(IL)-GLUT5(TM7 approximately C-terminus) chimera, which was observed at the basolateral membrane. Based on these results, the C-terminal domain does not determine isoform-specific targeting of GLUT1 and GLUT5. Rather, it is the intracellular loop in glucose transporters that appears to play a pivotal role in apical-basolateral sorting signals in Caco-2 cells.

Completion of cytokinesis in C. elegans requires a brefeldin A-sensitive membrane accumulation at the cleavage furrow apex

PubMed Central

Skop, Ahna R.; Bergmann, Dominique; Mohler, William A.; White, John G.

2013-01-01

Background The terminal phase of cytokinesis in eukaryotic cells involves breakage of the intercellular canal containing the spindle midzone and resealing of the daughter cells. Recent observations suggest that the spindle midzone is required for this process. In this study, we investigated the possibility that targeted secretion in the vicinity of the spindle midzone is required for the execution of the terminal phase of cytokinesis. Results We inhibited secretion in early C. elegans embryos by treatment with brefeldin A (BFA). Using 4D recordings of dividing cells, we showed that BFA induced stereotyped failures in the terminal phase of cytokinesis; although the furrow ingressed normally, after a few minutes the furrow completely regressed, even though spindle midzone and midbody microtubules appeared normal. In addition, using an FM1-43 membrane probe, we found that membrane accumulated locally at the apices of the late cleavage furrows that form the persisting intercellular canals between daughter cells. However, in BFA-treated embryos this membrane accumulation did not occur, which possibly accounts for the observed cleavage failures. Conclusions We have shown that BFA disrupts the terminal phase of cytokinesis in the embryonic blastomeres of C. elegans. We observed that membrane accumulates at the apices of the late cleavage furrow by means of a BFA-sensitive mechanism. We suggest that this local membrane accumulation is necessary for the completion of cytokinesis and speculate that the spindle midzone region of animal cells is functionally equivalent to the phragmoplast of plants and acts to target secretion to the equatorial plane of a cleaving cell. PMID:11378383

Altered Subcellular Localization of a Tobacco Membrane Raft-Associated Remorin Protein by Tobamovirus Infection and Transient Expression of Viral Replication and Movement Proteins

PubMed Central

Sasaki, Nobumitsu; Takashima, Eita; Nyunoya, Hiroshi

2018-01-01

Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexvirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by tomato mosaic virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamovirus movement. PMID:29868075

A Comparison of Water Diffusion in Polymer Based Fuel Cell and Reverse Osmosis Membrane Materials

NASA Astrophysics Data System (ADS)

Soles, Christopher; Frieberg, Bradley; Tarver, Jacob; Tyagi, Madhusudan; Jeong, Cheol; Chan, Edwin; Stafford, Christopher

Hydrated polymer membranes are critical in both fuel cells and water filtration and desalination. In both of these applications the membrane function (selectively transporting or separating ions) is coupled with the transport of water through the membrane. There is a significant need to understand the nature by which the water and ions distribute and move through these membranes. This presentation compares the transport mechanisms in in an ion containing block copolymer alkaline fuel cell membrane with that of a polyamide membrane that is used as the active layer in a reverse osmosis water desalination membrane. Small angle neutron scattering measurements are used to locally probe how water swells the different materials and quantitatively describe the distribution of water within the membrane microstructures. Quasielastic neutron scattering measurements are then used to separate the polymer dynamics of the host membranes from the dynamics of the water inside the membranes. This reveals that water moves at least an order of magnitude slower through the ion containing fuel cell membrane materials, consistent with a solution-diffusion model, while the water in the polyamide membranes moves faster, consistent with a pore-flow diffusion mechanism. These insights will be discussed in terms of a coupling of the water and polymer dynamics and design cues for high performance membrane materials.

Chemo-spectroscopic sensor for carboxyl terminus overexpressed in carcinoma cell membrane.

PubMed

Stanca, Sarmiza E; Matthäus, Christian; Neugebauer, Ute; Nietzsche, Sandor; Fritzsche, Wolfgang; Dellith, Jan; Heintzmann, Rainer; Weber, Karina; Deckert, Volker; Krafft, Christoph; Popp, Jürgen

2015-10-01

Certain carboxyl groups of the plasma membrane are involved in tumorgenesis processes. A gold core-hydroxyapatite shell (AuHA) nanocomposite is introduced as chemo-spectroscopic sensor to monitor these carboxyl groups of the cell membrane. Hydroxyapatite (HA) plays the role both of a chemical detector and of a biocompatible Raman marker. The principle of detection is based on chemical interaction between the hydroxyl groups of the HA and the carboxyl terminus of the proteins. The AuHA exhibits a surface enhanced Raman scattering (SERS) signal at 954 cm(-1) which can be used for its localization. The bio-sensing capacity of AuHA towards human skin epidermoid carcinoma (A431) and Chinese hamster ovary (CHO) cell lines is investigated using Raman microspectroscopic imaging. The localization of AuHA on cells is correlated with scanning electron microscopy, transmission electron microscopy and structured illumination fluorescence microscopy. This qualitative approach is a step towards a quantitative study of the proteins terminus. This method would enable further studies on the molecular profiling of the plasma membrane, in an attempt to provide accurate cell identification. Using a gold core-hydroxyapatite shell (AuHA) nanocomposite, the authors in this paper showed the feasibility of detecting and differentiating cell surface molecules by surface enhanced Raman scattering. Copyright © 2015 Elsevier Inc. All rights reserved.

Erythrocyte Membrane Failure by Electromechanical Stress.

PubMed

Du, E; Qiang, Yuhao; Liu, Jia

2018-01-01

We envision that electrodeformation of biological cells through dielectrophoresis as a new technique to elucidate the mechanistic details underlying membrane failure by electrical and mechanical stresses. Here we demonstrate the full control of cellular uniaxial deformation and tensile recovery in biological cells via amplitude-modified electric field at radio frequency by an interdigitated electrode array in microfluidics. Transient creep and cyclic experiments were performed on individually tracked human erythrocytes. Observations of the viscoelastic-to-viscoplastic deformation behavior and the localized plastic deformations in erythrocyte membranes suggest that electromechanical stress results in irreversible membrane failure. Examples of membrane failure can be separated into different groups according to the loading scenarios: mechanical stiffening, physical damage, morphological transformation from discocyte to echinocyte, and whole cell lysis. These results show that this technique can be potentially utilized to explore membrane failure in erythrocytes affected by other pathophysiological processes.

The N-terminal leucine-zipper motif in PTRF/cavin-1 is essential and sufficient for its caveolae-association

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Zhuang; Laboratory of System Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031; Zou, Xinle

2015-01-16

Highlight: • The N-terminal leucine-zipper motif in PTRF/cavin-1 determines caveolar association. • Different cellular localization of PTRF/cavin-1 influences its serine 389 and 391 phosphorylation state. • PTRF/cavin-1 regulates cell motility via its caveolar association. - Abstract: PTRF/cavin-1 is a protein of two lives. Its reported functions in ribosomal RNA synthesis and in caveolae formation happen in two different cellular locations: nucleus vs. plasma membrane. Here, we identified that the N-terminal leucine-zipper motif in PTRF/cavin-1 was essential for the protein to be associated with caveolae in plasma membrane. It could counteract the effect of nuclear localization sequence in the molecule (AAmore » 235–251). Deletion of this leucine-zipper motif from PTRF/cavin-1 caused the mutant to be exclusively localized in nuclei. The fusion of this leucine-zipper motif with histone 2A, which is a nuclear protein, could induce the fusion protein to be exported from nucleus. Cell migration was greatly inhibited in PTRF/cavin-1{sup −/−} mouse embryonic fibroblasts (MEFs). The inhibited cell motility could only be rescued by exogenous cavin-1 but not the leucine-zipper motif deleted cavin-1 mutant. Plasma membrane dynamics is an important factor in cell motility control. Our results suggested that the membrane dynamics in cell migration is affected by caveolae associated PTRF/cavin-1.« less

Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

PubMed

Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

2007-08-17

Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 < 1 min) between the plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

Segregation of two spectrin isoforms: polarized membrane-binding sites direct polarized membrane skeleton assembly.

PubMed

Dubreuil, R R; Maddux, P B; Grushko, T A; MacVicar, G R

1997-10-01

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and beta spectrin are recruited to sites of cell-cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (alpha beta H), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and alpha beta spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, alpha beta spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, alpha beta H spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell-cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells.

Exclusion of assembled MreB by anionic phospholipids at cell poles confers cell polarity for bidirectional growth.

PubMed

Kawazura, Takuma; Matsumoto, Kanon; Kojima, Koki; Kato, Fumiya; Kanai, Tomomi; Niki, Hironori; Shiomi, Daisuke

2017-05-01

Cell polarity determines the direction of cell growth in bacteria. MreB actin spatially regulates peptidoglycan synthesis to enable cells to elongate bidirectionally. MreB densely localizes in the cylindrical part of the rod cell and not in polar regions in Escherichia coli. When treated with A22, which inhibits MreB polymerization, rod-shaped cells became round and MreB was diffusely distributed throughout the cytoplasmic membrane. A22 removal resulted in restoration of the rod shape. Initially, diffuse MreB started to re-assemble, and MreB-free zones were subsequently observed in the cytoplasmic membrane. These MreB-free zones finally became cell poles, allowing the cells to elongate bidirectionally. When MreB was artificially located at the cell poles, an additional pole was created, indicating that artificial localization of MreB at the cell pole induced local peptidoglycan synthesis. It was found that the anionic phospholipids (aPLs), phosphatidylglycerol and cardiolipin, which were enriched in cell poles preferentially interact with monomeric MreB compared with assembled MreB in vitro. MreB tended to localize to cell poles in cells lacking both aPLs, resulting in production of Y-shaped cells. Their findings indicated that aPLs exclude assembled MreB from cell poles to establish cell polarity, thereby allowing cells to elongate in a particular direction. © 2017 John Wiley & Sons Ltd.

External push and internal pull forces recruit curvature sensing N-BAR domain proteins to the plasma membrane

PubMed Central

Galic, Milos; Jeong, Sangmoo; Tsai, Feng-Chiao; Joubert, Lydia-Marie; Wu, Yi I.; Hahn, Klaus M.; Cui, Yi; Meyer, Tobias

2012-01-01

Many of the more than 20 mammalian proteins with N-BAR domains1-2 control cell architecture3 and endocytosis4-5 by associating with curved sections of the plasma membrane (PM)6. It is not well understood whether N-BAR proteins are recruited directly by processes that mechanically curve the PM or indirectly by PM-associated adaptor proteins that recruit proteins with N-BAR domains that then induce membrane curvature. Here, we show that externally-induced inward deformation of the PM by cone-shaped nanostructures (Nanocones) and internally-induced inward deformation by contracting actin cables both trigger recruitment of isolated N-BAR domains to the curved PM. Markedly, live-cell imaging in adherent cells showed selective recruitment of full length N-BAR proteins and isolated N-BAR domains to PM sub-regions above Nanocone stripes. Electron microscopy confirmed that N-BAR domains are recruited to local membrane sites curved by Nanocones. We further showed that N-BAR domains are periodically recruited to curved PM sites during local lamellipodia retraction in the front of migrating cells. Recruitment required Myosin II-generated force applied to PM connected actin cables. Together, our study shows that N-BAR domains can be directly recruited to the PM by external push or internal pull forces that locally curve the PM. PMID:22750946

Localizing the Subunit Pool for the Temporally Regulated Polar Pili of Caulobacter crescentus.

DTIC Science & Technology

1987-01-01

was determined that the cellular location for un- assembled was the cell cytoplasm. All cell membranes and regions of muclear material were poorly...to colloidal gold. It was determined that the cellular location for unassembled pilin was the cell cytoplasm. All cell membranes and regions of nuclear...to determine the cellular location of the pilin pool. Because pilin is a small (8000 m.w. ) and hydrophobic molecule (3), problems with 3 non-specific

Contactless Stimulation and Control of Biomimetic Nanotubes by Calcium Ion Gradients.

PubMed

Kirejev, Vladimir; Ali Doosti, Baharan; Shaali, Mehrnaz; Jeffries, Gavin D M; Lobovkina, Tatsiana

2018-04-17

Membrane tubular structures are important communication pathways between cells and cellular compartments. Studying these structures in their native environment is challenging, due to the complexity of membranes and varying chemical conditions within and outside of the cells. This work demonstrates that a calcium ion gradient, applied to a synthetic lipid nanotube, triggers lipid flow directed toward the application site, resulting in the formation of a bulge aggregate. This bulge can be translated in a contactless manner by moving a calcium ion source along the lipid nanotube. Furthermore, entrapment of polystyrene nanobeads within the bulge does not tamper the bulge movement and allows transporting of the nanoparticle cargo along the lipid nanotube. In addition to the synthetic lipid nanotubes, the response of cell plasma membrane tethers to local calcium ion stimulation is investigated. The directed membrane transport in these tethers is observed, but with slower kinetics in comparison to the synthetic lipid nanotubes. The findings of this work demonstrate a novel and contactless mode of transport in lipid nanotubes, guided by local exposure to calcium ions. The observed lipid nanotube behavior can advance the current understanding of the cell membrane tubular structures, which are constantly reshaped during dynamic cellular processes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Uroplakins, specific membrane proteins of urothelial umbrella cells, as histological markers of metastatic transitional cell carcinomas.

PubMed Central

Moll, R.; Wu, X. R.; Lin, J. H.; Sun, T. T.

1995-01-01

Uroplakins (UPs) Ia, Ib, II, and III, transmembrane proteins constituting the asymmetrical unit membrane of urothelial umbrella cells, are the first specific urothelial differentiation markers described. We investigated the presence and localization patterns of UPs in various human carcinomas by applying immunohistochemistry (avidin-biotin-peroxidase complex method), using rabbit antibodies against UPs II and III, to paraffin sections. Positive reactions for UP III (sometimes very focal) were noted in 14 of the 16 papillary noninvasive transitional cell carcinomas (TCCs) (88%), 29 of the 55 invasive TCCs (53%), and 23 of the 35 TCC metastases (66%). Different localization patterns of UPs could be distinguished, including superficial membrane staining like that found in normal umbrella cells (in papillary carcinoma), luminal (microluminal) membrane staining (in papillary and invasive carcinoma), and, against expectations, peripheral membrane staining (in invasive carcinoma). Non-TCC carcinomas of various origins (n = 177) were consistently negative for UPs. The presence of UPs in metastatic TCCs represents a prime example of even advanced tumor progression being compatible with the (focal) expression of highly specialized differentiation repertoires. Although of only medium-grade sensitivity, UPs do seem to be highly specific urothelial lineage markers, thus operating up interesting histodiagnostic possibilities in cases of carcinoma metastases of uncertain origin. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:7485401

Mistargeting of a truncated Na-K-2Cl cotransporter in epithelial cells.

PubMed

Koumangoye, Rainelli; Omer, Salma; Delpire, Eric

2018-05-02

We recently reported the case of a young patient with multi-system failure carrying a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1. Heterologous expression studies in non-epithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using MDCK cells grown on glass coverslips, permeabilized support, and matrigel, we show that the fluorescently-tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na + /K + -ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 of Slc12a2. While the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.

Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells*

PubMed Central

An, Hyun Joo; Gip, Phung; Kim, Jaehan; Wu, Shuai; Park, Kun Wook; McVaugh, Cheryl T.; Schaffer, David V.; Bertozzi, Carolyn R.; Lebrilla, Carlito B.

2012-01-01

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation. PMID:22147732

A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

PubMed

Xu, Shuhua; Soroka, Carol J; Sun, An-Qiang; Backos, Donald S; Mennone, Albert; Suchy, Frederick J; Boyer, James L

2016-01-01

The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liberton, Michelle; Saha, Rajib; Jacobs, Jon M.

2016-04-07

Cyanobacteria are photosynthetic microbes with highly differentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes,more » many more proteins remain to be identified in these membrane systems, and a comprehensive catalog of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared to the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared to a more specialized role for the thylakoid membrane in cellular energetics. Overall, the protein composition of the Synechocystis 6803 plasma membrane and thylakoid membrane is quite similar to the E.coli plasma membrane and Arabidopsis thylakoid membrane, respectively. Synechocystis 6803 can therefore be described as a gram-negative bacterium that has an additional internal membrane system that fulfils the energetic requirements of the cell.« less

Real time monitoring of water distribution in an operando fuel cell during transient states

NASA Astrophysics Data System (ADS)

Martinez, N.; Peng, Z.; Morin, A.; Porcar, L.; Gebel, G.; Lyonnard, S.

2017-10-01

The water distribution of an operating proton exchange membrane fuel cell (PEMFC) was monitored in real time by using Small Angle Neutron Scattering (SANS). The formation of liquid water was obtained simultaneously with the evolution of the water content inside the membrane. Measurements were performed when changing current with a time resolution of 10 s, providing insights on the kinetics of water management prior to the stationary phase. We confirmed that water distribution is strongly heterogeneous at the scale at of the whole Membrane Electrode Assembly. As already reported, at the local scale there is no straightforward link between the amounts of water present inside and outside the membrane. However, we show that the temporal evolutions of these two parameters are strongly correlated. In particular, the local membrane water content is nearly instantaneously correlated to the total liquid water content, whether it is located at the anode or cathode side. These results can help in optimizing 3D stationary diphasic models used to predict PEMFC water distribution.

Resolving mixed mechanisms of protein subdiffusion at the T cell plasma membrane

NASA Astrophysics Data System (ADS)

Golan, Yonatan; Sherman, Eilon

2017-06-01

The plasma membrane is a complex medium where transmembrane proteins diffuse and interact to facilitate cell function. Membrane protein mobility is affected by multiple mechanisms, including crowding, trapping, medium elasticity and structure, thus limiting our ability to distinguish them in intact cells. Here we characterize the mobility and organization of a short transmembrane protein at the plasma membrane of live T cells, using single particle tracking and photoactivated-localization microscopy. Protein mobility is highly heterogeneous, subdiffusive and ergodic-like. Using mobility characteristics, we segment individual trajectories into subpopulations with distinct Gaussian step-size distributions. Particles of low-to-medium mobility consist of clusters, diffusing in a viscoelastic and fractal-like medium and are enriched at the centre of the cell footprint. Particles of high mobility undergo weak confinement and are more evenly distributed. This study presents a methodological approach to resolve simultaneous mixed subdiffusion mechanisms acting on polydispersed samples and complex media such as cell membranes.

Ras trafficking, localization and compartmentalized signalling

PubMed Central

Prior, Ian A.; Hancock, John F.

2012-01-01

Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes. PMID:21924373

Imaging the antimicrobial mechanism(s) of cathelicidin-2

PubMed Central

Schneider, Viktoria A. F.; Coorens, Maarten; Ordonez, Soledad R.; Tjeerdsma-van Bokhoven, Johanna L. M.; Posthuma, George; van Dijk, Albert; Haagsman, Henk P.; Veldhuizen, Edwin J. A.

2016-01-01

Host defence peptides (HDPs) have the potential to become alternatives to conventional antibiotics in human and veterinary medicine. The HDP chicken cathelicidin-2 (CATH-2) has immunomodulatory and direct killing activities at micromolar concentrations. In this study the mechanism of action of CATH-2 against Escherichia coli (E. coli) was investigated in great detail using a unique combination of imaging and biophysical techniques. Live-imaging with confocal fluorescence microscopy demonstrated that FITC-labelled CATH-2 mainly localized at the membrane of E. coli. Upon binding, the bacterial membrane was readily permeabilized as was shown by propidium iodide influx into the cell. Concentration- and time-dependent effects of the peptide on E. coli cells were examined by transmission electron microscopy (TEM). CATH-2 treatment was found to induce dose-dependent morphological changes in E. coli. At sub-minimal inhibitory concentrations (sub-MIC), intracellular granulation, enhanced vesicle release and wrinkled membranes were observed, while membrane breakage and cell lysis occurred at MIC values. These effects were visible within 1–5 minute of peptide exposure. Immuno-gold TEM showed CATH-2 binding to bacterial membranes. At sub-MIC values the peptide rapidly localized intracellularly without visible membrane permeabilization. It is concluded that CATH-2 has detrimental effects on E. coli at concentrations that do not immediately kill the bacteria. PMID:27624595

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters[S

PubMed Central

Sezgin, Erdinc; Can, Fatma Betul; Schneider, Falk; Clausen, Mathias P.; Galiani, Silvia; Stanly, Tess A.; Waithe, Dominic; Colaco, Alexandria; Honigmann, Alf; Wüstner, Daniel; Platt, Frances; Eggeling, Christian

2016-01-01

Cholesterol (Chol) is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. Thus, it is of utmost interest to develop tools for visualization of Chol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently labeled Chol analogs. Unfortunately, the introduction of the label may influence the characteristics of the analog, such as its localization, interaction, and trafficking in cells; hence, it is important to get knowledge of such bias. In this report, we compared different fluorescent lipid analogs for their performance in cellular assays: 1) plasma membrane incorporation, specifically the preference for more ordered membrane environments in phase-separated giant unilamellar vesicles and giant plasma membrane vesicles; 2) cellular trafficking, specifically subcellular localization in Niemann-Pick type C disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS)-based and super-resolution stimulated emission depletion-FCS-based measurements of membrane diffusion dynamics. The analogs exhibited strong differences, with some indicating positive performance in the membrane-based experiments and others in the intracellular trafficking assay. However, none showed positive performance in all assays. Our results constitute a concise guide for the careful use of fluorescent Chol analogs in visualizing cellular Chol dynamics. PMID:26701325

Expanded polyglutamine embedded in the endoplasmic reticulum causes membrane distortion and coincides with Bax insertion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ueda, Masashi; Li, Shimo; Itoh, Masanori

The endoplasmic reticulum (ER) is important in various cellular functions, such as secretary and membrane protein biosynthesis, lipid synthesis, and calcium storage. ER stress, including membrane distortion, is associated with many diseases such as Huntington's disease. In particular, nuclear envelope distortion is related to neuronal cell death associated with polyglutamine. However, the mechanism by which polyglutamine causes ER membrane distortion remains unclear. We used electron microscopy, fluorescence protease protection assay, and alkaline treatment to analyze the localization of polyglutamine in cells. We characterized polyglutamine embedded in the ER membrane and noted an effect on morphology, including the dilation of ERmore » luminal space and elongation of ER-mitochondria contact sites, in addition to the distortion of the nuclear envelope. The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. These results demonstrated that the ER membrane may be a target of polyglutamine, which triggers cell death through Bax. -- Highlights: •We characterized polyglutamine embedded in the ER membrane. •The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. •The ER membrane may be a target of polyglutamine, which triggers cell death.« less

Solubilization of human cells by the styrene-maleic acid copolymer: Insights from fluorescence microscopy.

PubMed

Dörr, Jonas M; van Coevorden-Hameete, Marleen H; Hoogenraad, Casper C; Killian, J Antoinette

2017-11-01

Extracting membrane proteins from biological membranes by styrene-maleic acid copolymers (SMAs) in the form of nanodiscs has developed into a powerful tool in membrane research. However, the mode of action of membrane (protein) solubilization in a cellular context is still poorly understood and potential specificity for cellular compartments has not been investigated. Here, we use fluorescence microscopy to visualize the process of SMA solubilization of human cells, exemplified by the immortalized human HeLa cell line. Using fluorescent protein fusion constructs that mark distinct subcellular compartments, we found that SMA solubilizes membranes in a concentration-dependent multi-stage process. While all major intracellular compartments were affected without a strong preference, plasma membrane solubilization was found to be generally slower than the solubilization of organelle membranes. Interestingly, some plasma membrane-localized proteins were more resistant against solubilization than others, which might be explained by their presence in specific membrane domains with differing properties. Our results support the general applicability of SMA for the isolation of membrane proteins from different types of (sub)cellular membranes. Copyright © 2017 Elsevier B.V. All rights reserved.

Hierarchy of stroma-derived factors in supporting growth of stroma-dependent hemopoietic cells: membrane-bound SCF is sufficient to confer stroma competence to epithelial cells.

PubMed

Friel, Jutta; Itoh, Katsuhiko; Bergholz, Ulla; Jücker, Manfred; Stocking, Carol; Harrison, Paul; Ostertag, Wolfram

2002-03-01

Hemopoiesis takes place in a microenvironment where hemopoietic cells are closely associated with stroma by various interactions. Stroma coregulates the proliferation and differentiation of hemopoietic cells. Stroma-hemopoietic-cell contact can be supported by locally produced membrane associated growth factors. The stroma derived growth factor, stem cell factor (SCF) is important in hemopoiesis. We examined the different biological interactions of membrane bound and soluble SCF with human hemopoietic cells expressing the SCF receptor, c-kit. To analyze the function of the SCF isoforms in inducing the proliferation of hemopoietic TF1 or Cord blood (CB) CD34+ cells we used stroma cell lines that differ in their presentation of no SCF, membrane SCF, or soluble SCF. We established a new coculture system using an epithelial cell line that excludes potential interfering effects with other known stroma encoded hemopoietic growth factors. We show that soluble SCF, in absence of membrane-bound SCF, inhibits long term clonal growth of primary or established CD34+ hemopoietic cells, whereas membrane-inserted SCF "dominantly" induces long term proliferation of these cells. We demonstrate a hierarchy of these SCF isoforms in the interaction of stroma with hemopoietic TF1 cells. Membrane-bound SCF is "dominant" over soluble SCF, whereas soluble SCF acts epistatically in interacting with hemopoietic cells compared with other stroma derived factors present in SCF deficient stroma. A hierarchy of stroma cell lines can be arranged according to their presentation of membrane SCF or soluble SCF. In our model system, membrane-bound SCF expression is sufficient to confer stroma properties to an epithelial cell line but soluble SCF does not.

A Concerted Action of Hepatitis C Virus P7 and Nonstructural Protein 2 Regulates Core Localization at the Endoplasmic Reticulum and Virus Assembly

PubMed Central

Boson, Bertrand; Granio, Ophélia; Bartenschlager, Ralf; Cosset, François-Loïc

2011-01-01

Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly. PMID:21814513

The SNARE Protein Syntaxin 3 Confers Specificity for Polarized Axonal Trafficking in Neurons

PubMed Central

Soo Hoo, Linda; Banna, Chris D.; Radeke, Carolyn M.; Sharma, Nikunj; Albertolle, Mary E.; Low, Seng Hui; Weimbs, Thomas; Vandenberg, Carol A.

2016-01-01

Cell polarity and precise subcellular protein localization are pivotal to neuronal function. The SNARE machinery underlies intracellular membrane fusion events, but its role in neuronal polarity and selective protein targeting remain unclear. Here we report that syntaxin 3 is involved in orchestrating polarized trafficking in cultured rat hippocampal neurons. We show that syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif, which causes mislocalization of syntaxin 3, results in coincident mistargeting of the axonal cargos neuron-glia cell adhesion molecule (NgCAM) and neurexin, but not transferrin receptor, a somatodendritic cargo. Similarly, RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 plays an important role in its targeting. Additionally, overexpression of syntaxin 3 results in increased axonal growth. Our findings suggest an important role for syntaxin 3 in maintaining neuronal polarity and in the critical task of selective trafficking of membrane protein to axons. PMID:27662481

The SNARE Protein Syntaxin 3 Confers Specificity for Polarized Axonal Trafficking in Neurons.

PubMed

Soo Hoo, Linda; Banna, Chris D; Radeke, Carolyn M; Sharma, Nikunj; Albertolle, Mary E; Low, Seng Hui; Weimbs, Thomas; Vandenberg, Carol A

Cell polarity and precise subcellular protein localization are pivotal to neuronal function. The SNARE machinery underlies intracellular membrane fusion events, but its role in neuronal polarity and selective protein targeting remain unclear. Here we report that syntaxin 3 is involved in orchestrating polarized trafficking in cultured rat hippocampal neurons. We show that syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif, which causes mislocalization of syntaxin 3, results in coincident mistargeting of the axonal cargos neuron-glia cell adhesion molecule (NgCAM) and neurexin, but not transferrin receptor, a somatodendritic cargo. Similarly, RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 plays an important role in its targeting. Additionally, overexpression of syntaxin 3 results in increased axonal growth. Our findings suggest an important role for syntaxin 3 in maintaining neuronal polarity and in the critical task of selective trafficking of membrane protein to axons.

Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane.

PubMed

Boassa, Daniela; Nguyen, Phuong; Hu, Junru; Ellisman, Mark H; Sosinsky, Gina E

2014-01-01

Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system.

Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed

NASA Astrophysics Data System (ADS)

Serebrovskaya, Ekaterina O.; Ryumina, Alina P.; Boulina, Maria E.; Shirmanova, Marina V.; Zagaynova, Elena V.; Bogdanova, Ekaterina A.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

2014-07-01

KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.

MreB filaments align along greatest principal membrane curvature to orient cell wall synthesis

PubMed Central

Szwedziak, Piotr; Wong, Felix; Schaefer, Kaitlin; Izoré, Thierry; Renner, Lars D; Holmes, Matthew J; Sun, Yingjie; Bisson-Filho, Alexandre W; Walker, Suzanne; Amir, Ariel; Löwe, Jan

2018-01-01

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape. PMID:29469806

Cooperative tumour cell membrane targeted phototherapy

NASA Astrophysics Data System (ADS)

Kim, Heegon; Lee, Junsung; Oh, Chanhee; Park, Ji-Ho

2017-06-01

The targeted delivery of therapeutics using antibodies or nanomaterials has improved the precision and safety of cancer therapy. However, the paucity and heterogeneity of identified molecular targets within tumours have resulted in poor and uneven distribution of targeted agents, thus compromising treatment outcomes. Here, we construct a cooperative targeting system in which synthetic and biological nanocomponents participate together in the tumour cell membrane-selective localization of synthetic receptor-lipid conjugates (SR-lipids) to amplify the subsequent targeting of therapeutics. The SR-lipids are first delivered selectively to tumour cell membranes in the perivascular region using fusogenic liposomes. By hitchhiking with extracellular vesicles secreted by the cells, the SR-lipids are transferred to neighbouring cells and further spread throughout the tumour tissues where the molecular targets are limited. We show that this tumour cell membrane-targeted delivery of SR-lipids leads to uniform distribution and enhanced phototherapeutic efficacy of the targeted photosensitizer.

Segregation of Two Spectrin Isoforms: Polarized Membrane-binding Sites Direct Polarized Membrane Skeleton Assembly

PubMed Central

Dubreuil, Ronald R.; Maddux, Pratumtip Boontrakulpoontawee; Grushko, Tanya A.; Macvicar, Gary R.

1997-01-01

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and β spectrin are recruited to sites of cell–cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (αβH), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and αβ spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, αβ spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, αβH spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell–cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells. PMID:9348534

High spatial resolution mass spectrometry imaging reveals the genetically programmed, developmental modification of the distribution of thylakoid membrane lipids among individual cells of maize leaf

DOE PAGES

Duenas, Maria Emilia; Klein, Adam T.; Alexander, Liza E.; ...

2016-11-17

Metabolism in plants is compartmentalized among different tissues, cells and subcellular organelles. Mass spectrometry imaging (MSI) with matrix-assisted laser desorption ionization (MALDI) has recently advanced to allow for the visualization of metabolites at single-cell resolution. Here we applied 5- and 10 μm high spatial resolution MALDI-MSI to the asymmetric Kranz anatomy of Zea mays (maize) leaves to study the differential localization of two major anionic lipids in thylakoid membranes, sulfoquinovosyldiacylglycerols (SQDG) and phosphatidylglycerols (PG). The quantification and localization of SQDG and PG molecular species, among mesophyll (M) and bundle sheath (BS) cells, are compared across the leaf developmental gradient frommore » four maize genotypes (the inbreds B73 and Mo17, and the reciprocal hybrids B73 × Mo17 and Mo17 × B73). SQDG species are uniformly distributed in both photosynthetic cell types, regardless of leaf development or genotype; however, PG shows photosynthetic cell-specific differential localization depending on the genotype and the fatty acyl chain constituent. Overall, 16:1-containing PGs primarily contribute to the thylakoid membranes of M cells, whereas BS chloroplasts are mostly composed of 16:0-containing PGs. Furthermore, PG 32:0 shows genotype-specific differences in cellular distribution, with preferential localization in BS cells for B73, but more uniform distribution between BS and M cells in Mo17. Maternal inheritance is exhibited within the hybrids, such that the localization of PG 32:0 in B73 × Mo17 is similar to the distribution in the B73 parental inbred, whereas that of Mo17 × B73 resembles the Mo17 parent. As a result, this study demonstrates the power of MALDI-MSI to reveal unprecedented insights on metabolic outcomes in multicellular organisms at single-cell resolution.« less

Polar transport in plants mediated by membrane transporters: focus on mechanisms of polar auxin transport.

PubMed

Naramoto, Satoshi

2017-12-01

Directional cell-to-cell transport of functional molecules, called polar transport, enables plants to sense and respond to developmental and environmental signals. Transporters that localize to plasma membranes (PMs) in a polar manner are key components of these systems. PIN-FORMED (PIN) auxin efflux carriers, which are the most studied polar-localized PM proteins, are implicated in the polar transport of auxin that in turn regulates plant development and tropic growth. In this review, the regulatory mechanisms underlying polar localization of PINs, control of auxin efflux activity, and PIN abundance at PMs are considered. Up to date information on polar-localized nutrient transporters that regulate directional nutrient movement from soil into the root vasculature is also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evidence that the modulation of membrane-associated protein kinase C activity by an endogenous inhibitor plays a role in N1E-115 murine neuroblastoma cell differentiation.

PubMed

Chakravarthy, B R; Wong, J; Durkin, J P

1995-10-01

Murine neuroblastoma cells, N1E-115, were induced to differentiate into neuron-like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro "direct" assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC-selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane-associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E-115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane-associated PKC activity in cells and thereby modulate PKC-related neuronal functions.

The N-Terminal Amphipathic Helix of the Topological Specificity Factor MinE Is Associated with Shaping Membrane Curvature

PubMed Central

Shih, Yu-Ling; Huang, Kai-Fa; Lai, Hsin-Mei; Liao, Jiahn-Haur; Lee, Chai-Siah; Chang, Chiao-Min; Mak, Huey-Ming; Hsieh, Cheng-Wei; Lin, Chu-Chi

2011-01-01

Pole-to-pole oscillations of the Min proteins in Escherichia coli are required for the proper placement of the division septum. Direct interaction of MinE with the cell membrane is critical for the dynamic behavior of the Min system. In vitro, this MinE-membrane interaction led to membrane deformation; however, the underlying mechanism remained unclear. Here we report that MinE-induced membrane deformation involves the formation of an amphipathic helix of MinE2–9, which, together with the adjacent basic residues, function as membrane anchors. Biochemical evidence suggested that the membrane association induces formation of the helix, with the helical face, consisting of A2, L3, and F6, inserted into the membrane. Insertion of this helix into the cell membrane can influence local membrane curvature and lead to drastic changes in membrane topology. Accordingly, MinE showed characteristic features of protein-induced membrane tubulation and lipid clustering in in vitro reconstituted systems. In conclusion, MinE shares common protein signatures with a group of membrane trafficking proteins in eukaryotic cells. These MinE signatures appear to affect membrane curvature. PMID:21738659

Membrane perturbing properties of toxin mycolactone from Mycobacterium ulcerans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez, Cesar A.; Unkefer, Clifford J.; Swanson, Basil I.

Mycolactone is the exotoxin produced by Mycobacterium ulcerans and is the virulence factor behind the neglected tropical disease Buruli ulcer. The toxin has a broad spectrum of biological effects within the host organism, stemming from its interaction with at least two molecular targets and the inhibition of protein uptake into the endoplasmic reticulum. Although it has been shown that the toxin can passively permeate into host cells, it is clearly lipophilic. Association with lipid carriers would have substantial implications for the toxin’s distribution within a host organism, delivery to cellular targets, diagnostic susceptibility, and mechanisms of pathogenicity. Yet the toxin’smore » interactions with, and distribution in, lipids are unknown. Herein we have used coarse-grained molecular dynamics simulations, guided by all-atom simulations, to study the interaction of mycolactone with pure and mixed lipid membranes. Using established techniques, we calculated the toxin’s preferential localization, membrane translocation, and impact on membrane physical and dynamical properties. The computed water-octanol partition coefficient indicates that mycolactone prefers to be in an organic phase rather than in an aqueous environment. Our results show that in a solvated membrane environment the exotoxin mainly localizes in the water-membrane interface, with a preference for the glycerol moiety of lipids, consistent with the reported studies that found it in lipid extracts of the cell. The calculated association constant to the model membrane is similar to the reported association constant for Wiskott-Aldrich syndrome protein. Mycolactone is shown to modify the physical properties of membranes, lowering the transition temperature, compressibility modulus, and critical line tension at which pores can be stabilized. It also shows a tendency to behave as a linactant, a molecule that localizes at the boundary between different fluid lipid domains in membranes and promotes inter-mixing of domains. This property has implications for the toxin’s cellular access, T-cell immunosuppression, and therapeutic potential.« less

Membrane perturbing properties of toxin mycolactone from Mycobacterium ulcerans

DOE PAGES

Lopez, Cesar A.; Unkefer, Clifford J.; Swanson, Basil I.; ...

2018-02-05

Mycolactone is the exotoxin produced by Mycobacterium ulcerans and is the virulence factor behind the neglected tropical disease Buruli ulcer. The toxin has a broad spectrum of biological effects within the host organism, stemming from its interaction with at least two molecular targets and the inhibition of protein uptake into the endoplasmic reticulum. Although it has been shown that the toxin can passively permeate into host cells, it is clearly lipophilic. Association with lipid carriers would have substantial implications for the toxin’s distribution within a host organism, delivery to cellular targets, diagnostic susceptibility, and mechanisms of pathogenicity. Yet the toxin’smore » interactions with, and distribution in, lipids are unknown. Herein we have used coarse-grained molecular dynamics simulations, guided by all-atom simulations, to study the interaction of mycolactone with pure and mixed lipid membranes. Using established techniques, we calculated the toxin’s preferential localization, membrane translocation, and impact on membrane physical and dynamical properties. The computed water-octanol partition coefficient indicates that mycolactone prefers to be in an organic phase rather than in an aqueous environment. Our results show that in a solvated membrane environment the exotoxin mainly localizes in the water-membrane interface, with a preference for the glycerol moiety of lipids, consistent with the reported studies that found it in lipid extracts of the cell. The calculated association constant to the model membrane is similar to the reported association constant for Wiskott-Aldrich syndrome protein. Mycolactone is shown to modify the physical properties of membranes, lowering the transition temperature, compressibility modulus, and critical line tension at which pores can be stabilized. It also shows a tendency to behave as a linactant, a molecule that localizes at the boundary between different fluid lipid domains in membranes and promotes inter-mixing of domains. This property has implications for the toxin’s cellular access, T-cell immunosuppression, and therapeutic potential.« less

Localization of Bacillus thuringiensis Cry1A toxin-binding molecules in gypsy moth larval gut sections using fluorescence microscopy

Treesearch

Algimantas P. Valaitis

2011-01-01

The microbial insecticide Bacillus thuringiensis (Bt) produces Cry toxins, proteins that bind to the brush border membranes of gut epithelial cells of insects that ingest it, disrupting the integrity of the membranes, and leading to cell lysis and insect death. In gypsy moth, Lymantria dispar, two toxin-binding molecules for the...

Cancer cell uptake behavior of Au nanoring and its localized surface plasmon resonance induced cell inactivation

NASA Astrophysics Data System (ADS)

Chu, Che-Kuan; Tu, Yi-Chou; Chang, Yu-Wei; Chu, Chih-Ken; Chen, Shih-Yang; Chi, Ting-Ta; Kiang, Yean-Woei; Yang, Chih-Chung

2015-02-01

Au nanorings (NRIs), which have the localized surface plasmon resonance (LSPR) wavelength around 1058 nm, either with or without linked antibodies, are applied to SAS oral cancer cells for cell inactivation through the LSPR-induced photothermal effect when they are illuminated by a laser of 1065 nm in wavelength. Different incubation times of cells with Au NRIs are considered for observing the variations of cell uptake efficiency of Au NRI and the threshold laser intensity for cell inactivation. In each case of incubation time, the cell sample is washed for evaluating the total Au NRI number per cell adsorbed and internalized by the cells based on inductively coupled plasma mass spectrometry measurement. Also, the Au NRIs remaining on cell membrane are etched with KI/I2 solution to evaluate the internalized Au NRI number per cell. The threshold laser intensities for cell inactivation before washout, after washout, and after KI/I2 etching are calibrated from the circular area sizes of inactivated cells around the illuminated laser spot center with various laser power levels. By using Au NRIs with antibodies, the internalized Au NRI number per cell increases monotonically with incubation time up to 24 h. However, the number of Au NRI remaining on cell membrane reaches a maximum at 12 h in incubation time. The cell uptake behavior of an Au NRI without antibodies is similar to that with antibodies except that the uptake NRI number is significantly smaller and the incubation time for the maximum NRI number remaining on cell membrane is delayed to 20 h. By comparing the threshold laser intensities before and after KI/I2 etching, it is found that the Au NRIs remaining on cell membrane cause more effective cancer cell inactivation, when compared with the internalized Au NRIs.

Wnt interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rappa, Germana; College of Pharmacy, Roseman University of Health Sciences, Henderson, NV 89104; Mercapide, Javier

2013-04-01

Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that threemore » distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1–positive structures appeared in three sizes (small, ≤40 nm; intermediates ∼40–80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1–containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma. - Highlights: ► First report of release of prominin-1–containing microvesicles from cancer cells. ► Pro-metastatic role of prominin-1–containing microvesicles in FEMX-I melanoma. ► Down-regulation of prominin-1 results in decreased nuclear localization of β-catenin. ► Wnt signaling as mediator of the pro-metastatic activity of prominin-1.« less

Estimating the magnitude of near-membrane PDE4 activity in living cells.

PubMed

Xin, Wenkuan; Feinstein, Wei P; Britain, Andrea L; Ochoa, Cristhiaan D; Zhu, Bing; Richter, Wito; Leavesley, Silas J; Rich, Thomas C

2015-09-15

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments. Copyright © 2015 the American Physiological Society.

Estimating the magnitude of near-membrane PDE4 activity in living cells

PubMed Central

Xin, Wenkuan; Feinstein, Wei P.; Britain, Andrea L.; Ochoa, Cristhiaan D.; Zhu, Bing; Richter, Wito; Leavesley, Silas J.

2015-01-01

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments. PMID:26201952

Vitreoscilla hemoglobin. Intracellular localization and binding to membranes.

PubMed

Ramandeep; Hwang, K W; Raje, M; Kim, K J; Stark, B C; Dikshit, K L; Webster, D A

2001-07-06

The obligate aerobic bacterium, Vitreoscilla, synthesizes elevated quantities of a homodimeric hemoglobin (VHb) under hypoxic growth conditions. Expression of VHb in heterologous hosts often enhances growth and product formation. A role in facilitating oxygen transfer to the respiratory membranes is one explanation of its cellular function. Immunogold labeling of VHb in both Vitreoscilla and recombinant Escherichia coli bearing the VHb gene clearly indicated that VHb has a cytoplasmic (not periplasmic) localization and is concentrated near the periphery of the cytosolic face of the cell membrane. OmpA signal-peptide VHb fusions were transported into the periplasm in E. coli, but this did not confer any additional growth advantage. The interaction of VHb with respiratory membranes was also studied. The K(d) values for the binding of VHb to Vitreoscilla and E. coli cell membranes were approximately 5-6 microm, a 4-8-fold higher affinity than those of horse myoglobin and hemoglobin for these same membranes. VHb stimulated the ubiquinol-1 oxidase activity of inverted Vitreoscilla membranes by 68%. The inclusion of Vitreoscilla cytochrome bo in proteoliposomes led to 2.4- and 6-fold increases in VHb binding affinity and binding site number, respectively, relative to control liposomes, suggesting a direct interaction between VHb and cytochrome bo.

Spectrophotometric and cytochemical analyses of phosphatase activity in Beta vulgaris L.

PubMed

Pesacreta, T C; Bennett, A B; Lucas, W J

1986-03-01

Spectrophotometric and cytochemical methods were used to investigate the localization and/or the sensitivity of phosphatase activities in aldehyde-fixed beet leaves and membrane fractions. The nonspecific acid phosphatase substrates, p-nitrophenyl phosphate and beta-glycerol phosphate, each exhibited unique spectrophotometric patterns of hydrolysis as a function of pH. Additionally, beta-glycerol phosphatase activity was primarily present on the tonoplast, whereas p-nitrophenyl phosphatase was present on the plasma membrane. Because of the unique pH response of each enzyme and their different localization, we conclude that they cannot be entirely "nonspecific." The spectrophotometric pattern of ATP hydrolysis differed from that of p-nitrophenol phosphate in that it decreased at pH 5.0-5.5 and was greatly inhibited by 10 mM sodium fluoride; however, both activities were on the plasma membrane. Therefore, we conclude that these activities represent either two enzymes or only one enzyme that differs in its ability to hydrolyze these two substrates. Generally, enzymatically produced lead deposits on the plasma membrane of non-vascular cells were as frequent and large as those on phloem cells; frequently, deposits on sieve element plasma membranes were relatively small. We therefore conclude that there is no evidence for the presence of relatively intense ATPase activity on the plasma membrane of phloem cells in beet leaf, in contrast to other species. Studies with membrane fractions indicated that formaldehyde could completely inhibit the inhibitor-sensitive phosphatase activities in mitochondrial and vacuolar fractions while preserving significant activity in the plasma membrane fraction.

Application of atomic force microscopy to microbial surfaces: from reconstituted cell surface layers to living cells.

