Dual effect of pseudorabies virus growth factor (PRGF) displayed on actin cytoskeleton.

PubMed

Urbancíková, M; Vozárová, G; Lesko, J; Golais, F

1999-10-01

Pseudorabies virus growth factor (PRGF) was shown to possess transforming activity as well as transformation repressing activity in in vitro systems. In order to better understand these phenomena we studied actin cytoskeleton and its alterations induced by PRGF using normal human fibroblasts VH-10 and transformed cell line HeLa. For specific detection of filamentous actin cells were stained with phalloidin conjugated with fluorescein isothiocyanate (FITC)-phalloidin. PRGF was applied to VH-10 cells for various length of time from 10 min up to 48 h. The effect was very fast and changes in actin filament composition could be detected already after 10 min. In comparison to untreated cells the staining of treated cells was more diffuse and a number of actin microfilaments in individual stress fibers became reduced. After 30 min thick short actin bundles appeared in the perinuclear region. A 24-h exposure resulted in a large reduction of actin bundles. After additional 24 h a partial restoration of actin cytoskeleton in cells was observed. In transformed HeLa cells PRGF induced opposite process than in normal cells: the number of filamentous actin structures increased. We hypothesise that PRGF may act as a transcription-like factor and may initiate changes in gene expression which consequently result in actin cytoskeleton alterations.

The Dynamic Actin Cytoskeleton in Smooth Muscle.

PubMed

Tang, Dale D

2018-01-01

Smooth muscle contraction requires both myosin activation and actin cytoskeletal remodeling. Actin cytoskeletal reorganization facilitates smooth muscle contraction by promoting force transmission between the contractile unit and the extracellular matrix (ECM), and by enhancing intercellular mechanical transduction. Myosin may be viewed to serve as an "engine" for smooth muscle contraction whereas the actin cytoskeleton may function as a "transmission system" in smooth muscle. The actin cytoskeleton in smooth muscle also undergoes restructuring upon activation with growth factors or the ECM, which controls smooth muscle cell proliferation and migration. Abnormal smooth muscle contraction, cell proliferation, and motility contribute to the development of vascular and pulmonary diseases. A number of actin-regulatory proteins including protein kinases have been discovered to orchestrate actin dynamics in smooth muscle. In particular, Abelson tyrosine kinase (c-Abl) is an important molecule that controls actin dynamics, contraction, growth, and motility in smooth muscle. Moreover, c-Abl coordinates the regulation of blood pressure and contributes to the pathogenesis of airway hyperresponsiveness and vascular/airway remodeling in vivo. Thus, c-Abl may be a novel pharmacological target for the development of new therapy to treat smooth muscle diseases such as hypertension and asthma. © 2018 Elsevier Inc. All rights reserved.

Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes.

PubMed

Kyozuka, Keiichiro; Chun, Jong T; Puppo, Agostina; Gragnaniello, Gianni; Garante, Ezio; Santella, Luigia

2008-08-15

Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca2+ release in the cell cortex. Here, we show that this Ca2+ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca2+ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca2+ release during meiotic maturation. The Ca2+ wave was inhibited by the classical antagonists of the InsP(3)-linked Ca2+ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca2+ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP(3), despite the massive release of Ca2+, implying that exocytosis of the cortical granules requires not only a Ca2+ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca2+ signals and in regulating cortical granule exocytosis.

Assembly and Function of the Actin Cytoskeleton of Yeast: Relationships between Cables and Patches

PubMed Central

Karpova, Tatiana S.; McNally, James G.; Moltz, Samuel L.; Cooper, John A.

1998-01-01

Actin in eukaryotic cells is found in different pools, with filaments being organized into a variety of supramolecular assemblies. To investigate the assembly and functional relationships between different parts of the actin cytoskeleton in one cell, we studied the morphology and dynamics of cables and patches in yeast. The fine structure of actin cables and the manner in which cables disassemble support a model in which cables are composed of a number of overlapping actin filaments. No evidence for intrinsic polarity of cables was found. To investigate to what extent different parts of the actin cytoskeleton depend on each other, we looked for relationships between cables and patches. Patches and cables were often associated, and their polarized distributions were highly correlated. Therefore, patches and cables do appear to depend on each other for assembly and function. Many cell types show rearrangements of the actin cytoskeleton, which can occur via assembly or movement of actin filaments. In our studies, dramatic changes in actin polarization did not include changes in filamentous actin. In addition, the concentration of actin patches was relatively constant as cells grew. Therefore, cells do not have bursts of activity in which new parts of the actin cytoskeleton are created. PMID:9744880

NHERF1 regulates actin cytoskeleton organization through modulation of α-actinin-4 stability.