PubMed

Dufrêne, Y F

2001-02-01

The application of atomic force microscopy (AFM) to probe the ultrastructure and physical properties of microbial cell surfaces is reviewed. The unique capabilities of AFM can be summarized as follows: imaging surface topography with (sub)nanometer lateral resolution; examining biological specimens under physiological conditions; measuring local properties and interaction forces. AFM is being used increasingly for: (i) visualizing the surface ultrastructure of microbial cell surface layers, including bacterial S-layers, purple membranes, porin OmpF crystals and fungal rodlet layers; (ii) monitoring conformational changes of individual membrane proteins; (iii) examining the morphology of bacterial biofilms, (iv) revealing the nanoscale structure of living microbial cells, including fungi, yeasts and bacteria, (v) mapping interaction forces at microbial surfaces, such as van der Waals and electrostatic forces, solvation forces, and steric/bridging forces; and (vi) probing the local mechanical properties of cell surface layers and of single cells.

EMP-1 is a junctional protein in a liver stem cell line and in the liver.

PubMed

Lee, Hsuan-Shu; Sherley, James L; Chen, Jeremy J W; Chiu, Chien-Chang; Chiou, Ling-Ling; Liang, Ja-Der; Yang, Pan-Chyr; Huang, Guan-Tarn; Sheu, Jin-Chuan

2005-09-09

In an attempt to discover cell markers for liver stem cells, a cDNA microarray analysis was carried out to compare the gene expression profiles between an adult liver stem cell line, Lig-8, and mature hepatocytes. Several genes in the categories of extracellular matrix, cell membrane, cell adhesion, transcription factor, signal molecule, transporter, and metabolic enzyme were shown to be differentially expressed in Lig-8 cells. Among them, epithelial membrane protein (EMP)-1 has been previously implicated with stem cell phenotypes. Antiserum to EMP-1 was produced to localize its expression. On monolayers of Lig-8 cells, EMP-1 was expressed along the intercellular border. In the liver harboring proliferating oval cells, the liver progenitors, EMP-1 was localized as ribbon bands, a staining pattern for epithelial junctions, all the way through bile duct epithelia, oval cell ductules, and into peri-hepatocytic regions. These peri-hepatocytic regions were proved to be bile canaliculi by co-localization of EMP-1 and dipeptidyl peptidase IV, an enzyme located on bile canaliculi. This report is the first to indicate EMP-1 to be a junctional protein in the liver.

Cell lines that support replication of a novel herpes simplex virus 1 U{sub L}31 deletion mutant can properly target U{sub L}34 protein to the nuclear rim in the absence of U{sub L}31

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang Li; Tanaka, Michiko; Kawaguchi, Yasushi

2004-11-10

Previous results indicated that the herpes simplex virus 1 (HSV-1) U{sub L}31 gene is necessary and sufficient for localization of the U{sub L}34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U{sub L}31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Veromore » cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U{sub L}31 gene. The replication of the U{sub L}31 deletion virus was restored on U{sub L}31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U{sub L}34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U{sub L}34 protein localized at the nuclear membrane in rabbit skin cells, and U{sub L}31 complementing CV1 cells infected with the U{sub L}31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U{sub L}34 protein to the nuclear membrane. We speculate that this function partially complements that of U{sub L}31 and may explain why U{sub L}31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.« less

The G protein alpha subunit (GP alpha1) is associated with the ER and the plasma membrane in meristematic cells of Arabidopsis and cauliflower.

PubMed

Weiss, C A; White, E; Huang, H; Ma, H

1997-05-05

Towards the elucidation of the cellular function(s) of GP alpha1, we have characterized its subcellular localization using immunofluorescence and cell fractionation. GP alpha1 is not present in nuclei or chloroplasts. It is a membrane-bound protein, and analysis of isolated endoplasmic and plasma membranes indicates a good correlation between GP alpha1 in both the plasma membrane and the ER compartment. Interestingly, these results may suggest more different functions for GP alpha1: it might be involved in transmission of extracellular signals across the plasma membrane and in the cytoplasm, and/or it may also be involved in regulating some aspects of the ER functions or membrane trafficking between both membranes.

Receptor for advanced glycation end-products is a marker of type I lung alveolar cells.

PubMed

Shirasawa, Madoka; Fujiwara, Naoyuki; Hirabayashi, Susumu; Ohno, Hideki; Iida, Junko; Makita, Koshi; Hata, Yutaka

2004-02-01

Lung alveolar epithelial cells are comprised of type I (ATI) and type II (ATII) cells. ATI cells are polarized, although they have very flat morphology. The identification of marker proteins for apical and basolateral membranes of ATI cells is important to investigate into the differentiation of ATI cells. In this paper, we characterized receptor for advanced glycation end-products (RAGE) as a marker for ATI cells. RAGE was localized on basolateral membranes of ATI cells in the immunoelectron microscopy and its expression was enhanced in a parallel manner to the differentiation of ATI cells in vivo and in primary cultures of ATII cells. RAGE and T1 alpha, a well-known ATI marker protein, were targeted to basolateral and apical membranes, respectively, when expressed in polarized Madine Darby canine kidney cells. Moreover, RAGE was expressed in ATI cells after T1 alpha in vivo and in ex in vivo organ cultures. In conclusion, RAGE is a marker for basolateral membranes of well-differentiated ATI cells. ATI cells require some signal provided by the in vivo environment to express RAGE.

Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens

PubMed Central

Zupan, John R.; Cameron, Todd A.; Anderson-Furgeson, James; Zambryski, Patricia C.

2013-01-01

Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes. PMID:23674672

Imaging plasma membrane deformations with pTIRFM.

PubMed

Passmore, Daniel R; Rao, Tejeshwar C; Peleman, Andrew R; Anantharam, Arun

2014-04-02

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress. Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or diD. DiD intercalates in the membrane plane with a "fixed" orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561 nm laser (p-pol, s-pol). A 488 nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Regulation of the vasopressin V2 receptor by vasopressin in polarized renal collecting duct cells.

PubMed

Robben, J H; Knoers, N V A M; Deen, P M T

2004-12-01

Binding of arginine-vasopressin (AVP) to its V2 receptor (V2R) in the basolateral membrane of principal cells induces Aquaporin-2-mediated water reabsorption in the kidney. To study the regulation of the V2R by dDAVP in a proper model, a polarized renal cell line stably-expressing V2R-GFP was generated. Labeled AVP-binding studies revealed an equal basolateral vs. apical membrane distribution for V2R-GFP and endogenous V2R. In these cells, GFP-V2R was expressed in its mature form and localized for 75% in the basolateral membrane and for 25% to late endosomes/lysosomes. dDAVP caused a dose- and time-dependent internalization of V2R-GFP, which was completed within 1 h with 100 nM dDAVP, was prevented by coincubation with a V2R antagonist, and which reduced its half-life from 11.5 to 2.8 h. Semiquantification of the V2R-GFP colocalization with E-cadherin (basolateral membrane), early endosomal antigen-1 (EEA-1) and lysosome-associated membrane protein-2 (LAMP-2) in time revealed that most dDAVP-bound V2R was internalized via early endosomes to late endosomes/lysosomes, where it was degraded. The dDAVP-internalized V2R did not recycle to the basolateral membrane. In conclusion, we established the itinerary of the V2R in a polarized cell model that likely resembles the in vivo V2R localization and regulation by AVP to a great extent.

NaCl-Induced Alterations in Both Cell Structure and Tissue-Specific Plasma Membrane H+ -ATPase Gene Expression.

PubMed Central

Niu, X.; Damsz, B.; Kononowicz, A. K.; Bressan, R. A.; Hasegawa, P. M.

1996-01-01

NaCl-induced plasma membrane H+-ATPase gene expression, which occurs in roots and fully expanded leaves of the halophyte Atriplex nummularia L. (X. Niu, M.L. Narasimhan, R.A. Salzman, R.A. Bressan, P.M. Hasegawa [1993] Plant Physiol 103: 713-718), has been differentially localized to specific tissues using in situ RNA hybridization techniques. Twenty-four-hour exposure of plants to 400 mM NaCl resulted in substantial accumulation of H+ pump message in the epidermis of the root tip and the endodermis of the root elongation/differentiation zone. In expanded leaves, NaCl induction of plasma membrane H+-ATPase message accumulation was localized to bundle-sheath cells. Ultrastructural analyses indicated that significant cytological adaptations in root cells included plasmolysis that is accompanied by plasma membrane invaginations, formation of Hechtian strands and vesiculation, and vacuolation. These results identify specific tissues that are involved in the regulation of Na+ and Cl- uptake into different organs of the halophyte A. nummularia and provide evidence of the intercellular and interorgan coordination that occurs in the mediation of NaCl adaptation. PMID:12226321

NaCl-Induced Alterations in Both Cell Structure and Tissue-Specific Plasma Membrane H+ -ATPase Gene Expression.

PubMed

Niu, X.; Damsz, B.; Kononowicz, A. K.; Bressan, R. A.; Hasegawa, P. M.

1996-07-01

NaCl-induced plasma membrane H+-ATPase gene expression, which occurs in roots and fully expanded leaves of the halophyte Atriplex nummularia L. (X. Niu, M.L. Narasimhan, R.A. Salzman, R.A. Bressan, P.M. Hasegawa [1993] Plant Physiol 103: 713-718), has been differentially localized to specific tissues using in situ RNA hybridization techniques. Twenty-four-hour exposure of plants to 400 mM NaCl resulted in substantial accumulation of H+ pump message in the epidermis of the root tip and the endodermis of the root elongation/differentiation zone. In expanded leaves, NaCl induction of plasma membrane H+-ATPase message accumulation was localized to bundle-sheath cells. Ultrastructural analyses indicated that significant cytological adaptations in root cells included plasmolysis that is accompanied by plasma membrane invaginations, formation of Hechtian strands and vesiculation, and vacuolation. These results identify specific tissues that are involved in the regulation of Na+ and Cl- uptake into different organs of the halophyte A. nummularia and provide evidence of the intercellular and interorgan coordination that occurs in the mediation of NaCl adaptation.

Timing is everything: Rac1 controls Net1A localization to regulate cell adhesion.

PubMed

Carr, Heather S; Frost, Jeffrey A

2013-01-01

Cell adhesion to the extracellular matrix elicits a temporal reorganization of the actin cytoskeleton that is regulated first by Rac1 and later by RhoA. The signaling mechanisms controlling late stage RhoA activation are incompletely understood. Net1A is a RhoA/RhoB-specific guanine nucleotide exchange factor that is required for cancer cell motility. The ability of Net1A to stimulate RhoA activation is negatively regulated by nuclear sequestration. However, mechanisms controlling the plasma membrane localization of Net1A had not previously been reported. Recently we have shown that Rac1 activation stimulates plasma membrane relocalization and activation of Net1A. Net1A relocalization is independent of its catalytic activity and does not require its C-terminal pleckstrin homology or PDZ interacting domains. Rac1 activation during cell adhesion stimulates a transient relocalization of Net1A that is terminated by proteasomal degradation of Net1A. Importantly, plasma membrane localization of Net1A is required for efficient myosin light chain phosphorylation, focal adhesion maturation, and cell spreading. These data show for the first time a physiological mechanism controlling Net1A relocalization from the nucleus. They also demonstrate a previously unrecognized role for Net1A in controlling actomyosin contractility and focal adhesion dynamics during cell adhesion.

Differential plasma membrane targeting of voltage-dependent calcium channel subunits expressed in a polarized epithelial cell line

PubMed Central

Brice, Nicola L; Dolphin, Annette C

1999-01-01

Voltage-dependent calcium channels (VDCCs) show a highly non-uniform distribution in many cell types, including neurons and other polarized secretory cells. We have examined whether this can be mimicked in a polarized epithelial cell line (Madin-Darby canine kidney), which has been used extensively to study the targeting of proteins. We expressed the VDCC α1A, α1B or α1C subunits either alone or in combination with accessory subunits α2-δ and the different β subunits, and examined their localization immunocytochemically. An α1 subunit was only targeted to the plasma membrane if co-expressed with the accessory subunits. The combination α1C/α2-δ and all β subunits was always localized predominantly to the basolateral membrane. It has been suggested that this is equivalent to somatodendritic targeting in neurons. In contrast, the α1B subunit was expressed at the apical membrane with all the accessory subunit combinations, by 24 h after microinjection. This membrane destination shows some parallels with axonal targeting in neurons. The α1A subunit was consistently observed at the apical membrane in the combinations α1A/α2-δ/β1b or β4. In contrast, when co-expressed with α2-δ/β2a, α1A was clearly targeted to the basolateral membrane. In conclusion, the VDCC α1 subunit appears to be the primary determinant for targeting the VDCC complex, but the β subunit can modify this destination, particularly for α1A. PMID:10066897

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

PubMed

Zhang, S X; Kobayashi, T; Okada, T; García del Saz, E; Seguchi, H

1991-07-01

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.

Dia-Interacting Protein (DIP) Imposes Migratory Plasticity in mDia2-Dependent Tumor Cells in Three-Dimensional Matrices

PubMed Central

Wyse, Meghan M.; Lei, Jun; Nestor-Kalinoski, Andrea L.; Eisenmann, Kathryn M.

2012-01-01

Tumor cells rely upon membrane pliancy to escape primary lesions and invade secondary metastatic sites. This process relies upon localized assembly and disassembly cycles of F-actin that support and underlie the plasma membrane. Dynamic actin generates both spear-like and bleb structures respectively characterizing mesenchymal and amoeboid motility programs utilized by metastatic cells in three-dimensional matrices. The molecular mechanism and physiological trigger(s) driving membrane plasticity are poorly understood. mDia formins are F-actin assembly factors directing membrane pliancy in motile cells. mDia2 is functionally coupled with its binding partner DIP, regulating cortical actin and inducing membrane blebbing in amoeboid cells. Here we show that mDia2 and DIP co-tether to nascent blebs and this linkage is required for bleb formation. DIP controls mesenchymal/amoeboid cell interconvertability, while CXCL12 induces assembly of mDia2:DIP complexes to bleb cortices in 3D matrices. These results demonstrate how DIP-directed mDia2-dependent F-actin dynamics regulate morphological plasticity in motile cancer cells. PMID:23024796

Adenylyl cyclase localization to the uropod of aggregating Dictyostelium cells requires RacC

PubMed Central

Wang, C.; Jung, D.; Cao, Z.; Chung, C. Y.

2015-01-01

The localization of adenylyl cyclase A (ACA) to uropod of cells is required for the stream formation during Dictyostelium development. RacC is a Dictyostelium orthologue of Cdc42. We identified a streaming defect of racC− cells as they are clearly less polarized and form smaller and fragmented streams. ACA-YFP is mainly associated with intracellular vesicular structures, but not with the plasma membrane in racC− cells. racC− cells have a slightly higher number of vesicles than Ax3 cells, suggesting that the defect of ACA trafficking is not simply due to the lack of vesicle formation. While the ACA-YFP vesicles traveled with an average velocity of 9.1 µm/min in Ax3 cells, a slow and diffusional movement without direction with an average velocity of 4 µm/min was maintained in racC− cells. Images acquired by using total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) analysis revealed that a significantly decreased number of ACA-YFP vesicles appeared near the cell membrane, indicating a defect in ACA-YFP vesicle trafficking. These results suggest an important role of RacC in the rapid and directional movements of ACA vesicles on microtubules to the plasma membrane, especially to the back of polarized cell. PMID:26315268

Reaction of cells to local, regional, and general low-intensive laser irradiation

NASA Astrophysics Data System (ADS)

Baibekov, Iskander M.; Kasymov, A. S.; Musaev, Erkin S.; Vorojeikin, V. M.; Artikov, S. N.

1993-07-01

Local influence of low intensive laser irradiation (LILI) of Helium-Neon (HNL), Copper vapor (CVL), Nitrogen (UVL) and Arsenic Gallium (AGL) lasers cause stimulation of processes of physiological and reparative regeneration in intact skin, and mucous membrane of stomach and duodenum, dermatome wounds and gastroduodenal ulcers. Structural bases of these effects are the acceleration of cell proliferation and differentiation and also the activation of intracellular structures and intensification of cell secretion. Regional influence of the pointed types of LILI on hepar in cirrhosis and hepatitis causes decreasing of the inflammatory and cirrhotic changes. After endo- and exo-vascular laser irradiations of blood the decreasing of the number of pathological forms of erythrocytes and the increasing of their catalase activity, are indicated. General (total) laser irradiation of the organism--laser shower, increases the bone marrow cells proliferation, especially myeloid series. It is accompanied with acceleration of their differentiation and migration in circulation. It was revealed, that HNL to a considerable extent influences the epithelial cells and CVL the connective tissue cells. UVL increases the amount of microorganisms on cell surfaces (membrane bound microorganisms). Regional irradiation of the LILI causes both direct and indirect influence of cells. Structural changes of bone marrow cells and gut mucous membrane cells indicate intersystemic interaction.

Neutron scattering to study membrane systems: from lipid vesicles to living cells.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nickels, Jonathan D.; Chatterjee, Sneha; Stanley, Christopher B.

The existence and role of lateral lipid organization in biological membranes has been studied and contested for more than 30 years. Lipid domains, or rafts, are hypothesized as scalable compartments in biological membranes, providing appropriate physical environments to their resident membrane proteins. This implies that lateral lipid organization is associated with a range of biological functions, such as protein co-localization, membrane trafficking, and cell signaling, to name just a few. Neutron scattering techniques have proven to be an excellent tool to investigate these structural features in model lipids, and more recently, in living cells. I will discuss our recent workmore » using neutrons to probe the structure and mechanical properties in model lipid systems and our current efforts in using neutrons to probe the structure and organization of the bilayer in a living cell. These efforts in living cells have used genetic and biochemical strategies to generate a large neutron scattering contrast, making the membrane visible. I will present our results showing in vivo bilayer structure and discuss the outlook for this approach.« less

Reverse membrane bioreactor: Introduction to a new technology for biofuel production.

PubMed

Mahboubi, Amir; Ylitervo, Päivi; Doyen, Wim; De Wever, Heleen; Taherzadeh, Mohammad J

2016-01-01

The novel concept of reverse membrane bioreactors (rMBR) introduced in this review is a new membrane-assisted cell retention technique benefiting from the advantageous properties of both conventional MBRs and cell encapsulation techniques to tackle issues in bioconversion and fermentation of complex feeds. The rMBR applies high local cell density and membrane separation of cell/feed to the conventional immersed membrane bioreactor (iMBR) set up. Moreover, this new membrane configuration functions on basis of concentration-driven diffusion rather than pressure-driven convection previously used in conventional MBRs. These new features bring along the exceptional ability of rMBRs in aiding complex bioconversion and fermentation feeds containing high concentrations of inhibitory compounds, a variety of sugar sources and high suspended solid content. In the current review, the similarities and differences between the rMBR and conventional MBRs and cell encapsulation regarding advantages, disadvantages, principles and applications for biofuel production are presented and compared. Moreover, the potential of rMBRs in bioconversion of specific complex substrates of interest such as lignocellulosic hydrolysate is thoroughly studied. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

DHA-fluorescent probe is sensitive to membrane order and reveals molecular adaptation of DHA in ordered lipid microdomains☆

PubMed Central

Teague, Heather; Ross, Ron; Harris, Mitchel; Mitchell, Drake C.; Shaikh, Saame Raza

2012-01-01

Docosahexaenoic acid (DHA) disrupts the size and order of plasma membrane lipid microdomains in vitro and in vivo. However, it is unknown how the highly disordered structure of DHA mechanistically adapts to increase the order of tightly packed lipid microdomains. Therefore, we studied a novel DHA-Bodipy fluorescent probe to address this issue. We first determined if the DHA-Bodipy probe localized to the plasma membrane of primary B and immortal EL4 cells. Image analysis revealed that DHA-Bodipy localized into the plasma membrane of primary B cells more efficiently than EL4 cells. We then determined if the probe detected changes in plasma membrane order. Quantitative analysis of time-lapse movies established that DHA-Bodipy was sensitive to membrane molecular order. This allowed us to investigate how DHA-Bodipy physically adapted to ordered lipid microdomains. To accomplish this, we employed steady-state and time-resolved fluorescence anisotropy measurements in lipid vesicles of varying composition. Similar to cell culture studies, the probe was highly sensitive to membrane order in lipid vesicles. Moreover, these experiments revealed, relative to controls, that upon incorporation into highly ordered microdomains, DHA-Bodipy underwent an increase in its fluorescence lifetime and molecular order. In addition, the probe displayed a significant reduction in its rotational diffusion compared to controls. Altogether, DHA-Bodipy was highly sensitive to membrane order and revealed for the first time that DHA, despite its flexibility, could become ordered with less rotational motion inside ordered lipid microdomains. Mechanistically, this explains how DHA acyl chains can increase order upon formation of lipid microdomains in vivo. PMID:22841541

A novel role for integrin-linked kinase in epithelial sheet morphogenesis.

PubMed

Vespa, Alisa; D'Souza, Sudhir J A; Dagnino, Lina

2005-09-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.

Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells

PubMed Central

1975-01-01

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane. PMID:163833

Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mukai, Atsushi; Hashimoto, Naohiro

2008-01-15

Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and themore » lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells.« less

WAVE2 targeting to phosphatidylinositol 3,4,5-triphosphate mediated by insulin receptor substrate p53 through a complex with WAVE2.

PubMed

Takahashi, Kazuhide; Suzuki, Katsuo

2010-11-01

Membrane targeting of WAVE2 along microtubules to phosphatidylinositol 3,4,5-triphosphate (PIP(3)) in response to an extracellular stimulus requires Rac1, Pak1, stathmin, and EB1. However, whether WAVE2 interacts directly with PIP(3) or not remains unclear. We demonstrate that insulin-like growth factor I (IGF-I) induces WAVE2 membrane targeting, accompanied by phosphorylation of Pak1 at serine 199/204 (Ser199/204) and stathmin at Ser38 in the inner cytoplasmic region. This is spatially independent of the membrane region where the IGF-I receptor (IGF-IR) is locally activated. WAVE2, phosphorylated Pak1, and phosphorylated stathmin located at the microtubule ends began to accumulate at the leading edge of cells in close proximity to PIP(3) that was produced in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. The PIP(3)-beads binding assay revealed that insulin receptor substrate p53 (IRSp53) and actin rather than WAVE2 bound to PIP(3). IRSp53 constitutively associated with WAVE2 and these two proteins colocalized with PIP(3) at the leading edge after IGF-I stimulation. Suppression of IRSp53 expression by two independent small interfering RNAs (siRNAs) completely inhibited IGF-I-induced membrane targeting and local accumulation of WAVE2 at the leading edge of cells. We propose that IRSp53 constitutively forms a complex with WAVE2 and is crucial for membrane targeting followed by local accumulation of WAVE2 at the leading edge of cells through linking WAVE2 to PIP(3) that is produced near locally activated IGF-IR in response to IGF-I. Copyright (c) 2010 Elsevier Inc. All rights reserved.

CALCIUM BINDING TO INTESTINAL MEMBRANES

PubMed Central

Oschman, James L.; Wall, Betty J.

1972-01-01

Flame photometry reveals that glutaraldehyde and buffer solutions in routine use for electron microscopy contain varying amounts of calcium. The presence of electron-opaque deposits adjacent to membranes in a variety of tissues can be correlated with the presence of calcium in the fixative. In insect intestine (midgut), deposits occur adjacent to apical and lateral plasma membranes. The deposits are particularly evident in tissues fixed in glutaraldehyde without postosmication. They are also observed in osmicated tissue if calcium is added to wash and osmium solutions. Deposits are absent when calcium-free fixatives are used, but are present when traces of CaCl2 (as low as 5 x 10-5 M) are added. The deposits occur at regular intervals along junctional membranes, providing images strikingly similar to those obtained by other workers who have used pyroantimonate in an effort to localize sodium. Other divalent cations (Mg++, Sr++, Ba++, Mn++, Fe++) appear to substitute for calcium, while sodium, potassium, lanthanum, and mercury do not. After postfixing with osmium with calcium added, the deposits can be resolved as patches along the inner leaflet of apical and lateral plasma membranes. The dense regions may thus localize membrane constituents that bind calcium. The results are discussed in relation to the role of calcium in control of cell-to-cell communication, intestinal calcium uptake, and the pyroantimonate technique for ion localization. PMID:4569411

Mapping of the Localization of Type 1 Angiotensin Receptor in Membrane Microdomains Using Bioluminescence Resonance Energy Transfer-based Sensors*

PubMed Central

Balla, András; Tóth, Dániel J.; Soltész-Katona, Eszter; Szakadáti, Gyöngyi; Erdélyi, László Sándor; Várnai, Péter; Hunyady, László

2012-01-01

Initiation and termination of signaling of the type I angiotensin receptor (AT1-R) can lead to dynamic changes in its localization in plasma membrane microdomains. Several markers were recently developed to investigate membrane microdomains. Here, we used several YFP-labeled fusion constructs (i.e. raft or non-raft plasma membrane markers) to analyze the agonist-induced changes in compartmentalization of AT1-R, including internalization or lateral movement between plasma membrane compartments in response to stimulation using bioluminescence resonance energy transfer measurements. Our data demonstrate that angiotensin II (AngII) stimulus changes the microdomain localization of wild type or mutated (DRY → AAY or TSTS → AAAA) AT1-Rs co-expressed with the fluorescent probes in HEK293 cells. The comparison of the trafficking of AT1-R upon AngII stimulus with those of [Sar1,Ile8]AngII or [Sar1,Ile4,Ile8]AngII stimulus revealed different types of changes, depending on the nature of the ligand. The observed changes in receptor compartmentalization of the AT1-R are strikingly different from those of 5HT-2C and EGF receptors, which demonstrate the usefulness of the bioluminescence resonance energy transfer-based measurements in the investigation of receptor trafficking in the plasma membrane in living cell experiments. PMID:22291018

Apical Plasma Membrane Mispolarization of NaK-ATPase in Polycystic Kidney Disease Epithelia Is Associated with Aberrant Expression of the β2 Isoform

PubMed Central

Wilson, Patricia D.; Devuyst, Olivier; Li, Xiaohong; Gatti, Laura; Falkenstein, Doris; Robinson, Shawn; Fambrough, Douglas; Burrow, Christopher R.

2000-01-01

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease of the kidney, characterized by cystic enlargement of renal tubules, aberrant epithelial proliferation, and ion and fluid secretion into the lumen. Previous studies have shown abnormalities in polarization of membrane proteins, including mislocalization of the NaK-ATPase to the apical plasma membranes of cystic epithelia. Apically located NaK-ATPase has previously been shown to be fully functional in vivo and in membrane-grown ADPKD epithelial cells in vitro, where basal-to-apical 22Na transport was inhibited by application of ouabain to the apical membrane compartment. Studies were conducted with polymerase chain reaction-generated specific riboprobes and polyclonal peptide antibodies against human sequences of α1, α3, β1, and β2 subunits of NaK-ATPase. High levels of expression of α1 and β1 messenger RNA were detected in ADPKD and age-matched normal adult kidneys in vivo, whereas β2 messenger RNA was detected only in ADPKD kidneys. Western blot analysis and immunocytochemical studies showed that, in normal adult kidneys, peptide subunit-specific antibodies against α1 and β1 localized to the basolateral membranes of normal renal tubules, predominantly thick ascending limbs of Henle’s loop. In ADPKD kidneys, α1 and β2 subunits were localized to the apical epithelial cell membranes, whereas β1 was distributed throughout the cytoplasm and predominantly in the endoplasmic reticulum, but was not seen associated with cystic epithelial cell membranes or in cell membrane fractions. Polarizing, renal-derived epithelial Madin Darby canine kidney cells, stably expressing normal or N-terminally truncated chicken β1 subunits, showed selective accumulation in the basolateral Madin Darby canine kidney cell surface, whereas c-myc epitope-tagged chicken β2 or human β2 subunits accumulated selectively in the apical cell surface. Similarly, human ADPKD epithelial cell lines, which endogenously expressed α1 and β2 NaK-ATPase subunits, showed colocalization at the apical cell surface and coassociation by immunoprecipitation analysis. These results are consistent with a model in which the additional transcription and translation of the β2 subunit of NaK-ATPase may result in the apical mislocalization of NaK-ATPase in ADPKD cystic epithelia. PMID:10623674

Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts.

PubMed

Ring, Axel; Le Lay, Soazig; Pohl, Juergen; Verkade, Paul; Stremmel, Wolfgang

2006-04-01

Several lines of evidence suggest that lipid rafts are involved in cellular fatty acid uptake and influence fatty acid translocase (FAT/CD36) function. However, it remains unknown whether caveolae, a specialized raft type, are required for this mechanism. Here, we show that wild-type (WT) mouse embryonic fibroblasts (MEFs) and caveolin-1 knockout (KO) MEFs, which are devoid of caveolae, have comparable overall expression of FAT/CD36 protein but altered subcellular FAT/CD36 localization and function. In WT MEFs, FAT/CD36 was isolated with both lipid raft enriched detergent-resistant membranes (DRMs) and detergent-soluble membranes (DSMs), whereas in cav-1 KO cells it was exclusively associated with DSMs. Subcellular fractionation demonstrated that FAT/CD36 in WT MEFs was localized intracellularly and at the plasma membrane level while in cav-1 KO MEFs it was absent from the plasma membrane. This mistargeting of FAT/CD36 in cav-1 KO cells resulted in reduced fatty acid uptake compared to WT controls. Adenoviral expression of caveolin-1 in KO MEFs induced caveolae formation, redirection of FAT/CD36 to the plasma membrane and rescue of fatty acid uptake. In conclusion, our data provide evidence that caveolin-1 is necessary to target FAT/CD36 to the plasma membrane. Caveolin-1 may influence fatty acid uptake by regulating surface availability of FAT/CD36.

Biophysical characterization of genistein-membrane interaction and its correlation with biological effect on cells - The case of EYPC liposomes and human erythrocyte membranes.

PubMed

Pawlikowska-Pawlęga, Bożena; Misiak, Lucjan E; Jarosz-Wilkołazka, Anna; Zarzyka, Barbara; Paduch, Roman; Gawron, Antoni; Gruszecki, Wieslaw I

2014-08-01

With application of EPR and (1)H NMR techniques genistein interaction with liposomes formed with egg yolk lecithin and with erythrocyte membranes was assessed. The present study addressed the problem of genistein localization and its effects on lipid membrane fluidity and protein conformation. The range of microscopic techniques was employed to study genistein effects on HeLa cells and human erythrocytes. Moreover, DPPH bioassay, superoxide anion radical test and enzymatic measurements were performed in HeLa cells subjected to genistein. The gathered results from both EPR and NMR techniques indicated strong ordering effect of genistein on the motional freedom of lipids in the head group region and the adjacent hydrophobic zone in liposomal as well as in red blood cell membranes. EPR study of human ghost showed also the changes in the erythrocyte membrane protein conformation. The membrane effects of genistein were correlated with the changes in internal membranes arrangement of HeLa cells as it was noticed using transmission electron microscopic and fluorescent techniques. Scanning electron and light microscopy methods showed that one of the aftermaths of genistein incorporation into membranes was creation of echinocytic form of the red blood cells with reduced diameter. Genistein improved redox status of HeLa cells treated with H2O2 by lowering radicals' level. In conclusion, the capacity of genistein to incorporate, to affect membrane organization and to change its biophysical properties is correlated with the changes inside the cells. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains[S

PubMed Central

Krager, Kimberly J.; Sarkar, Mitul; Twait, Erik C.; Lill, Nancy L.; Koland, John G.

2012-01-01

The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20–50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor. PMID:22822037

The Candida albicans stress response gene Stomatin-Like Protein 3 is implicated in ROS-induced apoptotic-like death of yeast phase cells

PubMed Central

Salcedo, Eugenia C.

2018-01-01

The ubiquitous presence of SPFH (Stomatin, Prohibitin, Flotillin, HflK/HflC) proteins in all domains of life suggests that their function would be conserved. However, SPFH functions are diverse with organism-specific attributes. SPFH proteins play critical roles in physiological processes such as mechanosensation and respiration. Here, we characterize the stomatin ORF19.7296/SLP3 in the opportunistic human pathogen Candida albicans. Consistent with the localization of stomatin proteins, a Slp3p-Yfp fusion protein formed visible puncta along the plasma membrane. We also visualized Slp3p within the vacuolar lumen. Slp3p primary sequence analyses identified four putative S-palmitoylation sites, which may facilitate membrane localization and are conserved features of stomatins. Plasma membrane insertion sequences are present in mammalian and nematode SPFH proteins, but are absent in Slp3p. Strikingly, Slp3p was present in yeast cells, but was absent in hyphal cells, thus categorizing it as a yeast-phase specific protein. Slp3p membrane fluorescence significantly increased in response to cellular stress caused by plasma membrane, cell wall, oxidative, or osmotic perturbants, implicating SLP3 as a general stress-response gene. A slp3Δ/Δ homozygous null mutant had no detected phenotype when slp3Δ/Δ mutants were grown in the presence of a variety of stress agents. Also, we did not observe a defect in ion accumulation, filamentation, endocytosis, vacuolar structure and function, cell wall structure, or cytoskeletal structure. However, SLP3 over-expression triggered apoptotic-like death following prolonged exposure to oxidative stress or when cells were induced to form hyphae. Our findings reveal the cellular localization of Slp3p, and for the first time associate Slp3p function with the oxidative stress response. PMID:29389961

Localization of Filipin-Sterol Complexes in the Membranes of Beta vulgaris Roots and Spinacia oleracea Chloroplasts 1

PubMed Central

Moeller, Curt H.; Mudd, J. Brian

1982-01-01

Filipin was used as a cytochemical probe for membrane sterols in the root storage tissue of the red beet Beta vulgaris L. and the chloroplasts of Spinacia oleracea L. In unfixed beet tissue, filipin lysed the cells. Freeze-fracture replicas revealed that the filipin-sterol complexes were tightly aggregated in the plasma membrane, while in thin section the complexes corrugated the plasma membrane. If the cells were fixed with glutaraldehyde prior to the filipin treatment, the cell structure was preserved. Filipin-induced lesions were dispersed or clustered loosely in the plasma membrane. A few filipin-sterol complexes were observed in the tonoplast. In spinach chloroplasts, filipin-sterol complexes were limited to the outer membrane of the envelope and were not found in the inner membrane of the envelope or in the lamellar membranes. If the filipin-sterol complexes accurately mapped the distribution of membrane sterols, then sterol was located predominantly in the plasma membrane of the red beet and in the outer membrane of the chloroplast envelope. Furthermore, the sterol may be heterogenously distributed laterally in both these membranes. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:16662716

Localization of palmitoylated and activated G protein α-subunit in Dictyostelium discoideum.

PubMed

Alamer, Sarah; Kageyama, Yusuke; Gundersen, Robert E

2018-06-01

Guanine nucleotide-binding proteins (G proteins) act as molecular switches to regulate many fundamental cellular processes. The lipid modification, palmitoylation, can be considered as a key factor for proper G protein function and plasma membrane localization. In Dictyostelium discoidum, Gα2 is essential for the chemotactic response to cAMP in their developmental life cycle. However, the regulation of Gα2 with respect to palmitoylation, activation and Gβγ association is less clear. In this study, Gα2 is shown to be palmitoylated on Cys-4 by [ 3 H]palmitate labeling. Loss of this palmitoylation site results in redistribution of Gα2 within the cell and poor D. discoideum development. Cellular re-localization is also observed for activated Gα2. In the membrane fraction, Gα2-wt (YFP) is highly enriched in a low-density membrane fraction, which is palmitoylation-dependent. Activated Gα2 monomer and heterotrimer are shifted to two different higher-density fractions. These results broaden our understanding of how G protein localization and function are regulated inside the cells. © 2018 Wiley Periodicals, Inc.

Dynamic activation of basilar membrane macrophages in response to chronic sensory cell degeneration in aging mouse cochleae

PubMed Central

Frye, Mitchell D.; Yang, Weiping; Zhang, Celia; Xiong, Binbin; Hu, Bo Hua

2016-01-01

In the sensory epithelium, macrophages have been identified on the scala tympani side of the basilar membrane. These basilar membrane macrophages are the spatially closest immune cells to sensory cells and are able to directly respond to and influence sensory cell pathogenesis. While basilar membrane macrophages have been studied in acute cochlear stresses, their behavior in response to chronic sensory cell degeneration is largely unknown. Here we report a systematic observation of the variance in phenotypes, the changes in morphology and distribution of basilar membrane tissue macrophages in different age groups of C57BL/6J mice, a mouse model of age-related sensory cell degeneration. This study reveals that mature, fully differentiated tissue macrophages, not recently infiltrated monocytes, are the major macrophage population for immune responses to chronic sensory cell death. These macrophages display dynamic changes in their numbers and morphologies as age increases, and the changes are related to the phases of sensory cell degeneration. Notably, macrophage activation precedes sensory cell pathogenesis, and strong macrophage activity is maintained until sensory cell degradation is complete. Collectively, these findings suggest that mature tissue macrophages on the basilar membrane are a dynamic group of cells that are capable of vigorous adaptation to changes in the local sensory epithelium environment influenced by sensory cell status. PMID:27837652

Dynamic activation of basilar membrane macrophages in response to chronic sensory cell degeneration in aging mouse cochleae.

PubMed

Frye, Mitchell D; Yang, Weiping; Zhang, Celia; Xiong, Binbin; Hu, Bo Hua

2017-02-01

In the sensory epithelium, macrophages have been identified on the scala tympani side of the basilar membrane. These basilar membrane macrophages are the spatially closest immune cells to sensory cells and are able to directly respond to and influence sensory cell pathogenesis. While basilar membrane macrophages have been studied in acute cochlear stresses, their behavior in response to chronic sensory cell degeneration is largely unknown. Here we report a systematic observation of the variance in phenotypes, the changes in morphology and distribution of basilar membrane tissue macrophages in different age groups of C57BL/6J mice, a mouse model of age-related sensory cell degeneration. This study reveals that mature, fully differentiated tissue macrophages, not recently infiltrated monocytes, are the major macrophage population for immune responses to chronic sensory cell death. These macrophages display dynamic changes in their numbers and morphologies as age increases, and the changes are related to the phases of sensory cell degeneration. Notably, macrophage activation precedes sensory cell pathogenesis, and strong macrophage activity is maintained until sensory cell degradation is complete. Collectively, these findings suggest that mature tissue macrophages on the basilar membrane are a dynamic group of cells that are capable of vigorous adaptation to changes in the local sensory epithelium environment influenced by sensory cell status. Copyright © 2016 Elsevier B.V. All rights reserved.

Multiphoton-generated localized electron plasma for membrane permeability modification in single cells

NASA Astrophysics Data System (ADS)

Merritt, T.; Leblanc, M.; McMillan, J.; Westwood, J.; Khodaparast, G. A.

2014-03-01

Successful incorporation of a specific macromolecule into a single cell would be ideal for characterizing trafficking dynamics through plasmodesmata or for studying intracellular localizations. Here, we demonstrate NIR femtosecond laser-mediated infiltration of a membrane impermeable dextran-conjugated dye into living cells of Arabidopsis thaliana seedling stems. Based on the reactions of fluorescing vacuoles of transgenic cells and artificial cell walls comprised of nanocellulose, laser intensity and exposure time were adjusted to avoid deleterious effects. Using these plant-tailored laser parameters, cells were injected with the fluorophores and long-term dye retention was observed, all while preserving vital cell functions. This method is ideal for studies concerning cell-to-cell interactions and potentially paves the way for introducing transgenes to specific cells. This work was supported by NSF award IOS-0843372 to JHW, with additional support from and U.S. Department of Agriculture Hatch Project no. 135997, and by the Institute of Critical Technology and Applied Sciences (ICTAS) at Virginia Tech.

In-situ Monitoring of Internal Local Temperature and Voltage of Proton Exchange Membrane Fuel Cells

PubMed Central

Lee, Chi-Yuan; Fan, Wei-Yuan; Hsieh, Wei-Jung

2010-01-01

The distribution of temperature and voltage of a fuel cell are key factors that influence performance. Conventional sensors are normally large, and are also useful only for making external measurements of fuel cells. Centimeter-scale sensors for making invasive measurements are frequently unable to accurately measure the interior changes of a fuel cell. This work focuses mainly on fabricating flexible multi-functional microsensors (for temperature and voltage) to measure variations in the local temperature and voltage of proton exchange membrane fuel cells (PEMFC) that are based on micro-electro-mechanical systems (MEMS). The power density at 0.5 V without a sensor is 450 mW/cm2, and that with a sensor is 426 mW/cm2. Since the reaction area of a fuel cell with a sensor is approximately 12% smaller than that without a sensor, but the performance of the former is only 5% worse. PMID:22163556

In-situ monitoring of internal local temperature and voltage of proton exchange membrane fuel cells.

PubMed

Lee, Chi-Yuan; Fan, Wei-Yuan; Hsieh, Wei-Jung

2010-01-01

The distribution of temperature and voltage of a fuel cell are key factors that influence performance. Conventional sensors are normally large, and are also useful only for making external measurements of fuel cells. Centimeter-scale sensors for making invasive measurements are frequently unable to accurately measure the interior changes of a fuel cell. This work focuses mainly on fabricating flexible multi-functional microsensors (for temperature and voltage) to measure variations in the local temperature and voltage of proton exchange membrane fuel cells (PEMFC) that are based on micro-electro-mechanical systems (MEMS). The power density at 0.5 V without a sensor is 450 mW/cm(2), and that with a sensor is 426 mW/cm(2). Since the reaction area of a fuel cell with a sensor is approximately 12% smaller than that without a sensor, but the performance of the former is only 5% worse.

Clathrin-Independent Endocytosis Suppresses Cancer Cell Blebbing and Invasion.

PubMed

Holst, Mikkel Roland; Vidal-Quadras, Maite; Larsson, Elin; Song, Jie; Hubert, Madlen; Blomberg, Jeanette; Lundborg, Magnus; Landström, Maréne; Lundmark, Richard

2017-08-22

Cellular blebbing, caused by local alterations in cell-surface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrin-independent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension-mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol-interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

PubMed

Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

2014-08-08

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Do mechanical forces contribute to nanoscale membrane organisation in T cells?

PubMed

Klotzsch, Enrico; Stiegler, Johannes; Ben-Ishay, Eldad; Gaus, Katharina

2015-04-01

Mechanotransduction describes how a cell senses and interacts with its environment. The concept originated in adhesion biology where adhesion receptors, integrins, facilitate force transmission between the extracellular matrix and the intracellular actin cytoskeleton. Indeed, during any adhesive contacts, cells do exert mechanical force. Hence, the probing of the local environment by cells results in mechanical cues that contribute to cellular functions and cell fate decisions such as migration, proliferation, differentiation and apoptosis. On the molecular level, mechanical forces can rearrange proteins laterally within the membrane, regulate their activity by inducing conformational changes and probe the mechanical properties and bond strength of receptor-ligands. From this point of view, it appears surprising that molecular forces have been largely overlooked in membrane organisation and ligand discrimination processes in lymphocytes. During T cell activation, the T cell receptor recognises and distinguishes antigenic from benign endogenous peptides to initiate the reorganisation of membrane proteins into signalling clusters within the immunological synapse. In this review, we asked whether characteristics of fibroblast force sensing could be applied to immune cell antigen recognition and signalling, and outline state-of-the-art experimental strategies for studying forces in the context of membrane organisation. This article is part of a Special Issue entitled: Nanoscale membrane orgainisation and signalling. Copyright © 2014 Elsevier B.V. All rights reserved.

ELECTRON MICROSCOPIC STUDY OF THE ATPASE ACTIVITY OF THE BAI STRAIN A (MYELOBLASTOSIS) AVIAN TUMOR VIRUS

PubMed Central

Novikoff, Alex B.; de Thé, Guy; Beard, D.; Beard, J. W.

1962-01-01

Thymus glands of chicks with leukemia induced by BAI strain A (myeloblastosis) virus were fixed in cold 4 per cent formaldehyde-sucrose. Frozen sections were incubated in the ATPase medium of Wachstein and Meisel and studied by light microscopy and electron microscopy. The ATPase activity of the virus is localized to the outermost membrane of the virus. The membrane of the blast-like cells of the thymus cortex from which the virus emerges, by budding, also possesses such activity. It appears likely that the outermost membrane of the virus is derived from the plasma membrane of these cells. PMID:13939125

Local induction of acetylcholine receptor clustering in myotube cultures using microfluidic application of agrin.

PubMed

Tourovskaia, Anna; Kosar, T Fettah; Folch, Albert

2006-03-15

During neuromuscular synaptogenesis, the exchange of spatially localized signals between nerve and muscle initiates the coordinated focal accumulation of the acetylcholine (ACh) release machinery and the ACh receptors (AChRs). One of the key first steps is the release of the proteoglycan agrin focalized at the axon tip, which induces the clustering of AChRs on the postsynaptic membrane at the neuromuscular junction. The lack of a suitable method for focal application of agrin in myotube cultures has limited the majority of in vitro studies to the application of agrin baths. We used a microfluidic device and surface microengineering to focally stimulate muscle cells with agrin at a small portion of their membrane and at a time and position chosen by the user. The device is used to verify the hypothesis that focal application of agrin to the muscle cell membrane induces local aggregation of AChRs in differentiated C2C12 myotubes.

Membrane Localization of Human Equilibrative Nucleoside Transporter 1 in Tumor Cells May Predict Response to Adjuvant Gemcitabine in Resected Cholangiocarcinoma Patients.

PubMed

Brandi, Giovanni; Deserti, Marzia; Vasuri, Francesco; Farioli, Andrea; Degiovanni, Alessio; Palloni, Andrea; Frega, Giorgio; Barbera, Maria A; de Lorenzo, Stefania; Garajova, Ingrid; Di Marco, Mariacristina; Pinna, Antonio D; Cescon, Matteo; Cucchetti, Alessandro; Ercolani, Giorgio; D'Errico-Grigioni, Antonietta; Pantaleo, Maria A; Biasco, Guido; Tavolari, Simona

2016-05-01

The use of gemcitabine as an adjuvant modality for cholangiocarcinoma (CC) is increasing, but limited data are available on predictive biomarkers of response. Human equilibrative nucleoside transporter 1 (hENT-1) is the major transporter involved in gemcitabine intracellular uptake. This study investigated the putative predictive role of hENT-1 localization in tumor cells of CC patients undergoing treatment with adjuvant gemcitabine. Seventy-one consecutive patients with resected CC receiving adjuvant gemcitabine at our center were retrospectively analyzed by immunohistochemistry for hENT-1 localization in tumor cells. The main outcome measure was disease-free survival (DFS). Hazard ratios (HRs) of relapse and associated 95% confidence intervals (CIs) were obtained from proportional hazards regression models stratified on quintiles of propensity score. Twenty-three (32.4%) cases were negative for hENT-1, 22 (31.0%) were positive in the cytoplasm only, and 26 (36.6%) showed concomitant cytoplasm/membrane staining. Patients with membrane hENT-1 had a longer DFS (HR 0.49, 95% CI 0.24-0.99, p = .046) than those who were negative or positive only in the cytoplasm of tumor cells. Notably, the association between DFS and membrane hENT-1 was dependent on the number of gemcitabine cycles (one to two cycles: HR 0.96, 95% CI 0.34-2.68; three to four cycles: HR 0.99, 95% CI 0.34-2.90; five to six cycles: HR 0.27, 95% CI 0.10-0.77). hENT-1 localization on tumor cell membrane may predict response to adjuvant gemcitabine in CC patients receiving more than four cycles of chemotherapy. Further prospective randomized trials on larger populations are required to confirm these preliminary results, so that optimal gemcitabine-based chemotherapy may be tailored for CC patients in the adjuvant setting. Gemcitabine is becoming an increasingly used adjuvant modality in cholangiocarcinoma (CC), but limited data are available on predictive biomarkers of response. In this study, patients receiving more than four cycles of adjuvant gemcitabine and harboring Human equilibrative nucleoside transporter 1 (hENT-1, the major transporter involved in gemcitabine intracellular uptake) on tumor cell membrane had a longer disease-free survival compared with patients negative or positive for hENT-1 only in the cytoplasm of tumor cells. Overall these results may lay the basis for further prospective randomized trials based on a larger population of patients and may prove useful for tailoring appropriate gemcitabine-based chemotherapy for CC patients in the adjuvant setting. ©AlphaMed Press.

Remodeling of the Host Cell Plasma Membrane by HIV-1 Nef and Vpu: A Strategy to Ensure Viral Fitness and Persistence.

PubMed

Sugden, Scott M; Bego, Mariana G; Pham, Tram N Q; Cohen, Éric A

2016-03-03

The plasma membrane protects the cell from its surroundings and regulates cellular communication, homing, and metabolism. Not surprisingly, the composition of this membrane is highly controlled through the vesicular trafficking of proteins to and from the cell surface. As intracellular pathogens, most viruses exploit the host plasma membrane to promote viral replication while avoiding immune detection. This is particularly true for the enveloped human immunodeficiency virus (HIV), which assembles and obtains its lipid shell directly at the plasma membrane. HIV-1 encodes two proteins, negative factor (Nef) and viral protein U (Vpu), which function primarily by altering the quantity and localization of cell surface molecules to increase virus fitness despite host antiviral immune responses. These proteins are expressed at different stages in the HIV-1 life cycle and employ a variety of mechanisms to target both unique and redundant surface proteins, including the viral receptor CD4, host restriction factors, immunoreceptors, homing molecules, tetraspanins and membrane transporters. In this review, we discuss recent progress in the study of the Nef and Vpu targeting of host membrane proteins with an emphasis on how remodeling of the cell membrane allows HIV-1 to avoid host antiviral immune responses leading to the establishment of systemic and persistent infection.

Select α-arrestins control cell-surface abundance of the mammalian Kir2.1 potassium channel in a yeast model.

PubMed

Hager, Natalie A; Krasowski, Collin J; Mackie, Timothy D; Kolb, Alexander R; Needham, Patrick G; Augustine, Andrew A; Dempsey, Alison; Szent-Gyorgyi, Christopher; Bruchez, Marcel P; Bain, Daniel J; Kwiatkowski, Adam V; O'Donnell, Allyson F; Brodsky, Jeffrey L

2018-05-21

Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K+ inwardly rectifying channel Kir2.1. Kir2.1 maintains potassium homeostasis in heart muscle cells, and Kir2.1 defects lead to human disease. By examining the ability of Kir2.1 to rescue the growth of yeast cells lacking endogenous potassium channels, we discovered that specific α-arrestins regulate Kir2.1 localization. Specifically, we found that the Ldb19/Art1, Aly1/Art6, and Aly2/Art3 α-arrestin adaptor proteins promote Kir2.1 trafficking to the cell surface, increase Kir2.1 activity at the plasma membrane, and raise intracellular potassium levels. To better quantify the intracellular and cell-surface populations of Kir2.1, we created fluorescence-activating protein fusions and for the first time used this technique to measure the cell-surface residency of a plasma membrane protein in yeast. Our experiments revealed that two α-arrestin effectors also control Kir2.1 localization. In particular, both the Rsp5 ubiquitin ligase and the protein phosphatase calcineurin facilitated the α-arrestin-mediated trafficking of Kir2.1. Together, our findings implicate α-arrestins in regulating an additional class of plasma membrane proteins and establish a new tool for dissecting the trafficking itinerary of any membrane protein in yeast. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Lipid packing determines protein-membrane interactions: challenges for apolipoprotein A–I and High Density Lipoproteins

PubMed Central

Sánchez, Susana A.; Tricerri, M. Alejandra; Ossato, Giulia; Gratton, Enrico

2010-01-01

Summary Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. FCS (Fluorescence Correlation Spectroscopy) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan GP (Generalized Polarization) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A–I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis, and offer a methodological design suited to different biological systems. PMID:20347719

A Novel Role for Integrin-linked Kinase in Epithelial Sheet Morphogenesis

PubMed Central

Vespa, Alisa; D'Souza, Sudhir J.A.; Dagnino, Lina

2005-01-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca2+ triggers formation of cell–cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca2+ treatment of keratinocytes induces rapid (≤1 h) translocation to the cell membrane of the adherens junction (AJ) proteins E-cadherin and β-catenin. This is followed by slower (>6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell–cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/β-catenin to cell borders, precluding Ca2+-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell–cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell–cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration. PMID:15975904

HER2 and β-catenin protein location: importance in the prognosis of breast cancer patients and their correlation when breast cancer cells suffer stressful situations.

PubMed

Cuello-Carrión, F Darío; Shortrede, Jorge E; Alvarez-Olmedo, Daiana; Cayado-Gutiérrez, Niubys; Castro, Gisela N; Zoppino, Felipe C M; Guerrero, Martín; Martinis, Estefania; Wuilloud, Rodolfo; Gómez, Nidia N; Biaggio, Verónica; Orozco, Javier; Gago, Francisco E; Ciocca, Leonardo A; Fanelli, Mariel A; Ciocca, Daniel R

2015-02-01

In human breast cancer, β-catenin localization has been related with disease prognosis. Since HER2-positive patients are an important subgroup, and that in breast cancer cells a direct interaction of β-catenin/HER2 has been reported, in the present study we have explored whether β-catenin location is related with the disease survival. The study was performed in a tumor bank from patients (n = 140) that did not receive specific anti-HER2 therapy. The proteins were detected by immunohistochemistry in serial sections, 47 (33.5%) patients were HER2-positive with a long follow-up. HER2-positive patients that displayed β-catenin at the plasma membrane (completely surrounding the tumour cells) showed a significant better disease-free survival and overall survival than the patients showing the protein on other locations. Then we explored the dynamics of the co-expression of β-catenin and HER2 in human MCF-7 and SKBR3 cells exposed to different stressful situations. In untreated conditions MCF-7 and SKBR3 cells showed very different β-catenin localization. In MCF-7 cells, cadmium administration caused a striking change in β-catenin localization driving it from plasma membrane to cytoplasmic and perinuclear areas and HER2 showed a similar localization patterns. The changes induced by cadmium were compared with heat shock, H2O2 and tamoxifen treatments. In conclusion, this study shows the dynamical associations of HER2 and β-catenin and their changes in subcellular localizations driven by stressful situations. In addition, we report for the first time the correlation between plasma membrane associated β-catenin in HER2-positive breast cancer and survival outcome, and the importance of the protein localization in breast cancer samples.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Venkatagopalan, Pavithra; School of Life Sciences, Arizona State University, Tempe, AZ 85287-5401; Microbiology Graduate Program, Arizona State University, Tempe, AZ 85287-5401

Coronaviruses (CoVs) assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and egress from cells in cargo vesicles. Only a few molecules of the envelope (E) protein are assembled into virions. The role of E in morphogenesis is not fully understood. The cellular localization and dynamics of mouse hepatitis CoV A59 (MHV) E protein were investigated to further understanding of its role during infection. E protein localized in the ERGIC and Golgi with the amino and carboxy termini in the lumen and cytoplasm, respectively. E protein does not traffic to the cell surface. MHV was genetically engineered with a tetracysteinemore » tag at the carboxy end of E. Fluorescence recovery after photobleaching (FRAP) showed that E is mobile in ERGIC/Golgi membranes. Correlative light electron microscopy (CLEM) confirmed the presence of E in Golgi cisternae. The results provide strong support that E proteins carry out their function(s) at the site of budding/assembly. - Highlights: • Mouse hepatitis coronavirus (MHV-CoV) E protein localizes in the ERGIC and Golgi. • MHV-CoV E does not transport to the cell surface. • MHV-CoV can be genetically engineered with a tetracysteine tag appended to E. • First FRAP and correlative light electron microscopy of a CoV E protein. • Live-cell imaging shows that E is mobile in ERGIC/Golgi membranes.« less

The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

PubMed

Cihil, Kristine M; Swiatecka-Urban, Agnieszka

2013-12-13

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Prominin-2 expression increases protrusions, decreases caveolae and inhibits Cdc42 dependent fluid phase endocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Raman Deep, E-mail: Takhter.Ramandeep@mayo.edu; Schroeder, Andreas S.; Scheffer, Luana

Highlights: •Prominin-2 expression induced protrusions that co-localized with lipid raft markers. •Prominin-2 expression decreased caveolae, caveolar endocytosis and increased pCav1. •Prominin-2 expression inhibited fluid phase endocytosis by inactivation of Cdc42. •These endocytic effects can be reversed by adding exogenous cholesterol. •Caveolin1 knockdown restored fluid phase endocytosis in Prominin2 expressing cells. -- Abstract: Background: Membrane protrusions play important roles in biological processes such as cell adhesion, wound healing, migration, and sensing of the external environment. Cell protrusions are a subtype of membrane microdomains composed of cholesterol and sphingolipids, and can be disrupted by cholesterol depletion. Prominins are pentaspan membrane proteins thatmore » bind cholesterol and localize to plasma membrane (PM) protrusions. Prominin-1 is of great interest as a marker for stem and cancer cells, while Prominin-2 (Prom2) is reportedly restricted to epithelial cells. Aim: To characterize the effects of Prom-2 expression on PM microdomain organization. Methods: Prom2-fluorescent protein was transfected in human skin fibroblasts (HSF) and Chinese hamster ovary (CHO) cells for PM raft and endocytic studies. Caveolae at PM were visualized using transmission electron microscopy. Cdc42 activation was measured and caveolin-1 knockdown was performed using siRNAs. Results: Prom2 expression in HSF and CHO cells caused extensive Prom2-positive protrusions that co-localized with lipid raft markers. Prom2 expression significantly decreased caveolae at the PM, reduced caveolar endocytosis and increased caveolin-1 phosphorylation. Prom2 expression also inhibited Cdc42-dependent fluid phase endocytosis via decreased Cdc42 activation. Effects on endocytosis were reversed by addition of cholesterol. Knockdown of caveolin-1 by siRNA restored Cdc42 dependent fluid phase endocytosis in Prom2-expressing cells. Conclusions: Prom2 protrusions primarily localize to lipid rafts and recruit cholesterol into protrusions and away from caveolae, leading to increased phosphorylation of caveolin-1, which inhibits Cdc42-dependent endocytosis. This study provides a new insight for the role for prominins in the regulation of PM lipid organization.« less

Wall to membrane linkers, stretch activated channels, and the detection of tension, voltage, temperature, auxin, and pH

NASA Technical Reports Server (NTRS)

Pickard, B. G.

1992-01-01

Introduction. The higher plant is a heterogeneous, mechanically prestressed structure continually subject to shifting forces. When a cell grows in a plant at gravitropic equilibrium, it must create localized maxima of shear in walls of neighboring cells. Such mechanical stress and strain are likely detected in a variety of ways. However, tension-sensitive ion channels are of particular interest because it appears that they are elaborately evolved for sensory function. We hypothesize that 1) the patchy patterns of high shear are focused via wall-to-membrane linkers onto the plasma membrane, where 2) they are translated by mechanosensory cation channels into corresponding patterns of high cytosolic Ca2+, which 3) initiate local enhancement of wall expansion. Further, we hypothesize that the local promotion of enhancement is achieved at least in part by local intensification of auxin transport across the plasma membrane. By implication, when an organ is asymmetrically pressed, rubbed, or bent or when it is displaced in the gravitational field, the net asymmetry of shear stress occurring across the organ would lead to asymmetric redistribution of auxin and corrective asymmetric growth. We shall describe a representative mechanosensitive Ca(2+) -selective cation channel (MCaC) with susceptibilities to xenobiotics implicating it as a force transducer in thigmo- and gravitropism. Then, we shall consider whether a putative wall-to-membrane linker (WML) could be a key feature of the molecular architecture permitting the stress distributed in the wall system to be focused on the channels.

Characterization of membrane association of Rinderpest virus matrix protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Subhashri, R.; Shaila, M.S.

2007-04-20

Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M proteinmore » gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein.« less

Models of dynamic extraction of lipid tethers from cell membranes.

PubMed

Nowak, Sarah A; Chou, Tom

2010-05-07

When a ligand that is bound to an integral membrane receptor is pulled, the membrane and the underlying cytoskeleton can deform before either the membrane delaminates from the cytoskeleton or the ligand detaches from the receptor. If the membrane delaminates from the cytoskeleton, it may be further extruded and form a membrane tether. We develop a phenomenological model for this process by assuming that deformations obey Hooke's law up to a critical force at which the cell membrane locally detaches from the cytoskeleton and a membrane tether forms. We compute the probability of tether formation and show that tethers can be extruded only within an intermediate range of force loading rates and pulling velocities. The mean tether length that arises at the moment of ligand detachment is computed as are the force loading rates and pulling velocities that yield the longest tethers.

Cell membrane reactivity of MIB-1 antibody to Ki67 in human tumors: fact or artifact?

PubMed

Leonardo, Eugenio; Volante, Marco; Barbareschi, Mattia; Cavazza, Alberto; Dei Tos, Angelo Paolo; Bussolati, Gianni; Papotti, Mauro

2007-06-01

Ki67 immunohistochemistry is a widely used marker of the tumor proliferative fraction. Apart from the nuclear staining of dividing cells, MIB-1 monoclonal antibody was also found to stain the cell membrane of some tumor types. Indeed, such membrane reactivity was proposed as a diagnostic feature of hyalinizing trabecular tumor (HTT) of the thyroid. To verify the diagnostic role of Ki67 membrane pattern, 6 HTTs, 8 pulmonary sclerosing hemangiomas (SH), and 6 other human tumors with MIB-1 cell membrane immunoreactivity were stained by immunoperoxidase with 5 different anti-Ki67 antibodies in different experimental conditions. We show here that the cell membrane reactivity reported in HTT is produced only by MIB-1 and not by other antibodies to Ki67 (including commercially available mouse and rabbit monoclonal antibodies). In addition, this peculiar pattern is obtained only if the reaction is performed at room temperature, because automated immunostainers which operate at 37 degrees C do not produce any MIB-1 membrane localization. The same findings were obtained in the other 6 tumors. Conversely, sclerosing hemangioma of the lung did not produce any MIB-1 cell membrane reactivity in our hands. A cross-reactivity of the MIB-1 monoclonal antibody with an epitope expressed at the cell membrane level (rather than an artifact) seems the most likely explanation for this finding, because the immunoreactivity is generally intense and uniform in the membrane positive tumors. We conclude that when Ki67 immunohistochemistry is used for diagnostic purposes in a suspected HTT, only MIB-1 clone at room temperature should be employed.

NHS-A isoform of the NHS gene is a novel interactor of ZO-1.

PubMed

Sharma, Shiwani; Koh, Katrina S Y; Collin, Caitlin; Dave, Alpana; McMellon, Amy; Sugiyama, Yuki; McAvoy, John W; Voss, Anne K; Gécz, Jozef; Craig, Jamie E

2009-08-15

Mutations in the NHS (Nance-Horan Syndrome) gene lead to severe congenital cataracts, dental defects and sometimes mental retardation. NHS encodes two protein isoforms, NHS-A and -1A that display cell-type dependent differential expression and localization. Here we demonstrate that of these two isoforms, the NHS-A isoform associates with the cell membrane in the presence of intercellular contacts and it immunoprecipitates with the tight junction protein ZO-1 in MDCK (Madin Darby Canine Kidney) epithelial cells and in neonatal rat lens. The NHS-1A isoform however is a cytoplasmic protein. Both Nhs isoforms are expressed during mouse development. Immunolabelling of developing mouse with the anti-NHS antibody that detects both isoforms revealed the protein in the developing head including the eye and brain. It was primarily expressed in epithelium including neural epithelium and certain vascular endothelium but only weakly expressed in mesenchymal cells. In the epithelium and vascular endothelium the protein associated with the cell membrane and co-localized with ZO-1, which indirectly indicates expression of the Nhs-A isoform in these structures. Membrane localization of the protein in the lens vesicle similarly supports Nhs-A expression. In conclusion, the NHS-A isoform of NHS is a novel interactor of ZO-1 and may have a role at tight junctions. This isoform is important in mammalian development especially of the organs in the head.

Comparative kinetics of damage to the plasma and mitochondrial membranes by intra-cellularly synthesized and externally-provided photosensitizers using multi-color FACS.

PubMed

Haupt, Sara; Malik, Zvi; Ehrenberg, Benjamin

2014-01-01

Photodynamic therapy (PDT) of cancer involves inflicting lethal damage to the cells of malignant tumors, primarily by singlet oxygen that is generated following light-absorption in a photosensitizer molecule. Dysfunction of cells is manifested in many ways, including peroxidation of cellular components, membrane rupture, depolarization of electric potentials, termination of mitochondrial activity, onset of apoptosis and necrosis and eventually cell lysis. These events do not necessarily occur in linear fashion and different types of damage to cell components occur, most probably, in parallel. In this report we measured the relative rates of damage to two cellular membranes: the plasma membrane and the mitochondrial membrane. We employed photosensitizers of diverse hydrophobicities and used different incubation procedures, which lead to their different intra-cellular localizations. We monitored the damage that was inflicted on these membranes, by employing optical probes of membrane integrity, in a multi-color FACS experiment. The potentiometric indicator JC-1 monitored the electric cross-membrane potential of the mitochondria and the fluorometric indicator Draq7 monitored the rupture of the plasma membrane. We show that the electric depolarization of the mitochondrial membrane and the damage to the enveloping plasma membrane proceed with different kinetics that reflect the molecular character and intracellular location of the sensitizer: PpIX that is synthesized in the cells from ALA causes rapid mitochondrial damage and very slow damage to the plasma membrane, while externally added PpIX has an opposite effect. The hydrophilic sensitizer HypS4 can be taken up by the cells by different incubation conditions, and these affect its intracellular location, and as a consequence either the plasma membrane or the mitochondria is damaged first. A similar correlation was found for additional extracellularly-provided photosensitizers HP and PpIX.

A review of reagents for fluorescence microscopy of cellular compartments and structures, Part III: reagents for actin, tubulin, cellular membranes, and whole cell and cytoplasm.

PubMed

Kilgore, Jason A; Dolman, Nick J; Davidson, Michael W

2014-01-02

Non-antibody commercial fluorescent reagents for imaging of cytoskeletal structures have been limited primarily to tubulin and actin, with the main factor in choice based mainly on whether cells are live or fixed and permeabilized. A wider range of options exist for cell membrane dyes, and the choice of reagent primarily depends on the preferred localization in the cell (i.e., all membranes or only the plasma membrane) and usage (i.e., whether the protocol involves fixation and permeabilization). For whole-cell or cytoplasmic imaging, the choice of reagent is determined mostly by the length of time that the cells need to be visualized (hours or days) and by fixation status. Presented here is a discussion on choosing commercially available reagents for these cellular structures, with an emphasis on use for microscopic imaging, with a featured reagent for each structure, a recommended protocol, troubleshooting guide, and example image. Copyright © 2014 John Wiley & Sons, Inc.

Specific membrane lipid composition is important for plasmodesmata function in Arabidopsis.

PubMed

Grison, Magali S; Brocard, Lysiane; Fouillen, Laetitia; Nicolas, William; Wewer, Vera; Dörmann, Peter; Nacir, Houda; Benitez-Alfonso, Yoselin; Claverol, Stéphane; Germain, Véronique; Boutté, Yohann; Mongrand, Sébastien; Bayer, Emmanuelle M

2015-04-01

Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of "native" PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes. © 2015 American Society of Plant Biologists. All rights reserved.

Specific Membrane Lipid Composition Is Important for Plasmodesmata Function in Arabidopsis

PubMed Central

Grison, Magali S.; Brocard, Lysiane; Fouillen, Laetitia; Nicolas, William; Wewer, Vera; Dörmann, Peter; Nacir, Houda; Benitez-Alfonso, Yoselin; Claverol, Stéphane; Germain, Véronique; Boutté, Yohann; Mongrand, Sébastien; Bayer, Emmanuelle M.

2015-01-01

Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of “native” PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes. PMID:25818623

ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity.

PubMed

Daley, William P; Gervais, Elise M; Centanni, Samuel W; Gulfo, Kathryn M; Nelson, Deirdre A; Larsen, Melinda

2012-01-01

The basement membrane is crucial for epithelial tissue organization and function. However, the mechanisms by which basement membrane is restricted to the basal periphery of epithelial tissues and the basement membrane-mediated signals that regulate coordinated tissue organization are not well defined. Here, we report that Rho kinase (ROCK) controls coordinated tissue organization by restricting basement membrane to the epithelial basal periphery in developing mouse submandibular salivary glands, and that ROCK inhibition results in accumulation of ectopic basement membrane throughout the epithelial compartment. ROCK-regulated restriction of PAR-1b (MARK2) localization in the outer basal epithelial cell layer is required for basement membrane positioning at the tissue periphery. PAR-1b is specifically required for basement membrane deposition, as inhibition of PAR-1b kinase activity prevents basement membrane deposition and disrupts overall tissue organization, and suppression of PAR-1b together with ROCK inhibition prevents interior accumulations of basement membrane. Conversely, ectopic overexpression of wild-type PAR-1b results in ectopic interior basement membrane deposition. Significantly, culture of salivary epithelial cells on exogenous basement membrane rescues epithelial organization in the presence of ROCK1 or PAR-1b inhibition, and this basement membrane-mediated rescue requires functional integrin β1 to maintain epithelial cell-cell adhesions. Taken together, these studies indicate that ROCK1/PAR-1b-dependent regulation of basement membrane placement is required for the coordination of tissue polarity and the elaboration of tissue structure in the developing submandibular salivary gland.

Endoplasmic Reticulum-Plasma Membrane Contact Sites.

PubMed

Saheki, Yasunori; De Camilli, Pietro

2017-06-20

The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca 2+ dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.

Flagellin peptide flg22 gains access to long-distance trafficking in Arabidopsis via its receptor, FLS2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jelenska, Joanna; Davern, Sandra M.; Standaert, Robert F.

Diverse pathogen-derived molecules, such as bacterial flagellin and its conserved peptide flg22, are recognized in plants via plasma membrane receptors and induce both local and systemic immune responses. The fate of such ligands was unknown: whether and by what mechanism(s) they enter plant cells and whether they are transported to distal tissues. We used biologically active fluorophore and radiolabeled peptides to establish that flg22 moves to distal organs with the closest vascular connections. Remarkably, entry into the plant cell via endocytosis together with the FLS2 receptor is needed for delivery to vascular tissue and long-distance transport of flg22. This contrastsmore » with known routes of long distance transport of other non-cell-permeant molecules in plants, which require membrane-localized transporters for entry to vascular tissue. Thus, a plasma membrane receptor acts as a transporter to enable access of its ligand to distal trafficking routes.« less

Flagellin peptide flg22 gains access to long-distance trafficking in Arabidopsis via its receptor, FLS2

DOE PAGES

Jelenska, Joanna; Davern, Sandra M.; Standaert, Robert F.; ...

2017-03-01

Diverse pathogen-derived molecules, such as bacterial flagellin and its conserved peptide flg22, are recognized in plants via plasma membrane receptors and induce both local and systemic immune responses. The fate of such ligands was unknown: whether and by what mechanism(s) they enter plant cells and whether they are transported to distal tissues. We used biologically active fluorophore and radiolabeled peptides to establish that flg22 moves to distal organs with the closest vascular connections. Remarkably, entry into the plant cell via endocytosis together with the FLS2 receptor is needed for delivery to vascular tissue and long-distance transport of flg22. This contrastsmore » with known routes of long distance transport of other non-cell-permeant molecules in plants, which require membrane-localized transporters for entry to vascular tissue. Thus, a plasma membrane receptor acts as a transporter to enable access of its ligand to distal trafficking routes.« less

ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells.

PubMed

Hinrichs, John W J; Klappe, Karin; Hummel, Ina; Kok, Jan W

2004-02-13

In this study we show that P-glycoprotein in multidrug-resistant 2780AD human ovarian carcinoma cells and multidrug resistance-associated protein 1 in multidrug-resistant HT29col human colon carcinoma cells are predominantly located in Lubrol-based detergent-insoluble glycosphingolipid-enriched membrane domains. This localization is independent of caveolae, since 2780AD cells do not express caveolin-1. Although HT29col cells do express caveolin-1, the ATP-binding cassette transporter and caveolin-1 were dissociated on the basis of differential solubility in Triton X-100 and absence of microscopical colocalization. While both the multidrug resistance-associated protein 1 and caveolin-1 are located in Lubrol-based membrane domains, they occupy different regions of these domains.

Analysis of Microtubule Mediated Functions of Prostate Specific Membrane Antigen

DTIC Science & Technology

2006-04-01

localization of vesicles containing these markers increased to approximately 43% (38/88) when cells are incubated with tunicamycin, indicating a role...PSMA at both plasma membrane domains following nocodazole treatment. Polarity of the basolateral marker Na,K- ATPase was unaffected by nocodazole...restricted to the apical surface facing the lumen. This staining was clearly distinct from that of the endothelial cell marker CD 34 and CD31

Trans-cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells.

PubMed

Chmyrov, Volodymyr; Spielmann, Thiemo; Hevekerl, Heike; Widengren, Jerker

2015-06-02

Membrane environment and fluidity can modulate the dynamics and interactions of membrane proteins and can thereby strongly influence the function of cells and organisms in general. In this work, we demonstrate that trans-cis isomerization of lipophilic dyes is a useful parameter to monitor packaging and fluidity of biomembranes. Fluorescence fluctuations, generated by trans-cis isomerization of the thiocarbocyanine dye Merocyanine 540 (MC540), were first analyzed by fluorescence correlation spectroscopy (FCS) in different alcohol solutions. Similar isomerization kinetics of MC540 in lipid vesicles could then also be monitored, and the influence of lipid polarity, membrane curvature, and cholesterol content was investigated. While no influence of membrane curvature and lipid polarity could be observed, a clear decrease in the isomerization rates could be observed with increasing cholesterol contents in the vesicle membranes. Finally, procedures to spatially map photoinduced and thermal isomerization rates on live cells by transient state (TRAST) imaging were established. On the basis of these procedures, MC540 isomerization was studied on live MCF7 cells, and TRAST images of the cells at different temperatures were found to reliably detect differences in the isomerization parameters. Our studies indicate that trans-cis isomerization is a useful parameter for probing membrane dynamics and that the TRAST imaging technique can provide spatial maps of photoinduced isomerization as well as both photoinduced and thermal back-isomerization, resolving differences in local membrane microviscosity in live cells.

Membrane Targeting of Grb2-associated Binder-1 (Gab1) Scaffolding Protein through Src Myristoylation Sequence Substitutes for Gab1 Pleckstrin Homology Domain and Switches an Epidermal Growth Factor Response to an Invasive Morphogenic Program

PubMed Central

Maroun, Christiane R.; Naujokas, Monica A.; Park, Morag

2003-01-01

The hepatocyte growth factor receptor tyrosine kinase Met promotes cell dissociation and the inherent morphogenic program of epithelial cells. In a search for substrates downstream from Met, we have previously identified the Grb2-associated binder-1 (Gab1) as critical for the morphogenic program. Gab1 is a scaffold protein that acts to diversify the signal downstream from the Met receptor through its ability to couple with multiple signal transduction pathways. Gab1 contains a pleckstrin homology (PH) domain with specificity for phosphatidylinositol 3,4,5-trisphosphate. The phospholipid binding capacity of the Gab1 PH domain is required for the localization of Gab1 at sites of cell-cell contact in colonies of epithelial cells and for epithelial morphogenesis, suggesting that PH domain-dependent subcellular localization of Gab1 is a prerequisite for function. We have investigated the requirement for membrane localization of Gab1 for biological activity. We show that substitution of the Gab1 PH domain with the myristoylation signal from the c-Src protein is sufficient to replace the Gab1 PH domain for epithelial morphogenesis. The membrane targeting of Gab1 enhances Rac activity in the absence of stimulation and switches a nonmorphogenic noninvasive response to epidermal growth factor to a morphogenic invasive program. These results suggest that the subcellular localization of Gab1 is a critical determinant for epithelial morphogenesis and invasiveness. PMID:12686619

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability.

PubMed

Smith, Alyson S; Nowak, Roberta B; Zhou, Sitong; Giannetto, Michael; Gokhin, David S; Papoin, Julien; Ghiran, Ionita C; Blanc, Lionel; Wan, Jiandi; Fowler, Velia M

2018-05-08

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.

Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation.

PubMed

Castellano, F; Montcourrier, P; Guillemot, J C; Gouin, E; Machesky, L; Cossart, P; Chavrier, P

1999-04-08

Cdc42, a GTP-binding protein of the Rho family, controls actin cytoskeletal organization and helps to generate actin-based protruding structures, such as filopodia. In vitro, Cdc42 regulates actin polymerization by facilitating the creation of free barbed ends - the more rapidly growing ends of actin filaments - and subsequent elongation at these ends. The Wiskott- Aldrich syndrome protein, WASP, which has a pleckstrin-homology domain and a Cdc42/Rac-binding motif, has been implicated in cell signaling and cytoskeleton reorganization. We have investigated the consequences of local recruitment of activated Cdc42 or WASP to the plasma membrane. We used an activated Cdc42 protein that could be recruited to an engineered membrane receptor by adding rapamycin as a bridge, and added antibody-coupled beads to aggregate these receptors. Inducible recruitment of Cdc42 to clusters of receptors stimulated actin polymerization, resulting in the formation of membrane protrusions. Cdc42-induced protrusions were enriched in the vasodilator-stimulated phosphoprotein VASP and the focal-adhesion-associated proteins zyxin and ezrin. The Cdc42 effector WASP could also induce the formation of protrusions, albeit of different morphology. This is the first demonstration that the local recruitment of activated Cdc42 or its downstream effector, WASP, to a membrane receptor in whole cells is sufficient to trigger actin polymerization that results in the formation of membrane protrusions. Our data suggest that Cdc42-induced actin-based protrusions result from the local and serial recruitment of cytoskeletal proteins including zyxin, VASP, and ezrin.

Spectral imaging toolbox: segmentation, hyperstack reconstruction, and batch processing of spectral images for the determination of cell and model membrane lipid order.

PubMed

Aron, Miles; Browning, Richard; Carugo, Dario; Sezgin, Erdinc; Bernardino de la Serna, Jorge; Eggeling, Christian; Stride, Eleanor

2017-05-12

Spectral imaging with polarity-sensitive fluorescent probes enables the quantification of cell and model membrane physical properties, including local hydration, fluidity, and lateral lipid packing, usually characterized by the generalized polarization (GP) parameter. With the development of commercial microscopes equipped with spectral detectors, spectral imaging has become a convenient and powerful technique for measuring GP and other membrane properties. The existing tools for spectral image processing, however, are insufficient for processing the large data sets afforded by this technological advancement, and are unsuitable for processing images acquired with rapidly internalized fluorescent probes. Here we present a MATLAB spectral imaging toolbox with the aim of overcoming these limitations. In addition to common operations, such as the calculation of distributions of GP values, generation of pseudo-colored GP maps, and spectral analysis, a key highlight of this tool is reliable membrane segmentation for probes that are rapidly internalized. Furthermore, handling for hyperstacks, 3D reconstruction and batch processing facilitates analysis of data sets generated by time series, z-stack, and area scan microscope operations. Finally, the object size distribution is determined, which can provide insight into the mechanisms underlying changes in membrane properties and is desirable for e.g. studies involving model membranes and surfactant coated particles. Analysis is demonstrated for cell membranes, cell-derived vesicles, model membranes, and microbubbles with environmentally-sensitive probes Laurdan, carboxyl-modified Laurdan (C-Laurdan), Di-4-ANEPPDHQ, and Di-4-AN(F)EPPTEA (FE), for quantification of the local lateral density of lipids or lipid packing. The Spectral Imaging Toolbox is a powerful tool for the segmentation and processing of large spectral imaging datasets with a reliable method for membrane segmentation and no ability in programming required. The Spectral Imaging Toolbox can be downloaded from https://uk.mathworks.com/matlabcentral/fileexchange/62617-spectral-imaging-toolbox .

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

PubMed Central

Adu-Gyamfi, Emmanuel; Johnson, Kristen A.; Fraser, Mark E.; Scott, Jordan L.; Soni, Smita P.; Jones, Keaton R.; Digman, Michelle A.; Gratton, Enrico; Tessier, Charles R.

2015-01-01

ABSTRACT Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. IMPORTANCE The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. PMID:26136573

Modulation of the caveolin-3 localization to caveolae and STAT3 to mitochondria by catecholamine-induced cardiac hypertrophy in H9c2 cardiomyoblasts

PubMed Central

Jeong, Kyuho; Kwon, Hayeong; Min, Chanhee

2009-01-01

We investigated the effect of phenylephrine (PE)- and isoproterenol (ISO)-induced cardiac hypertrophy on subcellular localization and expression of caveolin-3 and STAT3 in H9c2 cardiomyoblast cells. Caveolin-3 localization to plasma membrane was attenuated and localization of caveolin-3 to caveolae in the plasma membrane was 24.3% reduced by the catecholamine-induced hypertrophy. STAT3 and phospho-STAT3 were up-regulated but verapamil and cyclosporin A synergistically decreased the STAT3 and phospho-STAT3 levels in PE- and ISO-induced hypertrophic cells. Both expression and activation of STAT3 were increased in the nucleus by the hypertrophy. Immunofluorescence analysis revealed that the catecholamine-induced hypertrophy promoted nuclear localization of pY705-STAT3. Of interest, phosphorylation of pS727-STAT3 in mitochondria was significantly reduced by catecholamine-induced hypertrophy. In addition, mitochondrial complexes II and III were greatly down-regulated in the hypertrophic cells. Our data suggest that the alterations in nuclear and mitochondrial activation of STAT3 and caveolae localization of caveolin-3 are related to the development of the catecholamine-induced cardiac hypertrophy. PMID:19299911

Membrane topology of Golgi-localized probable S-adenosylmethionine-dependent methyltransferase in tobacco (Nicotiana tabacum) BY-2 cells.

PubMed

Liu, Jianping; Hayashi, Kyoko; Matsuoka, Ken

2015-01-01

S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) transfer methyl groups to substrates. In this study, a novel putative tobacco SAM-MTase termed Golgi-localized methyl transferase 1 (GLMT1) has been characterized. GLMT1 is comprised of 611 amino acids with short N-terminal region, putative transmembrane region, and C-terminal SAM-MTase domain. Expression of monomeric red fluorescence protein (mRFP)-tagged protein in tobacco BY-2 cell indicated that GLMT1 is a Golgi-localized protein. Analysis of the membrane topology by protease digestion suggested that both C-terminal catalytic region and N-terminal region seem to be located to the cytosolic side of the Golgi apparatus. Therefore, GLMT1 might have a different function than the previously studied SAM-MTases in plants.

Regulation of AMPA receptor localization in lipid rafts

PubMed Central

Hou, Qingming; Huang, Yunfei; Amato, Stephen; Snyder, Solomon H.; Huganir, Richard L.; Man, Heng-Ye

2009-01-01

Lipid rafts are special microdomains enriched in cholesterol, sphingolipids and certain proteins, and play important roles in a variety of cellular functions including signal transduction and protein trafficking. We report that in cultured cortical and hippocampal neurons the distribution of lipid rafts is development-dependent. Lipid rafts in mature neurons exist on the entire cell-surface and display a high degree of mobility. AMPA receptors co-localize and associate with lipid rafts in the plasma membrane. The association of AMPARs with rafts is under regulation; through the NOS–NO pathway, NMDA receptor activity increases AMPAR localization in rafts. During membrane targeting, AMPARs insert into or at close proximity of the surface raft domains. Perturbation of lipid rafts dramatically suppresses AMPA receptor exocytosis, resulting in significant reduction in AMPAR cell-surface expression. PMID:18411055

Neisseria gonorrhoeae PIII has a role on NG1873 outer membrane localization and is involved in bacterial adhesion to human cervical and urethral epithelial cells.

PubMed

Leuzzi, Rosanna; Nesta, Barbara; Monaci, Elisabetta; Cartocci, Elena; Serino, Laura; Soriani, Marco; Rappuoli, Rino; Pizza, Mariagrazia

2013-11-09

Protein PIII is one of the major outer membrane proteins of Neisseria gonorrhoeae, 95% identical to RmpM (reduction modifiable protein M) or class 4 protein of Neisseria meningitidis. RmpM is known to be a membrane protein associated by non-covalent bonds to the peptidoglycan layer and interacting with PorA/PorB porin complexes resulting in the stabilization of the bacterial membrane. The C-terminal domain of PIII (and RmpM) is highly homologous to members of the OmpA family, known to have a role in adhesion/invasion in many bacterial species. The contribution of PIII in the membrane architecture and its role in the interaction with epithelial cells has never been investigated. We generated a ΔpIII knock-out mutant strain and evaluated the effects of the loss of PIII expression on bacterial morphology and on outer membrane composition. Deletion of the pIII gene does not cause any alteration in bacterial morphology or sensitivity to detergents. Moreover, the expression profile of the main membrane proteins remains the same for the wild-type and knock-out strains, with the exception of the NG1873 which is not exported to the outer membrane and accumulates in the inner membrane in the ΔpIII knock-out mutant strain.We also show that purified PIII protein is able to bind human cervical and urethral cells and that the ΔpIII knock-out mutant strain has a lower ability to adhere to human cervical and urethral cells. Here we demonstrated that the PIII protein does not play a key structural role in the membrane organization of gonococcus and does not induce major effects on the expression of the main outer membrane proteins. However, in the PIII knock-out strain, the NG1873 protein is not localized in the outer membrane as it is in the wild-type strain suggesting a possible interaction of PIII with NG1873. The evidence that PIII binds to human epithelial cells derived from the female and male genital tract highlights a possible role of PIII in the virulence of gonococcus and suggests that the structural homology to OmpA is conserved also at functional level.

New type III effectors from Xanthomonas campestris pv. vesicatoria trigger plant reactions dependent on a conserved N-myristoylation motif.

PubMed

Thieme, Frank; Szczesny, Robert; Urban, Alexander; Kirchner, Oliver; Hause, Gerd; Bonas, Ulla

2007-10-01

Pathogenicity of the gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria depends on a type III secretion system, which translocates bacterial effector proteins into the plant cell. In this study, we identified two novel type III effectors, XopE1 and XopE2 (Xanthomonas outer proteins), using the AvrBs3 effector domain as reporter. XopE1 and XopE2 belong to the HopX family and possess a conserved putative N-myristoylation motif that is also present in the effector XopJ from X. campestris pv. vesicatoria 85-10. XopJ is a member of the YopJ/AvrRxv family of acetyltransferases. Confocal laser scanning microscopy and immunocytochemistry revealed that green fluorescent protein fusions of XopE1, XopE2, and XopJ localized to the plant cell plasma membrane. Targeting to the membrane is probably due to N-myristoylation, because a point mutation in the putative myristoylated glycine residue G2 in XopE1, XopE2, and XopJ resulted in cytoplasmic localization of the mutant proteins. Results of hydroxylamine treatments of XopE2 protein extracts suggest that the proteins are additionally anchored in the host cell plasma membrane by palmitoylation. The membrane localization of the effectors strongly influences the phenotypes they trigger in the plant. Agrobacterium-mediated expression of xopE1 and xopJ in Nicotiana benthamiana led to cell-death reactions that, for xopJ, were dependent on the N-myristoylation motif. In the case of xopE1(G2A), cell death was more pronounced with the mutant than with the wild-type protein. In addition, XopE2 has an avirulence activity in Solanum pseudocapsicum.

Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.

PubMed

Vosolsobě, Stanislav; Petrášek, Jan; Schwarzerová, Kateřina

2017-05-01

The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Magnetic domain wall tweezers: a new tool for mechanobiology studies on individual target cells.

PubMed

Monticelli, M; Conca, D V; Albisetti, E; Torti, A; Sharma, P P; Kidiyoor, G; Barozzi, S; Parazzoli, D; Ciarletta, P; Lupi, M; Petti, D; Bertacco, R

2016-08-07

In vitro tests are of fundamental importance for investigating cell mechanisms in response to mechanical stimuli or the impact of the genotype on cell mechanical properties. In particular, the application of controlled forces to activate specific bio-pathways and investigate their effects, mimicking the role of the cellular environment, is becoming a prominent approach in the emerging field of mechanobiology. Here, we present an on-chip device based on magnetic domain wall manipulators, which allows the application of finely controlled and localized forces on target living cells. In particular, we demonstrate the application of a magnetic force in the order of hundreds of pN on the membrane of HeLa cells cultured on-chip, via manipulation of 1 μm superparamagnetic beads. Such a mechanical stimulus produces a sizable local indentation of the cellular membrane of about 2 μm. Upon evaluation of the beads' position within the magnetic field originated by the domain wall, the force applied during the experiments is accurately quantified via micromagnetic simulations. The obtained value is in good agreement with that calculated by the application of an elastic model to the cellular membrane.

Proteomic Analysis of Detergent-resistant Membrane Microdomains in Trophozoite Blood Stage of the Human Malaria Parasite Plasmodium falciparum*

PubMed Central

Yam, Xue Yan; Birago, Cecilia; Fratini, Federica; Di Girolamo, Francesco; Raggi, Carla; Sargiacomo, Massimo; Bachi, Angela; Berry, Laurence; Fall, Gamou; Currà, Chiara; Pizzi, Elisabetta; Breton, Catherine Braun; Ponzi, Marta

2013-01-01

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum. PMID:24045696

Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayashi, Masami; Shimada, Yukiko; Inomata, Mitsushi

2006-12-22

The C-terminal domain (D4) of perfringolysin O binds selectively to cholesterol in cholesterol-rich microdomains. To address the issue of whether cholesterol-rich microdomains exist in the inner leaflet of the plasma membrane, we expressed D4 as a fusion protein with EGFP in MEF cells. More than half of the EGFP-D4 expressed in stable cell clones was bound to membranes in raft fractions. Depletion of membrane cholesterol with {beta}-cyclodextrin reduced the amount of EGFP-D4 localized in raft fractions, confirming EGFP-D4 binding to cholesterol-rich microdomains. Subfractionation of the raft fractions showed most of the EGFP-D4 bound to the plasma membrane rather than tomore » intracellular membranes. Taken together, these results strongly suggest the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane.« less

Cochlear potential difference between endolymph fluid and the hair cell's interior: a retold interpretation based on the Goldman equation.

PubMed

Kurbel, Sven; Borzan, Vladimir; Golem, Hilda; Dinjar, Kristijan

2017-02-01

Reported cochlear potential values of near 150 mV are often attributed to endolymph itself, although membrane potentials result from ion fluxes across the adjacent semipermeable membranes due to concentration gradients. Since any two fluids separated by a semipermeable membrane develop potential due to differences in solute concentrations, a proposed interpretation here is that positive potential emanates from the Reissner membrane due to small influx of sodium from perilymph to endolymph. Basolateral hair cell membranes leak potassium into the interstitial fluid and this negative potential inside hair cells further augments the electric gradient of cochlear potential. Taken together as a sum, these two potentials are near the reported values of cochlear potential. This is based on reported data for cochlear fluids used for the calculation of Nernst and Goldman potentials. The reported positive potential of Reissner membrane can be explained almost entirely by the traffic of Na+ that enters endolymph through this membrane. At the apical membrane of hair cells, acoustic stimulation modulates stereocillia permeability to potassium. Potassium concentration gradients on the apical membrane are low (the calculated Nernst value is <+3 mV), suggesting that the potassium current is not caused by the local potassium concentration gradient, but an electric field between the positive sodium generated potential on the Reissner membrane and negative inside hair cells. Potassium is forced by this overall electric field to enter hair cells when stereocilia are permeable due to mechanical bending. Copyright© by the Medical Assotiation of Zenica-Doboj Canton.

3D visualization of membrane failures in fuel cells

NASA Astrophysics Data System (ADS)

Singh, Yadvinder; Orfino, Francesco P.; Dutta, Monica; Kjeang, Erik

2017-03-01

Durability issues in fuel cells, due to chemical and mechanical degradation, are potential impediments in their commercialization. Hydrogen leak development across degraded fuel cell membranes is deemed a lifetime-limiting failure mode and potential safety issue that requires thorough characterization for devising effective mitigation strategies. The scope and depth of failure analysis has, however, been limited by the 2D nature of conventional imaging. In the present work, X-ray computed tomography is introduced as a novel, non-destructive technique for 3D failure analysis. Its capability to acquire true 3D images of membrane damage is demonstrated for the very first time. This approach has enabled unique and in-depth analysis resulting in novel findings regarding the membrane degradation mechanism; these are: significant, exclusive membrane fracture development independent of catalyst layers, localized thinning at crack sites, and demonstration of the critical impact of cracks on fuel cell durability. Evidence of crack initiation within the membrane is demonstrated, and a possible new failure mode different from typical mechanical crack development is identified. X-ray computed tomography is hereby established as a breakthrough approach for comprehensive 3D characterization and reliable failure analysis of fuel cell membranes, and could readily be extended to electrolyzers and flow batteries having similar structure.