PubMed

Sun, Licui; Zheng, Junfang; Wang, Qiqi; Song, Ran; Liu, Hua; Meng, Ran; Tao, Tao; Si, Yang; Jiang, Wenguo; He, Junqi

2016-02-01

The actin cytoskeleton is composed of a highly dynamic network of filamentous proteins, yet the molecular mechanism that regulates its organization and remodeling remains elusive. In this study, Na(+)/H(+) exchanger regulatory factor (NHERF)-1 loss-of-function and gain-of-function experiments reveal that polymerized actin cytoskeleton (F-actin) in HeLa cells is disorganized by NHERF1, whereas actin protein expression levels exhibit no detectable change. To elucidate the molecular mechanism underlying actin cytoskeleton disorganization by NHERF1, a combined 2-dimensional electrophoresis-matrix-assisted laser desorption/ionization-time of flight mass spectrometry approach was used to screen for proteins regulated by NHERF1 in HeLa cells. α-Actinin-4, an actin cross-linking protein, was identified. Glutathione S-transferase pull-down and coimmunoprecipitation studies showed the α-actinin-4 carboxyl-terminal region specifically interacted with the NHERF1 postsynaptic density 95/disc-large/zona occludens-1 domain. The NHERF1/α-actinin-4 interaction increased α-actinin-4 ubiquitination and decreased its expression levels, resulting in actin cytoskeleton disassembly. Our study identified α-actinin-4 as a novel NHERF1 interaction partner and provided new insights into the regulatory mechanism of the actin cytoskeleton by NHERF1. © FASEB.

Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.

PubMed

Walther, Christa G; Whitfield, Robert; James, David C

2016-04-01

The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.

The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration

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

Blom, Magdalena; Reis, Katarina; Heldin, Johan

RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as corticalmore » actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration. - Highlights: • Increased RhoD expression leads to loss of actin structures, e.g. stress fibers and gives rise to decreased actin dynamics. • RhoD knockdown induces various actin-containing structures such as edge ruffles, stress fibers and cortical actin, in a cell-type specific manner. • RhoD induces specific actin rearrangements depending on its subcellular localization. • RhoD knockdown has effects on cellular processes, such as directed cell migration and proliferation.« less

The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

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

Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton

2010-08-15

Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmicmore » tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger.« less

The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

PubMed Central

Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton; Moncman, Carole L.; Dutch, Rebecca Ellis; McCann, Richard O.

2010-01-01

Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmic tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger. PMID:20537366

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

PubMed Central

Torreno-Pina, Juan A.; Manzo, Carlo; Salio, Mariolina; Aichinger, Michael C.; Oddone, Anna; Lakadamyali, Melike; Shepherd, Dawn; Besra, Gurdyal S.; Cerundolo, Vincenzo

2016-01-01

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters. PMID:26798067

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. ...

Control of the actin cytoskeleton in root hair development.

PubMed

Pei, Weike; Du, Fei; Zhang, Yi; He, Tian; Ren, Haiyun

2012-05-01

The development of root hair includes four stages: bulge site selection, bulge formation, tip growth, and maturation. The actin cytoskeleton is involved in all of these stages and is organized into distinct arrangements in the different stages. In addition to the actin configuration, actin isoforms also play distinct roles in the different stages. The actin cytoskeleton is regulated by actin-binding proteins, such as formin, Arp2/3 complex, profilin, actin depolymerizing factor, and villin. Some upstream signals, i.e. calcium, phospholipids, and small GTPase regulate the activity of these actin-binding proteins to produce the proper actin configuration. We constructed a working model on how the actin cytoskeleton is controlled by actin-binding proteins and upstream signaling in root hair development based on the current literature: at the tip of hairs, actin polymerization appears to be facilitated by Arp2/3 complex that is activated by small GTPase, and profilin that is regulated by phosphatidylinositol 4,5-bisphosphate. Meanwhile, actin depolymerization and turnover are likely mediated by villin and actin depolymerizing factor, which are stimulated by calcium. At the shank, actin cables are produced by formin and villin. Under the complicated interaction, the actin cytoskeleton is controlled spatially and temporally during root hair development. © 2012 Elsevier Ireland Ltd. All rights reserved.

Phosphoinositide-specific phospholipase C in oat roots: association with the actin cytoskeleton.