Propagating Cell-Membrane Waves Driven by Curved Activators of Actin Polymerization

PubMed Central

Peleg, Barak; Disanza, Andrea; Scita, Giorgio; Gov, Nir

2011-01-01

Cells exhibit propagating membrane waves which involve the actin cytoskeleton. One type of such membranal waves are Circular Dorsal Ruffles (CDR) which are related to endocytosis and receptor internalization. Experimentally, CDRs have been associated with membrane bound activators of actin polymerization of concave shape. We present experimental evidence for the localization of convex membrane proteins in these structures, and their insensitivity to inhibition of myosin II contractility in immortalized mouse embryo fibroblasts cell cultures. These observations lead us to propose a theoretical model which explains the formation of these waves due to the interplay between complexes that contain activators of actin polymerization and membrane-bound curved proteins of both types of curvature (concave and convex). Our model predicts that the activity of both types of curved proteins is essential for sustaining propagating waves, which are abolished when one type of curved activator is removed. Within this model waves are initiated when the level of actin polymerization induced by the curved activators is higher than some threshold value, which allows the cell to control CDR formation. We demonstrate that the model can explain many features of CDRs, and give several testable predictions. This work demonstrates the importance of curved membrane proteins in organizing the actin cytoskeleton and cell shape. PMID:21533032

ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants.

PubMed

Isono, Erika; Kalinowska, Kamila

2017-12-01

To control the abundance of plasma membrane receptors and transporters is crucial for proper perception and response to extracellular signals from surrounding cells and the environment. Posttranslational modification of plasma membrane proteins, especially ubiquitin conjugation or ubiquitylation, is key for the determination of stability for many transmembrane proteins localized on the cell surface. The targeted degradation is ensured by a complex network of proteins among which the endosomal sorting complex required for transport (ESCRT) plays a central role. This review focuses on progresses made in recent years on the understanding of the function of the ESCRT machinery in the degradation of ubiquitylated plasma membrane proteins in plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cell recruitment by amnion chorion grafts promotes neovascularization.

PubMed

Maan, Zeshaan N; Rennert, Robert C; Koob, Thomas J; Januszyk, Michael; Li, William W; Gurtner, Geoffrey C

2015-02-01

Nonhealing wounds are a significant health burden. Stem and progenitor cells can accelerate wound repair and regeneration. Human amniotic membrane has demonstrated efficacy in promoting wound healing, though the underlying mechanisms remain unknown. A dehydrated human amnion chorion membrane (dHACM) was tested for its ability to recruit hematopoietic progenitor cells to a surgically implanted graft in a murine model of cutaneous ischemia. dHACM was subcutaneously implanted under elevated skin (ischemic stimulus) in either wild-type mice or mice surgically parabiosed to green fluorescent protein (GFP) + reporter mice. A control acellular dermal matrix, elevated skin without an implant, and normal unwounded skin were used as controls. Wound tissue was harvested and processed for histology and flow cytometric analysis. Implanted dHACMs recruited significantly more progenitor cells compared with controls (*P < 0.05) and displayed in vivo SDF-1 expression with incorporation of CD34 + progenitor cells within the matrix. Parabiosis modeling confirmed the circulatory origin of recruited cells, which coexpressed progenitor cell markers and were localized to foci of neovascularization within implanted matrices. In summary, dHACM effectively recruits circulating progenitor cells, likely because of stromal derived factor 1 (SDF-1) expression. The recruited cells express markers of "stemness" and localize to sites of neovascularization, providing a partial mechanism for the clinical efficacy of human amniotic membrane in the treatment of chronic wounds. Copyright © 2015 Elsevier Inc. All rights reserved.

Cell recruitment by amnion chorion grafts promotes neovascularization

PubMed Central

Koob, Thomas J.; Januszyk, Michael; Li, William W.; Gurtner, Geoffrey C.

2015-01-01

Background Nonhealing wounds are a significant health burden. Stem and progenitor cells can accelerate wound repair and regeneration. Human amniotic membrane has demonstrated efficacy in promoting wound healing, though the underlying mechanisms remain unknown. A dehydrated human amnion chorion membrane (dHACM) was tested for its ability to recruit hematopoietic progenitor cells to a surgically implanted graft in a murine model of cutaneous ischemia. Methods dHACM was subcutaneously implanted under elevated skin (ischemic stimulus) in either wild-type mice or mice surgically parabiosed to green fluorescent protein (GFP) + reporter mice. A control acellular dermal matrix, elevated skin without an implant, and normal unwounded skin were used as controls. Wound tissue was harvested and processed for histology and flow cytometric analysis. Results Implanted dHACMs recruited significantly more progenitor cells compared with controls (*P < 0.05) and displayed in vivo SDF-1 expression with incorporation of CD34 + progenitor cells within the matrix. Parabiosis modeling confirmed the circulatory origin of recruited cells, which coexpressed progenitor cell markers and were localized to foci of neovascularization within implanted matrices. Conclusions In summary, dHACM effectively recruits circulating progenitor cells, likely because of stromal derived factor 1 (SDF-1) expression. The recruited cells express markers of “stemness” and localize to sites of neovascularization, providing a partial mechanism for the clinical efficacy of human amniotic membrane in the treatment of chronic wounds. PMID:25266600

Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane.

PubMed

Röper, K; Corbeil, D; Huttner, W B

2000-09-01

Membrane cholesterol-sphingolipid 'rafts', which are characterized by their insolubility in the non-ionic detergent Triton X-100 in the cold, have been implicated in the sorting of certain membrane proteins, such as placental alkaline phosphatase (PLAP), to the apical plasma membrane domain of epithelial cells. Here we show that prominin, an apically sorted pentaspan membrane protein, becomes associated in the trans-Golgi network with a lipid raft that is soluble in Triton X-100 but insoluble in another non-ionic detergent, Lubrol WX. At the cell surface, prominin remains insoluble in Lubrol WX and is selectively associated with microvilli, being largely segregated from the membrane subdomains containing PLAP. Cholesterol depletion results in the loss of prominin's microvillus-specific localization but does not lead to its complete intermixing with PLAP. We propose the coexistence within a membrane domain, such as the apical plasma membrane, of different cholesterol-based lipid rafts, which underlie the generation and maintenance of membrane subdomains.

Migration of the guinea pig sperm membrane protein PH-20 from one localized surface domain to another does not occur by a simple diffusion-trapping mechanism.

PubMed

Cowan, A E; Myles, D G; Koppel, D E

1991-03-01

The redistribution of membrane proteins on the surface of cells is a prevalent feature of differentiation in a variety of cells. In most cases the mechanism responsible for such redistribution is poorly understood. Two potential mechanisms for the redistribution of surface proteins are: (1) passive diffusion coupled with trapping, and (2) active translocation. We have studied the process of membrane protein redistribution for the PH-20 protein of guinea pig sperm, a surface protein required for sperm binding to the egg zona pellucida (P. Primakoff, H. Hyatt, and D. G. Myles (1985). J. Cell Biol. 101, 2239-2244). PH-20 protein is localized to the posterior head plasma menbrane of the mature sperm cell. Following the exocytotic acrosome reaction, PH-20 protein moves into the newly incorporated inner acrosomal membrane (IAM), placing it in a position favorable for a role in binding sperm to the egg zona pellucida (D. G. Myles, and P. Primakoff (1984), J. Cell Biol. 99, 1634-1641). To analyze the mechanistic basis for this protein migration, we have used fluorescence microscopy and digital image processing to characterize PH-20 protein migration in individual cells. PH-20 protein was observed to move against a concentration gradient in the posterior head plasma membrane. This result argues strongly against a model of passive diffusion followed by trapping in the IAM, and instead suggests that an active process serves to concentrate PH-20 protein toward the boundary separating the posterior head and IAM regions. A transient gradient of PH-20 concentration observed in the IAM suggests that once PH-20 protein reaches the IAM, it is freely diffusing. Additionally, we observed that migration of PH-20 protein was calcium dependent.

Arenavirus budding resulting from viral-protein-associated cell membrane curvature

PubMed Central

Schley, David; Whittaker, Robert J.; Neuman, Benjamin W.

2013-01-01

Viral replication occurs within cells, with release (and onward infection) primarily achieved through two alternative mechanisms: lysis, in which virions emerge as the infected cell dies and bursts open; or budding, in which virions emerge gradually from a still living cell by appropriating a small part of the cell membrane. Virus budding is a poorly understood process that challenges current models of vesicle formation. Here, a plausible mechanism for arenavirus budding is presented, building on recent evidence that viral proteins embed in the inner lipid layer of the cell membrane. Experimental results confirm that viral protein is associated with increased membrane curvature, whereas a mathematical model is used to show that localized increases in curvature alone are sufficient to generate viral buds. The magnitude of the protein-induced curvature is calculated from the size of the amphipathic region hypothetically removed from the inner membrane as a result of translation, with a change in membrane stiffness estimated from observed differences in virion deformation as a result of protein depletion. Numerical results are based on experimental data and estimates for three arenaviruses, but the mechanisms described are more broadly applicable. The hypothesized mechanism is shown to be sufficient to generate spontaneous budding that matches well both qualitatively and quantitatively with experimental observations. PMID:23864502

Interaction of Munc18c and syntaxin4 facilitates invadopodium formation and extracellular matrix invasion of tumor cells.

PubMed

Brasher, Megan I; Martynowicz, David M; Grafinger, Olivia R; Hucik, Andrea; Shanks-Skinner, Emma; Uniacke, James; Coppolino, Marc G

2017-09-29

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

USDA-ARS?s Scientific Manuscript database

Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>500) known and predicted TA proteins in Arabidopsis for those annotated, based on Gene Ontology, to possess mitochondrial...

Peroxisomal membrane ascorbate peroxidase is sorted to a membranous network that resembles a subdomain of the endoplasmic reticulum.

PubMed Central

Mullen, R T; Lisenbee, C S; Miernyk, J A; Trelease, R N

1999-01-01

The peroxisomal isoform of ascorbate peroxidase (APX) is a novel membrane isoform that functions in the regeneration of NAD(+) and protection against toxic reactive oxygen species. The intracellular localization and sorting of peroxisomal APX were examined both in vivo and in vitro. Epitope-tagged peroxisomal APX, which was expressed transiently in tobacco BY-2 cells, localized to a reticular/circular network that resembled endoplasmic reticulum (ER; 3,3'-dihexyloxacarbocyanine iodide-stained membranes) and to peroxisomes. The reticular network did not colocalize with other organelle marker proteins, including three ER reticuloplasmins. However, in vitro, peroxisomal APX inserted post-translationally into the ER but not into other purified organelle membranes (including peroxisomal membranes). Insertion into the ER depended on the presence of molecular chaperones and ATP. These results suggest that regions of the ER serve as a possible intermediate in the sorting pathway of peroxisomal APX. Insight into this hypothesis was obtained from in vivo experiments with brefeldin A (BFA), a toxin that blocks vesicle-mediated protein export from ER. A transiently expressed chloramphenicol acetyltransferase-peroxisomal APX (CAT-pAPX) fusion protein accumulated only in the reticular/circular network in BFA-treated cells; after subsequent removal of BFA from these cells, the CAT-pAPX was distributed to preexisting peroxisomes. Thus, plant peroxisomal APX, a representative enzymatic peroxisomal membrane protein, is sorted to peroxisomes through an indirect pathway involving a preperoxisomal compartment with characteristics of a distinct subdomain of the ER, possibly a peroxisomal ER subdomain. PMID:10559442

Plasma membrane organization and dynamics is probe and cell line dependent.

PubMed

Huang, Shuangru; Lim, Shi Ying; Gupta, Anjali; Bag, Nirmalya; Wohland, Thorsten

2017-09-01

The action and interaction of membrane receptor proteins take place within the plasma membrane. The plasma membrane, however, is not a passive matrix. It rather takes an active role and regulates receptor distribution and function by its composition and the interaction of its lipid components with embedded and surrounding proteins. Furthermore, it is not a homogenous fluid but contains lipid and protein domains of various sizes and characteristic lifetimes which are important in regulating receptor function and signaling. The precise lateral organization of the plasma membrane, the differences between the inner and outer leaflet, and the influence of the cytoskeleton are still debated. Furthermore, there is a lack of comparisons of the organization and dynamics of the plasma membrane of different cell types. Therefore, we used four different specific membrane markers to test the lateral organization, the differences between the inner and outer membrane leaflet, and the influence of the cytoskeleton of up to five different cell lines, including Chinese hamster ovary (CHO-K1), Human cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) cells by Imaging Total Internal Reflection (ITIR)-Fluorescence Correlation Spectroscopy (FCS). We measure diffusion in the temperature range of 298-310K to measure the Arrhenius activation energy (E Arr ) of diffusion and apply the FCS diffusion law to obtain information on the spatial organization of the probe molecules on the various cell membranes. Our results show clear differences of the FCS diffusion law and E Arr for the different probes in dependence of their localization. These differences are similar in the outer and inner leaflet of the membrane. However, these values can differ significantly between different cell lines raising the question how molecular plasma membrane events measured in different cell lines can be compared. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2016 Elsevier B.V. All rights reserved.

Intracellular redox status controls membrane localization of pro- and anti-migratory signaling molecules.

PubMed

Hempel, Nadine; Melendez, J Andres

2014-01-01

Shifts in intracellular Reactive Oxygen Species (ROS) have been shown to contribute to carcinogenesis and to tumor progression. In addition to DNA and cell damage by surges in ROS, sub-lethal increases in ROS are implicated in regulating cellular signaling that enhances pro-metastatic behavior. We previously showed that subtle increases in endogenous H2O2 regulate migratory and invasive behavior of metastatic bladder cancer cells through phosphatase inhibition and consequential phosphorylation of p130cas, an adapter of the FAK signaling pathway. We further showed that enhanced redox status contributed to enhanced localization of p130cas to the membrane of metastatic cells. Here we show that this signaling complex can similarly be induced in a redox-engineered cell culture model that enables regulation of intracellular steady state H2O2 level by enforced expression of superoxide dismutase 2 (Sod2) and catalase. Expression of Sod2 leads to enhanced p130cas phosphorylation in HT-1080 fibrosarcoma and UM-UC-6 bladder cancer cells. These changes are mediated by H2O2, as co-expression of Catalase abrogates p130cas phosphorylation and its interaction with the adapter protein Crk. Importantly, we establish that the redox environment influence the localization of the tumor suppressor and phosphatase PTEN, in both redox-engineered and metastatic bladder cancer cells that display endogenous increases in H2O2. Importantly, PTEN oxidation leads to its dissociation from the plasma membrane. This indicates that oxidation of PTEN not only influences its activity, but also regulates its cellular localization, effectively removing it from its primary site of lipid phosphatase activity. These data introduce hitherto unappreciated paradigms whereby ROS can reciprocally regulate the cellular localization of pro- and anti-migratory signaling molecules, p130cas and PTEN, respectively. These data further confirm that altering antioxidant status and the intracellular ROS environment can have profound effects on pro-metastatic signaling pathways.

Network and neuronal membrane properties in hybrid networks reciprocally regulate selectivity to rapid thalamocortical inputs.

PubMed

Pesavento, Michael J; Pinto, David J

2012-11-01

Rapidly changing environments require rapid processing from sensory inputs. Varying deflection velocities of a rodent's primary facial vibrissa cause varying temporal neuronal activity profiles within the ventral posteromedial thalamic nucleus. Local neuron populations in a single somatosensory layer 4 barrel transform sparsely coded input into a spike count based on the input's temporal profile. We investigate this transformation by creating a barrel-like hybrid network with whole cell recordings of in vitro neurons from a cortical slice preparation, embedding the biological neuron in the simulated network by presenting virtual synaptic conductances via a conductance clamp. Utilizing the hybrid network, we examine the reciprocal network properties (local excitatory and inhibitory synaptic convergence) and neuronal membrane properties (input resistance) by altering the barrel population response to diverse thalamic input. In the presence of local network input, neurons are more selective to thalamic input timing; this arises from strong feedforward inhibition. Strongly inhibitory (damping) network regimes are more selective to timing and less selective to the magnitude of input but require stronger initial input. Input selectivity relies heavily on the different membrane properties of excitatory and inhibitory neurons. When inhibitory and excitatory neurons had identical membrane properties, the sensitivity of in vitro neurons to temporal vs. magnitude features of input was substantially reduced. Increasing the mean leak conductance of the inhibitory cells decreased the network's temporal sensitivity, whereas increasing excitatory leak conductance enhanced magnitude sensitivity. Local network synapses are essential in shaping thalamic input, and differing membrane properties of functional classes reciprocally modulate this effect.

Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells.

PubMed

Migocka, Magdalena; Papierniak, Anna; Kosatka, Ewelina; Klobus, Grazyna

2011-10-01

The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.

Sidedness of Carbamazepine Accessibility to Voltage-Gated Sodium Channels

PubMed Central

Jo, Sooyeon

2014-01-01

Voltage-gated sodium channels are inhibited by many local anesthetics, antiarrhythmics, and antiepileptic drugs. The local anesthetic lidocaine appears to be able to access its binding site in the sodium channel only from the membrane phase or from the internal face of the channel. In contrast, the antiepileptic drug carbamazepine was found to inhibit voltage-gated sodium channels only with external, but not internal, application, implying a major difference. We investigated this point using both whole-cell and inside-out patch recordings from human Nav1.7 channels in a stable cell line. In the whole-cell configuration, carbamazepine inhibited sodium current within seconds when applied externally, but had little or no effect when applied internally for up to 15 minutes, confirming previous results. However, carbamazepine inhibited sodium channels effectively and rapidly when applied to the internal face of the membrane using inside-out patch recording. We found that lidocaine also has little or no effect when applied intracellularly in whole-cell recording, but blocks effectively and rapidly when applied to the internal surface using inside-out patches. In contrast, the cationic lidocaine derivative QX-314 (N-ethyl-lidocaine) blocks effectively when applied internally with whole-cell dialysis, as well as when applied to inside-out patches. We conclude that carbamazepine and lidocaine access the sodium channel in similar ways and hypothesize that their lack of effect with internal dialysis in whole-cell recording reflects rapid exit through membrane near the pipette recording site. This effect likely limits the ability of any compound with significant membrane permeability to be applied intracellularly by whole-cell dialysis. PMID:24319110

Targeting Thromboxane A2 Receptor for Antimetastasis Therapy of Breast Cancer

DTIC Science & Technology

2012-09-01

Figure 1B, right). The predominant localization of TP in the cytosol of MDA-MB-231 cells led us to examine whether a subset of TP is expressed at cell...in the Figure 1C, MDA-MB-231 cells had positive staining at cell surface, suggesting that a subset of TP was localized at plasma membrane. We...34, ISBN 979- 953-307-183-0. 2011. Robbins GT, Nie D. PPAR gamma, bioactive lipids, and cancer progression. Front Biosci. 17:1816-34, 2012. Grants

Bleb Expansion in Migrating Cells Depends on Supply of Membrane from Cell Surface Invaginations.

PubMed

Goudarzi, Mohammad; Tarbashevich, Katsiaryna; Mildner, Karina; Begemann, Isabell; Garcia, Jamie; Paksa, Azadeh; Reichman-Fried, Michal; Mahabaleshwar, Harsha; Blaser, Heiko; Hartwig, Johannes; Zeuschner, Dagmar; Galic, Milos; Bagnat, Michel; Betz, Timo; Raz, Erez

2017-12-04

Cell migration is essential for morphogenesis, organ formation, and homeostasis, with relevance for clinical conditions. The migration of primordial germ cells (PGCs) is a useful model for studying this process in the context of the developing embryo. Zebrafish PGC migration depends on the formation of cellular protrusions in form of blebs, a type of protrusion found in various cell types. Here we report on the mechanisms allowing the inflation of the membrane during bleb formation. We show that the rapid expansion of the protrusion depends on membrane invaginations that are localized preferentially at the cell front. The formation of these invaginations requires the function of Cdc42, and their unfolding allows bleb inflation and dynamic cell-shape changes performed by migrating cells. Inhibiting the formation and release of the invaginations strongly interfered with bleb formation, cell motility, and the ability of the cells to reach their target. Copyright © 2017 Elsevier Inc. All rights reserved.

Integrated light and scanning electron microscopy of GFP-expressing cells.

PubMed

Peddie, Christopher J; Liv, Nalan; Hoogenboom, Jacob P; Collinson, Lucy M

2014-01-01

Integration of light and electron microscopes provides imaging tools in which fluorescent proteins can be localized to cellular structures with a high level of precision. However, until recently, there were few methods that could deliver specimens with sufficient fluorescent signal and electron contrast for dual imaging without intermediate staining steps. Here, we report protocols that preserve green fluorescent protein (GFP) in whole cells and in ultrathin sections of resin-embedded cells, with membrane contrast for integrated imaging. Critically, GFP is maintained in a stable and active state within the vacuum of an integrated light and scanning electron microscope. For light microscopists, additional structural information gives context to fluorescent protein expression in whole cells, illustrated here by analysis of filopodia and focal adhesions in Madin Darby canine kidney cells expressing GFP-Paxillin. For electron microscopists, GFP highlights the proteins of interest within the architectural space of the cell, illustrated here by localization of the conical lipid diacylglycerol to cellular membranes. © 2014 Elsevier Inc. All rights reserved.

Cell surface localization of the 78 kD glucose regulated protein (GRP 78) induced by thapsigargin.

PubMed

Delpino, A; Piselli, P; Vismara, D; Vendetti, S; Colizzi, V

1998-01-01

In the present study it was found that the synthesis of the 78 kD glucose-regulated protein (GRP 78 or BIP) is vigorously induced in human rabdomiosarcoma cells (TE 671/RD) following both short-term (1 h) and prolonged (18 h) exposure to 100 nM thapsigargin (Tg). Flow cytometric analysis with a specific anti-GRP 78 polyclonal antibody showed that Tg-treated cells express the GRP 78 on the plasma membrane. Cell surface localization of the Tg-induced GRP 78 was confirmed by biotinylation of membrane-exposed proteins and subsequent isolation of the biotin-labelled proteins by streptavidin/agarose affinity chromatography. It was found that a fraction of the Tg-induced GRP 78 is present among the biotin-labelled, surface-exposed, proteins. Conversely, the GRP 78 immunoprecipitated from unfractionated lysates of Tg-treated and biotin-reacted cells was found to be biotinylated. This is the first report demonstrating surface expression of GRP 78 in cells exposed to a specific GRP 78-inducing stimulus.

Localization of cholesterol in sphingomyelinase-treated fibroblasts.

PubMed Central

Pörn, M I; Slotte, J P

1995-01-01

The distribution of cellular unesterified cholesterol was studied in fibroblasts, which had been depleted of plasma membrane sphingomyelin by exposure to exogenous sphingomyelinase. This treatment has previously been shown to induce an increase in cholesterol esterification, a decrease in the biosynthesis of cholesterol, and a decreased susceptibility of cell cholesterol to oxidation with cholesterol oxidase. When the cellular localization of cholesterol was studied with fluorescent filipin staining, sphingomyelin depletion did not cause any visible changes in the filipin-cholesterol staining pattern, suggesting that the major part of cellular cholesterol was retained in the plasma membrane after sphingomyelinase treatment. After the oxidation of cell-surface cholesterol with cholesterol oxidase, the plasma membrane was no longer stained by filipin, but the plasma membrane cholesterol of sphingomyelin-depleted cells appeared to be resistant to oxidation with cholesterol oxidase when sphingomyelinase was used as an oxidation-promoting agent. However, the use of hypotonic buffer or phosphatidylcholine-specific phospholipase C together with cholesterol oxidase resulted in a complete oxidation of the cell-surface cholesterol in sphingomyelin-depleted cells, as evidenced by the filipin-cholesterol staining pattern. Similar results were obtained when [3H]cholesterol-labelled fibroblasts were used for determination of the susceptibility to cholesterol oxidation. The kinetics of [3H]cholesterol oxidation in sphingomyelin-depleted cells with cholesterol oxidase in hypotonic buffer indicated that approximately 85% of the cellular cholesterol still resided in the plasma membrane after sphingomyelin depletion. These results are contradictory to earlier reports on sphingomyelinase-induced changes in cellular cholesterol distribution and suggest that minor changes in the kinetics of cholesterol transport from the plasma membrane to the endoplasmic reticulum may be responsible for the sphingomyelinase-induced changes in the rates of cholesterol metabolism. Whereas the use of phospholipases to promote the oxidation of cholesterol in some instances might lead to misinterpretations, the use of hypotonic buffer together with cholesterol oxidase proved to be a more reliable method for the determination of cellular cholesterol distribution. Images Figure 1 Figure 2 PMID:7755574

Local protein dynamics during microvesicle exocytosis in neuroendocrine cells.

PubMed

Somasundaram, Agila; Taraska, Justin

2018-06-06

Calcium triggered exocytosis is key to many physiological processes, including neurotransmitter and hormone release by neurons and endocrine cells. Dozens of proteins regulate exocytosis, yet the temporal and spatial dynamics of these factors during vesicle fusion remain unclear. Here we use total internal reflection fluorescence microscopy to visualize local protein dynamics at single sites of exocytosis of small synaptic-like microvesicles in live cultured neuroendocrine PC12 cells. We employ two-color imaging to simultaneously observe membrane fusion (using vesicular acetylcholine transporter (VAChT) tagged to pHluorin) and the dynamics of associated proteins at the moments surrounding exocytosis. Our experiments show that many proteins, including the SNAREs syntaxin1 and VAMP2, the SNARE modulator tomosyn, and Rab proteins, are pre-clustered at fusion sites and rapidly lost at fusion. The ATPase NSF is locally recruited at fusion. Interestingly, the endocytic BAR domain-containing proteins amphiphysin1, syndapin2, and endophilins are dynamically recruited to fusion sites, and slow the loss of vesicle membrane-bound cargo from fusion sites. A similar effect on vesicle membrane protein dynamics was seen with the over-expression of the GTPases dynamin1 and dynamin2. These results suggest that proteins involved in classical clathrin-mediated endocytosis can regulate exocytosis of synaptic-like microvesicles. Our findings provide insights into the dynamics, assembly, and mechanistic roles of many key factors of exocytosis and endocytosis at single sites of microvesicle fusion in live cells.

Nuclear Lipids in the Nervous System: What they do in Health and Disease.

PubMed

Garcia-Gil, Mercedes; Albi, Elisabetta

2017-02-01

In the last 20 years it has been widely demonstrated that cell nucleus contains neutral and polar lipids localized in nuclear membranes, nucleoli, nuclear matrix and chromatin. Nuclear lipids may show specific organization forming nuclear lipid microdomains and have both structural and functional roles. Depending on their localization, nuclear lipids play different roles such as the regulation of nuclear membrane and nuclear matrix fluidity but they also can act as platforms for vitamin and hormone function, for active chromatin anchoring, and for the regulation of gene expression, DNA duplication and transcription. Crosstalk among different kinds of lipid signalling pathways influence the physiopathology of numerous cell types. In neural cells the nuclear lipids are involved in cell proliferation, differentiation, inflammation, migration and apoptosis. Abnormal metabolism of nuclear lipids might be closely associated with tumorigenesis and neurodegenerative diseases such as Alzheimer disease and Parkinson disease among others.

Essential Function of Protein 4.1G in Targeting of Membrane Protein Palmitoylated 6 into Schmidt-Lanterman Incisures in Myelinated Nerves

PubMed Central

Saitoh, Yurika; Ohno, Nobuhiko; Komada, Masayuki; Saitoh, Sei; Peles, Elior; Ohno, Shinichi

2012-01-01

Protein 4.1G is a membrane skeletal protein found in specific subcellular structures in myelinated Schwann cells and seminiferous tubules. Here, we show that in the mouse sciatic nerve, protein 4.1G colocalized at Schmidt-Lanterman incisures (SLI) and the paranodes with a member of the membrane-associated guanylate kinase (MAGUK) family, membrane protein palmitoylated 6 (MPP6). Coimmunoprecipitation experiments revealed that MPP6 was interacting with protein 4.1G. In contrast to wild-type nerves, in 4.1G knockout mice, MPP6 was found largely in the cytoplasm near Schwann cell nuclei, indicating an abnormal protein transport. Although the SLI remained in the 4.1G knockout sciatic nerves, as confirmed by E-cadherin immunostaining, their shape was altered in aged 4.1G knockout nerves compared to their shape in wild-type nerves. In the seminiferous tubules, MPP6 was localized similarly to protein 4.1G along cell membranes of the spermatogonium and early spermatocytes. However, in contrast to myelinated peripheral nerves, the specific localization of MPP6 in the seminiferous tubules was unaltered in the absence of protein 4.1G. These results indicate that 4.1G has a specific role in the targeting of MPP6 to the SLI and the assembly of these subcellular structures. PMID:22025680

Prevention of Ca(2+)-mediated action potentials in GABAergic local circuit neurones of rat thalamus by a transient K+ current.

PubMed Central

Pape, H C; Budde, T; Mager, R; Kisvárday, Z F

1994-01-01

1. Neurones enzymatically dissociated from the rat dorsal lateral geniculate nucleus (LGN) were identified as GABAergic local circuit interneurones and geniculocortical relay cells, based upon quantitative analysis of soma profiles, immunohistochemical detection of GABA or glutamic acid decarboxylase, and basic electrogenic behaviour. 2. During whole-cell current-clamp recording, isolated LGN neurones generated firing patterns resembling those in intact tissue, with the most striking difference relating to the presence in relay cells of a Ca2+ action potential with a low threshold of activation, capable of triggering fast spikes, and the absence of a regenerative Ca2+ response with a low threshold of activation in local circuit cells. 3. Whole-cell voltage-clamp experiments demonstrated that both classes of LGN neurones possess at least two voltage-dependent membrane currents which operate in a range of membrane potentials negative to the threshold for generation of Na(+)-K(+)-mediated spikes: the T-type Ca2+ current (IT) and an A-type K+ current (IA). Taking into account the differences in membrane surface area, the average size of IT was similar in the two types of neurones, and interneurones possessed a slightly larger A-conductance. 4. In local circuit neurones, the ranges of steady-state inactivation and activation of IT and IA were largely overlapping (VH = 81.1 vs. -82.8 mV), both currents activated at around -70 mV, and they rapidly increased in amplitude with further depolarization. In relay cells, the inactivation curve of IT was negatively shifted along the voltage axis by about 20 mV compared with that of IA (Vh = -86.1 vs. -69.2 mV), and the activation threshold for IT (at -80 mV) was 20 mV more negative than that for IA. In interneurones, the activation range of IT was shifted to values more positive than that in relay cells (Vh = -54.9 vs. -64.5 mV), whereas the activation range of IA was more negative (Vh = -25.2 vs. -14.5 mV). 5. Under whole-cell voltage-clamp conditions that allowed the combined activation of Ca2+ and K+ currents, depolarizing voltage steps from -110 mV evoked inward currents resembling IT in relay cells and small outward currents indicative of IA in local circuit neurones. After blockade of IA with 4-aminopyridine (4-AP), the same pulse protocol produced IT in both types of neurones. Under current clamp, 4-AP unmasked a regenerative membrane depolarization with a low threshold of activation capable of triggering fast spikes in local circuit neurones.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 1 PMID:7965855

Membrane Localization of Human Equilibrative Nucleoside Transporter 1 in Tumor Cells May Predict Response to Adjuvant Gemcitabine in Resected Cholangiocarcinoma Patients

PubMed Central

Deserti, Marzia; Vasuri, Francesco; Farioli, Andrea; Degiovanni, Alessio; Palloni, Andrea; Frega, Giorgio; Barbera, Maria A.; de Lorenzo, Stefania; Garajova, Ingrid; Di Marco, Mariacristina; Pinna, Antonio D.; Cescon, Matteo; Cucchetti, Alessandro; Ercolani, Giorgio; D’Errico-Grigioni, Antonietta; Pantaleo, Maria A.; Biasco, Guido; Tavolari, Simona

2016-01-01

Background. The use of gemcitabine as an adjuvant modality for cholangiocarcinoma (CC) is increasing, but limited data are available on predictive biomarkers of response. Human equilibrative nucleoside transporter 1 (hENT-1) is the major transporter involved in gemcitabine intracellular uptake. This study investigated the putative predictive role of hENT-1 localization in tumor cells of CC patients undergoing treatment with adjuvant gemcitabine. Methods. Seventy-one consecutive patients with resected CC receiving adjuvant gemcitabine at our center were retrospectively analyzed by immunohistochemistry for hENT-1 localization in tumor cells. The main outcome measure was disease-free survival (DFS). Hazard ratios (HRs) of relapse and associated 95% confidence intervals (CIs) were obtained from proportional hazards regression models stratified on quintiles of propensity score. Results. Twenty-three (32.4%) cases were negative for hENT-1, 22 (31.0%) were positive in the cytoplasm only, and 26 (36.6%) showed concomitant cytoplasm/membrane staining. Patients with membrane hENT-1 had a longer DFS (HR 0.49, 95% CI 0.24–0.99, p = .046) than those who were negative or positive only in the cytoplasm of tumor cells. Notably, the association between DFS and membrane hENT-1 was dependent on the number of gemcitabine cycles (one to two cycles: HR 0.96, 95% CI 0.34–2.68; three to four cycles: HR 0.99, 95% CI 0.34–2.90; five to six cycles: HR 0.27, 95% CI 0.10–0.77). Conclusion. hENT-1 localization on tumor cell membrane may predict response to adjuvant gemcitabine in CC patients receiving more than four cycles of chemotherapy. Further prospective randomized trials on larger populations are required to confirm these preliminary results, so that optimal gemcitabine-based chemotherapy may be tailored for CC patients in the adjuvant setting. Implications for Practice: Gemcitabine is becoming an increasingly used adjuvant modality in cholangiocarcinoma (CC), but limited data are available on predictive biomarkers of response. In this study, patients receiving more than four cycles of adjuvant gemcitabine and harboring Human equilibrative nucleoside transporter 1 (hENT-1, the major transporter involved in gemcitabine intracellular uptake) on tumor cell membrane had a longer disease-free survival compared with patients negative or positive for hENT-1 only in the cytoplasm of tumor cells. Overall these results may lay the basis for further prospective randomized trials based on a larger population of patients and may prove useful for tailoring appropriate gemcitabine-based chemotherapy for CC patients in the adjuvant setting. PMID:27032872

Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.

PubMed Central

Xiao, Z; Devreotes, P N

1997-01-01

Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1, adenylate cyclase (ACA), were specifically localized in these structures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures. Images PMID:9168471

Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.

PubMed

Xiao, Z; Devreotes, P N

1997-05-01

Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1, adenylate cyclase (ACA), were specifically localized in these structures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures.

Role of host protein Ebp1 in influenza virus growth: intracellular localization of Ebp1 in virus-infected and uninfected cells.

PubMed

Honda, Ayae

2008-01-20

The cellular protein Ebp1 was identified to interact with PB1 protein of influenza virus RNA polymerase, and inhibit both RNA synthesis in vitro and influenza virus replication in vivo [Honda, A., Okamoto, T., Ishihama, A., 2007. Host factor Ebp1: selective inhibitor of influenza virus transcriptase. Genes Cells 12, 133-142]. The intracellular localization of Ebp1 that is involved in cell proliferation control was analyzed by direct immunostaining of cells before and after influenza virus infection. Ebp1 was found to localize in the nuclear membrane of uninfected cells, and to form nuclear aggregates with viral P proteins in virus-infected cells.

[Expression and localization of transmembrane protein CMTM2 in human testis and sperm].

PubMed

Zhang, X W; Lan, K; Yang, W B; Li, Q; Zhao, Y P; Yin, H Q; Kite, B; Bai, W J; Xu, T

2017-08-18

To study the expression of transmembrane protein CMTM2 in the testis and sperm of adult males and to approach the potential function of the protein in the male reproductive system. The expression of CMTM2 in human testis and sperm was confirmed by Western blot. Immunohistochemical staining was used for detecting CMTM2 localization in the testis tissue, TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction, that is, immunofluorescent staining was used for detecting CMTM2 localization in both the testis and sperm before and after the acrosome reaction. CMTM2 was presented in both human testis and sperm. In the testis, CMTM2 immunoreactive particles were observed mainly in the membrane of the different stages of spermatogenic cells. In the human sperm, its immunoreactivity was restrictively localized to the posterior head where sperm-egg fusion occurred, and the CMTM2 localization was not affected by sperm acrosome reaction. CMTM2 was widely expressed in seminiferous tubules of the human testis, mainly in the cell membranes of spermatogenic cells, which was consistent with the previous reports. The immunofluorescence performed on frozen human testis slides showed similar findings with immunohistochemistry, which gave weight to the localization of CMTM2 in the cell membranes of spermatogenic cells at different stages. TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction. CMTM2 was localized at the posterior head of sperm before and after acrosome reaction. The localization and expression of CMTM2 were not affected by sperm acrosome reaction. Expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. The expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. However, it still remains to be further elucidated about the definite role of CMTM2 in male reproductive system and the process of spermatogenesis. And in vitro fertilization experiments are needed to confirm the role of CMTM2 in fertilization in future.

RanGTPase regulates the interaction between the inner nuclear membrane proteins, Samp1 and Emerin.

PubMed

Vijayaraghavan, Balaje; Figueroa, Ricardo A; Bergqvist, Cecilia; Gupta, Amit J; Sousa, Paulo; Hallberg, Einar

2018-06-01

Samp1, spindle associated membrane protein 1, is a type II integral membrane protein localized in the inner nuclear membrane. Recent studies have shown that the inner nuclear membrane protein, Emerin and the small monomeric GTPase, Ran are direct binding partners of Samp1. Here we addressed the question whether Ran could regulate the interaction between Samp1 and Emerin in the inner nuclear membrane. To investigate the interaction between Samp1 and Emerin in live cells, we performed FRAP experiments in cells overexpressing YFP-Emerin. We compared the mobility of YFP-Emerin in Samp1 knock out cells and cells overexpressing Samp1. The results showed that the mobility of YFP-Emerin was higher in Samp1 knock out cells and lower in cells overexpressing Samp1, suggesting that Samp1 significantly attenuates the mobility of Emerin in the nuclear envelope. FRAP experiments using tsBN2 cells showed that the mobility of Emerin depends on RanGTP. Consistently, in vitro binding experiments showed that the affinity between Samp1 and Emerin is decreased in the presence of Ran, suggesting that Ran attenuates the interaction between Samp1 and Emerin. This is the first demonstration that Ran can regulate the interaction between two proteins in the nuclear envelope. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

PKCε Phosphorylates and Mediates the Cell Membrane Localization of RhoA

PubMed Central

Su, Tizhi; Bao, Liwei; Xie, Xiujie; Lehner, Caryn L.; Cavey, Greg S.; Teknos, Theodoros N.

2013-01-01

Protein kinase Cε (PKCε) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKCε and RhoA was defined. Phosphopeptide mapping revealed that PKCε phosphorylates RhoA at T127 and S188. Recombinant PKCε bound to recombinant RhoA in the absence of ATP indicating that the association between PKCε and RhoA does not require an active ATP-docked PKCε conformation. Activation of PKCε resulted in a dramatic coordinated translocation of PKCε and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKCε and RhoA is a biphasic event, an initial peak at the cytoplasm and a gradual prolonged increase at the cell membrane for the entire time-course (12.5 minutes). These results suggest that the PKCε-RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKCε is able to recruit RhoA to the cell membrane indicating that the association between PKCε and RhoA is proximal to the active catalytic site and perhaps independent of a PKCε-RhoA phosphorylation event. This work demonstrates, for the first time, that PKCε phosphorylates and modulates the cell membrane translocation of RhoA. PMID:24191200

PASTA repeats of the protein kinase StkP interconnect cell constriction and separation of Streptococcus pneumoniae.

PubMed

Zucchini, Laure; Mercy, Chryslène; Garcia, Pierre Simon; Cluzel, Caroline; Gueguen-Chaignon, Virginie; Galisson, Frédéric; Freton, Céline; Guiral, Sébastien; Brochier-Armanet, Céline; Gouet, Patrice; Grangeasse, Christophe

2018-02-01

Eukaryotic-like serine/threonine kinases (eSTKs) with extracellular PASTA repeats are key membrane regulators of bacterial cell division. How PASTA repeats govern eSTK activation and function remains elusive. Using evolution- and structural-guided approaches combined with cell imaging, we disentangle the role of each PASTA repeat of the eSTK StkP from Streptococcus pneumoniae. While the three membrane-proximal PASTA repeats behave as interchangeable modules required for the activation of StkP independently of cell wall binding, they also control the septal cell wall thickness. In contrast, the fourth and membrane-distal PASTA repeat directs StkP localization at the division septum and encompasses a specific motif that is critical for final cell separation through interaction with the cell wall hydrolase LytB. We propose a model in which the extracellular four-PASTA domain of StkP plays a dual function in interconnecting the phosphorylation of StkP endogenous targets along with septal cell wall remodelling to allow cell division of the pneumococcus.

KSHV cell attachment sites revealed by ultra sensitive tyramide signal amplification (TSA) localize to membrane microdomains that are up-regulated on mitotic cells.

PubMed

Garrigues, H Jacques; Rubinchikova, Yelena E; Rose, Timothy M

2014-03-01

Cell surface structures initiating attachment of Kaposi's sarcoma-associated herpesvirus (KSHV) were characterized using purified hapten-labeled virions visualized by confocal microscopy with a sensitive fluorescent enhancement using tyramide signal amplification (TSA). KSHV attachment sites were present in specific cellular domains, including actin-based filopodia, lamellipodia, ruffled membranes, microvilli and intercellular junctions. Isolated microdomains were identified on the dorsal surface, which were heterogeneous in size with a variable distribution that depended on cellular confluence and cell cycle stage. KSHV binding domains ranged from scarce on interphase cells to dense and continuous on mitotic cells, and quantitation of bound virus revealed a significant increase on mitotic compared to interphase cells. KSHV also bound to a supranuclear domain that was distinct from microdomains in confluent and interphase cells. These results suggest that rearrangement of the cellular membrane during mitosis induces changes in cell surface receptors implicated in the initial attachment stage of KSHV entry. Copyright © 2014 Elsevier Inc. All rights reserved.

ADP-Ribosylation Factor 6 Regulates a Novel Plasma Membrane Recycling Pathway

PubMed Central

Radhakrishna, Harish; Donaldson, Julie G.

1997-01-01

ADP-ribosylation factor (ARF) 6 localizes to the plasma membrane (PM) in its GTP state and to a tubulovesicular compartment in its GDP state in HeLa cells that express wild-type or mutant forms of this GTPase. Aluminum fluoride (AlF) treatment of ARF6-transfected cells redistributes ARF6 to the PM and stimulates the formation of actin-rich surface protrusions. Here we show that cytochalasin D (CD) treatment inhibited formation of the AlF-induced protrusions and shifted the distribution of ARF6 to a tubular membrane compartment emanating from the juxtanuclear region of cells, which resembled the compartment where the GTP-binding defective mutant of ARF6 localized. This membrane compartment was distinct from transferrin-positive endosomes, could be detected in the absence of ARF6 overexpression or CD treatment, and was accessible to loading by PM proteins lacking clathrin/AP-2 cytoplasmic targeting sequences, such as the IL-2 receptor α subunit Tac. ARF6 and surface Tac moved into this compartment and back out to the PM in the absence of pharmacologic treatment. Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively. Thus, the ARF6 GTP cycle regulates this membrane traffic pathway. The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton. PMID:9314528

The acceleration of boron neutron capture therapy using multi-linked mercaptoundecahydrododecaborate (BSH) fused cell-penetrating peptide.

PubMed

Michiue, Hiroyuki; Sakurai, Yoshinori; Kondo, Natsuko; Kitamatsu, Mizuki; Bin, Feng; Nakajima, Kiichiro; Hirota, Yuki; Kawabata, Shinji; Nishiki, Tei-ichi; Ohmori, Iori; Tomizawa, Kazuhito; Miyatake, Shin-ichi; Ono, Koji; Matsui, Hideki

2014-03-01

New anti-cancer therapy with boron neutron capture therapy (BNCT) is based on the nuclear reaction of boron-10 with neutron irradiation. The median survival of BNCT patients with glioblastoma was almost twice as long as those receiving standard therapy in a Japanese BNCT clinical trial. In this clinical trial, two boron compounds, BPA (boronophenylalanine) and BSH (sodium borocaptate), were used for BNCT. BPA is taken up into cells through amino acid transporters that are expressed highly in almost all malignant cells, but BSH cannot pass through the cell membrane and remains outside the cell. We simulated the energy transfer against the nucleus at different locations of boron from outside the cell to the nuclear region with neutron irradiation and concluded that there was a marked difference between inside and outside the cell in boron localization. To overcome this disadvantage of BSH in BNCT, we used a cell-penetrating peptide system for transduction of BSH. CPP (cell-membrane penetrating peptide) is very common peptide domains that transduce many physiologically active substances into cells in vitro and in vivo. BSH-fused CPPs can penetrate the cell membrane and localize inside a cell. To increase the boron ratio in one BSH-peptide molecule, 8BSH fused to 11R with a dendritic lysine structure was synthesized and administrated to malignant glioma cells and a brain tumor mouse model. 8BSH-11R localized at the cell nucleus and showed a very high boron value in ICP results. With neutron irradiation, the 8BSH-11R administrated group showed a significant cancer killing effect compared to the 100 times higher concentration of BSH-administrated group. We concluded that BSH-fused CPPs were one of the most improved and potential boron compounds in the next-stage BNCT trial and 8BSH-11R may be applied in the clinical setting. Copyright © 2013 Elsevier Ltd. All rights reserved.

Fixed charge in the cell membrane

PubMed Central

Elul, R.

1967-01-01

1. Focal electric field was generated by passing a current of 5 × 10-7 to 1 × 10-5 A from a micropipette into the culture medium. Movement of cells at a distance of 5-50 μ from the electrode tip was observed. In case of cells embedded in the culture only local deformation of the membrane was observed. 2. The cell species explored included neurones, glia, muscle fibres, connective cells, malignant cells and erythrocytes. All cells responded in a similar manner to the electric field, and the current required was in the same range. 3. Cells were attracted to a positive micropipette and repelled from a negative one: the only exception was observed in certain malignant cells which moved in the opposite direction. 4. Movement and membrane deformation could be obtained with electrodes filled with various concentrated and isotonic solutions. The composition of the culture medium also had no qualitative influence on these effects. 5. Metabolic poisons or rupture of the cell membrane had no effect on the movement. Isolated membrane fragments showed movement similar to that of intact cells. 6. The possibility of artifacts due to proximity of the focal electrode is considered. It is shown that electro-osmosis cannot account for the present observations. Some other artifacts are also excluded. 7. It is proposed that the most satisfactory way to account for the present observations is by a membrane carrying negative fixed charge of the order of 2·5 × 103 e.s.u./cm2. Some physiological consequences of presence of negative charge in the membrane are briefly discussed. ImagesFig. 1Fig. 2Fig. 3 PMID:6040152

Functional characterization of Autographa californica multiple nucleopolyhedrovirus gp16 (ac130)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Ming; Huang, Cui; Qian, Duo-Duo

2014-09-15

To investigate the function of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp16, multiple gp16-knockout and repair mutants were constructed and characterized. No obvious difference in productivity of budded virus, DNA synthesis, late gene expression and morphogenesis was observed between gp16-knockout and repair viruses, but gp16 deletion resulted in six hours of lengthening in ST{sub 50} to the third instar Spodoptera exigua larvae in bioassays. GP16 was fractionated mainly in the light membrane fraction, by subcellular fractionation. A GP16-EGFP fusion protein was predominantly localized close around the nuclear membrane in infected cells, being coincident with formation of the vesicles associated with themore » nuclear membrane, which hosted nucleocapsids released from the nucleus. These data suggest that gp16 is not required for viral replication, but may be involved in membrane trafficking associated with the envelopment/de-envelopment of budded viruses when they cross over the nuclear membrane and pass through cytoplasm. - Highlights: • gp16 knockout and repair mutants of AcMNPV were constructed and characterized. • AcMNPV gp16 is not essential to virus replication. • Deletion of gp16 resulted in time lengthening to kill S. exigua larvae. • GP16 was localized close around the nuclear membrane of infected cells. • GP16 was fractionated in the light membrane fraction in subcellular fractionation.« less

Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.

PubMed

Sorrenson, Brie; Suetani, Rachel J; Williams, Michael J A; Bickley, Vivienne M; George, Peter M; Jones, Gregory T; McCormick, Sally P A

2013-01-01

Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

Loss of membranous Ep-CAM in budding colorectal carcinoma cells.

PubMed

Gosens, Marleen J E M; van Kempen, Léon C L; van de Velde, Cornelis J H; van Krieken, J Han J M; Nagtegaal, Iris D

2007-02-01

Tumor budding is a histological feature that reflects loss of adhesion of tumor cells and is associated with locoregional metastasis of colorectal carcinoma. Although nuclear localization of beta-catenin is associated with tumor budding, the molecular mechanism remains largely elusive. In this study, we hypothesize that the epithelial cell adhesion molecule (Ep-CAM) is involved in tumor budding. In order to address this question, we performed immunohistochemistry on Ep-CAM using three different antibodies (monoclonal antibodies Ber-ep4 and 311-1K1 and a polyclonal antibody) and a double staining on beta-catenin and Ep-CAM. In addition, Ep-CAM mRNA was monitored with mRNA in situ hybridization. Subsequently, we determined the effect of Ep-CAM staining patterns on tumor spread in rectal cancer. In contrast to the tumor mass, budding cells of colorectal carcinoma displayed lack of membranous but highly increased cytoplasmic Ep-CAM staining and nuclear translocation of beta-catenin. mRNA in situ hybridization suggested no differences in Ep-CAM expression between the invasive front and the tumor mass. Importantly, reduced Ep-CAM staining at the invasive margin of rectal tumor specimens (n=133) correlated significantly with tumor budding, tumor grade and an increased risk of local recurrence (P=0.001, P=0.04 and P=0.03, respectively). These data demonstrate abnormal processing of Ep-CAM at the invasive margin of colorectal carcinomas. Our observations indicate that loss of membranous Ep-CAM is associated with nuclear beta-catenin localization and suggest that this contributes to reduced cell-cell adhesions, increased migratory potential and tumor budding.

The DEP domain-containing protein TOE-2 promotes apoptosis in the Q lineage of C. elegans through two distinct mechanisms

PubMed Central

Gurling, Mark; Talavera, Karla; Garriga, Gian

2014-01-01

Neuroblast divisions in the nematode Caenorhabditis elegans often give rise to a larger neuron and a smaller cell that dies. We have previously identified genes that, when mutated, result in neuroblast divisions that generate daughter cells that are more equivalent in size. This effect correlates with the survival of daughter cells that would normally die. We now describe a role for the DEP domain-containing protein TOE-2 in promoting the apoptotic fate in the Q lineage. TOE-2 localized at the plasma membrane and accumulated in the cleavage furrow of the Q.a and Q.p neuroblasts, suggesting that TOE-2 might position the cleavage furrow asymmetrically to generate daughter cells of different sizes. This appears to be the case for Q.a divisions where loss of TOE-2 led to a more symmetric division and to survival of the smaller Q.a daughter. Localization of TOE-2 to the membrane is required for this asymmetry, but, surprisingly, the DEP domain is dispensable. By contrast, loss of TOE-2 led to loss of the apoptotic fate in the smaller Q.p daughter but did not affect the size asymmetry of the Q.p daughters. This function of TOE-2 required the DEP domain but not localization to the membrane. We propose that TOE-2 ensures an apoptotic fate for the small Q.a daughter by promoting asymmetry in the daughter cell sizes of the Q.a neuroblast division but by a mechanism that is independent of cell size in the Q.p division. PMID:24961802

Coexistence of a two-states organization for a cell-penetrating peptide in lipid bilayer.

PubMed

Plénat, Thomas; Boichot, Sylvie; Dosset, Patrice; Milhiet, Pierre-Emmanuel; Le Grimellec, Christian

2005-12-01

Primary amphipathic cell-penetrating peptides transport cargoes across cell membranes with high efficiency and low lytic activity. These primary amphipathic peptides were previously shown to form aggregates or supramolecular structures in mixed lipid-peptide monolayers, but their behavior in lipid bilayers remains to be characterized. Using atomic force microscopy, we have examined the interactions of P(alpha), a primary amphipathic cell-penetrating peptide which remains alpha-helical whatever the environment, with dipalmitoylphosphatidylcholine (DPPC) bilayers. Addition of P(alpha) at concentrations up to 5 mol % markedly modified the supported bilayers topography. Long and thin filaments lying flat at the membrane surface coexisted with deeply embedded peptides which induced a local thinning of the bilayer. On the other hand, addition of P(alpha) only exerted very limited effects on the corresponding liposome's bilayer physical state, as estimated from differential scanning calorimetry and diphenylhexatriene fluorescence anisotropy experiments. The use of a gel-fluid phase separated supported bilayers made of a dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine mixture confirmed both the existence of long filaments, which at low peptide concentration were preferentially localized in the fluid phase domains and the membrane disorganizing effects of 5 mol % P(alpha). The simultaneous two-states organization of P(alpha), at the membrane surface and deeply embedded in the bilayer, may be involved in the transmembrane carrier function of this primary amphipathic peptide.

The F-BAR domains from srGAP1, srGAP2 and srGAP3 regulate membrane deformation differently

PubMed Central

Coutinho-Budd, Jaeda; Ghukasyan, Vladimir; Zylka, Mark J.; Polleux, Franck

2012-01-01

Summary Coordination of membrane deformation and cytoskeletal dynamics lies at the heart of many biological processes critical for cell polarity, motility and morphogenesis. We have recently shown that Slit-Robo GTPase-activating protein 2 (srGAP2) regulates neuronal morphogenesis through the ability of its F-BAR domain to regulate membrane deformation and induce filopodia formation. Here, we demonstrate that the F-BAR domains of two closely related family members, srGAP1 and srGAP3 [designated F-BAR(1) and F-BAR(3), respectively] display significantly different membrane deformation properties in non-neuronal COS7 cells and in cortical neurons. F-BAR(3) induces filopodia in both cell types, though less potently than F-BAR(2), whereas F-BAR(1) prevents filopodia formation in cortical neurons and reduces plasma membrane dynamics. These three F-BAR domains can heterodimerize, and they act synergistically towards filopodia induction in COS7 cells. As measured by fluorescence recovery after photobleaching, F-BAR(2) displays faster molecular dynamics than F-BAR(3) and F-BAR(1) at the plasma membrane, which correlates well with its increased potency to induce filopodia. We also show that the molecular dynamic properties of F-BAR(2) at the membrane are partially dependent on F-Actin. Interestingly, acute phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] depletion in cells does not interfere with plasma membrane localization of F-BAR(2), which is compatible with our result showing that F-BAR(2) binds to a broad range of negatively-charged phospholipids present at the plasma membrane, including phosphatidylserine (PtdSer). Overall, our results provide novel insights into the functional diversity of the membrane deformation properties of this subclass of F-BAR-domains required for cell morphogenesis. PMID:22467852

Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

PubMed

Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

2016-03-29

Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions on protein activity and the roles of membrane proteins in disease pathways.

Membrane permeable local anesthetics modulate NaV1.5 mechanosensitivity

PubMed Central

Beyder, Arthur; Strege, Peter R.; Bernard, Cheryl; Farrugia, Gianrico

2012-01-01

Voltage-gated sodium selective ion channel NaV1.5 is expressed in the heart and the gastrointestinal tract, which are mechanically active organs. NaV1.5 is mechanosensitive at stimuli that gate other mechanosensitive ion channels. Local anesthetic and antiarrhythmic drugs act upon NaV1.5 to modulate activity by multiple mechanisms. This study examined whether NaV1.5 mechanosensitivity is modulated by local anesthetics. NaV1.5 channels wereexpressed in HEK-293 cells, and mechanosensitivity was tested in cell-attached and excised inside-out configurations. Using a novel protocol with paired voltage ladders and short pressure pulses, negative patch pressure (-30 mmHg) in both configurations produced a hyperpolarizing shift in the half-point of the voltage-dependence of activation (V1/2a) and inactivation (V1/2i) by about -10 mV. Lidocaine (50 µM) inhibited the pressure-induced shift of V1/2a but not V1/2i. Lidocaine inhibited the tonic increase in pressure-induced peak current in a use-dependence protocol, but it did not otherwise affect use-dependent block. The local anesthetic benzocaine, which does not show use-dependent block, also effectively blocked a pressure-induced shift in V1/2a. Lidocaine inhibited mechanosensitivity in NaV1.5 at the local anesthetic binding site mutated (F1760A). However, a membrane impermeable lidocaine analog QX-314 did not affect mechanosensitivity of F1760A NaV1.5 when applied from either side of the membrane. These data suggest that the mechanism of lidocaine inhibition of the pressure-induced shift in the half-point of voltage-dependence of activation is separate from the mechanisms of use-dependent block. Modulation of NaV1.5 mechanosensitivity by the membrane permeable local anesthetics may require hydrophobic access and may involve membrane-protein interactions. PMID:22874086

Organelle-localized potassium transport systems in plants.

PubMed

Hamamoto, Shin; Uozumi, Nobuyuki

2014-05-15

Some intracellular organelles found in eukaryotes such as plants have arisen through the endocytotic engulfment of prokaryotic cells. This accounts for the presence of plant membrane intrinsic proteins that have homologs in prokaryotic cells. Other organelles, such as those of the endomembrane system, are thought to have evolved through infolding of the plasma membrane. Acquisition of intracellular components (organelles) in the cells supplied additional functions for survival in various natural environments. The organelles are surrounded by biological membranes, which contain membrane-embedded K(+) transport systems allowing K(+) to move across the membrane. K(+) transport systems in plant organelles act coordinately with the plasma membrane intrinsic K(+) transport systems to maintain cytosolic K(+) concentrations. Since it is sometimes difficult to perform direct studies of organellar membrane proteins in plant cells, heterologous expression in yeast and Escherichia coli has been used to elucidate the function of plant vacuole K(+) channels and other membrane transporters. The vacuole is the largest organelle in plant cells; it has an important task in the K(+) homeostasis of the cytoplasm. The initial electrophysiological measurements of K(+) transport have categorized three classes of plant vacuolar cation channels, and since then molecular cloning approaches have led to the isolation of genes for a number of K(+) transport systems. Plants contain chloroplasts, derived from photoautotrophic cyanobacteria. A novel K(+) transport system has been isolated from cyanobacteria, which may add to our understanding of K(+) flux across the thylakoid membrane and the inner membrane of the chloroplast. This chapter will provide an overview of recent findings regarding plant organellar K(+) transport proteins. Copyright © 2014 Elsevier GmbH. All rights reserved.

Investigation of subcellular localization and dynamics of membrane proteins in living bacteria by combining optical micromanipulation and high-resolution microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Barroso Peña, Álvaro; Nieves, Marcos; Teper, Konrad; Wedlich-Soldner, Roland; Denz, Cornelia

2016-09-01

The plasma membrane serves as protective interface between cells and their environment. It also constitutes a hub for selective nutrient uptake and signal transduction. Increasing evidence over the last years indicates that, similar to eukaryotic cells, lateral membrane organization plays an important role in the regulation of prokaryotic signaling pathways. However, the mechanisms underlying this phenomenon are still poorly understood. Spatiotemporal characterization of bacterial signal transduction demands very sensitive high-resolution microscopy techniques due to the low expression levels of most signaling proteins and the small size of bacterial cells. In addition, direct study of subcellular confinement and dynamics of bacterial signaling proteins during the different stages of the signal transduction also requires immobilization in order to avoid cell displacement caused by Brownian motion, local fluid flows and bacterial self-propulsion. In this work we present a novel approach based on the combination of high resolution imaging and optical manipulation that enables the investigation of the distribution and dynamics of proteins at the bacterial plasma membrane. For this purpose, we combine the versatility of holographic optical tweezers (HOT) with the sensitivity and resolution of total internal reflection fluorescence (TIRF) microscopy. Furthermore, we discuss the implementation of microfluidic devices in our integrated HOT+TIRF system for the control of growth conditions of bacterial cells. The capabilities of our workstation provides thus new valuable insights into the fundamental cellular and physical mechanisms underlying the regulation of bacterial signal transduction.

Acylation-dependent protein export in Leishmania.

PubMed

Denny, P W; Gokool, S; Russell, D G; Field, M C; Smith, D F

2000-04-14

The surface of the protozoan parasite Leishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologous Aequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a "non-classical" pathway involving N-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism.

Unconventional Transport Routes of Soluble and Membrane Proteins and Their Role in Developmental Biology

PubMed Central

Pompa, Andrea; De Marchis, Francesca; Pallotta, Maria Teresa; Benitez-Alfonso, Yoselin; Jones, Alexandra; Schipper, Kerstin; Moreau, Kevin; Žárský, Viktor; Di Sansebastiano, Gian Pietro; Bellucci, Michele

2017-01-01

Many proteins and cargoes in eukaryotic cells are secreted through the conventional secretory pathway that brings proteins and membranes from the endoplasmic reticulum to the plasma membrane, passing through various cell compartments, and then the extracellular space. The recent identification of an increasing number of leaderless secreted proteins bypassing the Golgi apparatus unveiled the existence of alternative protein secretion pathways. Moreover, other unconventional routes for secretion of soluble or transmembrane proteins with initial endoplasmic reticulum localization were identified. Furthermore, other proteins normally functioning in conventional membrane traffic or in the biogenesis of unique plant/fungi organelles or in plasmodesmata transport seem to be involved in unconventional secretory pathways. These alternative pathways are functionally related to biotic stress and development, and are becoming more and more important in cell biology studies in yeast, mammalian cells and in plants. The city of Lecce hosted specialists working on mammals, plants and microorganisms for the inaugural meeting on “Unconventional Protein and Membrane Traffic” (UPMT) during 4–7 October 2016. The main aim of the meeting was to include the highest number of topics, summarized in this report, related to the unconventional transport routes of protein and membranes. PMID:28346345

Induction of stable ER–plasma-membrane junctions by Kv2.1 potassium channels

PubMed Central

Fox, Philip D.; Haberkorn, Christopher J.; Akin, Elizabeth J.; Seel, Peter J.; Krapf, Diego; Tamkun, Michael M.

2015-01-01

ABSTRACT Junctions between cortical endoplasmic reticulum (cER) and the plasma membrane are a subtle but ubiquitous feature in mammalian cells; however, very little is known about the functions and molecular interactions that are associated with neuronal ER–plasma-membrane junctions. Here, we report that Kv2.1 (also known as KCNB1), the primary delayed-rectifier K+ channel in the mammalian brain, induces the formation of ER–plasma-membrane junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, indicating that they have a function that is unrelated to membrane-potential regulation. Accordingly, Kv2.1 clusters function as membrane-trafficking hubs, providing platforms for delivery and retrieval of multiple membrane proteins. Using both total internal reflection fluorescence and electron microscopy we demonstrate that the clustered Kv2.1 plays a direct structural role in the induction of stable ER–plasma-membrane junctions in both transfected HEK 293 cells and cultured hippocampal neurons. Glutamate exposure results in a loss of Kv2.1 clusters in neurons and subsequent retraction of the cER from the plasma membrane. We propose Kv2.1-induced ER–plasma-membrane junctions represent a new macromolecular plasma-membrane complex that is sensitive to excitotoxic insult and functions as a scaffolding site for both membrane trafficking and Ca2+ signaling. PMID:25908859

Glucose recruits K(ATP) channels via non-insulin-containing dense-core granules.

PubMed

Yang, Shao-Nian; Wenna, Nancy Dekki; Yu, Jia; Yang, Guang; Qiu, Hua; Yu, Lina; Juntti-Berggren, Lisa; Köhler, Martin; Berggren, Per-Olof

2007-09-01

beta cells rely on adenosine triphosphate-sensitive potassium (K(ATP)) channels to initiate and end glucose-stimulated insulin secretion through changes in membrane potential. These channels may also act as a constituent of the exocytotic machinery to mediate insulin release independent of their electrical function. However, the molecular mechanisms whereby the beta cell plasma membrane maintains an appropriate number of K(ATP) channels are not known. We now show that glucose increases K(ATP) current amplitude by increasing the number of K(ATP) channels in the beta cell plasma membrane. The effect was blocked by inhibition of protein kinase A (PKA) as well as by depletion of extracellular or intracellular Ca(2+). Furthermore, glucose promoted recruitment of the potassium inward rectifier 6.2 to the plasma membrane, and intracellular K(ATP) channels localized in chromogranin-positive/insulin-negative dense-core granules. Our data suggest that glucose can recruit K(ATP) channels to the beta cell plasma membrane via non-insulin-containing dense-core granules in a Ca(2+)- and PKA-dependent manner.

Disruption of adherens junction and alterations in YAP-related proliferation behavior as part of the underlying cell transformation process of alcohol-induced oral carcinogenesis.

PubMed

Husari, Ayman; Hülter-Hassler, Diana; Steinberg, Thorsten; Schulz, Simon Daniel; Tomakidi, Pascal

2018-01-01

Accumulating evidences indicate that alcohol might play a causative in oral cancer. Unfortunately, in vitro cell systems, uncovering the molecular background of the underlying cell transformation process, are rare. Therefore, this study was conducted, to identify molecular changes and characterize their putative cell behavioral consequences in epitheloid (EPI) and fibroblastoid (FIB) oral keratinocyte phenotypes, arising from chronical alcohol treatment. Concerning adherens junctions (AJs), both EPI and FIB showed membrane-bound β-catenin, but exhibited differences for E-cadherin and zyxin. While EPI revealed E-cadherin/β-catenin membrane co-localization, which in parts also applied for zyxin, FIB membranes were devoid of E-cadherin and exhibited marginal zyxin expression. Fetal calf serum (FCS) administration in starved cells promoted proliferation in both keratinocyte phenotypes, whereat EPI and FIB yielded a strikingly modified FCS sensitivity on the temporal scale. Impedance measurement-based cell index detection yielded proliferation stimulation occurring much earlier in FIB (<20h) compared to EPI (>45h). Nuclear preference of the proliferation-associated YAP co-transcription factor in FIB was FCS independent, while it required FCS in EPI. Taken together, the lack of membrane-inherent E-cadherin/β-catenin co-localization together with low zyxin - reveals perturbation of AJ integrity in FIB. Regarding cell behavior, perturbed AJs in FIB correlate with temporal proliferation sensitivity towards FCS. CYF of 5.6 strongly suggests involvement of chromatin-bound YAP in FIB's proliferation temperosensitivity. These molecular differences detected for EPI and FIB are part of the underlying cell transformation process of alcohol-induced oral carcinogenesis, and indicate FIB being in a more advanced transformation stage. Copyright © 2017 Elsevier B.V. All rights reserved.

Co-localization of endogenous Arf6 and its activator EFA6D in the granular convoluted tubule cells of mouse submandibular glands under normal conditions and when stimulated by isoproterenol, noradrenaline and carbachol.

PubMed

Tachow, Apussara; Thoungseabyoun, Wipawee; Phuapittayalert, Laorrat; Petcharat, Kanoktip; Sakagami, Hiroyuki; Kondo, Hisatake; Hipkaeo, Wiphawi

2017-10-01

This study proposed to investigate the localization at light and electron microscopic levels of Arf6 and its activator EFA6D in the mouse submandibular gland (SMG) under normal conditions and when stimulated by adrenergic or cholinergic agonists. SMGs of male adult mice were utilized for immunoblotting and immuno-light and -electron microscopic analyses. Isoproterenol and noradrenalin were used as adrenergics, while carbachol was used for the cholinergic stimulant. SMGs were examined at 15, 30, 60 and 120min after intraperitoneal injection of these agents. Immunoreactivities for both Arf6 and its activator EFA6D were similarly intense in the basolateral domain of GCTs, but no significant immunoreactivities were seen in the apical domain of GCT cells or any domain of acinar cells under normal conditions. In immuno-electron microscopy, the immunoreactive materials were mainly deposited on the basolateral plasma membranes and subjacent cytoplasm. Shortly after injection of isoproterenol and noradrenaline, but not carbachol, the immunoreactivities for both molecules were additionally seen on the apical plasmalemma of most, if not all, GCT cells, but not acinar cells. The present findings suggest that the direct involvement of Arf6/EFA6D in regulatory exocytosis at the apical plasma membrane of acinar and GCT cells is apparently to be smaller, if present, than that of endocytosis at the basolateral membranes of GCT cells under normal conditions. This also suggests that the two molecules function additionally at the apical membrane of GCT cells for modulation of saliva secretion under β-adrenoceptor stimulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Machine learning to design integral membrane channelrhodopsins for efficient eukaryotic expression and plasma membrane localization.

PubMed

Bedbrook, Claire N; Yang, Kevin K; Rice, Austin J; Gradinaru, Viviana; Arnold, Frances H

2017-10-01

There is growing interest in studying and engineering integral membrane proteins (MPs) that play key roles in sensing and regulating cellular response to diverse external signals. A MP must be expressed, correctly inserted and folded in a lipid bilayer, and trafficked to the proper cellular location in order to function. The sequence and structural determinants of these processes are complex and highly constrained. Here we describe a predictive, machine-learning approach that captures this complexity to facilitate successful MP engineering and design. Machine learning on carefully-chosen training sequences made by structure-guided SCHEMA recombination has enabled us to accurately predict the rare sequences in a diverse library of channelrhodopsins (ChRs) that express and localize to the plasma membrane of mammalian cells. These light-gated channel proteins of microbial origin are of interest for neuroscience applications, where expression and localization to the plasma membrane is a prerequisite for function. We trained Gaussian process (GP) classification and regression models with expression and localization data from 218 ChR chimeras chosen from a 118,098-variant library designed by SCHEMA recombination of three parent ChRs. We use these GP models to identify ChRs that express and localize well and show that our models can elucidate sequence and structure elements important for these processes. We also used the predictive models to convert a naturally occurring ChR incapable of mammalian localization into one that localizes well.

Machine learning to design integral membrane channelrhodopsins for efficient eukaryotic expression and plasma membrane localization

PubMed Central

Rice, Austin J.; Gradinaru, Viviana; Arnold, Frances H.

2017-01-01

There is growing interest in studying and engineering integral membrane proteins (MPs) that play key roles in sensing and regulating cellular response to diverse external signals. A MP must be expressed, correctly inserted and folded in a lipid bilayer, and trafficked to the proper cellular location in order to function. The sequence and structural determinants of these processes are complex and highly constrained. Here we describe a predictive, machine-learning approach that captures this complexity to facilitate successful MP engineering and design. Machine learning on carefully-chosen training sequences made by structure-guided SCHEMA recombination has enabled us to accurately predict the rare sequences in a diverse library of channelrhodopsins (ChRs) that express and localize to the plasma membrane of mammalian cells. These light-gated channel proteins of microbial origin are of interest for neuroscience applications, where expression and localization to the plasma membrane is a prerequisite for function. We trained Gaussian process (GP) classification and regression models with expression and localization data from 218 ChR chimeras chosen from a 118,098-variant library designed by SCHEMA recombination of three parent ChRs. We use these GP models to identify ChRs that express and localize well and show that our models can elucidate sequence and structure elements important for these processes. We also used the predictive models to convert a naturally occurring ChR incapable of mammalian localization into one that localizes well. PMID:29059183

Opposing effects of estradiol- and testosterone-membrane binding sites on T47D breast cancer cell apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kampa, Marilena; Nifli, Artemissia-Phoebe; Charalampopoulos, Ioannis

Classical steroid mode of action involves binding to intracellular receptors, the later acting as ligand-activated nuclear transcription factors. Recently, membrane sites for different steroids have been also identified, mediating rapid, non-genomic, steroid actions. Membrane sites for estrogen and androgen have been found in a number of different cell types, bearing or not classical intracellular receptors. In the present study, with the use of radioligand binding, flow cytometry and confocal laser microscopy, we report that T47D human breast cancer cells express specific and saturable membrane receptors for both estrogen (K {sub D} 4.06 {+-} 3.31 nM) and androgen (K {sub D}more » 7.64 {+-} 3.15 nM). Upon activation with BSA-conjugated, non-permeable ligands (E{sub 2}-BSA and testosterone-BSA), membrane estrogen receptors protect cells from serum-deprivation-induced apoptosis, while androgen receptors induce apoptosis in serum-supplemented T47D cells. In addition, co-incubation of cells with a fixed concentration of one steroid and varying concentrations of the other reversed the abovementioned effect (apoptosis for androgen, and anti-apoptosis for E{sub 2}), suggesting that the fate of the cell depends on the relative concentration of either steroid in the culture medium. We also report the identification of membrane receptors for E{sub 2} and androgen in biopsy slides from breast cancer patients. Both sites are expressed, with the staining for membrane E{sub 2} being strongly present in ER-negative, less differentiated, more aggressive tumors. These findings suggest that aromatase inhibitors may exert their beneficial effects on breast cancer by also propagating the metabolism of local steroids towards androgen, inducing thus cell apoptosis through membrane androgen receptor activation.« less

Mechanisms of bacterial membrane permeabilization by crotalicidin (Ctn) and its fragment Ctn(15-34), antimicrobial peptides from rattlesnake venom.

PubMed

Pérez-Peinado, Clara; Dias, Susana Almeida; Domingues, Marco M; Benfield, Aurélie H; Freire, João Miguel; Rádis-Baptista, Gandhi; Gaspar, Diana; Castanho, Miguel A R B; Craik, David J; Henriques, Sónia Troeira; Veiga, Ana Salomé; Andreu, David

2018-02-02

Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system.

PubMed

Bortfeld, M; Rius, M; König, J; Herold-Mende, C; Nies, A T; Keppler, D

2006-01-01

Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 microM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.

Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

USDA-ARS?s Scientific Manuscript database

Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>600) known and predicted TA proteins in Arabidopsis thaliana for those annotated, based on Gene Ontology, to possess mitoc...

Fission Yeast Sec3 and Exo70 Are Transported on Actin Cables and Localize the Exocyst Complex to Cell Poles

PubMed Central

Martin, Sophie G.

2012-01-01

The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP2 and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk. PMID:22768263

MapA, an iron-regulated, cytoplasmic membrane protein in the cyanobacterium Synechococcus sp. strain PCC7942.

PubMed

Webb, R; Troyan, T; Sherman, D; Sherman, L A

1994-08-01

Growth of Synechococcus sp. strain PCC 7942 in iron-deficient media leads to the accumulation of an approximately 34-kDa protein. The gene encoding this protein, mapA (membrane-associated protein A), has been cloned and sequenced (GenBank accession number, L01621). The mapA transcript is not detectable in normally grown cultures but is stably accumulated by cells grown in iron-deficient media. However, the promoter sequence for this gene does not resemble other bacterial iron-regulated promoters described to date. The carboxyl-terminal region of the derived amino acid sequence of MapA resembles bacterial proteins involved in iron acquisition, whereas the amino-terminal end of MapA has a high degree of amino acid identity with the abundant, chloroplast envelope protein E37. An approach employing improved cellular fractionation techniques as well as electron microscopy and immunocytochemistry was essential in localizing MapA protein to the cytoplasmic membrane of Synechococcus sp. strain PCC 7942. When these cells were grown under iron-deficient conditions, a significant fraction of MapA could also be localized to the thylakoid membranes.

Hepatocellular differentiation status is characterized by distinct subnuclear localization and form of the chanzyme TRPM7.

PubMed

Ogunrinde, Adenike; Pereira, Robyn D; Beaton, Natalie; Lam, D Hung; Whetstone, Christiane; Hill, Ceredwyn E

The channel-kinase TRPM7 is important for the survival, proliferation, and differentiation, of many cell types. Both plasma membrane channel activity and kinase function are implicated in these roles. Channel activity is greater in less differentiated hepatoma cells compared with non-dividing, terminally differentiated adult hepatocytes, suggesting differences in protein expression and/or localization. We used electrophysiological and immunofluorescence approaches to establish whether hepatocellular differentiation is associated with altered TRPM7 expression. Mean outward current decreased by 44% in WIF-B hepatoma cells incubated with the established hepatic differentiating factors oncostatin M/dexamethasone for 1-8 days. Pre-incubation with pyridone 6, a pan-JAK inhibitor, blocked the current reduction. An antibody targeted to the C-terminus of TRPM7 labelled the cytoplasm in WIF-B cells and intact rat liver. Significant label also localized to the nuclear envelope (NE), with relatively more detected in adult hepatocytes compared with WIF-B cells. Hepatoma cells also exhibited nucleoplasmic labelling with intense signal in the nucleolus. The endogenous labelling pattern closely resembles that of HEK293T cells heterologously expressing a TRPM7 kinase construct containing a putative nucleolar localization sequence. These results suggest that TRPM7 form and distribution between the plasma membrane and nucleus, rather than expression, is altered in parallel with differentiation status in rat hepatic cells. Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion

PubMed Central

Gui, Long; Ebner, Jamie L.; Mileant, Alexander; Williams, James A.

2016-01-01

ABSTRACT Protein-mediated membrane fusion is an essential step in many fundamental biological events, including enveloped virus infection. The nature of protein and membrane intermediates and the sequence of membrane remodeling during these essential processes remain poorly understood. Here we used cryo-electron tomography (cryo-ET) to image the interplay between influenza virus and vesicles with a range of lipid compositions. By following the population kinetics of membrane fusion intermediates imaged by cryo-ET, we found that membrane remodeling commenced with the hemagglutinin fusion protein spikes grappling onto the target membrane, followed by localized target membrane dimpling as local clusters of hemagglutinin started to undergo conformational refolding. The local dimples then transitioned to extended, tightly apposed contact zones where the two proximal membrane leaflets were in most cases indistinguishable from each other, suggesting significant dehydration and possible intermingling of the lipid head groups. Increasing the content of fusion-enhancing cholesterol or bis-monoacylglycerophosphate in the target membrane led to an increase in extended contact zone formation. Interestingly, hemifused intermediates were found to be extremely rare in the influenza virus fusion system studied here, most likely reflecting the instability of this state and its rapid conversion to postfusion complexes, which increased in population over time. By tracking the populations of fusion complexes over time, the architecture and sequence of membrane reorganization leading to efficient enveloped virus fusion were thus resolved. IMPORTANCE Enveloped viruses employ specialized surface proteins to mediate fusion of cellular and viral membranes that results in the formation of pores through which the viral genetic material is delivered to the cell. For influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike mediates host cell attachment and membrane fusion. While structures of a subset of conformations and parts of the fusion machinery have been characterized, the nature and sequence of membrane deformations during fusion have largely eluded characterization. Building upon studies that focused on early stages of HA-mediated membrane remodeling, here cryo-electron tomography (cryo-ET) was used to image the three-dimensional organization of intact influenza virions at different stages of fusion with liposomes, leading all the way to completion of the fusion reaction. By monitoring the evolution of fusion intermediate populations over the course of acid-induced fusion, we identified the progression of membrane reorganization that leads to efficient fusion by an enveloped virus. PMID:27226364

How curvature-generating proteins build scaffolds on membrane nanotubes

PubMed Central

Evergren, Emma; Golushko, Ivan; Prévost, Coline; Renard, Henri-François; Johannes, Ludger; McMahon, Harvey T.; Lorman, Vladimir; Voth, Gregory A.; Bassereau, Patricia

2016-01-01

Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex subcellular structures, and many other cellular phenomena. They form 3D assemblies that act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we use various experimental, theoretical, and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube’s length. Our work implies that the nature of local protein–membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30–40% of a tube’s surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes. PMID:27655892

Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.

PubMed

Stone, Matthew B; Shelby, Sarah A; Núñez, Marcos F; Wisser, Kathleen; Veatch, Sarah L

2017-02-01

Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.

A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells.

PubMed

Park, Sungjin; Szumlanski, Amy L; Gu, Fangwei; Guo, Feng; Nielsen, Erik

2011-07-17

In plants, cell shape is defined by the cell wall, and changes in cell shape and size are dictated by modification of existing cell walls and deposition of newly synthesized cell-wall material. In root hairs, expansion occurs by a process called tip growth, which is shared by root hairs, pollen tubes and fungal hyphae. We show that cellulose-like polysaccharides are present in root-hair tips, and de novo synthesis of these polysaccharides is required for tip growth. We also find that eYFP-CSLD3 proteins, but not CESA cellulose synthases, localize to a polarized plasma-membrane domain in root hairs. Using biochemical methods and genetic complementation of a csld3 mutant with a chimaeric CSLD3 protein containing a CESA6 catalytic domain, we provide evidence that CSLD3 represents a distinct (1→4)-β-glucan synthase activity in apical plasma membranes during tip growth in root-hair cells.

Tuning B cell responses to antigens by cell polarity and membrane trafficking.

PubMed

Del Valle Batalla, Felipe; Lennon-Dumenil, Ana-María; Yuseff, María-Isabel

2018-06-20

The capacity of B lymphocytes to produce specific antibodies, particularly broadly neutralizing antibodies that provide immunity to viral pathogens has positioned them as valuable therapeutic targets for immunomodulation. To become competent as antibody secreting cells, B cells undergo a series of activation steps, which are triggered by the recognition of antigens frequently displayed on the surface of other presenting cells. Such antigens elicit the formation of an immune synapse (IS), where local cytoskeleton rearrangements coupled to mechanical forces and membrane trafficking orchestrate the extraction and processing of antigens in B cells. In this review, we discuss the molecular mechanisms that regulate polarized membrane trafficking and mechanical properties of the immune synapse, as well as the potential extracellular cues from the environment, which may impact the ability of B cells to sense and acquire antigens at the immune synapse. An integrated view of the diverse cellular mechanisms that shape the immune synapse will provide a better understanding on how B cells are efficiently activated. Copyright © 2018 Elsevier Ltd. All rights reserved.

In-Situ Measurement of High-Temperature Proton Exchange Membrane Fuel Cell Stack Using Flexible Five-in-One Micro-Sensor

PubMed Central

Lee, Chi-Yuan; Weng, Fang-Bor; Kuo, Yzu-Wei; Tsai, Chao-Hsuan; Cheng, Yen-Ting; Cheng, Chih-Kai; Lin, Jyun-Ting

2016-01-01

In the chemical reaction that proceeds in a high-temperature proton exchange membrane fuel cell stack (HT-PEMFC stack), the internal local temperature, voltage, pressure, flow and current nonuniformity may cause poor membrane material durability and nonuniform fuel distribution, thus influencing the performance and lifetime of the fuel cell stack. In this paper micro-electro-mechanical systems (MEMS) are utilized to develop a high-temperature electrochemical environment-resistant five-in-one micro-sensor embedded in the cathode channel plate of an HT-PEMFC stack, and materials and process parameters are appropriately selected to protect the micro-sensor against failure or destruction during long-term operation. In-situ measurement of the local temperature, voltage, pressure, flow and current distributions in the HT-PEMFC stack is carried out. This integrated micro-sensor has five functions, and is favorably characterized by small size, good acid resistance and temperature resistance, quick response, real-time measurement, and the goal is being able to be put in any place for measurement without affecting the performance of the battery. PMID:27763559

In-Situ Measurement of High-Temperature Proton Exchange Membrane Fuel Cell Stack Using Flexible Five-in-One Micro-Sensor.

PubMed

Lee, Chi-Yuan; Weng, Fang-Bor; Kuo, Yzu-Wei; Tsai, Chao-Hsuan; Cheng, Yen-Ting; Cheng, Chih-Kai; Lin, Jyun-Ting

2016-10-18

In the chemical reaction that proceeds in a high-temperature proton exchange membrane fuel cell stack (HT-PEMFC stack), the internal local temperature, voltage, pressure, flow and current nonuniformity may cause poor membrane material durability and nonuniform fuel distribution, thus influencing the performance and lifetime of the fuel cell stack. In this paper micro-electro-mechanical systems (MEMS) are utilized to develop a high-temperature electrochemical environment-resistant five-in-one micro-sensor embedded in the cathode channel plate of an HT-PEMFC stack, and materials and process parameters are appropriately selected to protect the micro-sensor against failure or destruction during long-term operation. In-situ measurement of the local temperature, voltage, pressure, flow and current distributions in the HT-PEMFC stack is carried out. This integrated micro-sensor has five functions, and is favorably characterized by small size, good acid resistance and temperature resistance, quick response, real-time measurement, and the goal is being able to be put in any place for measurement without affecting the performance of the battery.

Local potential evolutions during proton exchange membrane fuel cell operation with dead-ended anode - Part I: Impact of water diffusion and nitrogen crossover

NASA Astrophysics Data System (ADS)

Abbou, S.; Dillet, J.; Maranzana, G.; Didierjean, S.; Lottin, O.

2017-02-01

Operating a PEMFC with a dead-ended anode may lead to local fuel-starvation because of water and possibly nitrogen accumulation in the anode compartment. In previous works, we used a segmented linear cell with reference electrodes to monitor simultaneously the local potentials and current densities during dead-ended anode operation. The results indicated that water transport as well as nitrogen crossover through the membrane were most probably the two key factors governing fuel starvation. In this first from a set of two papers, we evaluated with more details the contributions of nitrogen crossover and water transport to hydrogen starvation. To assess nitrogen contribution, the fuel cell cathode compartment was first supplied with pure oxygen instead of air. The results showed that in the absence of nitrogen (in the cathode side) the fuel starvation was much slower than with air, suggesting that nitrogen contribution cannot be neglected. On the other hand, the contribution of water flooding to hydrogen starvation was investigated by using different cooling temperature on the cathode and anode sides in order to drive water toward the colder plate. The results showed that with a colder anode side, fuel starvation was faster. In the opposite case of a hotter anode plate, water accumulation in the anode compartment was limited, nitrogen crossover through the membrane was the main reason for hydrogen starvation in this case. To fully assess the impact of the thermal configurations on membrane-electrode assembly (MEA) degradation, aging protocols with a dead-ended anode and a fixed closing time were also performed. The results showed that operation with a hotter anode could help to limit significantly cathode ElectroChemical Surface Area (ECSA) losses along the cell area and performance degradation induced by hydrogen starvation.

Long clinostation influence on the localization of free and weakly bound calcium in cell walls of Funaria hygrometrica moss protonema cells

NASA Astrophysics Data System (ADS)

Nedukha, E. M.

The pyroantimonate method was used to study the localization of free and weakly bound calcium in cells of moss protonema of Funaria hygrometrica Hedw. cultivated on a clinostat (2 rev/min). Electroncytochemical study of control cells cultivated at 1 g revealed that granular precipitate marked chloroplasts, mitochondria, Golgi apparatus, lipid drops, nucleoplasma, nucleolus, nucleus membranes, cell walls and endoplasmic reticulum. In mitochondria the precipitate was revealed in stroma, in chloroplast it was found on thylakoids and envelope membranes. The cultivation of protonema on clinostat led to the intensification in cytochemical reaction product deposit. A considerable intensification of the reaction was noted in endomembranes, vacuoles, periplasmic space and cell walls. At the same time analysis of pectinase localization was made using the electroncytochemical method. A high reaction intensity in walls in comparison to that in control was found out to be a distinctive pecularity of the cells cultivated on clinostat. It testifies to the fact that increasing of freee calcium concentrations under conditions of clinostation is connected with pectinic substances hydrolysis and breaking of methoxy groups of pectins. Data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance of plant cells and the role of cell walls in gomeostasis of cell grown under conditions of simulated weighlessness.

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

PubMed

Adu-Gyamfi, Emmanuel; Johnson, Kristen A; Fraser, Mark E; Scott, Jordan L; Soni, Smita P; Jones, Keaton R; Digman, Michelle A; Gratton, Enrico; Tessier, Charles R; Stahelin, Robert V

2015-09-01

Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Motion of variable-length MreB filaments at the bacterial cell membrane influences cell morphology

PubMed Central

Reimold, Christian; Defeu Soufo, Herve Joel; Dempwolff, Felix; Graumann, Peter L.

2013-01-01

The maintenance of rod-cell shape in many bacteria depends on actin-like MreB proteins and several membrane proteins that interact with MreB. Using superresolution microscopy, we show that at 50-nm resolution, Bacillus subtilis MreB forms filamentous structures of length up to 3.4 μm underneath the cell membrane, which run at angles diverging up to 40° relative to the cell circumference. MreB from Escherichia coli forms at least 1.4-μm-long filaments. MreB filaments move along various tracks with a maximal speed of 85 nm/s, and the loss of ATPase activity leads to the formation of extended and static filaments. Suboptimal growth conditions lead to formation of patch-like structures rather than extended filaments. Coexpression of wild-type MreB with MreB mutated in the subunit interface leads to formation of shorter MreB filaments and a strong effect on cell shape, revealing a link between filament length and cell morphology. Thus MreB has an extended-filament architecture with the potential to position membrane proteins over long distances, whose localization in turn may affect the shape of the cell wall. PMID:23783036

Motion of variable-length MreB filaments at the bacterial cell membrane influences cell morphology.

PubMed

Reimold, Christian; Defeu Soufo, Herve Joel; Dempwolff, Felix; Graumann, Peter L

2013-08-01

The maintenance of rod-cell shape in many bacteria depends on actin-like MreB proteins and several membrane proteins that interact with MreB. Using superresolution microscopy, we show that at 50-nm resolution, Bacillus subtilis MreB forms filamentous structures of length up to 3.4 μm underneath the cell membrane, which run at angles diverging up to 40° relative to the cell circumference. MreB from Escherichia coli forms at least 1.4-μm-long filaments. MreB filaments move along various tracks with a maximal speed of 85 nm/s, and the loss of ATPase activity leads to the formation of extended and static filaments. Suboptimal growth conditions lead to formation of patch-like structures rather than extended filaments. Coexpression of wild-type MreB with MreB mutated in the subunit interface leads to formation of shorter MreB filaments and a strong effect on cell shape, revealing a link between filament length and cell morphology. Thus MreB has an extended-filament architecture with the potential to position membrane proteins over long distances, whose localization in turn may affect the shape of the cell wall.

The interaction of the carbon nanoparticles with human cell plasma membrane

NASA Astrophysics Data System (ADS)

Overchuk, M.; Prylutska, S.; Bilyy, Rostyslav; Prylutsky, Yu.; Ritter, U.

2013-09-01

The study of carbon nanostructures is a highly topical branch of bionanotechnology because of their potential application in biomedicine. Carbon nanotubes (CNTs) are known for their ability to kill tumor cells causing hyperthermia shock and can be used in photothermal therapy respectively. Also chemically modified CNTs can be used for drug delivery. The needle-like shape of CNTs allows them to penetrate into the cell plasma membrane without killing the cell. C60 fullerenes are regarded as valuable nanocarriers for different hydrophobic molecules as well as potential antiviral agents or photosensitizers. In our previous studies we have demonstrated that all types of carbon nanoparticles cause externalization of phosphatidylserine (PS) from the inner to the outer layer of the cell membrane in the small local patches (points of contact), leaving the other parts of plasma membrane PS-negative. In the current work there were studied the interactions of pristine C60 fullerenes and different types of CNTs with human blood cells (erythrocytes and Jurkat T-cells). We have shown, that carbon nanoparticles do not have any hemolytic effects, if judged by the dynamics of acidic hemolysis, although they are capable of permeabilizating the cells and facilitating the internalization of propidium iodide into the nuclei.