PubMed

Huang, Chiung-Hua; Crain, Richard C

2009-10-01

Phosphoinositide-specific phospholipase C (PI-PLC) activities are involved in mediating plant cell responses to environmental stimuli. Two variants of PI-PLC have been partially purified from the roots of oat seedlings; one cytosolic and one particulate. Although the cytosolic enzyme was significantly purified, the activity still co-migrated with a number of other proteins on heparin HPLC and also on size-exclusion chromatography. The partially purified PI-PLC was tested by Western blotting, and we found that actin and actin-binding proteins, profilin and tropomyosin, co-purified with cytosolic phospholipase C. After a non-ionic detergent (Triton X-100) treatment, PI-PLC activities still remained with the actin cytoskeleton. The effects of phalloidin and F-buffer confirmed this association; these conditions, which favor actin polymerization, decreased the release of PI-PLC from the cytoskeleton. The treatments of latrunculin and G-buffer, the conditions that favor actin depolymerization, increased the release of PI-PLC from the cytoskeleton. These results suggest that oat PI-PLC associates with the actin cytoskeleton.

Regulation of Retinoschisin Secretion in Weri-Rb1 Cells by the F-Actin and Microtubule Cytoskeleton

PubMed Central

Kitamura, Eiko; Gribanova, Yekaterina E.; Farber, Debora B.

2011-01-01

Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process. PMID:21738583

Microcompartmentation of cytosolic aldolase by interaction with the actin cytoskeleton in Arabidopsis.

PubMed

Garagounis, Constantine; Kostaki, Kalliopi-Ioanna; Hawkins, Tim J; Cummins, Ian; Fricker, Mark D; Hussey, Patrick J; Hetherington, Alistair M; Sweetlove, Lee J

2017-02-01

Evidence is accumulating for molecular microcompartments formed when proteins interact in localized domains with the cytoskeleton, organelle surfaces, and intracellular membranes. To understand the potential functional significance of protein microcompartmentation in plants, we studied the interaction of the glycolytic enzyme fructose bisphosphate aldolase with actin in Arabidopsis thaliana. Homology modelling of a major cytosolic isozyme of aldolase, FBA8, suggested that the tetrameric holoenzyme has two actin binding sites and could therefore act as an actin-bundling protein, as was reported for animal aldolases. This was confirmed by in vitro measurements of an increase in viscosity of F-actin polymerized in the presence of recombinant FBA8. Simultaneously, interaction with F-actin caused non-competitive inhibition of aldolase activity. We did not detect co-localization of an FBA8-RFP fusion protein, expressed in an fba8-knockout background, with the actin cytoskeleton using confocal laser-scanning microscopy. However, we did find evidence for a low level of interaction using FRET-FLIM analysis of FBA8-RFP co-expressed with the actin-binding protein GFP-Lifeact. Furthermore, knockout of FBA8 caused minor alterations of guard cell actin cytoskeleton morphology and resulted in a reduced rate of stomatal closure in response to decreased humidity. We conclude that cytosolic aldolase can be microcompartmented in vivo by interaction with the actin cytoskeleton and may subtly modulate guard cell behaviour as a result. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

WASp-dependent actin cytoskeleton stability at the dendritic cell immunological synapse is required for extensive, functional T cell contacts.

PubMed

Malinova, Dessislava; Fritzsche, Marco; Nowosad, Carla R; Armer, Hannah; Munro, Peter M G; Blundell, Michael P; Charras, Guillaume; Tolar, Pavel; Bouma, Gerben; Thrasher, Adrian J

2016-05-01

The immunological synapse is a highly structured and molecularly dynamic interface between communicating immune cells. Although the immunological synapse promotes T cell activation by dendritic cells, the specific organization of the immunological synapse on the dendritic cell side in response to T cell engagement is largely unknown. In this study, confocal and electron microscopy techniques were used to investigate the role of dendritic cell actin regulation in immunological synapse formation, stabilization, and function. In the dendritic cell-restricted absence of the Wiskott-Aldrich syndrome protein, an important regulator of the actin cytoskeleton in hematopoietic cells, the immunological synapse contact with T cells occupied a significantly reduced surface area. At a molecular level, the actin network localized to the immunological synapse exhibited reduced stability, in particular, of the actin-related protein-2/3-dependent, short-filament network. This was associated with decreased polarization of dendritic cell-associated ICAM-1 and MHC class II, which was partially dependent on Wiskott-Aldrich syndrome protein phosphorylation. With the use of supported planar lipid bilayers incorporating anti-ICAM-1 and anti-MHC class II antibodies, the dendritic cell actin cytoskeleton organized into recognizable synaptic structures but interestingly, formed Wiskott-Aldrich syndrome protein-dependent podosomes within this area. These findings demonstrate that intrinsic dendritic cell cytoskeletal remodeling is a key regulatory component of normal immunological synapse formation, likely through consolidation of adhesive interaction and modulation of immunological synapse stability. © The Author(s).