Palmitoylated SCP1 is targeted to the plasma membrane and negatively regulates angiogenesis

PubMed Central

Liao, Peng; Wang, Weichao; Li, Yu; Wang, Rui; Jin, Jiali; Pang, Weijuan; Chen, Yunfei; Shen, Mingyue; Wang, Xinbo; Jiang, Dongyang; Pang, Jinjiang; Liu, Mingyao; Lin, Xia; Feng, Xin-Hua; Wang, Ping; Ge, Xin

2017-01-01

SCP1 as a nuclear transcriptional regulator acts globally to silence neuronal genes and to affect the dephosphorylation of RNA Pol ll. However, we report the first finding and description of SCP1 as a plasma membrane-localized protein in various cancer cells using EGFP- or other epitope-fused SCP1. Membrane-located SCP1 dephosphorylates AKT at serine 473, leading to the abolishment of serine 473 phosphorylation that results in suppressed angiogenesis and a decreased risk of tumorigenesis. Consistently, we observed increased AKT phosphorylation and angiogenesis followed by enhanced tumorigenesis in Ctdsp1 (which encodes SCP1) gene - knockout mice. Importantly, we discovered that the membrane localization of SCP1 is crucial for impeding angiogenesis and tumor growth, and this localization depends on palmitoylation of a conserved cysteine motif within its NH2 terminus. Thus, our study discovers a novel mechanism underlying SCP1 shuttling between the plasma membrane and nucleus, which constitutes a unique pathway in transducing AKT signaling that is closely linked to angiogenesis and tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.22058.001 PMID:28440748

The antimicrobial peptide, lactoferricin B, is cytotoxic to neuroblastoma cells in vitro and inhibits xenograft growth in vivo.

PubMed

Eliassen, Liv Tone; Berge, Gerd; Leknessund, Arild; Wikman, Mari; Lindin, Inger; Løkke, Cecilie; Ponthan, Frida; Johnsen, John Inge; Sveinbjørnsson, Baldur; Kogner, Per; Flaegstad, Trond; Rekdal, Øystein

2006-08-01

Antimicrobial peptides have been shown to exert cytotoxic activity towards cancer cells through their ability to interact with negatively charged cell membranes. In this study the cytotoxic effect of the antimicrobial peptide, LfcinB was tested in a panel of human neuroblastoma cell lines. LfcinB displayed a selective cytotoxic activity against both MYCN-amplified and non-MYCN-amplified cell lines. Non-transformed fibroblasts were not substantially affected by LfcinB. Treatment of neuroblastoma cells with LfcinB induced rapid destabilization of the cytoplasmic membrane and formation of membrane blebs. Depolarization of the mitochondria membranes and irreversible changes in the mitochondria morphology was also evident. Immuno- and fluorescence-labeled LfcinB revealed that the peptide co-localized with mitochondria. Furthermore, treatment of neuroblastoma cells with LfcinB induced cleavage of caspase-6, -7 and -9 followed by cell death. However, neither addition of the pan-caspase inhibitor, zVAD-fmk, or specific caspase inhibitors could reverse the cytotoxic effect induced by LfcinB. Treatment of established SH-SY-5Y neuroblastoma xenografts with repeated injections of LfcinB resulted in significant tumor growth inhibition. These results revealed a selective destabilizing effect of LfcinB on two important targets in the neuroblastoma cells, the cytoplasmic- and the mitochondria membrane. Copyright (c) 2006 Wiley-Liss, Inc.

Principles and biophysical applications of single particle super-localization and rotational tracking

NASA Astrophysics Data System (ADS)

Gu, Yan

While conventional Single Particle Tracking (SPT) techniques acquire 2D or 3D trajectories of particle probes, we have developed Single Particle Orientation and Rotational Tracking (SPORT) techniques to extract orientation and rotational information. Combined with DIC microscopy, the SPORT technique has been applied in biophysical studies, including membrane diffusion and intracellular transport. The rotational dynamics of nanoparticle vectors on live cell membranes was recorded and its influence on the fate of these nanoparticle vectors was elucidated. The rotational motions of gold nanorods with various surface modifiers were tracked continuously at a temporal resolution of 5 ms under a DIC microscope. We found that the rotational behaviors of gold nanorod vectors are strongly related to their surface charge, specific surface functional groups, and the availability of receptors on cell membranes. The study of rotational Brownian motion of nanoparticles on cell membranes will lead to a better understanding of the mechanisms of drug delivery and provide guidance in designing surface modification strategies for drug delivery vectors under various circumstances. To characterize the rotation mode of surface functionalized gold nanorods on cell membranes, the SPORT technique is combined with the correlation analysis of the bright and dark DIC intensities. The unique capabilities of visualizing and understanding rotational motions of functionalized nanoparticles on live cell membranes allow us to correlate rotational and translational dynamics in unprecedented detail and provide new insights for complex membrane processes, including electrostatic interactions, ligand-receptor binding, and lateral (confined and hopping) diffusion of membrane receptors. Surface-functionalized nanoparticles interact with the membrane in fundamentally different ways and exhibit distinct rotational modes. The early events of particle-membrane approach and attachment are directly visualized for the first time. The rotational dynamics of cargos in both active directional transport and pausing stages of axonal transport was also visualized using high-speed SPORT with a temporal resolution of 2 ms. Both long and short pauses are imaged, and the correlations between the pause duration, the rotational behaviour of the cargo at the pause, and the moving direction after the pause are established. Furthermore, the rotational dynamics leading to switching tracks are visualized in detail. These first-time observations of cargo's rotational dynamics provide new insights on how kinesin and dynein motors take the cargo through the alternating stages of active directional transport and pause. To improve the localization precision of the SPT technique with DIC microscopy, a precise three-dimensional (3D) localization method of spherical gold nanoparticle probes using model-based correlation coefficient mapping was introduced. To accomplish this, a stack of sample images at different z-positions are acquired, and a 3D intensity profile of the probe serving as the model is used to map out the positions of nanoparticles in the sample. By using this model-based correlation imaging method, precise localization can be achieved in imaging techniques with complicated point spread functions (PSF) such as differential interference contrast (DIC) microscopy. The 3D superlocalization method was applied to tracking gold nanospheres during live endocytosis events. Finally, a novel dual-modality imaging technique has been developed to super-localize a single gold nanorod while providing its orientation and rotational information. The super-localization of the gold nanorod can be accomplished by curve fitting the modified bright-field images generated by one of the two beams laterally shifted by the first Nomarski prism in a DIC microscope. The orientation and rotational information is derived from the DIC images of gold nanorods. The new imaging setup has been applied to study the steric hindrance induced by relatively large cargos in the microtubule gliding assay and to track nanocargos in the crowded cellular environment.

Principles and biophysical applications of single particle super-localization and rotational tracking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gu, Yan

While conventional Single Particle Tracking (SPT) techniques acquire 2D or 3D trajectories of particle probes, we have developed Single Particle Orientation and Rotational Tracking (SPORT) techniques to extract orientation and rotational information. Combined with DIC microscopy, the SPORT technique has been applied in biophysical studies, including membrane diffusion and intracellular transport. The rotational dynamics of nanoparticle vectors on live cell membranes was recorded and its influence on the fate of these nanoparticle vectors was elucidated. The rotational motions of gold nanorods with various surface modifiers were tracked continuously at a temporal resolution of 5 ms under a DIC microscope. Wemore » found that the rotational behaviors of gold nanorod vectors are strongly related to their surface charge, specific surface functional groups, and the availability of receptors on cell membranes. The study of rotational Brownian motion of nanoparticles on cell membranes will lead to a better understanding of the mechanisms of drug delivery and provide guidance in designing surface modification strategies for drug delivery vectors under various circumstances. To characterize the rotation mode of surface functionalized gold nanorods on cell membranes, the SPORT technique is combined with the correlation analysis of the bright and dark DIC intensities. The unique capabilities of visualizing and understanding rotational motions of functionalized nanoparticles on live cell membranes allow us to correlate rotational and translational dynamics in unprecedented detail and provide new insights for complex membrane processes, including electrostatic interactions, ligand-receptor binding, and lateral (confined and hopping) diffusion of membrane receptors. Surface-functionalized nanoparticles interact with the membrane in fundamentally different ways and exhibit distinct rotational modes. The early events of particle-membrane approach and attachment are directly visualized for the first time. The rotational dynamics of cargos in both active directional transport and pausing stages of axonal transport was also visualized using high-speed SPORT with a temporal resolution of 2 ms. Both long and short pauses are imaged, and the correlations between the pause duration, the rotational behaviour of the cargo at the pause, and the moving direction after the pause are established. Furthermore, the rotational dynamics leading to switching tracks are visualized in detail. These first-time observations of cargo's rotational dynamics provide new insights on how kinesin and dynein motors take the cargo through the alternating stages of active directional transport and pause. To improve the localization precision of the SPT technique with DIC microscopy, a precise three-dimensional (3D) localization method of spherical gold nanoparticle probes using model-based correlation coefficient mapping was introduced. To accomplish this, a stack of sample images at different z-positions are acquired, and a 3D intensity profile of the probe serving as the model is used to map out the positions of nanoparticles in the sample. By using this model-based correlation imaging method, precise localization can be achieved in imaging techniques with complicated point spread functions (PSF) such as differential interference contrast (DIC) microscopy. The 3D superlocalization method was applied to tracking gold nanospheres during live endocytosis events. Finally, a novel dual-modality imaging technique has been developed to super-localize a single gold nanorod while providing its orientation and rotational information. The super-localization of the gold nanorod can be accomplished by curve fitting the modified bright-field images generated by one of the two beams laterally shifted by the first Nomarski prism in a DIC microscope. The orientation and rotational information is derived from the DIC images of gold nanorods. The new imaging setup has been applied to study the steric hindrance induced by relatively large cargos in the microtubule gliding assay and to track nanocargos in the crowded cellular environment.« less

PrPC Undergoes Basal to Apical Transcytosis in Polarized Epithelial MDCK Cells

PubMed Central

Arkhipenko, Alexander; Syan, Sylvie; Victoria, Guiliana Soraya

2016-01-01

The Prion Protein (PrP) is an ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of prion disease, the cellular isoform (PrPC) is an enigmatic protein with unclear function. Of interest, PrP localization in polarized MDCK cells is controversial and its mechanism of trafficking is not clear. Here we investigated PrP traffic in MDCK cells polarized on filters and in three-dimensional MDCK cysts, a more physiological model of polarized epithelia. We found that, unlike other GPI-anchored proteins (GPI-APs), PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells. Following this event full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures. PMID:27389581

Isolation of basal membrane proteins from BeWo cells and their expression in placentas from fetal growth-restricted pregnancies.

PubMed

Oh, Soo-Young; Hwang, Jae Ryoung; Lee, Yoonna; Choi, Suk-Joo; Kim, Jung-Sun; Kim, Jong-Hwa; Sadovsky, Yoel; Roh, Cheong-Rae

2016-03-01

The syncytiotrophoblast, a key barrier between the mother and fetus, is a polarized epithelium composed of a microvillus and basal membrane (BM). We sought to characterize BM proteins of BeWo cells in relation to hypoxia and to investigate their expression in placentas from pregnancies complicated by fetal growth restriction (FGR). We isolated the BM fraction of BeWo cells by the cationic colloidal silica method and identified proteins enriched in this fraction by mass spectrometry. We evaluated the effect of hypoxia on the expression and intracellular localization of identified proteins and compared their expression in BM fractions of FGR placentas to those from normal pregnancies. We identified BM proteins from BeWo cells. Among BM proteins, we further characterized heme oxygenase-1 (HO-1), voltage-dependent anion channel-1 (VDAC1), and ribophorin II (RPN2), based on their relevance to placental biology. Hypoxia enhanced the localization of these proteins to the BM of BeWo cells. HO-1, VDAC1, and RPN2 were selectively expressed in the human placental BM fraction. C-terminally truncated HO-1 was identified in placental BM fractions, and its BM expression was significantly reduced in FGR placentas than in normal placentas. Interestingly, a truncated HO-1 construct was predominantly localized in the BM in response to hypoxia and co-localized with VDAC1 in BeWo cells. Hypoxia increased the BM localization of HO-1, VDAC1, and RPN2 proteins. FGR significantly reduced the expression of truncated HO-1, which was surmised to co-localize with VDAC1 in hypoxic BeWo cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Timing is everything

PubMed Central

Carr, Heather S; Frost, Jeffrey A

2013-01-01

Cell adhesion to the extracellular matrix elicits a temporal reorganization of the actin cytoskeleton that is regulated first by Rac1 and later by RhoA. The signaling mechanisms controlling late stage RhoA activation are incompletely understood. Net1A is a RhoA/RhoB-specific guanine nucleotide exchange factor that is required for cancer cell motility. The ability of Net1A to stimulate RhoA activation is negatively regulated by nuclear sequestration. However, mechanisms controlling the plasma membrane localization of Net1A had not previously been reported. Recently we have shown that Rac1 activation stimulates plasma membrane relocalization and activation of Net1A. Net1A relocalization is independent of its catalytic activity and does not require its C-terminal pleckstrin homology or PDZ interacting domains. Rac1 activation during cell adhesion stimulates a transient relocalization of Net1A that is terminated by proteasomal degradation of Net1A. Importantly, plasma membrane localization of Net1A is required for efficient myosin light chain phosphorylation, focal adhesion maturation, and cell spreading. These data show for the first time a physiological mechanism controlling Net1A relocalization from the nucleus. They also demonstrate a previously unrecognized role for Net1A in controlling actomyosin contractility and focal adhesion dynamics during cell adhesion. PMID:23792411

The eighth fibronectin type III domain of protein tyrosine phosphatase receptor J influences the formation of protein complexes and cell localization.

PubMed

Iuliano, Rodolfo; Raso, Cinzia; Quintiero, Alfina; Pera, Ilaria Le; Pichiorri, Flavia; Palumbo, Tiziana; Palmieri, Dario; Pattarozzi, Alessandra; Florio, Tullio; Viglietto, Giuseppe; Trapasso, Francesco; Croce, Carlo Maria; Fusco, Alfredo

2009-03-01

Regulation of receptor-type phosphatases can involve the formation of higher-order structures, but the exact role played in this process by protein domains is not well understood. In this study we show the formation of different higher-order structures of the receptor-type phosphatase PTPRJ, detected in HEK293A cells transfected with different PTPRJ expression constructs. In the plasma membrane PTPRJ forms dimers detectable by treatment with the cross-linking reagent BS(3) (bis[sulfosuccinimidyl]suberate). However, other PTPRJ complexes, dependent on the formation of disulfide bonds, are detected by treatment with the oxidant agent H(2)O(2) or by a mutation Asp872Cys, located in the eighth fibronectin type III domain of PTPRJ. A deletion in the eighth fibronectin domain of PTPRJ impairs its dimerization in the plasma membrane and increases the formation of PTPRJ complexes dependent on disulfide bonds that remain trapped in the cytoplasm. The deletion mutant maintains the catalytic activity but is unable to carry out inhibition of proliferation on HeLa cells, achieved by the wild type form, since it does not reach the plasma membrane. Therefore, the intact structure of the eighth fibronectin domain of PTPRJ is critical for its localization in plasma membrane and biological function.

Regulation of aquaporins in plants under stress.

PubMed

Kapilan, Ranganathan; Vaziri, Maryam; Zwiazek, Janusz J

2018-01-16

Aquaporins (AQP) are channel proteins belonging to the Major Intrinsic Protein (MIP) superfamily that play an important role in plant water relations. The main role of aquaporins in plants is transport of water and other small neutral molecules across cellular biological membranes. AQPs have remarkable features to provide an efficient and often, specific water flow and enable them to transport water into and out of the cells along the water potential gradient. Plant AQPs are classified into five main subfamilies including the plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26 like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and X intrinsic proteins (XIPs). AQPs are localized in the cell membranes and are found in all living cells. However, most of the AQPs that have been described in plants are localized to the tonoplast and plasma membranes. Regulation of AQP activity and gene expression, are also considered as a part of the adaptation mechanisms to stress conditions and rely on complex processes and signaling pathways as well as complex transcriptional, translational and posttranscriptional factors. Gating of AQPs through different mechanisms, such as phosphorylation, tetramerization, pH, cations, reactive oxygen species, phytohormones and other chemical agents, may play a key role in plant responses to environmental stresses by maintaining the uptake and movement of water in the plant body.

Aptamer-recognized carbohydrates on the cell membrane revealed by super-resolution microscopy.

PubMed

Jing, Yingying; Cai, Mingjun; Xu, Haijiao; Zhou, Lulu; Yan, Qiuyan; Gao, Jing; Wang, Hongda

2018-04-26

Carbohydrates are one of the most important components on the cell membrane, which participate in various physiological activities, and their aberrant expression is a consequence of pathological changes. In previous studies, carbohydrate analysis basically relied on lectins. However, discrimination between lectins still exists due to their multivalent character. Furthermore, the structures obtained by carbohydrate-lectin crosslinking confuse our direct observation to some extent. Fortunately, the emergence of aptamers, which are smaller and more flexible, has provided us an unprecedented choice. Herein, an aptamer recognition method with high precise localization was developed for imaging membrane-bound N-acetylgalactosamine (GalNAc). By using direct stochastic optical reconstruction microscopy (dSTORM), we compared this aptamer recognition method with the lectin recognition method for visualizing the detailed structure of GalNAc at the nanometer scale. The results indicated that GalNAc forms irregular clusters on the cell membrane with a resolution of 23 ± 7 nm by aptamer recognition. Additionally, when treated with N-acetylgalactosidase, the aptamer-recognized GalNAc shows a more significant decrease in cluster size and localization density, thus verifying better specificity of aptamers than lectins. Collectively, our study suggests that aptamers can act as perfect substitutes for lectins in carbohydrate labeling, which will be of great potential value in the field of super-resolution fluorescence imaging.

A quinolinol-based small molecule with anti-MRSA activity that targets bacterial membrane and promotes fermentative metabolism.

PubMed

Nair, Dhanalakshmi R; Chen, Ji; Monteiro, João M; Josten, Michaele; Pinho, Mariana G; Sahl, Hans-Georg; Wu, Jimmy; Cheung, Ambrose

2017-10-01

In a loss-of-viability screen of small molecules against methicillin-resistant Staphylococcus aureus (MRSA) USA300, we found a small molecule, designated DNAC-2, which has an MIC of 8 μg ml -1 . DNAC-2 is a quinolinol derivative that is bactericidal at 2X MIC. Macromolecular synthesis assays at 2 × MIC of DNAC-2 revealed inhibition of DNA, cell wall, RNA and protein synthesis within fifteen to thirty minutes of treatment when compared to the untreated control. Transmission electron microscopy of DNAC-2-treated cells revealed a significantly thicker cell wall and impaired daughter cell separation. Exposure of USA300 cells to 1 × MIC of DNAC-2 resulted in mislocalization of PBP2 away from the septum in an FtsZ-independent manner. In addition, membrane localization with FM4-64, as well as depolarization study with DiOC 2 and lipophilic cation TPP+ displayed membrane irregularities and rapid membrane depolarization, respectively, in DNAC-2-treated cells vs -untreated control. However, DNAC-2 exhibited almost no toxicity toward eukaryotic membranes. Notably, DNAC-2 drives energy generation toward substrate level phosphorylation and the bacteria become more sensitive to DNAC-2 under anaerobic conditions. We propose that DNAC-2 affects USA300 by targeting the membrane, leading to partial membrane depolarization and subsequently affecting aerobic respiration and energy-dependent functional organization of macromolecular biosynthetic pathways. The multiple effects may have the desirable consequence of limiting the emergence of resistance to DNAC-2.

Immunocytochemical localization of actin in epithelial cells of rat small intestine by light and electron microscopy.

PubMed

Hagen, S J; Trier, J S

1988-07-01

We used post-embedding immunocytochemical techniques and affinity-purified anti-actin antibody to evaluate localization of actin in epithelial cells of small intestine by fluorescence and electron microscopy. Small intestine was fixed with 2% formaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M. One-micron or thin sections were stained with antibody followed by rhodamine- or colloidal gold-labeled goat anti-rabbit IgG, respectively. Label was present overlying microvilli, the apical terminal web, and the cytoplasm directly adjacent to occluding and intermediate junctions. Label was associated with outer mitochondrial membranes of all cells and the supranuclear Golgi region of goblet cells. Lateral cytoplasmic interdigitations between mature cells and subplasmalemmal filaments next to intrusive cells were densely labeled. The cytoplasm adjacent to unplicated domains of lateral membrane was focally labeled. Label was prominent over organized filament bundles within the subplasmalemmal web at the base of mature cells, whereas there was focal labeling of the cytoplasm adjacent to the basal membrane of undifferentiated cells. Basolateral epithelial cell processes were labeled. Label was focally present overlying the cellular ground substance. Our results demonstrate that actin is distributed in a distinctive fashion within intestinal epithelial cells. This distribution suggests that in addition to its function as a structural protein, actin may participate in regulation of epithelial tight junction permeability, in motile processes including migration of cells from the crypt to the villus tip, in accommodation of intrusive intraepithelial cells and in adhesion of cells to one another and to their substratum.

Procyanidins can interact with Caco-2 cell membrane lipid rafts: involvement of cholesterol.

PubMed

Verstraeten, Sandra V; Jaggers, Grayson K; Fraga, Cesar G; Oteiza, Patricia I

2013-11-01

Large procyanidins (more than three subunits) are not absorbed at the gastrointestinal tract but could exert local effects through their interactions with membranes. We previously showed that hexameric procyanidins (Hex), although not entering cells, interact with membranes modulating cell signaling and fate. This paper investigated if Hex, as an example of large procyanidins, can selectively interact with lipid rafts which could in part explain its biological actions. This mechanism was studied in both synthetic membranes (liposomes) and Caco-2 cells. Hex promoted Caco-2 cell membrane rigidification and dehydration, effects that were abolished upon cholesterol depletion with methyl-β-cyclodextrin (MCD). Hex prevented lipid raft structure disruption induced by cholesterol depletion/redistribution by MCD or sodium deoxycholate. Supporting the involvement of cholesterol-Hex bonding in Hex interaction with lipid rafts, the absence of cholesterol markedly decreased the capacity of Hex to prevent deoxycholate- and Triton X-100-mediated disruption of lipid raft-like liposomes. Stressing the functional relevance of this interaction, Hex mitigated lipid raft-associated activation of the extracellular signal-regulated kinases (ERK) 1/2. Results support the capacity of a large procyanidin (Hex) to interact with membrane lipid rafts mainly through Hex-cholesterol bondings. Procyanidin-lipid raft interactions can in part explain the capacity of large procyanidins to modulate cell physiology. © 2013 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duenas, Maria Emilia; Klein, Adam T.; Alexander, Liza E.

Metabolism in plants is compartmentalized among different tissues, cells and subcellular organelles. Mass spectrometry imaging (MSI) with matrix-assisted laser desorption ionization (MALDI) has recently advanced to allow for the visualization of metabolites at single-cell resolution. Here we applied 5- and 10 μm high spatial resolution MALDI-MSI to the asymmetric Kranz anatomy of Zea mays (maize) leaves to study the differential localization of two major anionic lipids in thylakoid membranes, sulfoquinovosyldiacylglycerols (SQDG) and phosphatidylglycerols (PG). The quantification and localization of SQDG and PG molecular species, among mesophyll (M) and bundle sheath (BS) cells, are compared across the leaf developmental gradient frommore » four maize genotypes (the inbreds B73 and Mo17, and the reciprocal hybrids B73 × Mo17 and Mo17 × B73). SQDG species are uniformly distributed in both photosynthetic cell types, regardless of leaf development or genotype; however, PG shows photosynthetic cell-specific differential localization depending on the genotype and the fatty acyl chain constituent. Overall, 16:1-containing PGs primarily contribute to the thylakoid membranes of M cells, whereas BS chloroplasts are mostly composed of 16:0-containing PGs. Furthermore, PG 32:0 shows genotype-specific differences in cellular distribution, with preferential localization in BS cells for B73, but more uniform distribution between BS and M cells in Mo17. Maternal inheritance is exhibited within the hybrids, such that the localization of PG 32:0 in B73 × Mo17 is similar to the distribution in the B73 parental inbred, whereas that of Mo17 × B73 resembles the Mo17 parent. As a result, this study demonstrates the power of MALDI-MSI to reveal unprecedented insights on metabolic outcomes in multicellular organisms at single-cell resolution.« less

Fluorinated colloidal gold immunolabels for imaging select proteins in parallel with lipids using high-resolution secondary ion mass spectrometry

PubMed Central

Wilson, Robert L.; Frisz, Jessica F.; Hanafin, William P.; Carpenter, Kevin J.; Hutcheon, Ian D.; Weber, Peter K.; Kraft, Mary L.

2014-01-01

The local abundance of specific lipid species near a membrane protein is hypothesized to influence the protein’s activity. The ability to simultaneously image the distributions of specific protein and lipid species in the cell membrane would facilitate testing these hypotheses. Recent advances in imaging the distribution of cell membrane lipids with mass spectrometry have created the desire for membrane protein probes that can be simultaneously imaged with isotope labeled lipids. Such probes would enable conclusive tests of whether specific proteins co-localize with particular lipid species. Here, we describe the development of fluorine-functionalized colloidal gold immunolabels that facilitate the detection and imaging of specific proteins in parallel with lipids in the plasma membrane using high-resolution SIMS performed with a NanoSIMS. First, we developed a method to functionalize colloidal gold nanoparticles with a partially fluorinated mixed monolayer that permitted NanoSIMS detection and rendered the functionalized nanoparticles dispersible in aqueous buffer. Then, to allow for selective protein labeling, we attached the fluorinated colloidal gold nanoparticles to the nonbinding portion of antibodies. By combining these functionalized immunolabels with metabolic incorporation of stable isotopes, we demonstrate that influenza hemagglutinin and cellular lipids can be imaged in parallel using NanoSIMS. These labels enable a general approach to simultaneously imaging specific proteins and lipids with high sensitivity and lateral resolution, which may be used to evaluate predictions of protein co-localization with specific lipid species. PMID:22284327

Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mader, Jamie S.; Richardson, Angela; Salsman, Jayme

2007-07-15

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused themore » death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB.« less

Sensing Phosphatidylserine in Cellular Membranes

PubMed Central

Kay, Jason G.; Grinstein, Sergio

2011-01-01

Phosphatidylserine, a phospholipid with a negatively charged head-group, is an important constituent of eukaryotic cellular membranes. On the plasma membrane, rather than being evenly distributed, phosphatidylserine is found preferentially in the inner leaflet. Disruption of this asymmetry, leading to the appearance of phosphatidylserine on the surface of the cell, is known to play a central role in both apoptosis and blood clotting. Despite its importance, comparatively little is known about phosphatidylserine in cells: its precise subcellular localization, transmembrane topology and intracellular dynamics are poorly characterized. The recent development of new, genetically-encoded probes able to detect phosphatidylserine within live cells, however, is leading to a more in-depth understanding of the biology of this phospholipid. This review aims to give an overview of the current methods for phosphatidylserine detection within cells, and some of the recent realizations derived from their use. PMID:22319379

Sensing phosphatidylserine in cellular membranes.

PubMed

Kay, Jason G; Grinstein, Sergio

2011-01-01

Phosphatidylserine, a phospholipid with a negatively charged head-group, is an important constituent of eukaryotic cellular membranes. On the plasma membrane, rather than being evenly distributed, phosphatidylserine is found preferentially in the inner leaflet. Disruption of this asymmetry, leading to the appearance of phosphatidylserine on the surface of the cell, is known to play a central role in both apoptosis and blood clotting. Despite its importance, comparatively little is known about phosphatidylserine in cells: its precise subcellular localization, transmembrane topology and intracellular dynamics are poorly characterized. The recent development of new, genetically-encoded probes able to detect phosphatidylserine within live cells, however, is leading to a more in-depth understanding of the biology of this phospholipid. This review aims to give an overview of the current methods for phosphatidylserine detection within cells, and some of the recent realizations derived from their use.

Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

PubMed Central

Palma-Guerrero, Javier; Zhao, Jiuhai; Gonçalves, A. Pedro; Starr, Trevor L.

2015-01-01

The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1 or Δlfd-1 germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, for late fusion defect-2) that showed a calcium-dependent cell lysis phenotype. lfd-2 encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter, fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion. PMID:25595444

pH and Ion Homeostasis on Plant Endomembrane Dynamics: Insights from structural models and mutants of K+/H+ antiporters.

PubMed

Sze, Heven; Chanroj, Salil

2018-04-24

Plants remodel their cells through the dynamic endomembrane system. Intracellular pH is important for membrane trafficking, but the determinants of pH homeostasis are poorly defined in plants. Electrogenic proton (H+) pumps depend on counter-ion fluxes to establish transmembrane pH gradients at the plasma membrane and endomembranes. Vacuolar-type H+-ATPase-mediated acidification of the trans-Golgi network (TGN) is crucial for secretion and membrane recycling. Pump and counter-ion fluxes are unlikely to fine-tune pH; rather, alkali cation/H+ antiporters, which can alter pH and/or cation homeostasis locally and transiently, are prime candidates. Plants have a large family of predicted cation/H+ exchangers (CHX) of obscure function, in addition to the well-studied K+(Na+)/H+ exchangers (NHX). Here, we review the regulation of cytosolic and vacuolar pH, highlighting the similarities and distinctions of NHX and CHX members. In planta, alkalinization of the TGN or vacuole by NHXs promotes membrane trafficking, endocytosis, cell expansion, and growth. CHXs localize to endomembranes and/or the plasma membrane, contribute to male fertility, pollen tube guidance, pollen wall construction, stomatal opening, and in soybean (Glycine max), tolerance to salt stress. Three-dimensional structural models and mutagenesis of Arabidopsis thaliana genes have allowed us to infer that AtCHX17 and AtNHX1 share a global architecture and a translocation core like bacterial Na+/H+ antiporters. Yet the presence of distinct residues suggests some CHXs differ from NHXs in pH sensing and electrogenicity. How H+ pumps, counter-ion fluxes, and cation/H+ antiporters are linked with signaling and membrane trafficking to remodel membranes and cell walls awaits further investigation. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Subcellular localization and expression pattern of the neurofibromatosis type 2 protein merlin/schwannomin.

PubMed

Schmucker, B; Ballhausen, W G; Kressel, M

1997-01-01

To elucidate the physiological function of the neurofibromatosis type 2 (NF2) tumor suppressor protein merlin/schwannomin, we studied the expression pattern and subcellular localization in human fibroblasts by Western blot analyses and immunofluorescence using a polyclonal antibody raised against the C-terminus of merlin. Three of the six merlin isoforms identified in this study (75 kDa, 58 kDa, 45 kDa) have been reported earlier and can be explained by alternative splicing. In addition, we detected higher molecular weight bands of about 110 kDa, 100 kDa and 84 kDa. Although the merlin bands of 100 kDa and 110 kDa may represent homo- or heterodimers, oligomerization due to formation of disulfide bonds was excluded. Furthermore, the isoforms of 84 kDa and 58 kDa were quantitatively extractable in Lubrol WX, indicating a localization in or close to the plasma membrane. The 45 kDa band, however, was not soluble in Lubrol WX compatible with a localization of this NF2 isoform in the endoplasmic reticulum. Applying confocal laser scanning microscopy, merlin was shown to be located in four subcellular compartments: (i) perinuclear in a compartment resembling endoplasmic reticulum, (ii) in ruffling membranes and at the leading edges, (iii) in filopodia, and (iv) at cell/substrate adhesion points. Codistribution of merlin and F-actin filaments was found in filopodia, ruffling membranes and at the insertion points of stress fibers at cell/substrate adhesion junctions as shown by phalloidin-rhodamine staining. Double immunofluorescence analyses of merlin and moesin revealed a colocalization in filopodia and ruffling membranes. The localization of merlin in the actin-rich cortical cytoskeleton corresponds to the ezrin-radixin-moesin family of proteins suggesting the NF2 protein to contribute to the regulation of cell growth by interaction with cytoskeleton-associated proteins.

Zoledronic acid inhibits vasculogenic mimicry in murine osteosarcoma cell line in vitro.

PubMed

Fu, Dehao; He, Xianfeng; Yang, Shuhua; Xu, Weihua; Lin, Tao; Feng, Xiaobo

2011-06-30

To study the effects of zoledronic acid (ZA) on the vasculogenic mimicry of osteosarcoma cells in vitro. A Three-dimensional culture of LM8 osteosarcoma cells on a type I collagen matrix was used to investigate whether osteosarcoma cells can develop vasculogenic mimicry, and to determine the effects of ZA on this process. In addition, the cellular ultrastructural changes were observed using scanning electron microscopy and laser confocal microscopy. The effects of ZA on the translocation of RhoA protein from the cytosol to the membrane in LM8 cells were measured via immunoblotting. ZA inhibited the development of vasculogenic mimicry by the LM8 osteosarcoma cells, decreased microvilli formation on the cell surface, and disrupted the F-actin cytoskeleton. ZA prevented translocation of RhoA protein from the cytosol to the membrane in LM8 cells. ZA can impair RhoA membrane localization in LM8 cells, causing obvious changes in the ultrastructure of osteosarcoma cells and induce cell apoptosis, which may be one of the underlying mechanisms by which the agent inhibits the development of vasculogenic mimicry by the LM8 cells.

The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins.

PubMed

Kawada, Daiki; Kobayashi, Hiromu; Tomita, Tsuyoshi; Nakata, Eisuke; Nagano, Makoto; Siekhaus, Daria Elisabeth; Toshima, Junko Y; Toshima, Jiro

2015-01-01

Small GTP-binding proteins of the Ras superfamily play diverse roles in intracellular trafficking. Among them, the Rab, Arf, and Rho families function in successive steps of vesicle transport, in forming vesicles from donor membranes, directing vesicle trafficking toward target membranes and docking vesicles onto target membranes. These proteins act as molecular switches that are controlled by a cycle of GTP binding and hydrolysis regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In this study we explored the role of GAPs in the regulation of the endocytic pathway using fluorescently labeled yeast mating pheromone α-factor. Among 25 non-essential GAP mutants, we found that deletion of the GLO3 gene, encoding Arf-GAP protein, caused defective internalization of fluorescently labeled α-factor. Quantitative analysis revealed that glo3Δ cells show defective α-factor binding to the cell surface. Interestingly, Ste2p, the α-factor receptor, was mis-localized from the plasma membrane to the vacuole in glo3Δ cells. Domain deletion mutants of Glo3p revealed that a GAP-independent function, as well as the GAP activity, of Glo3p is important for both α-factor binding and Ste2p localization at the cell surface. Additionally, we found that deletion of the GLO3 gene affects the size and number of Arf1p-residing Golgi compartments and causes a defect in transport from the TGN to the plasma membrane. Furthermore, we demonstrated that glo3Δ cells were defective in the late endosome-to-TGN transport pathway, but not in the early endosome-to-TGN transport pathway. These findings suggest novel roles for Arf-GAP Glo3p in endocytic recycling of cell surface proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells

PubMed Central

Fretz, Marjan M.; Penning, Neal A.; Al-Taei, Saly; Futaki, Shiroh; Takeuchi, Toshihide; Nakase, Ikuhiko; Storm, Gert; Jones, Arwyn T.

2007-01-01

Delineating the mechanisms by which cell-penetrating peptides, such as HIV-Tat peptide, oligoarginines and penetratin, gain access to cells has recently received intense scrutiny. Heightened interest in these entities stems from their ability to enhance cellular delivery of associated macromolecules, such as genes and proteins, suggesting that they may have widespread applications as drug-delivery vectors. Proposed uptake mechanisms include energy-independent plasma membrane translocation and energy-dependent vesicular uptake and internalization through endocytic pathways. In the present study, we investigated the effects of temperature, peptide concentration and plasma membrane cholesterol levels on the uptake of a model cell-penetrating peptide, L-octa-arginine (L-R8) and its D-enantiomer (D-R8) in CD34+ leukaemia cells. We found that, at 4–12 °C, L-R8 uniformly labels the cytoplasm and nucleus, but in cells incubated with D-R8 there is additional labelling of the nucleolus which is still prominent at 30 °C incubations. At temperatures between 12 and 30 °C, the peptides are also localized to endocytic vesicles which consequently appear as the only labelled structures in cells incubated at 37 °C. Small increases in the extracellular peptide concentration in 37 °C incubations result in a dramatic increase in the fraction of the peptide that is localized to the cytosol and promoted the binding of D-R8 to the nucleolus. Enhanced labelling of the cytosol, nucleus and nucleolus was also achieved by extraction of plasma membrane cholesterol with methyl-β-cyclodextrin. The data argue for two, temperature-dependent, uptake mechanism for these peptides and for the existence of a threshold concentration for endocytic uptake that when exceeded promotes direct translocation across the plasma membrane. PMID:17217340

Uptake and localization mechanisms of fluorescent and colored lipid probes. Part 2. QSAR models that predict localization of fluorescent probes used to identify ("specifically stain") various biomembranes and membranous organelles.

PubMed

Horobin, R W; Stockert, J C; Rashid-Doubell, F

2015-05-01

We discuss a variety of biological targets including generic biomembranes and the membranes of the endoplasmic reticulum, endosomes/lysosomes, Golgi body, mitochondria (outer and inner membranes) and the plasma membrane of usual fluidity. For each target, we discuss the access of probes to the target membrane, probe uptake into the membrane and the mechanism of selectivity of the probe uptake. A statement of the QSAR decision rule that describes the required physicochemical features of probes that enable selective staining also is provided, followed by comments on exceptions and limits. Examples of probes typically used to demonstrate each target structure are noted and decision rule tabulations are provided for probes that localize in particular targets; these tabulations show distribution of probes in the conceptual space defined by the relevant structure parameters ("parameter space"). Some general implications and limitations of the QSAR models for probe targeting are discussed including the roles of certain cell and protocol factors that play significant roles in lipid staining. A case example illustrates the predictive ability of QSAR models. Key limiting values of the head group hydrophilicity parameter associated with membrane-probe interactions are discussed in an appendix.

The PDZ domain of the guanine nucleotide exchange factor PDZGEF directs binding to phosphatidic acid during brush border formation.

PubMed

Consonni, Sarah V; Brouwer, Patricia M; van Slobbe, Eleonora S; Bos, Johannes L

2014-01-01

PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid.

The PDZ Domain of the Guanine Nucleotide Exchange Factor PDZGEF Directs Binding to Phosphatidic Acid during Brush Border Formation

PubMed Central

Consonni, Sarah V.; Brouwer, Patricia M.; van Slobbe, Eleonora S.; Bos, Johannes L.

2014-01-01

PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid. PMID:24858808

HIP1 exhibits an early recruitment and a late stage function in the maturation of coated pits.

PubMed

Gottfried, Irit; Ehrlich, Marcelo; Ashery, Uri

2009-09-01

Huntingtin interacting protein 1 (HIP1) is an accessory protein of the clathrin-mediated endocytosis (CME) pathway, yet its precise role and the step at which it becomes involved are unclear. We employed live-cell imaging techniques to focus on the early steps of CME and characterize HIP1 dynamics. We show that HIP1 is highly colocalized with clathrin at the plasma membrane and shares similar dynamics with a subpopulation of clathrin assemblies. Employing transferrin receptor fused to pHluorin, we distinguished between open pits to which HIP1 localizes and newly internalized vesicles that are devoid of HIP1. Moreover, shRNA knockdown of clathrin compromised HIP1 membranal localization, unlike the reported behavior of Sla2p. HIP1 fragment, lacking its ANTH and Talin-like domains, inhibits internalization of transferrin, but retains colocalization with membranal clathrin assemblies. These data demonstrate HIP1's role in pits maturation and formation of the coated vesicle, and its strong dependence on clathrin for membranal localization.

Expression patterns of genes encoding plasma membrane aquaporins during fruit development in cucumber (Cucumis sativus L.).

PubMed

Shi, Jin; Wang, Jinfang; Li, Ren; Li, Dianbo; Xu, Fengfeng; Sun, Qianqian; Zhao, Bin; Mao, Ai-Jun; Guo, Yang-Dong

2015-11-01

Aquaporins are membrane channels precisely regulating water movement through cell membranes in most living organisms. Despite the advances in the physiology of fruit development, their participation during fruit development in cucumber still barely understood. In this paper, the expressions of 12 genes encoding plasma membrane intrinsic proteins (PIPs) were analyzed during cucumber fruit development in our work. Based on the homology search with known PIPs from rice, Arabidopsis and strawberry, 12 cucumber PIP genes subfamily members were identified. Cellular localization assays indicated that CsPIPs were localized in the plasma membrane. The qRT-PCR analysis of CsPIPs showed that 12 CsPIPs were differentially expressed during fruit development. These results suggest that 12 genes encoding plasma membrane intrinsic proteins (CsPIPs) play very important roles in cucumber life cycle and the data generated will be helpful in understanding their precise roles during fruit development in cucumber. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Dissecting the role of conformational change and membrane binding by the bacterial cell division regulator MinE in the stimulation of MinD ATPase activity.

PubMed

Ayed, Saud H; Cloutier, Adam D; McLeod, Laura J; Foo, Alexander C Y; Damry, Adam M; Goto, Natalie K

2017-12-15

The bacterial cell division regulators MinD and MinE together with the division inhibitor MinC localize to the membrane in concentrated zones undergoing coordinated pole-to-pole oscillation to help ensure that the cytokinetic division septum forms only at the mid-cell position. This dynamic localization is driven by MinD-catalyzed ATP hydrolysis, stimulated by interactions with MinE's anti-MinCD domain. This domain is buried in the 6-β-stranded MinE "closed" structure, but is liberated for interactions with MinD, giving rise to a 4-β-stranded "open" structure through an unknown mechanism. Here we show that MinE-membrane interactions induce a structural change into a state resembling the open conformation. However, MinE mutants lacking the MinE membrane-targeting sequence stimulated higher ATP hydrolysis rates than the full-length protein, indicating that binding to MinD is sufficient to trigger this conformational transition in MinE. In contrast, conformational change between the open and closed states did not affect stimulation of ATP hydrolysis rates in the absence of membrane binding, although the MinD-binding residue Ile-25 is critical for this conformational transition. We therefore propose an updated model where MinE is brought to the membrane through interactions with MinD. After stimulation of ATP hydrolysis, MinE remains bound to the membrane in a state that does not catalyze additional rounds of ATP hydrolysis. Although the molecular basis for this inhibited state is unknown, previous observations of higher-order MinE self-association may explain this inhibition. Overall, our findings have general implications for Min protein oscillation cycles, including those that regulate cell division in bacterial pathogens. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

SU-E-J-102: Separation of Metabolic Supply and Demand: From Power Grid Economics to Cancer Metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Epstein, T; Xu, L; Gillies, R

2014-06-01

Purpose: To study a new model of glucose metabolism which is primarily governed by the timescale of the energetic demand and not by the oxygen level, and its implication on cancer metabolism (Warburg effect) Methods: 1) Metabolic profiling of membrane transporters activity in several cell lines, which represent the spectrum from normal breast epithelium to aggressive, metastatic cancer, using Seahorse XF reader.2) Spatial localization of oxidative and non-oxidative metabolic components using immunocytochemical imaging of the glycolytic ATP-producing enzyme, pyruvate kinase and mitochondria. 3) Finite element simulations of coupled partial differential equations using COMSOL and MATLAB. Results: Inhibition or activation ofmore » pumps on the cell membrane led to reduction or increase in aerobic glycolysis, respectively, while oxidative phosphorylation remained unchanged. These results were consistent with computational simulations of changes in short-timescale demand for energy by cell membrane processes. A specific model prediction was that the spatial distribution of ATP-producing enzymes in the glycolytic pathway must be primarily localized adjacent to the cell membrane, while mitochondria should be predominantly peri-nuclear. These predictions were confirmed experimentally. Conclusion: The results in this work support a new model for glucose metabolism in which glycolysis and oxidative phosphorylation supply different types of energy demand. Similar to power grid economics, optimal metabolic control requires the two pathways, even in normoxic conditions, to match two different types of energy demands. Cells use aerobic metabolism to meet baseline, steady energy demand and glycolytic metabolism to meet short-timescale energy demands, mainly from membrane transport activities, even in the presence of oxygen. This model provides a mechanism for the origin of the Warburg effect in cancer cells. Here, the Warburg effect emerges during carcinogenesis is a physiological response to an increase in energy demands from membrane transporters, required for cell division, growth, and migration. This work is supported by the NIH Physical Sciences in Oncology Center grant 1U54CA143970-03 and NIH R01 CA077575-10.« less

Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells.

PubMed

Wang, Zhen; Schey, Kevin L

2015-12-01

Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids-key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis. A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains. These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells.

Intercellular ultrafast Ca2+ wave in vascular smooth muscle cells: numerical and experimental study

NASA Astrophysics Data System (ADS)

Quijano, J. C.; Raynaud, F.; Nguyen, D.; Piacentini, N.; Meister, J. J.

2016-08-01

Vascular smooth muscle cells exhibit intercellular Ca2+ waves in response to local mechanical or KCl stimulation. Recently, a new type of intercellular Ca2+ wave was observed in vitro in a linear arrangement of smooth muscle cells. The intercellular wave was denominated ultrafast Ca2+ wave and it was suggested to be the result of the interplay between membrane potential and Ca2+ dynamics which depended on influx of extracellular Ca2+, cell membrane depolarization and its intercel- lular propagation. In the present study we measured experimentally the conduction velocity of the membrane depolarization and performed simulations of the ultrafast Ca2+ wave along coupled smooth muscle cells. Numerical results reproduced a wide spectrum of experimental observations, including Ca2+ wave velocity, electrotonic membrane depolarization along the network, effects of inhibitors and independence of the Ca2+ wave speed on the intracellular stores. The numerical data also provided new physiological insights suggesting ranges of crucial model parameters that may be altered experimentally and that could significantly affect wave kinetics allowing the modulation of the wave characteristics experimentally. Numerical and experimental results supported the hypothesis that the propagation of membrane depolarization acts as an intercellular messenger mediating intercellular ultrafast Ca2+ waves in smooth muscle cells.

Hampering Effect of Cholesterol on the Permeation of Reactive Oxygen Species through Phospholipids Bilayer: Possible Explanation for Plasma Cancer Selectivity

NASA Astrophysics Data System (ADS)

van der Paal, Jonas; Verheyen, Claudia; Neyts, Erik C.; Bogaerts, Annemie

2017-01-01

In recent years, the ability of cold atmospheric pressure plasmas (CAPS) to selectively induce cell death in cancer cells has been widely established. This selectivity has been assigned to the reactive oxygen and nitrogen species (RONS) created in CAPs. To provide new insights in the search for an explanation for the observed selectivity, we calculate the transfer free energy of multiple ROS across membranes containing a varying amount of cholesterol. The cholesterol fraction is investigated as a selectivity parameter because membranes of cancer cells are known to contain lower fractions of cholesterol compared to healthy cells. We find that cholesterol has a significant effect on the permeation of reactive species across a membrane. Indeed, depending on the specific reactive species, an increasing cholesterol fraction can lead to (i) an increase of the transfer free energy barrier height and width, (ii) the formation of a local free energy minimum in the center of the membrane and (iii) the creation of extra free energy barriers due to the bulky sterol rings. In the context of plasma oncology, these observations suggest that the increased ingress of RONS in cancer cells can be explained by the decreased cholesterol fraction of their cell membrane.

Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization

PubMed Central

Kaneko, Toshiro; Sasaki, Shota; Takashima, Keisuke; Kanzaki, Makoto

2017-01-01

Gas-liquid interfacial atmospheric-pressure plasma jets (GLI-APPJ) are used medically for plasma-induced cell-membrane permeabilization. In an attempt to identify the dominant factors induced by GLI-APPJ responsible for enhancing cell-membrane permeability, the concentration and distribution of plasma-produced reactive species in the gas and liquid phase regions are measured. These reactive species are classified in terms of their life-span: long-lived (e.g., H2O2), short-lived (e.g., O2•−), and extremely-short-lived (e.g., •OH). The concentration of plasma-produced •OHaq in the liquid phase region decreases with an increase in solution thickness (<1 mm), and plasma-induced cell-membrane permeabilization is found to decay markedly as the thickness of the solution increases. Furthermore, the horizontally center-localized distribution of •OHaq, resulting from the center-peaked distribution of •OH in the gas phase region, corresponds with the distribution of the permeabilized cells upon APPJ irradiation, whereas the overall plasma-produced oxidizing species such as H2O2aq in solution exhibit a doughnut-shaped horizontal distribution. These results suggest that •OHaq is likely one of the dominant factors responsible for plasma-induced cell-membrane permeabilization. PMID:28163376

Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization.

PubMed

Kaneko, Toshiro; Sasaki, Shota; Takashima, Keisuke; Kanzaki, Makoto

2017-01-01

Gas-liquid interfacial atmospheric-pressure plasma jets (GLI-APPJ) are used medically for plasma-induced cell-membrane permeabilization. In an attempt to identify the dominant factors induced by GLI-APPJ responsible for enhancing cell-membrane permeability, the concentration and distribution of plasma-produced reactive species in the gas and liquid phase regions are measured. These reactive species are classified in terms of their life-span: long-lived (e.g., H 2 O 2 ), short-lived (e.g., O 2 •- ), and extremely-short-lived (e.g., • OH). The concentration of plasma-produced • OH aq in the liquid phase region decreases with an increase in solution thickness (<1 mm), and plasma-induced cell-membrane permeabilization is found to decay markedly as the thickness of the solution increases. Furthermore, the horizontally center-localized distribution of • OH aq , resulting from the center-peaked distribution of • OH in the gas phase region, corresponds with the distribution of the permeabilized cells upon APPJ irradiation, whereas the overall plasma-produced oxidizing species such as H 2 O 2aq in solution exhibit a doughnut-shaped horizontal distribution. These results suggest that • OH aq is likely one of the dominant factors responsible for plasma-induced cell-membrane permeabilization.

Evolution and development of model membranes for physicochemical and functional studies of the membrane lateral heterogeneity.

PubMed

Morigaki, Kenichi; Tanimoto, Yasushi

2018-03-14

One of the main questions in the membrane biology is the functional roles of membrane heterogeneity and molecular localization. Although segregation and local enrichment of protein/lipid components (rafts) have been extensively studied, the presence and functions of such membrane domains still remain elusive. Along with biochemical, cell observation, and simulation studies, model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information on the physicochemical properties of membrane proteins and lipids. Segregation of fluid lipid bilayer into liquid-ordered (Lo) and liquid-disordered (Ld) phases has been studied as a simplified model of raft in model membranes, including giant unilamellar vesicles (GUVs), giant plasma membrane vesicles (GPMVs), and supported lipid bilayers (SLB). Partition coefficients of membrane proteins between Lo and Ld phases were measured to gauze their affinities to lipid rafts (raftophilicity). One important development in model membrane is patterned SLB based on the microfabrication technology. Patterned Lo/Ld phases have been applied to study the partition and function of membrane-bound molecules. Quantitative information of individual molecular species attained by model membranes is critical for elucidating the molecular functions in the complex web of molecular interactions. The present review gives a short account of the model membranes developed for studying the lateral heterogeneity, especially focusing on patterned model membranes on solid substrates. Copyright © 2018 Elsevier B.V. All rights reserved.

The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells

PubMed Central

Bach, Thomas J

2013-01-01

We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL. PMID:24555083

PI(4,5)P2 regulates myoblast fusion through Arp2/3 regulator localization at the fusion site

PubMed Central

Bothe, Ingo; Deng, Su; Baylies, Mary

2014-01-01

Cell-cell fusion is a regulated process that requires merging of the opposing membranes and underlying cytoskeletons. However, the integration between membrane and cytoskeleton signaling during fusion is not known. Using Drosophila, we demonstrate that the membrane phosphoinositide PI(4,5)P2 is a crucial regulator of F-actin dynamics during myoblast fusion. PI(4,5)P2 is locally enriched and colocalizes spatially and temporally with the F-actin focus that defines the fusion site. PI(4,5)P2 enrichment depends on receptor engagement but is upstream or parallel to actin remodeling. Regulators of actin branching via Arp2/3 colocalize with PI(4,5)P2 in vivo and bind PI(4,5)P2 in vitro. Manipulation of PI(4,5)P2 availability leads to impaired fusion, with a reduction in the F-actin focus size and altered focus morphology. Mechanistically, the changes in the actin focus are due to a failure in the enrichment of actin regulators at the fusion site. Moreover, improper localization of these regulators hinders expansion of the fusion interface. Thus, PI(4,5)P2 enrichment at the fusion site encodes spatial and temporal information that regulates fusion progression through the localization of activators of actin polymerization. PMID:24821989

Adenoviral vector gene delivery via the round window membrane in guinea pigs.

PubMed

Suzuki, Mitsuya; Yamasoba, Tatsuya; Suzukawa, Keigo; Kaga, Kimitaka

2003-10-27

We have found that damage from a local anesthetic solution containing phenol permitted beta-galactosidase (beta-gal) gene delivery to the guinea pig inner ear via the round window membrane (RWM). RWM damage was evident as degeneration of the outer epithelium. After adenovirus lacZ vector was applied to the damaged RWM, immunohistochemistry showed strong beta-gal expression in the RWM, mesothelial cells, organ of Corti, spiral limbus, spiral ligament and spiral ganglion. In the vestibular labyrinth, expression was seen in the sensory and supporting cells, transitional cells, and the dark-cell area. Thus, adenovirus can transfect a variety of inner ear cells in the guinea pig through a damaged RWM.

Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies

PubMed Central

Caiolfa, Valeria R.; Zamai, Moreno; Malengo, Gabriele; Andolfo, Annapaola; Madsen, Chris D.; Sutin, Jason; Digman, Michelle A.; Gratton, Enrico; Blasi, Francesco; Sidenius, Nicolai

2007-01-01

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents. PMID:18056417

Syndecan-4 Regulates Muscle Differentiation and Is Internalized from the Plasma Membrane during Myogenesis.

PubMed

Rønning, Sissel B; Carlson, Cathrine R; Stang, Espen; Kolset, Svein O; Hollung, Kristin; Pedersen, Mona E

2015-01-01

The cell surface proteoglycan syndecan-4 has been reported to be crucial for muscle differentiation, but the molecular mechanisms still remain to be fully understood. During in vitro differentiation of bovine muscle cells immunocytochemical analyses showed strong labelling of syndecan-4 intracellularly, in close proximity with Golgi structures, in membranes of intracellular vesicles and finally, in the nuclear area including the nuclear envelope. Chase experiments showed that syndecan-4 was internalized from the plasma membrane during this process. Furthermore, when syndecan-4 was knocked down by siRNA more myotubes were formed, and the expression of myogenic transcription factors, β1-integrin and actin was influenced. However, when bovine muscle cells were treated with a cell-penetrating peptide containing the cytoplasmic region of syndecan-4, myoblast fusion and thus myotube formation was blocked, both in normal cells and in syndecan-4 knock down cells. Altogether this suggests that the cytoplasmic domain of syndecan-4 is important in regulation of myogenesis. The internalization of syndecan-4 from the plasma membrane during muscle differentiation and the nuclear localization of syndecan-4 in differentiated muscle cells may be part of this regulation, and is a novel aspect of syndecan biology which merits further studies.

Synaptotagmin-Like Proteins Control Formation of a Single Apical Membrane Domain in Epithelial Cells

PubMed Central

Gálvez-Santisteban, Manuel; Rodriguez-Fraticelli, Alejo E.; Bryant, David M.; Vergarajauregui, Silvia; Yasuda, Takao; Bañón-Rodríguez, Inmaculada; Bernascone, Ilenia; Datta, Anirban; Spivak, Natalie; Young, Kitty; Slim, Christiaan L.; Brakeman, Paul R.; Fukuda, Mitsunori; Mostov, Keith E.; Martín-Belmonte, Fernando

2012-01-01

SUMMARY The formation of epithelial tissues requires both the generation of apical-basal polarity and the co-ordination of this polarity between neighboring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to formation of a singular apical membrane, resulting in contribution of each cell to only a single lumen. Here, from a functional screen for genes required for 3D epithelial architecture we identify key roles for Synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PI(4,5)P2-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE Syntaxin-3. Together, Slp2-a/4-a co-ordinate the spatiotemporal organization of vectorial apical transport to ensure only a single apical surface, and thus formation of a single lumen, occurs per cell. PMID:22820376

Rga6 is a fission yeast Rho GAP involved in Cdc42 regulation of polarized growth

PubMed Central

Revilla-Guarinos, M. T.; Martín-García, Rebeca; Villar-Tajadura, M. Antonia; Estravís, Miguel; Coll, Pedro M.; Pérez, Pilar

2016-01-01

Active Cdc42 is essential for the establishment of polarized growth. This GTPase is negatively regulated by the GTPase-activating proteins (GAPs), which are important for the spatial specificity of Cdc42 function. Rga4 is the only GAP described as negative regulator of fission yeast Cdc42. We report here that Rga6, another fission yeast Cdc42 GAP, shares some functions with Rga4. Cells lacking Rga6 are viable but slightly shorter and broader than wild type, and cells lacking Rga6 and Rga4 simultaneously are rounded. In these cells, active Cdc42 is observed all around the membrane. These additive effects indicate that both GAPs collaborate in the spatial regulation of active Cdc42. Rga6 localizes to the plasma membrane, forming clusters different from those formed by Rga4. A polybasic region at the Rga6 C-terminus is responsible for its membrane localization. Rga6-GFP fluorescence decreases considerably at the growing tips, and this decrease is dependent on the actin cables. Of note, in the absence of Rga6, the amplitude of active Cdc42 oscillations at the tips decreases, and less GTP-Cdc42 accumulates at the new end of the cells. We propose that Rga6 collaborates with Rga4 to spatially restrict active Cdc42 at the cell tips and maintain cell dimensions. PMID:26960792

Characteristics of the Localization of Connexin 43 in Satellite Cells during Skeletal Muscle Regeneration In Vivo

PubMed Central

Ishido, Minenori; Kasuga, Norikatsu

2015-01-01

For myogenesis, new myotubes are formed by the fusion of differentiated myoblasts. In the sequence of events for myotube formation, intercellular communication through gap junctions composed of connexin 43 (Cx43) plays critical roles in regulating the alignment and fusion of myoblasts in advances of myotube formation in vitro. On the other hand, the relationship between the expression patterns of Cx43 and the process of myotube formation in satellite cells during muscle regeneration in vivo remains poorly understood. The present study investigated the relationship between Cx43 and satellite cells in muscle regeneration in vivo. The expression of Cx43 was detected in skeletal muscles on day 1 post-muscle injury, but not in control muscles. Interestingly, the expression of Cx43 was not localized on the inside of the basement membrane of myofibers in the regenerating muscles. Moreover, although the clusters of differentiated satellite cells, which represent a more advanced stage of myotube formation, were observed on the inside of the basement membrane of myofibers in regenerating muscles, the expression of Cx43 was not localized in the clusters of these satellite cells. Therefore, in the present study, it was suggested that Cx43 may not directly contribute to muscle regeneration via satellite cells. PMID:26019374

Local potential evolutions during proton exchange membrane fuel cell operation with dead-ended anode - Part II: Aging mitigation strategies based on water management and nitrogen crossover

NASA Astrophysics Data System (ADS)

Abbou, S.; Dillet, J.; Maranzana, G.; Didierjean, S.; Lottin, O.

2017-02-01

Proton exchange membrane (PEM) fuel cells operate with dead-ended anode in order to reduce system cost and complexity when compared with hydrogen re-circulation systems. In the first part of this work, we showed that localized fuel starvation events may occur, because of water and nitrogen accumulation in the anode side, which could be particularly damaging to the cell performance. To prevent these degradations, the anode compartment must be purged which may lead to an overall system efficiency decrease because of significant hydrogen waste. In the second part, we present several purge strategies in order to minimize both hydrogen waste and membrane-electrode assembly degradations during dead-ended anode operation. A linear segmented cell with reference electrodes was used to monitor simultaneously the current density distribution along the gas channel and the time evolution of local anode and cathode potentials. To asses MEA damages, Platinum ElectroChemical Surface Area (ECSA) and cell performance were periodically measured. The results showed that dead-end mode operation with an anode plate maintained at a temperature 5 °C hotter than the cathode plate limits water accumulation in the anode side, reducing significantly purge frequency (and thus hydrogen losses) as well as MEA damages. As nitrogen contribution to hydrogen starvation is predominant in this thermal configuration, we also tested a microleakage solution to discharge continuously most the nitrogen accumulating in the anode side while ensuring low hydrogen losses and minimum ECSA losses provided the right microleakage flow rate is chosen.

Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.

PubMed

Okuhira, Keiichiro; Shoda, Takuji; Omura, Risa; Ohoka, Nobumichi; Hattori, Takayuki; Shibata, Norihito; Demizu, Yosuke; Sugihara, Ryo; Ichino, Asato; Kawahara, Haruka; Itoh, Yukihiro; Ishikawa, Minoru; Hashimoto, Yuichi; Kurihara, Masaaki; Itoh, Susumu; Saito, Hiroyuki; Naito, Mikihiko

2017-03-01

Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Computing Curvature Sensitivity of Biomolecules in Membranes by Simulated Buckling.

PubMed

Elías-Wolff, Federico; Lindén, Martin; Lyubartsev, Alexander P; Brandt, Erik G

2018-03-13

Membrane curvature sensing, where the binding free energies of membrane-associated molecules depend on the local membrane curvature, is a key factor to modulate and maintain the shape and organization of cell membranes. However, the microscopic mechanisms are not well understood, partly due to absence of efficient simulation methods. Here, we describe a method to compute the curvature dependence of the binding free energy of a membrane-associated probe molecule that interacts with a buckled membrane, which has been created by lateral compression of a flat bilayer patch. This buckling approach samples a wide range of curvatures in a single simulation, and anisotropic effects can be extracted from the orientation statistics. We develop an efficient and robust algorithm to extract the motion of the probe along the buckled membrane surface, and evaluate its numerical properties by extensive sampling of three coarse-grained model systems: local lipid density in a curved environment for single-component bilayers, curvature preferences of individual lipids in two-component membranes, and curvature sensing by a homotrimeric transmembrane protein. The method can be used to complement experimental data from curvature partition assays and provides additional insight into mesoscopic theories and molecular mechanisms for curvature sensing.

Local calcium signalling is mediated by mechanosensitive ion channels in mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chubinskiy-Nadezhdin, Vladislav I., E-mail: vchubinskiy@gmail.com; Vasileva, Valeria Y.; Pugovkina, Natalia A.

Mechanical forces are implicated in key physiological processes in stem cells, including proliferation, differentiation and lineage switching. To date, there is an evident lack of understanding of how external mechanical cues are coupled with calcium signalling in stem cells. Mechanical reactions are of particular interest in adult mesenchymal stem cells because of their promising potential for use in tissue remodelling and clinical therapy. Here, single channel patch-clamp technique was employed to search for cation channels involved in mechanosensitivity in mesenchymal endometrial-derived stem cells (hMESCs). Functional expression of native mechanosensitive stretch-activated channels (SACs) and calcium-sensitive potassium channels of different conductances inmore » hMESCs was shown. Single current analysis of stretch-induced channel activity revealed functional coupling of SACs and BK channels in plasma membrane. The combination of cell-attached and inside-out experiments have indicated that highly localized Ca{sup 2+} entry via SACs triggers BK channel activity. At the same time, SK channels are not coupled with SACs despite of high calcium sensitivity as compared to BK. Our data demonstrate novel mechanism controlling BK channel activity in native cells. We conclude that SACs and BK channels are clusterized in functional mechanosensitive domains in the plasma membrane of hMESCs. Co-clustering of ion channels may significantly contribute to mechano-dependent calcium signalling in stem cells. - Highlights: • Stretch-induced channel activity in human mesenchymal stem cells was analyzed. • Functional expression of SACs and Ca{sup 2+}-sensitive BK and SK channels was shown. • Local Ca{sup 2+} influx via stretch-activated channels triggers BK channel activity. • SK channels are not coupled with SACs despite higher sensitivity to [Ca{sup 2+}]{sub i}. • Functional clustering of SACs and BK channels in stem cell membrane is proposed.« less

Expression of TM4SF10, a Claudin/EMP/PMP22 family cell junction protein, during mouse kidney development and podocyte differentiation.

PubMed

Bruggeman, Leslie A; Martinka, Scott; Simske, Jeffrey S

2007-02-01

Cell junctions in the nephron are highly specialized to perform specific and distinct filtration and reabsorption functions. The mature kidney forms complex cell junctions including slit diaphragms that prevent the passage of serum proteins into the filtrate, and tubule cell junctions that regulate specific paracellular ion reuptake. We have investigated the expression of TM4SF10 (Trans-Membrane tetra(4)-Span Family 10) in mouse kidneys. TM4SF10 is the vertebrate orthologue of Caenorhabditis elegans VAB-9, a tetraspan adherens junction protein in the PMP22/EMP/Claudin family of proteins. We found that TM4SF10 localizes at the basal-most region of podocyte precursors before the capillary loop stage, at some tubule precursors, and at the ureteric bud junction with S-shaped bodies. Overall expression of TM4SF10 peaked at postnatal day 4 and was virtually absent in adult kidneys. The very limited expression of TM4SF10 protein that persisted into adulthood was restricted to a few tubule segments but remained localized to the basal region of lateral membranes. In undifferentiated cultured podocytes, TM4SF10 localized to the perinuclear region and translocated to the cell membrane after Cadherin appearance at cell-cell contacts. TM4SF10 colocalized with ZO1 and p120ctn in undifferentiated confluent podocytes and also colocalized with the tips of actin filaments at cell contacts. Upon differentiation of cultured podocytes, TM4SF10 protein disappeared from cell contacts and expression ceased. These results suggest that TM4SF10 functions during differentiation of podocytes and may participate in the maturation of cell junctions from simple adherens junctions to elaborate slit diaphragms. TM4SF10 may define a new class of Claudin-like proteins that function during junctional development.

The MARVEL transmembrane motif of occludin mediates oligomerization and targeting to the basolateral surface in epithelia.

PubMed

Yaffe, Yakey; Shepshelovitch, Jeanne; Nevo-Yassaf, Inbar; Yeheskel, Adva; Shmerling, Hedva; Kwiatek, Joanna M; Gaus, Katharina; Pasmanik-Chor, Metsada; Hirschberg, Koret

2012-08-01

Occludin (Ocln), a MARVEL-motif-containing protein, is found in all tight junctions. MARVEL motifs are comprised of four transmembrane helices associated with the localization to or formation of diverse membrane subdomains by interacting with the proximal lipid environment. The functions of the Ocln MARVEL motif are unknown. Bioinformatics sequence- and structure-based analyses demonstrated that the MARVEL domain of Ocln family proteins has distinct evolutionarily conserved sequence features that are consistent with its basolateral membrane localization. Live-cell microscopy, fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) were used to analyze the intracellular distribution and self-association of fluorescent-protein-tagged full-length human Ocln or the Ocln MARVEL motif excluding the cytosolic C- and N-termini (amino acids 60-269, FP-MARVEL-Ocln). FP-MARVEL-Ocln efficiently arrived at the plasma membrane (PM) and was sorted to the basolateral PM in filter-grown polarized MDCK cells. A series of conserved aromatic amino acids within the MARVEL domain were found to be associated with Ocln dimerization using BiFC. FP-MARVEL-Ocln inhibited membrane pore growth during Triton-X-100-induced solubilization and was shown to increase the membrane-ordered state using Laurdan, a lipid dye. These data demonstrate that the Ocln MARVEL domain mediates self-association and correct sorting to the basolateral membrane.

Subcellular localization, interactions and dynamics of the phage-shock protein-like Lia response in Bacillus subtilis.

PubMed

Domínguez-Escobar, Julia; Wolf, Diana; Fritz, Georg; Höfler, Carolin; Wedlich-Söldner, Roland; Mascher, Thorsten

2014-05-01

The liaIH operon of Bacillus subtilis is the main target of the envelope stress-inducible two-component system LiaRS. Here, we studied the localization, interaction and cellular dynamics of Lia proteins to gain insights into the physiological role of the Lia response. We demonstrate that LiaI serves as the membrane anchor for the phage-shock protein A homologue LiaH. Under non-inducing conditions, LiaI locates in highly motile membrane-associated foci, while LiaH is dispersed throughout the cytoplasm. Under stress conditions, both proteins are strongly induced and colocalize in numerous distinct static spots at the cytoplasmic membrane. This behaviour is independent of MreB and does also not correlate with the stalling of the cell wall biosynthesis machinery upon antibiotic inhibition. It can be induced by antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis, while compounds that inhibit the cytoplasmic or extracytoplasmic steps do not trigger this response. Taken together, our data are consistent with a model in which the Lia system scans the cytoplasmic membrane for envelope perturbations. Upon their detection, LiaS activates the cognate response regulator LiaR, which in turn strongly induces the liaIH operon. Simultaneously, LiaI recruits LiaH to the membrane, presumably to protect the envelope and counteract the antibiotic-induced damage. © 2014 John Wiley & Sons Ltd.

Arabidopsis SYT1 maintains stability of cortical endoplasmic reticulum networks and VAP27-1-enriched endoplasmic reticulum-plasma membrane contact sites.

PubMed

Siao, Wei; Wang, Pengwei; Voigt, Boris; Hussey, Patrick J; Baluska, Frantisek

2016-11-01

Arabidopsis synaptotagmin 1 (SYT1) is localized on the endoplasmic reticulum-plasma membrane (ER-PM) contact sites in leaf and root cells. The ER-PM localization of Arabidopsis SYT1 resembles that of the extended synaptotagmins (E-SYTs) in animal cells. In mammals, E-SYTs have been shown to regulate calcium signaling, lipid transfer, and endocytosis. Arabidopsis SYT1 was reported to be essential for maintaining cell integrity and virus movement. This study provides detailed insight into the subcellular localization of SYT1 and VAP27-1, another ER-PM-tethering protein. SYT1 and VAP27-1 were shown to be localized on distinct ER-PM contact sites. The VAP27-1-enriched ER-PM contact sites (V-EPCSs) were always in contact with the SYT1-enriched ER-PM contact sites (S-EPCSs). The V-EPCSs still existed in the leaf epidermal cells of the SYT1 null mutant; however, they were less stable than those in the wild type. The polygonal networks of cortical ER disassembled and the mobility of VAP27-1 protein on the ER-PM contact sites increased in leaf cells of the SYT1 null mutant. These results suggest that SYT1 is responsible for stabilizing the ER network and V-EPCSs. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.

PubMed

Möhn, H; Le Cabec, V; Fischer, S; Maridonneau-Parini, I

1995-07-15

The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process.

Physiological properties of anatomically identified basket and bistratified cells in the CA1 area of the rat hippocampus in vitro.

PubMed

Buhl, E H; Szilágyi, T; Halasy, K; Somogyi, P

1996-01-01

Basket and bistratified cells form two anatomically distinct classes of GABAergic local-circuit neurons in the CA1 region of the rat hippocampus. A physiological comparison was made of intracellularly recorded basket (n = 13) and bistratified neurons (n = 6), all of which had been anatomically defined by their efferent target profile (Halasy et al., 1996). Basket cells had an average resting membrane potential of -64.2 +/- 7.2 vs. -69.2 +/- 4.6 mV in bistratified cells. The latter had considerably higher mean input resistances (60.2 +/- 42.1 vs. 31.3 +/- 10.9 M Ohms) and longer membrane time constants (18.6 +/- 8.1 vs. 9.8 +/- 4.5 ms) than basket cells. Differences were also apparent in the duration of action potentials, those of basket cells being 364 +/- 77 and those of bistratified cells being 527 +/- 138 microseconds at half-amplitude. Action potentials were generally followed by prominent, fast after-hyperpolarizing potentials which in basket cells were 13.5 +/- 6.7 mV in amplitude vs. 10.5 +/- 5.1 in bistratified cells. The differences in membrane time constant, resting membrane potential, and action potential duration reached statistical significance (P < 0.05). Extracellular stimulation of Schaffer collateral/commissural afferents elicited short-latency excitatory postsynaptic potentials (EPSPs) in both cell types. The average 10-90% rise time and duration (at half-amplitude) of subthreshold EPSPs in basket cells were 1.9 +/- 0.5 and 10.7 +/- 5.6 ms, compared to 3.3 +/- 1.3 and 20.1 +/- 9.7 ms in bistratified cells, the difference in EPSP rise times being statistically significant. Basket and bistratified EPSPs were highly sensitive to a bath applied antagonist of non-N-methyl-D-aspartate (NMDA) receptors, whereas the remaining slow-rise EPSP could be abolished by an NMDA receptor antagonist. Increasing stimulation intensity elicited biphasic inhibitory postsynaptic potentials (IPSPs) in both basket and bistratified cells. In conclusion, basket and bistratified cells in the CA1 area show prominent differences in several of their membrane and firing properties. Both cell classes are activated by Schaffer collateral/commissural axons in a feedforward manner and receive inhibitory input from other, as yet unidentified, local-circuit neurons.

Metallated porphyrin-doped conjugated polymer nanoparticles for efficient photodynamic therapy of brain and colorectal tumor cells.

PubMed

Ibarra, Luis Exequiel; Porcal, Gabriela Valeria; Macor, Lorena Paola; Ponzio, Rodrigo Andrés; Spada, Ramiro Martin; Lorente, Carolina; Chesta, Carlos Alberto; Rivarola, Viviana Alicia; Palacios, Rodrigo Emiliano

2018-03-01

 Assess biocompatibility, uptake and photodynamic therapy (PDT) mechanism of metallated porphyrin doped conjugated polymer nanoparticles (CPNs) in human brain and colorectal tumor cells and macrophages. CPNs were developed employing 9,9-dioctylfluorene-alt-benzothiadiazole, an amphiphilic polymer (PS-PEG-COOH),  and platinum octaethylporphyrin. T98G, SW480 and RAW 264.7 cell lines were exposed to CPNs to assess uptake and intracellular localization. Additionally, a PDT protocol using CPNs was employed for the in vitro killing of cancer and macrophage cell lines. CPNs were well incorporated into glioblastoma and macrophage cells with localization in lysosomes. SW480 cells were less efficient incorporating CPNs with localization in the plasma membrane. In all cell lines PDT treatment was efficient inducing oxidative stress that triggered apoptosis.

Controlled permeation of cell membrane by single bubble acoustic cavitation

PubMed Central

Zhou, Y.; Yang, K.; Cui, J.; Ye, J. Y.; Deng, C. X.

2011-01-01

Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustic, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency (7.44 MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5 MHz) ultrasound pulse (duration 13.3 or 40 µs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d = 0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106 ± 0.032 µm (n = 70) for acoustic pressure of 1.5 MPa (duration 13.3 µs), and increased to 0.171 ± 0.030 µm (n = 125) for acoustic pressure of 1.7 MPa and to 0.182 ± 0.052 µm (n=112) for a pulse duration of 40 µs (1.5 MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level. PMID:21945682

Differential subcellular membrane recruitment of Src may specify its downstream signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diesbach, Philippe de; Medts, Thierry; Carpentier, Sarah

2008-04-15

Most Src family members are diacylated and constitutively associate with membrane 'lipid rafts' that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at 'rafts' remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to 'lipid rafts'; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 deg. C and floated into sucrose density gradients like caveolin-1 andmore » flotillin-2, i.e. 'lipid rafts'. By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe ({approx} 70%) cholesterol extraction with methyl-{beta}-cyclodextrin (M{beta}CD) did not abolish 'rafts' floatation, but strongly decreased Src association with floating 'rafts' and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to M{beta}CD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at 'non-raft' domains on endosomes, then via PI3-kinase-Akt on a distinct set of 'rafts' at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.« less

Investigation of dynamic driving cycle effect on the degradation of proton exchange membrane fuel cell by segmented cell technology

NASA Astrophysics Data System (ADS)

Lin, R.; Xiong, F.; Tang, W. C.; Técher, L.; Zhang, J. M.; Ma, J. X.

2014-08-01

Durability is one of the most important limiting factors for the commercialization of proton exchange membrane fuel cell (PEMFC). Fuel cells are more vulnerable to degradation under operating conditions as dynamic load cycle or start up/shut down. The purpose of this study is to evaluate influences of driving cycles on the durability of fuel cells through analyzing the degradation mechanism of a segmented cell in real time. This study demonstrates that the performance of the fuel cell significantly decreases after 200 cycles. The segmented cell technology is used to measure the local current density distribution, which shows that the current density at the exit region and the inlet region declines much faster than the other parts. Meanwhile, electro-chemical impedance spectroscopy (EIS) reveals that after 200 cycles the ohmic resistance of fuel cell increases, especially at the cathode, and electro-chemical surface area (ESA) decreases from 392 to 307 cm2 mg-1. Furthermore, scanning electron microscopy (SEM) images of the membrane-electrode assembly (MEA) in cross-section demonstrate crackle flaw on the surface of the catalyst layer and the delamination of the electrodes from the membrane. Transmission electron microscope (TEM) results also show that the Pt particle size increases distinctly after driving cycles.

IFITM3 Restricts Human Metapneumovirus Infection.

PubMed

McMichael, Temet M; Zhang, Yu; Kenney, Adam D; Zhang, Lizhi; Zani, Ashley; Lu, Mijia; Chemudupati, Mahesh; Li, Jianrong; Yount, Jacob S

2018-06-15

Human metapneumovirus (hMPV) utilizes a bifurcated cellular entry strategy, fusing either with the plasma membrane or, after endocytosis, with the endosome membrane. Whether cellular factors restrict or enhance either entry pathway is largely unknown. We found that the interferon-induced transmembrane protein 3 (IFITM3) inhibits hMPV infection to an extent similar to endocytosis-inhibiting drugs, and an IFITM3 variant that accumulates at the plasma membrane in addition to its endosome localization provided increased virus restriction. Mechanistically, IFITM3 blocks hMPV F protein-mediated membrane fusion, and inhibition of infection was reversed by the membrane destabilizing drug amphotericin B. Conversely, we found that infection by some hMPV strains is enhanced by the endosomal protein Toll-like receptor 7 (TLR7), and that IFITM3 retains the ability to restrict hMPV infection even in cells expressing TLR7. Overall, our results identify IFITM3 as an endosomal restriction factor that limits hMPV infection of cells.

Interactions with the actin cytoskeleton are required for cell wall localization of barley stripe mosaic virus TGB proteins

USDA-ARS?s Scientific Manuscript database

The host cytoskeleton and membrane system are the main routes by which plant viruses move within or between cells. Barley stripe mosaic virus (BSMV) -induced actin filament thickening was visualized in the cytoskeleton of agroinfiltrated Nicotiana benthamiana epidermal cells expressing DsRed:Talin. ...

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis

PubMed Central

Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko

2015-01-01

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor. PMID:25398910

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis.

PubMed

Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko

2015-01-02

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor. © 2014 The Authors.

Recovery of Na-glucose cotransport activity after renal ischemia is impaired in mice lacking vimentin.

PubMed

Runembert, Isabelle; Couette, Sylviane; Federici, Pierre; Colucci-Guyon, Emma; Babinet, Charles; Briand, Pascale; Friedlander, Gérard; Terzi, Fabiola

2004-11-01

Vimentin, an intermediate filament protein mainly expressed in mesenchyma-derived cells, is reexpressed in renal tubular epithelial cells under many pathological conditions, characterized by intense cell proliferation. Whether vimentin reexpression is only a marker of cell dedifferentiation or is instrumental in the maintenance of cell structure and/or function is still unknown. Here, we used vimentin knockout mice (Vim(-/-)) and an experimental model of acute renal injury (30-min bilateral renal ischemia) to explore the role of vimentin. Bilateral renal ischemia induced an initial phase of acute tubular necrosis that did not require vimentin and was similar, in terms of morphological and functional changes, in Vim(+/+) and Vim(-/-) mice. However, vimentin was essential to favor Na-glucose cotransporter 1 localization to brush-border membranes and to restore Na-glucose cotransport activity in regenerating tubular cells. We show that the effect of vimentin inactivation is specific and results in persistent glucosuria. We propose that vimentin is part of a structural network that favors carrier localization to plasma membranes to restore transport activity in injured kidneys.

Functional characterization and cellular dynamics of the CDC-42 - RAC - CDC-24 module in Neurospora crassa.

PubMed

Araujo-Palomares, Cynthia L; Richthammer, Corinna; Seiler, Stephan; Castro-Longoria, Ernestina

2011-01-01

Rho-type GTPases are key regulators that control eukaryotic cell polarity, but their role in fungal morphogenesis is only beginning to emerge. In this study, we investigate the role of the CDC-42 - RAC - CDC-24 module in Neurospora crassa. rac and cdc-42 deletion mutants are viable, but generate highly compact colonies with severe morphological defects. Double mutants carrying conditional and loss of function alleles of rac and cdc-42 are lethal, indicating that both GTPases share at least one common essential function. The defects of the GTPase mutants are phenocopied by deletion and conditional alleles of the guanine exchange factor (GEF) cdc-24, and in vitro GDP-GTP exchange assays identify CDC-24 as specific GEF for both CDC-42 and RAC. In vivo confocal microscopy shows that this module is organized as membrane-associated cap that covers the hyphal apex. However, the specific localization patterns of the three proteins are distinct, indicating different functions of RAC and CDC-42 within the hyphal tip. CDC-42 localized as confined apical membrane-associated crescent, while RAC labeled a membrane-associated ring excluding the region labeled by CDC42. The GEF CDC-24 occupied a strategic position, localizing as broad apical membrane-associated crescent and in the apical cytosol excluding the Spitzenkörper. RAC and CDC-42 also display distinct localization patterns during branch initiation and germ tube formation, with CDC-42 accumulating at the plasma membrane before RAC. Together with the distinct cellular defects of rac and cdc-42 mutants, these localizations suggest that CDC-42 is more important for polarity establishment, while the primary function of RAC may be maintaining polarity. In summary, this study identifies CDC-24 as essential regulator for RAC and CDC-42 that have common and distinct functions during polarity establishment and maintenance of cell polarity in N. crassa.

Synaptic activity-related classical protein kinase C isoform localization in the adult rat neuromuscular synapse.

PubMed

Besalduch, Núria; Tomàs, Marta; Santafé, Manel M; Garcia, Neus; Tomàs, Josep; Lanuza, Maria Angel

2010-01-10

Protein kinase C (PKC) is essential for signal transduction in a variety of cells, including neurons and myocytes, and is involved in both acetylcholine release and muscle fiber contraction. Here, we demonstrate that the increases in synaptic activity by nerve stimulation couple PKC to transmitter release in the rat neuromuscular junction and increase the level of alpha, betaI, and betaII isoforms in the membrane when muscle contraction follows the stimulation. The phosphorylation activity of these classical PKCs also increases. It seems that the muscle has to contract in order to maintain or increase classical PKCs in the membrane. We use immunohistochemistry to show that PKCalpha and PKCbetaI were located in the nerve terminals, whereas PKCalpha and PKCbetaII were located in the postsynaptic and the Schwann cells. Stimulation and contraction do not change these cellular distributions, but our results show that the localization of classical PKC isoforms in the membrane is affected by synaptic activity.

Deformable micro torque swimmer

NASA Astrophysics Data System (ADS)

Ishikawa, Takuji; Tanaka, Tomoyuki; Omori, Toshihiro; Imai, Yohsuke

2015-11-01

We investigated the deformation of a ciliate swimming freely in a fluid otherwise at rest. The cell body was modeled as a capsule with a hyper elastic membrane enclosing Newtonian fluid. Thrust forces due to the ciliary beat were modeled as torques distributed above the cell body. Effects of the membrane elasticity, the aspect ratio of cell's reference shape and the density difference between the cell and the surrounding fluid were investigated. The results showed that the cell deformed like heart shape when Capillary number (Ca) was sufficiently large, and the swimming velocity decreased as Ca was increased. The gravity effect on the membrane tension suggested that the upwards and downwards swimming velocities of Paramecium might be reglated by the calcium ion channels distributed locally around the anterior end. Moreover, the gravity induced deformation made a cell directed vertically downwards, which resulted in a positive geotaxis like behavior with physical origin. These results are important to understand physiology of ciliate's biological responses to mechanical stimuli.

Ultrastructural findings in Hashimoto's thyroiditis and focal lymphocytic thyroiditis with reference to giant cell formation.

PubMed

Knecht, H; Hedinger, C E

1982-09-01

Ultrastructural findings in two cases of Hashimoto's disease and two cases of focal lymphocytic thyroiditis are reported. Stimulated thyrocytes, oncocytes and degenerating thyrocytes were observed in all cases. Multinucleated thyrocytes and epithelial pseudogiant cells were identified in Hashimoto's disease only. Infiltrating lymphocytes, plasma cells, monocytes and macrophages were present in all cases. The ultrastructure of germinal centres was similar to that seen in lymphatic organs. Giant cells of both intra- and extrafollicular localization were seen in Hashimoto's disease. Most of the giant cells were macrophage-derived. Two different ways of giant cell formation were identified: besides the familiar dissolution of plasma membranes of adjacent macrophages, another mechanism of fusion was observed. At sites of contact, peculiar membrane structures were developed and disintegration of plasma membranes occurred in parts adjacent to these structures. These are not identical to desmosomes and are different from Langerhans' granules. They probably represent special organelles for the initiation of cellular fusion.

Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom.

PubMed

Xu, Yanjie; Xia, Jixiang; Liu, Suxuan; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

2017-03-01

Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via its binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complexes and their intracellular trafficking based on protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair.

Nanoscale architecture of the Schizosaccharomyces pombe contractile ring.

PubMed

McDonald, Nathan A; Lind, Abigail L; Smith, Sarah E; Li, Rong; Gould, Kathleen L

2017-09-15

The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of Schizosaccharomyces pombe contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0-80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80-160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160-350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function.

Nanoscale architecture of the Schizosaccharomyces pombe contractile ring

PubMed Central

McDonald, Nathan A; Lind, Abigail L; Smith, Sarah E; Li, Rong

2017-01-01

The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of Schizosaccharomyces pombe contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0–80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80–160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160–350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function. PMID:28914606

A Glycosylphosphatidylinositol Anchor Is Required for Membrane Localization but Dispensable for Cell Wall Association of Chitin Deacetylase 2 in Cryptococcus neoformans

PubMed Central

Gilbert, Nicole M.; Baker, Lorina G.; Specht, Charles A.; Lodge, Jennifer K.

2012-01-01

ABSTRACT Cell wall proteins (CWPs) mediate important cellular processes in fungi, including adhesion, invasion, biofilm formation, and flocculation. The current model of fungal cell wall organization includes a major class of CWPs covalently bound to β-1,6-glucan via a remnant of a glycosylphosphatidylinositol (GPI) anchor. This model was established by studies of ascomycetes more than a decade ago, and relatively little work has been done with other fungi, although the presumption has been that proteins identified in the cell wall which contain a predicted GPI anchor are covalently linked to cell wall glucans. The pathogenic basidiomycete Cryptococcus neoformans encodes >50 putatively GPI-anchored proteins, some of which have been identified in the cell wall. One of these proteins is chitin deacetylase 2 (Cda2), an enzyme responsible for converting chitin to chitosan, a cell wall polymer recently established as a virulence factor for C. neoformans infection of mammalian hosts. Using a combination of biochemistry, molecular biology, and genetics, we show that Cda2 is GPI anchored to membranes but noncovalently associated with the cell wall by means independent of both its GPI anchor and β-1,6-glucan. We also show that Cda2 produces chitosan when localized to the plasma membrane, but association with the cell wall is not essential for this process, thereby providing insight into the mechanism of chitosan biosynthesis. These results increase our understanding of the surface of C. neoformans and provide models of cell walls likely applicable to other undercharacterized basidiomycete pathogenic fungi. PMID:22354955

Overexpression of BAX INHIBITOR-1 Links Plasma Membrane Microdomain Proteins to Stress.

PubMed

Ishikawa, Toshiki; Aki, Toshihiko; Yanagisawa, Shuichi; Uchimiya, Hirofumi; Kawai-Yamada, Maki

2015-10-01

BAX INHIBITOR-1 (BI-1) is a cell death suppressor widely conserved in plants and animals. Overexpression of BI-1 enhances tolerance to stress-induced cell death in plant cells, although the molecular mechanism behind this enhancement is unclear. We recently found that Arabidopsis (Arabidopsis thaliana) BI-1 is involved in the metabolism of sphingolipids, such as the synthesis of 2-hydroxy fatty acids, suggesting the involvement of sphingolipids in the cell death regulatory mechanism downstream of BI-1. Here, we show that BI-1 affects cell death-associated components localized in sphingolipid-enriched microdomains of the plasma membrane in rice (Oryza sativa) cells. The amount of 2-hydroxy fatty acid-containing glucosylceramide increased in the detergent-resistant membrane (DRM; a biochemical counterpart of plasma membrane microdomains) fraction obtained from BI-1-overexpressing rice cells. Comparative proteomics analysis showed quantitative changes of DRM proteins in BI-1-overexpressing cells. In particular, the protein abundance of FLOTILLIN HOMOLOG (FLOT) and HYPERSENSITIVE-INDUCED REACTION PROTEIN3 (HIR3) markedly decreased in DRM of BI-1-overexpressing cells. Loss-of-function analysis demonstrated that FLOT and HIR3 are required for cell death by oxidative stress and salicylic acid, suggesting that the decreased levels of these proteins directly contribute to the stress-tolerant phenotypes in BI-1-overexpressing rice cells. These findings provide a novel biological implication of plant membrane microdomains in stress-induced cell death, which is negatively modulated by BI-1 overexpression via decreasing the abundance of a set of key proteins involved in cell death. © 2015 American Society of Plant Biologists. All Rights Reserved.

Mobility of human immunodeficiency virus type 1 Pr55Gag in living cells.

PubMed

Gomez, Candace Y; Hope, Thomas J

2006-09-01

Human immunodeficiency virus type 1 (HIV-1) assembly requires the converging of thousands of structural proteins on cellular membranes to form a tightly packed immature virion. The Gag polyprotein contains all of the determinants important for viral assembly and must move around in the cell in order to form particles. This work has focused on Gag mobility in order to provide more insights into the dynamics of particle assembly. Key to these studies was the use of several fluorescently labeled Gag derivatives. We used fluorescence recovery after photobleaching as well as photoactivation to determine Gag mobility. Upon expression, Gag can be localized diffusely in the cytoplasm, associated with the plasma membrane, or in virus-like particles (VLPs). Here we show that Gag VLPs are primarily localized in the plasma membrane and do not colocalize with CD63. We have shown using full-length Gag as well as truncation mutants fused to green fluorescent protein that Gag is highly mobile in live cells when it is not assembled into VLPs. Results also showed that this mobility is highly dependent upon cholesterol. When cholesterol is depleted from cells expressing Gag, mobility is significantly decreased. Once cholesterol was replenished, Gag mobility returned to wild-type levels. Taken together, results from these mobility studies suggest that Gag is highly mobile and that as the assembly process proceeds, mobility decreases. These studies also suggest that Gag assembly must occur in cholesterol-rich domains in the plasma membrane.

C 60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Russ, K. A.; Elvati, P.; Parsonage, T. L.

There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C 60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. Themore » evidence suggests marginal uptake of C 60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Compu-tational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal defor-mation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. Lastly, the surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.« less

C 60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages

DOE PAGES

Russ, K. A.; Elvati, P.; Parsonage, T. L.; ...

2016-01-01

There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C 60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. Themore » evidence suggests marginal uptake of C 60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Compu-tational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal defor-mation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. Lastly, the surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.« less

Physical Model of the Dynamic Instability in an Expanding Cell Culture

PubMed Central

Mark, Shirley; Shlomovitz, Roie; Gov, Nir S.; Poujade, Mathieu; Grasland-Mongrain, Erwan; Silberzan, Pascal

2010-01-01

Abstract Collective cell migration is of great significance in many biological processes. The goal of this work is to give a physical model for the dynamics of cell migration during the wound healing response. Experiments demonstrate that an initially uniform cell-culture monolayer expands in a nonuniform manner, developing fingerlike shapes. These fingerlike shapes of the cell culture front are composed of columns of cells that move collectively. We propose a physical model to explain this phenomenon, based on the notion of dynamic instability. In this model, we treat the first layers of cells at the front of the moving cell culture as a continuous one-dimensional membrane (contour), with the usual elasticity of a membrane: curvature and surface-tension. This membrane is active, due to the forces of cellular motility of the cells, and we propose that this motility is related to the local curvature of the culture interface; larger convex curvature correlates with a stronger cellular motility force. This shape-force relation gives rise to a dynamic instability, which we then compare to the patterns observed in the wound healing experiments. PMID:20141748

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family1[C][W

PubMed Central

Roppolo, Daniele; Boeckmann, Brigitte; Pfister, Alexandre; Boutet, Emmanuel; Rubio, Maria C.; Dénervaud-Tendon, Valérie; Vermeer, Joop E.M.; Gheyselinck, Jacqueline; Xenarios, Ioannis; Geldner, Niko

2014-01-01

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells. PMID:24920445

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family.