Evidence for actin cytoskeleton-dependent and -independent pathways for RelA/p65 nuclear translocation in endothelial cells.

PubMed

Fazal, Fabeha; Minhajuddin, Mohd; Bijli, Kaiser M; McGrath, James L; Rahman, Arshad

2007-02-09

Activation of the transcription factor NF-kappaB involves its release from the inhibitory protein IkappaBalpha in the cytoplasm and subsequently, its translocation to the nucleus. Whereas the events responsible for its release have been elucidated, mechanisms regulating the nuclear transport of NF-kappaB remain elusive. We now provide evidence for actin cytoskeleton-dependent and -independent mechanisms of RelA/p65 nuclear transport using the proinflammatory mediators, thrombin and tumor necrosis factor alpha, respectively. We demonstrate that thrombin alters the actin cytoskeleton in endothelial cells and interfering with these alterations, whether by stabilizing or destabilizing the actin filaments, prevents thrombin-induced NF-kappaB activation and consequently, expression of its target gene, ICAM-1. The blockade of NF-kappaB activation occurs downstream of IkappaBalpha degradation and is associated with impaired RelA/p65 nuclear translocation. Importantly, thrombin induces association of RelA/p65 with actin and this interaction is sensitive to stabilization/destabilization of the actin filaments. In parallel studies, stabilizing or destabilizing the actin filaments fails to inhibit RelA/p65 nuclear accumulation and ICAM-1 expression by tumor necrosis factor alpha, consistent with its inability to induce actin filament formation comparable with thrombin. Thus, these studies reveal the existence of actin cytoskeleton-dependent and -independent pathways that may be engaged in a stimulus-specific manner to facilitate RelA/p65 nuclear import and thereby ICAM-1 expression in endothelial cells.

Building bridges: formin1 of Arabidopsis forms a connection between the cell wall and the actin cytoskeleton.

PubMed

Martinière, Alexandre; Gayral, Philippe; Hawes, Chris; Runions, John

2011-04-01

Actin microfilament (MF) organization and remodelling is critical to cell function. The formin family of actin binding proteins are involved in nucleating MFs in Arabidopsis thaliana. They all contain formin homology domains in the intracellular, C-terminal half of the protein that interacts with MFs. Formins in class I are usually targeted to the plasma membrane and this is true of Formin1 (AtFH1) of A. thaliana. In this study, we have investigated the extracellular domain of AtFH1 and we demonstrate that AtFH1 forms a bridge from the actin cytoskeleton, across the plasma membrane and is anchored within the cell wall. AtFH1 has a large, extracellular domain that is maintained by purifying selection and that contains four conserved regions, one of which is responsible for immobilising the protein. Protein anchoring within the cell wall is reduced in constructs that express truncations of the extracellular domain and in experiments in protoplasts without primary cell walls. The 18 amino acid proline-rich extracellular domain that is responsible for AtFH1 anchoring has homology with cell-wall extensins. We also have shown that anchoring of AtFH1 in the cell wall promotes actin bundling within the cell and that overexpression of AtFH1 has an inhibitory effect on organelle actin-dependant dynamics. Thus, the AtFH1 bridge provides stable anchor points for the actin cytoskeleton and is probably a crucial component of the signalling response and actin-remodelling mechanisms. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

From Cytoskeleton to Gene Expression: Actin in the Nucleus.

PubMed

Viita, Tiina; Vartiainen, Maria K

2017-01-01

Although most people still associate actin mainly with the cytoskeleton, several lines of evidence, with the earliest studies dating back to decades ago, have emphasized the importance of actin also inside the cell nucleus. Actin has been linked to many gene expression processes from gene activation to chromatin remodeling, but also to maintenance of genomic integrity and intranuclear movement of chromosomes and chromosomal loci. Recent advances in visualizing different forms and dynamic properties of nuclear actin have clearly advanced our understanding of the basic concepts by which actin operates in the nucleus. In this chapter we address the different breakthroughs in nuclear actin studies, as well as discuss the regulation nuclear actin and the importance of nuclear actin dynamics in relation to its different nuclear functions. Our aim is to highlight the fact that actin should be considered as an essential component of the cell nucleus, and its nuclear actions should be taken into account also in experiments on cytoplasmic actin networks.