PubMed

Roppolo, Daniele; Boeckmann, Brigitte; Pfister, Alexandre; Boutet, Emmanuel; Rubio, Maria C; Dénervaud-Tendon, Valérie; Vermeer, Joop E M; Gheyselinck, Jacqueline; Xenarios, Ioannis; Geldner, Niko

2014-08-01

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells. © 2014 American Society of Plant Biologists. All Rights Reserved.

A PDZ-interacting domain in CFTR is an apical membrane polarization signal

PubMed Central

Moyer, Bryan D.; Denton, Jerod; Karlson, Katherine H.; Reynolds, Donna; Wang, Shusheng; Mickle, John E.; Milewski, Michal; Cutting, Garry R.; Guggino, William B.; Li, Min; Stanton, Bruce A.

1999-01-01

Polarization of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, to the apical plasma membrane of epithelial cells is critical for vectorial transport of chloride in a variety of epithelia, including the airway, pancreas, intestine, and kidney. However, the motifs that localize CFTR to the apical membrane are unknown. We report that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. We also demonstrate that CFTR binds to ezrin-radixin-moesin–binding phosphoprotein 50 (EBP50), an apical membrane PDZ domain–containing protein. We propose that COOH-terminal deletions of CFTR, which represent about 10% of CFTR mutations, result in defective vectorial chloride transport, partly by altering the polarized distribution of CFTR in epithelial cells. Moreover, our data demonstrate that PDZ-interacting domains and PDZ domain–containing proteins play a key role in the apical polarization of ion channels in epithelial cells. J. Clin. Invest. 104:1353–1361 (1999). PMID:10562297

Probing platinum degradation in polymer electrolyte membrane fuel cells by synchrotron X-ray microscopy.

PubMed

Berejnov, Viatcheslav; Martin, Zulima; West, Marcia; Kundu, Sumit; Bessarabov, Dmitri; Stumper, Jürgen; Susac, Darija; Hitchcock, Adam P

2012-04-14

Synchrotron-based scanning transmission X-ray spectromicroscopy (STXM) was used to characterize the local chemical environment at and around the platinum particles in the membrane (PTIM) which form in operationally tested (end-of-life, EOL) catalyst coated membranes (CCMs) of polymer electrolyte membrane fuel cells (PEM-FC). The band of metallic Pt particles in operationally tested CCM membranes was imaged using transmission electron microscopy (TEM). The cathode catalyst layer in the beginning-of-life (BOL) CCMs was fabricated using commercially available catalysts created from Pt precursors with and without nitrogen containing ligands. The surface composition of these catalyst powders was measured by X-ray Photoelectron Spectroscopy (XPS). The local chemical environment of the PTIM in EOL CCMs was found to be directly related to the Pt precursor used in CCM fabrication. STXM chemical mapping at the N 1s edge revealed a characteristic spectrum at and around the dendritic Pt particles in CCMs fabricated with nitrogen containing Pt-precursors. This N 1s spectrum was identical to that of the cathode and different from the membrane. For CCM samples fabricated without nitrogen containing Pt-precursors the N 1s spectrum at the Pt particles was indistinguishable from that of the adjacent membrane. We interpret these observations to indicate that nitrogenous ligands in the nitrogen containing precursors, or decomposition product(s) from that source, are transported together with the dissolved Pt from the cathode into the membrane as a result of the catalyst degradation process. This places constraints on possible mechanisms for the PTIM band formation process.

Maize plasma membrane aquaporin ZmPIP2;5, but not ZmPIP1;2, facilitates transmembrane diffusion of hydrogen peroxide.

PubMed

Bienert, Gerd P; Heinen, Robert B; Berny, Marie C; Chaumont, François

2014-01-01

Plant aquaporins play important roles in transmembrane water transport processes, but some also facilitate the diffusion of other small uncharged solutes ranging from gases to metalloids. Recent evidence suggests that the transmembrane movement of hydrogen peroxide, an intra- and intercellular multifunctional signaling and defense compound, can be regulated by aquaporins. We addressed the question whether maize aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily facilitate hydrogen peroxide diffusion using heterologous expression in the yeast Saccharomyces cerevisiae. We showed that ZmPIP proteins belonging to the PIP1 and PIP2 groups were significantly expressed in yeast cells only after codon optimization of their cDNA. In accordance with previous localization studies in oocytes and plants, ZmPIP1;2 was mainly retained in intracellular membranes, while ZmPIP2;5 was localized to the plasma membrane. However, upon co-expression with ZmPIP2;5, ZmPIP1;2 was re-localized to the plasma membrane. Using a non-functional plasma membrane-localized ZmPIP2;5 mutant to deliver ZmPIP1;2 to the plasma membrane, we demonstrated that, in contrast to wild type ZmPIP2;5, ZmPIP1;2 was not permeable to hydrogen peroxide. Our study further highlighted the fact that, when using the yeast system, which is widely employed to study substrates for plant aquaporins and other transporters, although positive transport assay results allow direct conclusions to be drawn regarding solute permeability, negative results require additional control experiments to show that the protein is expressed and localized correctly before concluding on the lack of transport activity. © 2013.

ZIFL1.1 transporter modulates polar auxin transport by stabilizing membrane abundance of multiple PINs in Arabidopsis root tip

PubMed Central

Remy, Estelle; Baster, Pawel; Friml, Jiří; Duque, Paula

2013-01-01

Cell-to-cell directional flow of the phytohormone auxin is primarily established by polar localization of the PIN auxin transporters, a process tightly regulated at multiple levels by auxin itself. We recently reported that, in the context of strong auxin flows, activity of the vacuolar ZIFL1.1 transporter is required for fine-tuning of polar auxin transport rates in the Arabidopsis root. In particular, ZIFL1.1 function protects plasma-membrane stability of the PIN2 carrier in epidermal root tip cells under conditions normally triggering PIN2 degradation. Here, we show that ZIFL1.1 activity at the root tip also promotes PIN1 plasma-membrane abundance in central cylinder cells, thus supporting the notion that ZIFL1.1 acts as a general positive modulator of polar auxin transport in roots. PMID:23857365

Endocytosis of Nanoscale Systems for Cancer Treatments.

PubMed

Chen, Kai; Li, Xue; Zhu, Hongyan; Gong, Qiyong; Luo, Kui

2017-04-28

Advances of nanoscale systems for cancer treatment have been involved in enabling highly regulated site-specific localization to sub cellular organelles hidden beneath cell membranes. Thus far, the cellular entry of these nanoscale systems has been not fully understood. Endocytosisis a form of active transport in which cell transports elected extracellular molecules (such as proteins, viruses, micro-organisms and nanoscale systems) are allowed into cell interiors by engulfing them in an energy-dependent process. This process appears at the plasma membrane surface and contains internalization of the cell membrane as well as the membrane proteins and lipids of cell. There are multiform pathways of endocytosis for nanoscale systems. Further comprehension for the mechanisms of endocytosis is achieved with a combination of efficient genetic manipulations, cell dynamic imaging, and chemical endocytosis inhibitors. This review provides an account of various endocytic pathways, itemizes current methods to study endocytosis of nanoscale systems, discusses some factors associated with cellular uptake for nanoscale systems and introduces the trafficking behavior for nanoscale systems with active targeting. An insight into the endocytosis mechanism is urgent and significant for developing safe and efficient nanoscale systems for cancer diagnosis and therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Clinical indices of in vivo biocompatibility: the role of ex vivo cell function studies and effluent markers in peritoneal dialysis patients.

PubMed

Mackenzie, Ruth; Holmes, Clifford J; Jones, Suzanne; Williams, John D; Topley, Nicholas

2003-12-01

Clinical indices of in vivo biocompatibility: The role of ex vivo cell function studies and effluent markers in peritoneal dialysis patients. Over the past 20 years, studies of the biocompatibility profile of peritoneal dialysis solutions (PDF) have evolved from initial in vitro studies assessing the impact of solutions on leukocyte function to evaluations of mesothelial cell behavior. More recent biocompatibility evaluations have involved assessments of the impact of PDF on membrane integrity and cell function in peritoneal dialysis (PD) patients. The development of ex vivo systems for the evaluation of in vivo cell function, and effluent markers of membrane integrity and inflammation in patients exposed both acutely and chronically to conventional and new PDF will be interpreted in the context of our current understanding of the biology of the dialyzed peritoneum. The available data indicate that exposure of the peritoneal environment to more biocompatible PDF is associated with improvements in peritoneal cell function, alterations in markers of membrane integrity, and reduced local inflammation. These data suggest that more biocompatible PDF will have a positive impact on host defense, peritoneal homeostasis, and the long-term preservation of peritoneal membrane function in PD patients.

Polarization of IRON-REGULATED TRANSPORTER 1 (IRT1) to the plant-soil interface plays crucial role in metal homeostasis.

PubMed

Barberon, Marie; Dubeaux, Guillaume; Kolb, Cornelia; Isono, Erika; Zelazny, Enric; Vert, Grégory

2014-06-03

In plants, the controlled absorption of soil nutrients by root epidermal cells is critical for growth and development. IRON-REGULATED TRANSPORTER 1 (IRT1) is the main root transporter taking up iron from the soil and is also the main entry route in plants for potentially toxic metals such as manganese, zinc, cobalt, and cadmium. Previous work demonstrated that the IRT1 protein localizes to early endosomes/trans-Golgi network (EE/TGN) and is constitutively endocytosed through a monoubiquitin- and clathrin-dependent mechanism. Here, we show that the availability of secondary non-iron metal substrates of IRT1 (Zn, Mn, and Co) controls the localization of IRT1 between the outer polar domain of the plasma membrane and EE/TGN in root epidermal cells. We also identify FYVE1, a phosphatidylinositol-3-phosphate-binding protein recruited to late endosomes, as an important regulator of IRT1-dependent metal transport and metal homeostasis in plants. FYVE1 controls IRT1 recycling to the plasma membrane and impacts the polar delivery of this transporter to the outer plasma membrane domain. This work establishes a functional link between the dynamics and the lateral polarity of IRT1 and the transport of its substrates, and identifies a molecular mechanism driving polar localization of a cell surface protein in plants.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

NASA Astrophysics Data System (ADS)

Duman, M.; Pfleger, M.; Zhu, R.; Rankl, C.; Chtcheglova, L. A.; Neundlinger, I.; Bozna, B. L.; Mayer, B.; Salio, M.; Shepherd, D.; Polzella, P.; Moertelmaier, M.; Kada, G.; Ebner, A.; Dieudonne, M.; Schütz, G. J.; Cerundolo, V.; Kienberger, F.; Hinterdorfer, P.

2010-03-01

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ~ 25 to ~ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

PubMed

Duman, M; Pfleger, M; Zhu, R; Rankl, C; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Mayer, B; Salio, M; Shepherd, D; Polzella, P; Moertelmaier, M; Kada, G; Ebner, A; Dieudonne, M; Schütz, G J; Cerundolo, V; Kienberger, F; Hinterdorfer, P

2010-03-19

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

An AGEF-1/Arf GTPase/AP-1 Ensemble Antagonizes LET-23 EGFR Basolateral Localization and Signaling during C. elegans Vulva Induction

PubMed Central

Skorobogata, Olga; Escobar-Restrepo, Juan M.; Rocheleau, Christian E.

2014-01-01

LET-23 Epidermal Growth Factor Receptor (EGFR) signaling specifies the vulval cell fates during C. elegans larval development. LET-23 EGFR localization on the basolateral membrane of the vulval precursor cells (VPCs) is required to engage the LIN-3 EGF-like inductive signal. The LIN-2 Cask/LIN-7 Veli/LIN-10 Mint (LIN-2/7/10) complex binds LET-23 EGFR, is required for its basolateral membrane localization, and therefore, vulva induction. Besides the LIN-2/7/10 complex, the trafficking pathways that regulate LET-23 EGFR localization have not been defined. Here we identify vh4, a hypomorphic allele of agef-1, as a strong suppressor of the lin-2 mutant Vulvaless (Vul) phenotype. AGEF-1 is homologous to the mammalian BIG1 and BIG2 Arf GTPase guanine nucleotide exchange factors (GEFs), which regulate secretory traffic between the Trans-Golgi network, endosomes and the plasma membrane via activation of Arf GTPases and recruitment of the AP-1 clathrin adaptor complex. Consistent with a role in trafficking we show that AGEF-1 is required for protein secretion and that AGEF-1 and the AP-1 complex regulate endosome size in coelomocytes. The AP-1 complex has previously been implicated in negative regulation of LET-23 EGFR, however the mechanism was not known. Our genetic data indicate that AGEF-1 is a strong negative regulator of LET-23 EGFR signaling that functions in the VPCs at the level of the receptor. In line with AGEF-1 being an Arf GEF, we identify the ARF-1.2 and ARF-3 GTPases as also negatively regulating signaling. We find that the agef-1(vh4) mutation results in increased LET-23 EGFR on the basolateral membrane in both wild-type and lin-2 mutant animals. Furthermore, unc-101(RNAi), a component of the AP-1 complex, increased LET-23 EGFR on the basolateral membrane in lin-2 and agef-1(vh4); lin-2 mutant animals. Thus, an AGEF-1/Arf GTPase/AP-1 ensemble functions opposite the LIN-2/7/10 complex to antagonize LET-23 EGFR basolateral membrane localization and signaling. PMID:25329472

An AGEF-1/Arf GTPase/AP-1 ensemble antagonizes LET-23 EGFR basolateral localization and signaling during C. elegans vulva induction.

PubMed

Skorobogata, Olga; Escobar-Restrepo, Juan M; Rocheleau, Christian E

2014-10-01

LET-23 Epidermal Growth Factor Receptor (EGFR) signaling specifies the vulval cell fates during C. elegans larval development. LET-23 EGFR localization on the basolateral membrane of the vulval precursor cells (VPCs) is required to engage the LIN-3 EGF-like inductive signal. The LIN-2 Cask/LIN-7 Veli/LIN-10 Mint (LIN-2/7/10) complex binds LET-23 EGFR, is required for its basolateral membrane localization, and therefore, vulva induction. Besides the LIN-2/7/10 complex, the trafficking pathways that regulate LET-23 EGFR localization have not been defined. Here we identify vh4, a hypomorphic allele of agef-1, as a strong suppressor of the lin-2 mutant Vulvaless (Vul) phenotype. AGEF-1 is homologous to the mammalian BIG1 and BIG2 Arf GTPase guanine nucleotide exchange factors (GEFs), which regulate secretory traffic between the Trans-Golgi network, endosomes and the plasma membrane via activation of Arf GTPases and recruitment of the AP-1 clathrin adaptor complex. Consistent with a role in trafficking we show that AGEF-1 is required for protein secretion and that AGEF-1 and the AP-1 complex regulate endosome size in coelomocytes. The AP-1 complex has previously been implicated in negative regulation of LET-23 EGFR, however the mechanism was not known. Our genetic data indicate that AGEF-1 is a strong negative regulator of LET-23 EGFR signaling that functions in the VPCs at the level of the receptor. In line with AGEF-1 being an Arf GEF, we identify the ARF-1.2 and ARF-3 GTPases as also negatively regulating signaling. We find that the agef-1(vh4) mutation results in increased LET-23 EGFR on the basolateral membrane in both wild-type and lin-2 mutant animals. Furthermore, unc-101(RNAi), a component of the AP-1 complex, increased LET-23 EGFR on the basolateral membrane in lin-2 and agef-1(vh4); lin-2 mutant animals. Thus, an AGEF-1/Arf GTPase/AP-1 ensemble functions opposite the LIN-2/7/10 complex to antagonize LET-23 EGFR basolateral membrane localization and signaling.

Antibodies against Clonorchis sinensis LDH could cross-react with LDHB localizing on the plasma membrane of human hepatocarcinoma cell SMMC-7721 and induce apoptosis.

PubMed

Song, Tianzhang; Gan, Wenjia; Chen, Jintao; Huang, Lilin; Yin, Hongling; He, Tailong; Huang, Huaiqiu; Hu, Xuchu

2016-04-01

Lactate dehydrogenase (LDH) is a terminal enzyme in anaerobic glycolytic pathway. It widely exists in various organisms and is in charge of converting the glycolysis product pyruvic acid to lactic acid. Most parasites, including Clonorchis sinensis, predominantly depend on glycolysis to provide energy. Bioinformatic analysis predicts that the LDHs from many species have more than one transmembrane region, suggesting that it may be a membrane protein. C. sinensis LDH (CsLDH) has been confirmed as a transmembrane protein mainly located in the tegument. The antibodies against CsLDH can inhibit the worm's energy metabolism, kill the worm, and may have the same effects on human cancer cells. In this study, we cloned and characterized human LDHA (HsLDHA), HsLDHB, and CsLDH. Semi-quantitative real-time RCP showed that HsLDHB only existed in hepatocarcinoma cell SMMC-7721. Confocal microscopy and Western blot experiments revealed that HsLDHB was localized in the plasma membrane of SMMC-7721 cells, and the antibodies against CsLDH could cross-react with it. This cross-reaction could inhibit the enzymatic activity of HsLDHB. The cancer cells co-cultured with anti-CsLDH sera showed a significant decrease in cell proliferation rate and increases in caspase 9 and reactive oxygen species (ROS) levels. Therefore, anti-CsLDH antibodies can induce the apoptosis of cancer cells SMMC-7721 and may serve as a new tool to inhibit tumor.

Cell signalling and phospholipid metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boss, W.F.

1989-01-01

Our research for the past two years has involved the study of phosphoinositides and their potential role in regulating plant growth and development. Our initial goal was to document the sequence of events involved in inositol phospholipid metabolism in response to external stimuli. Our working hypothesis was that phosphatidylinositol bisphosphate (PIP/sub 2/) was in the plasma membrane of plants cells and would be hydrolyzed by phospholipase C to yield the second messengers inositol triphosphate (IP/sub 3/) and diacyglycerol (DAG) and that IP/sub 3/ would mobilize intracellular calcium as has been shown for animal cells. Our results with both carrot suspensionmore » culture cells and sunflower hypocotyl indicate that this paradigm is not the primary mechanism of signal transduction in these systems. We have observed very rapid, within 5 sec, stimulation of phosphatidylinositol monophosphate (PIP) kinase which resulted in an increase in PIP/sub 2/. However, there was no evidence for activation of phospholipase C. In addition, we have shown that PIP and PIP/sub 2/ can activate the plasma membrane ATPase. The results of these studies are described briefly in the paragraphs below. Inositol phospholipids are localized in distinct membrane fractions. If PIP and PIP/sub 2/ play a role in the transduction of external signals, they should be present in the plasma membrane. We used the fusogenic carrot suspension culture cells as a model system to study the distribution of inositol phospholipids in various membrane fractions and organelles. Cells were labeled 12 to 18 h with myo(2-/sup 3/H) inositol and the membranes were isolated by aqueous two-phase partitioning. The plasma membrane was enriched in PIP and PIP/sub 2/ compared to the intracellular membranes.« less

Exploring Membrane Dynamics during Electric Pulse Exposure with Second Harmonic Generation

NASA Astrophysics Data System (ADS)

Moen, Erick; Ibey, Bennett; Beier, Hope; Armani, Andrea

Optical second harmonic generation (SHG) is a powerful tool for investigating the nanostructure of symmetry-breaking materials and interfacial layers. Recently, we developed an imaging technique based on SHG for quantifying and localizing nanoporation in the plasma membrane of living cells. Nanosecond pulsed electric fields (nsPEF) were used to controllably disrupt the membrane, and the observed changes were validated against an extensible cell circuit model. In this talk, I will discuss the development of this method and its application to various cell types and stimuli, with a specific focus on bipolar (BP) nsPEF. BP nsPEF hold special interest as a cellular insult because they allow for a unique exposition of transmembrane potential and membrane charging/relaxation. Using this approach, we examine the structural response of the membrane as the temporal spacing between pulse phases was varied over several orders of magnitude and compare these results to the response when the cell is exposed to a monopolar (MP) nsPEF. Disagreement of the experimental results with the model demonstrates that biological processes may play a larger role than previously thought. These findings could lead to a greater understanding of the fundamental processes essential to all electroporation.

N-Glycosylation of the alpha subunit does not influence trafficking or functional activity of the human organic solute transporter alpha/beta

PubMed Central

Soroka, Carol J; Xu, Shuhua; Mennone, Albert; Lam, Ping; Boyer, James L

2008-01-01

Background The organic solute transporter (OSTα-OSTβ) is a heteromeric transporter that is expressed on the basolateral membrane of epithelium in intestine, kidney, liver, testis and adrenal gland and facilitates efflux of bile acids and other steroid solutes. Both subunits are required for plasma membrane localization of the functional transporter but it is unclear how and where the subunits interact and whether glycosylation is required for functional activity. We sought to examine these questions for the human OSTα-OSTβ transporter using the human hepatoma cell line, HepG2, and COS7 cells transfected with constructs of human OSTα-FLAG and OSTβ-Myc. Results Tunicamycin treatment demonstrated that human OSTα is glycosylated. In COS7 cells Western blotting identified the unglycosylated form (~31 kD), the core precursor form (~35 kD), and the mature, complex glycoprotein (~40 kD). Immunofluorescence of both cells indicated that, in the presence of OSTβ, the alpha subunit could still be expressed on the plasma membrane after tunicamycin treatment. Furthermore, the functional uptake of 3H-estrone sulfate was unchanged in the absence of N-glycosylation. Co-immunoprecipitation indicates that the immature form of OSTα interact with OSTβ. However, immunoprecipitation of OSTβ using an anti-Myc antibody did not co-precipitate the mature, complex glycosylated form of OSTα, suggesting that the primary interaction occurs early in the biosynthetic pathway and may be transient. Conclusion In conclusion, human OSTα is a glycoprotein that requires interaction with OSTβ to reach the plasma membrane. However, glycosylation of OSTα is not necessary for interaction with the beta subunit or for membrane localization or function of the heteromeric transporter. PMID:18847488

The basic route of the nuclear translocation porcine growth hormone (GH)-growth hormone receptor (GHR) complex (pGH/GHR) in porcine hepatocytes.

PubMed

Hainan, Lan; Huilin, Liu; Khan, Mahamad; Xin, Zheng; YuJiang, Yang; Hui, Zhang; Naiquan, Yao

2018-06-08

Traditional views suggest that growth hormone and the growth hormone receptor (GH/GHR complex) exert their functions only on the plasma membrane. This paradigm, however, has been challenged by recent new findings that the GH/GHR complex could translocate into cell nuclei where they could still exhibit important physiological functions. We also reported the nuclear localization of porcine GH/GHR and their potential functions in porcine hepatocytes. However, the basic path of pGH/GHR's nuclear translocation remains unclear. Combining previous research results and our current findings, we proposed two basic routes of pGH/GHR's nuclear transportation as follows: 1) after pGH binding to GHR, pGH/GHR enters into the cytoplasm though clathrin- or caveolin-mediated endocytosis, then the pGH/GHR complex enters into early endosomes (Rab5-positive), and the endosome carries the GH/GHR complex to the endoplasmic reticulum (ER). After endosome docking on the ER, the endosome starts fission, and the pGH/GHR complex enters into the ER lumen. Then the pGH/GHR complex transports into the cytoplasm, possibly by the ERAD pathway. Subsequently, the pGH/GHR complex interacts with IMPα/β, which, in turn, mediates GH/GHR nuclear localization; 2) pGH binds with the GHR on the cell membrane and, subsequently, pGH/GHR internalizes into the cell and enters into the endosome (this endosome may belong to a class of endosomes called envelope-associated endosomes (NAE)). Then, the endosome carries the pGH/GHR to the nuclear membrane. After docking on the nuclear membrane, the pGH/GHR complex fuses with the nuclear membrane and then enters into the cell nucleus. Copyright © 2018 Elsevier Inc. All rights reserved.

Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane

PubMed Central

Park, Se Won; Schonhoff, Christopher M.; Webster, Cynthia R. L.

2012-01-01

Cyclic AMP stimulates translocation of Na+/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation. PMID:22744337

Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane.

PubMed

Park, Se Won; Schonhoff, Christopher M; Webster, Cynthia R L; Anwer, M Sawkat

2012-09-01

Cyclic AMP stimulates translocation of Na(+)/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.

Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation.

PubMed

Carquin, Mélanie; Conrard, Louise; Pollet, Hélène; Van Der Smissen, Patrick; Cominelli, Antoine; Veiga-da-Cunha, Maria; Courtoy, Pierre J; Tyteca, Donatienne

2015-12-01

Although cholesterol is essential for membrane fluidity and deformability, the level of its lateral heterogeneity at the plasma membrane of living cells is poorly understood due to lack of appropriate probe. We here report on the usefulness of the D4 fragment of Clostridium perfringens toxin fused to mCherry (theta*), as specific, non-toxic, sensitive and quantitative cholesterol-labeling tool, using erythrocyte flat membrane. By confocal microscopy, theta* labels cholesterol-enriched submicrometric domains in coverslip-spread but also gel-suspended (non-stretched) fresh erythrocytes, suggesting in vivo relevance. Cholesterol domains on spread erythrocytes are stable in time and space, restricted by membrane:spectrin anchorage via 4.1R complexes, and depend on temperature and sphingomyelin, indicating combined regulation by extrinsic membrane:cytoskeleton interaction and by intrinsic lipid packing. Cholesterol domains partially co-localize with BODIPY-sphingomyelin-enriched domains. In conclusion, we show that theta* is a useful vital probe to study cholesterol organization and demonstrate that cholesterol forms submicrometric domains in living cells.

Anomalous Dynamics of a Lipid Recognition Protein on a Membrane Surface

PubMed Central

Yamamoto, Eiji; Kalli, Antreas C.; Akimoto, Takuma; Yasuoka, Kenji; Sansom, Mark S. P.

2015-01-01

Pleckstrin homology (PH) domains are lipid-binding modules present in peripheral membrane proteins which interact with phosphatidyl-inositol phosphates (PIPs) in cell membranes. We use multiscale molecular dynamics simulations to characterize the localization and anomalous dynamics of the DAPP1 PH domain on the surface of a PIP-containing lipid bilayer. Both translational and rotational diffusion of the PH domain on the lipid membrane surface exhibit transient subdiffusion, with an exponent α ≈ 0.5 for times of less than 10 ns. In addition to a PIP3 molecule at the canonical binding site of the PH domain, we observe additional PIP molecules in contact with the protein. Fluctuations in the number of PIPs associated with the PH domain exhibit 1/f noise. We suggest that the anomalous diffusion and long-term correlated interaction of the PH domain with the membrane may contribute to an enhanced probability of encounter with target complexes on cell membrane surfaces. PMID:26657413

Small cationic antimicrobial peptides delocalize peripheral membrane proteins

PubMed Central

Wenzel, Michaela; Chiriac, Alina Iulia; Otto, Andreas; Zweytick, Dagmar; May, Caroline; Schumacher, Catherine; Gust, Ronald; Albada, H. Bauke; Penkova, Maya; Krämer, Ute; Erdmann, Ralf; Metzler-Nolte, Nils; Straus, Suzana K.; Bremer, Erhard; Becher, Dörte; Brötz-Oesterhelt, Heike; Sahl, Hans-Georg; Bandow, Julia Elisabeth

2014-01-01

Short antimicrobial peptides rich in arginine (R) and tryptophan (W) interact with membranes. To learn how this interaction leads to bacterial death, we characterized the effects of the minimal pharmacophore RWRWRW-NH2. A ruthenium-substituted derivative of this peptide localized to the membrane in vivo, and the peptide also integrated readily into mixed phospholipid bilayers that resemble Gram-positive membranes. Proteome and Western blot analyses showed that integration of the peptide caused delocalization of peripheral membrane proteins essential for respiration and cell-wall biosynthesis, limiting cellular energy and undermining cell-wall integrity. This delocalization phenomenon also was observed with the cyclic peptide gramicidin S, indicating the generality of the mechanism. Exogenous glutamate increases tolerance to the peptide, indicating that osmotic destabilization also contributes to antibacterial efficacy. Bacillus subtilis responds to peptide stress by releasing osmoprotective amino acids, in part via mechanosensitive channels. This response is triggered by membrane-targeting bacteriolytic peptides of different structural classes as well as by hypoosmotic conditions. PMID:24706874

Essential role of flotillin-1 palmitoylation in the intracellular localization and signaling function of IGF-1 receptor.

PubMed

Jang, Donghwan; Kwon, Hayeong; Jeong, Kyuho; Lee, Jaewoong; Pak, Yunbae

2015-06-01

Here, we explored flotillin-1-mediated regulation of insulin-like growth factor-1 (IGF-1) signaling. Flotillin-1-deficient cells exhibited a reduction in the activation of IGF-1 receptor (IGF-1R), ERK1/2 and Akt pathways, and the transcriptional activation of Elk-1 and the proliferation in response to IGF-1 were reduced in these cells. We found that IGF-1-independent flotillin-1 palmitoylation at Cys34 in the endoplasmic reticulum (ER) was required for the ER exit and the plasma membrane localization of flotillin-1 and IGF-1R. IGF-1-dependent depalmitoylation and repalmitoylation of flotillin-1 sustained tyrosine kinase activation of the plasma-membrane-targeted IGF-1R. Dysfunction and blocking the turnover of flotillin-1 palmitoylation abrogated cancer cell proliferation after IGF-1R signaling activation. Our data show that flotillin-1 palmitoylation is a new mechanism by which the intracellular localization and activation of IGF-1R are controlled. © 2015. Published by The Company of Biologists Ltd.

Distribution Profile of Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channels in α and β Cells of Pancreas: Dominant Localization in Secretory Granules and Common Error in Identification of Secretory Granule Membranes.

PubMed

Hur, Yong Suk; Yoo, Seung Hyun

2015-01-01

The α and β cells of pancreatic islet release important hormones in response to intracellular Ca increases that result from Ca releases through the inositol 1,4,5-trisphoshate receptor (IP3R)/Ca channels. Yet no systematic studies on distribution of IP3R/Ca channels have been done, prompting us to investigate the distribution of all 3 IP3R isoforms. Immunogold electron microscopy was performed to determine the presence and the relative concentrations of all 3 IP3R isoforms in 2 major organelles secretory granules (SGs) and the endoplasmic reticulum of α and β cells of rat pancreas. All 3 IP3R isoforms were present in SG membranes of both cells, and the IP3R concentrations in SGs were ∼2-fold higher than those in the endoplasmic reticulum. Moreover, large halos shown in the electron microscope images of insulin-containing SGs of β cells were gap spaces that resulted from separation of granule membranes from the surrounding cytoplasm. These results strongly suggest the important roles of SGs in IP3-induced, Ca-dependent regulatory secretory pathway in pancreas. Moreover, the accurate location of SG membranes of β cells was further confirmed by the location of another integral membrane protein synaptotagmin V and of membrane phospholipid PI(4,5)P2.

Aspects of nuclear envelope dynamics in mitotic cells.

PubMed

Burke, Brian; Shanahan, Catherine; Salina, Davide; Crisp, Melissa

2005-01-01

Major features of the nuclear envelope (NE) are a pair of inner and outer nuclear membranes (INM, ONM) spanned by nuclear pore complexes. While the composition of the ONM resembles that of the endoplasmic reticulum, the INM contains a unique spectrum of proteins. Localization of INM proteins involves a mechanism of selective retention whereby integral proteins are immobilized and concentrated by virtue of interactions with nuclear components. In the case of emerin, INM localization involves interaction with A-type lamins. Interactions between membrane proteins may also play a significant role in INM localization. This conclusion stems from studies on nesprins, a family of membrane proteins that feature a large cytoplasmic domain, a single C-terminal membrane-spanning domain and a small lumenal domain. The nesprin membrane anchor and lumenal (KASH) domains are related to the Drosophila Klarsicht protein. Evidence is emerging that this KASH region interacts with other NE proteins and may influence their distributions. Overexpression of GFP-KASH causes loss of emerin and LAP2 from the NE. This is not due to global reorganization of the NE since LAP1 as well as lamins and NPCs remain unaffected. Our results suggest that interactions between NE membrane components are far more extensive and complex than current models suggest.

X11/Mint Genes Control Polarized Localization of Axonal Membrane Proteins in Vivo

PubMed Central

Gross, Garrett G.; Lone, G. Mohiddin; Leung, Lok Kwan; Hartenstein, Volker

2013-01-01

Mislocalization of axonal proteins can result in misassembly and/or miswiring of neural circuits, causing disease. To date, only a handful of genes that control polarized localization of axonal membrane proteins have been identified. Here we report that Drosophila X11/Mint proteins are required for targeting several proteins, including human amyloid precursor protein (APP) and Drosophila APP-like protein (APPL), to axonal membranes and for their exclusion from dendrites of the mushroom body in Drosophila, a brain structure involved in learning and memory. Axonal localization of APP is mediated by an endocytic motif, and loss of X11/Mint results in a dramatic increase in cell-surface levels of APPL, especially on dendrites. Mutations in genes required for endocytosis show similar mislocalization of these proteins to dendrites, and strongly enhance defects seen in X11/Mint mutants. These results suggest that X11/Mint-dependent endocytosis in dendrites may serve to promote the axonal localization of membrane proteins. Since X11/Mint binds to APP, and abnormal trafficking of APP contributes to Alzheimer's disease, deregulation of X11/Mint may be important for Alzheimer's disease pathogenesis. PMID:23658195

Abnormal N-Glycosylation of a Novel Missense Creatine Transporter Mutant, G561R, Associated with Cerebral Creatine Deficiency Syndromes Alters Transporter Activity and Localization.

PubMed

Uemura, Tatsuki; Ito, Shingo; Ohta, Yusuke; Tachikawa, Masanori; Wada, Takahito; Terasaki, Tetsuya; Ohtsuki, Sumio

2017-01-01

Cerebral creatine deficiency syndromes (CCDSs) are caused by loss-of-function mutations in creatine transporter (CRT, SLC6A8), which transports creatine at the blood-brain barrier and into neurons of the central nervous system (CNS). This results in low cerebral creatine levels, and patients exhibit mental retardation, poor language skills and epilepsy. We identified a novel human CRT gene missense mutation (c.1681 G>C, G561R) in Japanese CCDSs patients. The purpose of the present study was to evaluate the reduction of creatine transport in G561R-mutant CRT-expressing 293 cells, and to clarify the mechanism of its functional attenuation. G561R-mutant CRT exhibited greatly reduced creatine transport activity compared to wild-type CRT (WT-CRT) when expressed in 293 cells. Also, the mutant protein is localized mainly in intracellular membrane fraction, while WT-CRT is localized in plasma membrane. Western blot analysis revealed a 68 kDa band of WT-CRT protein in plasma membrane fraction, while G561R-mutant CRT protein predominantly showed bands at 55, 110 and 165 kDa in crude membrane fraction. The bands of both WT-CRT and G561R-mutant CRT were shifted to 50 kDa by N-glycosidase treatment. Our results suggest that the functional impairment of G561R-mutant CRT was probably caused by incomplete N-linked glycosylation due to misfolding during protein maturation, leading to oligomer formation and changes of cellular localization.

Lipid - Motor Interactions: Soap Opera or Symphony?

PubMed

Pathak, Divya; Mallik, Roop

2017-02-01

Intracellular transport of organelles can be driven by multiple motor proteins that bind to the lipid membrane of the organelle and work as a team. We review present knowledge on how lipids orchestrate the recruitment of motors to a membrane. Looking beyond recruitment, we also discuss how heterogeneity and local mechanical properties of the membrane may influence function of motor-teams. These issues gain importance because phagocytosed pathogens use lipid-centric strategies to manipulate motors and survive in host cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

An activating mutant of Rac1 that fails to interact with Rho GDP-dissociation inhibitor stimulates membrane ruffling in mammalian cells.

PubMed Central

Gandhi, Payal N; Gibson, Richard M; Tong, Xiaofeng; Miyoshi, Jun; Takai, Yoshimi; Konieczkowski, Martha; Sedor, John R; Wilson-Delfosse, Amy L

2004-01-01

Rac1, a member of the Rho family of small GTP-binding proteins, is involved in the regulation of the actin cytoskeleton via activation of lamellipodia and membrane ruffle formation. RhoGDI (Rho-family-specific GDP-dissociation inhibitor) forms a complex with Rho proteins in the cytosol of mammalian cells. It not only regulates guanine nucleotide binding to Rho proteins, but may also function as a molecular shuttle to carry Rho proteins from an inactive cytosolic pool to the membrane for activation. These studies tested if RhoGDI is necessary for the translocation of Rac1 from the cytosol to the plasma membrane for the formation of membrane ruffles. We describe a novel mutant of Rac1, R66E (Arg66-->Glu), that fails to bind RhoGDI. This RhoGDI-binding-defective mutation is combined with a Rac1-activating mutation G12V, resulting in a double-mutant [Rac1(G12V/R66E)] that fails to interact with RhoGDI in COS-7 cells, but remains constitutively activated. This double mutant stimulates membrane ruffling to a similar extent as that observed after epidermal growth factor treatment of non-transfected cells. To confirm that Rac1 can signal ruffle formation in the absence of interaction with RhoGDI, Rac1(G12V) was overexpressed in cultured mesangial cells derived from a RhoGDI knockout mouse. Rac1-mediated membrane ruffling was indistinguishable between the RhoGDI(-/-) and RhoGDI(+/+) cell lines. In both the COS-7 and cultured mesangial cells, Rac1(G12V) and Rac1(G12V/R66E) co-localize with membrane ruffles. These findings suggest that interaction with RhoGDI is not essential in the mechanism by which Rac1 translocates to the plasma membrane to stimulate ruffle formation. PMID:14629200

Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells

PubMed Central

Wang, Zhen; Schey, Kevin L.

2015-01-01

Purpose Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids—key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. Methods Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis. Results A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains. Conclusions These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells. PMID:26747763

MapA, an iron-regulated, cytoplasmic membrane protein in the cyanobacterium Synechococcus sp. strain PCC7942.

PubMed Central

Webb, R; Troyan, T; Sherman, D; Sherman, L A

1994-01-01

Growth of Synechococcus sp. strain PCC 7942 in iron-deficient media leads to the accumulation of an approximately 34-kDa protein. The gene encoding this protein, mapA (membrane-associated protein A), has been cloned and sequenced (GenBank accession number, L01621). The mapA transcript is not detectable in normally grown cultures but is stably accumulated by cells grown in iron-deficient media. However, the promoter sequence for this gene does not resemble other bacterial iron-regulated promoters described to date. The carboxyl-terminal region of the derived amino acid sequence of MapA resembles bacterial proteins involved in iron acquisition, whereas the amino-terminal end of MapA has a high degree of amino acid identity with the abundant, chloroplast envelope protein E37. An approach employing improved cellular fractionation techniques as well as electron microscopy and immunocytochemistry was essential in localizing MapA protein to the cytoplasmic membrane of Synechococcus sp. strain PCC 7942. When these cells were grown under iron-deficient conditions, a significant fraction of MapA could also be localized to the thylakoid membranes. Images PMID:8051004

Impact of Morphological Changes on the Motility of Amoeba proteus

NASA Astrophysics Data System (ADS)

Shroff N, Sunitha

2016-11-01

Bio-mechanical properties of cell membrane, actin and cytoskeleton have influence on the cell locomotion. To explore, morphological changes were induced in Amoeba proteus by depriving nutrition, also either through ATP mediated or through KCl mediated membrane depolarization. We observed that, membrane depolarization leads to complete loss/reduction of pseudopodia in a dose dependent manner, gradually A. proteus becomes globular. We also report that with depravation of its nutrition (Chilomonas) A. proteus transforms them into tube/filament like structure and this transformation is reversible with the supply of Chilomonas. Results indicate that the structural and locomotion variation of A. proteus through nucleotides may not be just a membrane phenomenon, but may involve signaling mechanisms. Further, we carried out immunostaining of A. proteus with P2X2 and P2Y2 antibodies to analyze their localization and the extent of expression. The result indicated that in normal A. proteus receptors are dispersed uniformly, whereas in filament shaped A. proteus P2X2-receptor was found to be localized, unlike P2Y2 receptor. As nucleotides are known to cause structural changes in the organism, we report corresponding changes in their locomotion. Assistant Professor, Department of Biotechnology. Mount Carmel College, Bangalore 560 052.

AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2

PubMed Central

Cho, Kwang-jin; Casteel, Darren E.; Prakash, Priyanka; Tan, Lingxiao; van der Hoeven, Dharini; Salim, Angela A.; Kim, Choel; Capon, Robert J.; Lacey, Ernest; Cunha, Shane R.; Gorfe, Alemayehu A.

2016-01-01

K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function. PMID:27697864

Induction of filopodia-like protrusions in N1E-115 neuroblastoma cells by diacylglycerol kinase γ independent of its enzymatic activity: potential novel function of the C-terminal region containing the catalytic domain of diacylglycerol kinase γ.

PubMed

Tanino, Fumihiko; Maeda, Yuki; Sakai, Hiromichi; Sakane, Fumio

2013-01-01

Type I diacylglycerol kinase (DGK) isozymes (α, β, and γ) contain recoverin homology domains and calcium-binding EF-hand motifs at their N-termini. The γ-isoform of DGK is abundantly expressed in retinal and Purkinje cells; however, its function in neuronal cells remains unknown. Here, we report that the mRNA and protein levels of DGKγ, but not DGKα or β, were markedly increased in N1E-115 neuroblastoma cells upon cellular differentiation by serum starvation. Interestingly, overexpression of wild-type DGKγ, which was partially located at the plasma membrane, considerably induced the formation of slender, filopodia-like cytoplasmic projections from N1E-115 cell bodies. Deletion of the recoverin homology domain and the EF-hand motifs, which potentiated the plasma membrane localization of the isozyme, significantly enhanced the formation of the filopodia-like protrusions. Intriguingly, the catalytic activity of the isozyme is not essential for the protrusion formation. The N-terminal half of the catalytic domain and a short stretch of amino acid residues at the C-terminus are responsible for plasma membrane localization and filopodia-like process formation. Taken together, we have described a potentially novel morphological function of the C-terminal DGKγ catalytic region that is independent of its enzymatic activity.

Functional Sieve Element Protoplasts1[OA

PubMed Central

Hafke, Jens B.; Furch, Alexandra C.U.; Reitz, Marco U.; van Bel, Aart J.E.

2007-01-01

Sieve element (SE) protoplasts were liberated by exposing excised phloem strands of Vicia faba to cell wall-degrading enzyme mixtures. Two types of SE protoplasts were found: simple protoplasts with forisome inclusions and composite twin protoplasts—two protoplasts intermitted by a sieve plate—of which one protoplast often includes a forisome. Forisomes are giant protein inclusions of SEs in Fabaceae. Membrane integrity of SE protoplasts was tested by application of CFDA, which was sequestered in the form of carboxyfluorescein. Further evidence for membrane intactness was provided by swelling of SE protoplasts and forisome dispersion in reaction to abrupt lowering of medium osmolarity. The absence of cell wall remnants as demonstrated by negative Calcofluor White staining allowed patch-clamp studies. At negative membrane voltages, the current-voltage relations of the SE protoplasts were dominated by a weak inward-rectifying potassium channel that was active at physiological membrane voltages of the SE plasma membrane. This channel had electrical properties that are reminiscent of those of the AKT2/3 channel family, localized in phloem cells of Arabidopsis (Arabidopsis thaliana). All in all, SE protoplasts promise to be a powerful tool in studying the membrane biology of SEs with inherent implications for the understanding of long-distance transport and signaling. PMID:17885083

Enquiry into the Topology of Plasma Membrane-Localized PIN Auxin Transport Components.

PubMed

Nodzyński, Tomasz; Vanneste, Steffen; Zwiewka, Marta; Pernisová, Markéta; Hejátko, Jan; Friml, Jiří

2016-11-07

Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regardless of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immunolocalization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane (TM) bundles of five α-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

DOE PAGES

Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; ...

2015-06-16

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referredmore » to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRas G12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas G12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.« less

Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

PubMed Central

Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li-Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

2015-01-01

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors. PMID:26080442

Oxygen depletion speeds and simplifies diffusion in HeLa cells.

PubMed

Edwald, Elin; Stone, Matthew B; Gray, Erin M; Wu, Jing; Veatch, Sarah L

2014-10-21

Many cell types undergo a hypoxic response in the presence of low oxygen, which can lead to transcriptional, metabolic, and structural changes within the cell. Many biophysical studies to probe the localization and dynamics of single fluorescently labeled molecules in live cells either require or benefit from low-oxygen conditions. In this study, we examine how low-oxygen conditions alter the mobility of a series of plasma membrane proteins with a range of anchoring motifs in HeLa cells at 37°C. Under high-oxygen conditions, diffusion of all proteins is heterogeneous and confined. When oxygen is reduced with an enzymatic oxygen-scavenging system for ≥ 15 min, diffusion rates increase by > 2-fold, motion becomes unconfined on the timescales and distance scales investigated, and distributions of diffusion coefficients are remarkably consistent with those expected from Brownian motion. More subtle changes in protein mobility are observed in several other laboratory cell lines examined under both high- and low-oxygen conditions. Morphological changes and actin remodeling are observed in HeLa cells placed in a low-oxygen environment for 30 min, but changes are less apparent in the other cell types investigated. This suggests that changes in actin structure are responsible for increased diffusion in hypoxic HeLa cells, although superresolution localization measurements in chemically fixed cells indicate that membrane proteins do not colocalize with F-actin under either experimental condition. These studies emphasize the importance of controls in single-molecule imaging measurements, and indicate that acute response to low oxygen in HeLa cells leads to dramatic changes in plasma membrane structure. It is possible that these changes are either a cause or consequence of phenotypic changes in solid tumor cells associated with increased drug resistance and malignancy.