Dynamics of the actin cytoskeleton mediates receptor cross talk: An emerging concept in tuning receptor signaling

PubMed Central

Mattila, Pieta K.; Batista, Facundo D.

2016-01-01

Recent evidence implicates the actin cytoskeleton in the control of receptor signaling. This may be of particular importance in the context of immune receptors, such as the B cell receptor, where dysregulated signaling can result in autoimmunity and malignancy. Here, we discuss the role of the actin cytoskeleton in controlling receptor compartmentalization, dynamics, and clustering as a means to regulate receptor signaling through controlling the interactions with protein partners. We propose that the actin cytoskeleton is a point of integration for receptor cross talk through modulation of protein dynamics and clustering. We discuss the implication of this cross talk via the cytoskeleton for both ligand-induced and low-level constitutive (tonic) signaling necessary for immune cell survival. PMID:26833785

Duplication and segregation of the actin (MreB) cytoskeleton during the prokaryotic cell cycle.

PubMed

Vats, Purva; Rothfield, Lawrence

2007-11-06

The bacterial actin homolog MreB exists as a single-copy helical cytoskeletal structure that extends between the two poles of rod-shaped bacteria. In this study, we show that equipartition of the MreB cytoskeleton into daughter cells is accomplished by division and segregation of the helical MreB array into two equivalent structures located in opposite halves of the predivisional cell. This process ensures that each daughter cell inherits one copy of the MreB cytoskeleton. The process is triggered by the membrane association of the FtsZ cell division protein. The cytoskeletal division and segregation events occur before and independently of cytokinesis and involve specialized MreB structures that appear to be intermediates in this process.

Integrity of the actin cytoskeleton of host macrophages is essential for Leishmania donovani infection.

PubMed

Roy, Saptarshi; Kumar, G Aditya; Jafurulla, Md; Mandal, Chitra; Chattopadhyay, Amitabha

2014-08-01

Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis. Copyright © 2014 Elsevier B.V. All rights reserved.

The Plant Actin Cytoskeleton Responds to Signals from Microbe-Associated Molecular Patterns

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

Henty-Ridilla, Jessica L.; Shimono, Masaki; Li, Jiejie

2013-04-04

Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence thatmore » the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.« less

Membrane tension and cytoskeleton organization in cell motility.

PubMed

Sens, Pierre; Plastino, Julie

2015-07-15

Cell membrane shape changes are important for many aspects of normal biological function, such as tissue development, wound healing and cell division and motility. Various disease states are associated with deregulation of how cells move and change shape, including notably tumor initiation and cancer cell metastasis. Cell motility is powered, in large part, by the controlled assembly and disassembly of the actin cytoskeleton. Much of this dynamic happens in close proximity to the plasma membrane due to the fact that actin assembly factors are membrane-bound, and thus actin filaments are generally oriented such that their growth occurs against or near the membrane. For a long time, the membrane was viewed as a relatively passive scaffold for signaling. However, results from the last five years show that this is not the whole picture, and that the dynamics of the actin cytoskeleton are intimately linked to the mechanics of the cell membrane. In this review, we summarize recent findings concerning the role of plasma membrane mechanics in cell cytoskeleton dynamics and architecture, showing that the cell membrane is not just an envelope or a barrier for actin assembly, but is a master regulator controlling cytoskeleton dynamics and cell polarity.

Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis

PubMed Central

Tang, Elizabeth I.; Lee, Will M.

2016-01-01

Germ cell transport across the seminiferous epithelium during spermatogenesis requires the intricate coordination of cell junctions, signaling proteins, and both actin- and microtubule (MT)-based cytoskeletons. Although the involvement of cytoskeletons in germ cell transport has been suggested, the precise mechanism(s) remains elusive. Based on growing evidence that actin and MT interactions underlie fundamental cellular processes, such as cell motility, it is unlikely that actin- and MT-based cytoskeletons work independently to regulate germ cell transport in the testis. Using rats treated with adjudin, a potential male contraceptive that disrupts spermatid adhesion and transport in the testis, as a study model, we show herein that actin- and MT-based cytoskeletons are both necessary for transport of spermatids and residual bodies/phagosomes across the seminiferous epithelium in adult rat testes. Analysis of intratubular expression of F-actin and tubulin revealed disruption of both actin and MT networks, concomitant with misdirected spermatids and phagosomes in rats treated with adjudin. Actin regulatory proteins, epidermal growth factor receptor pathway substrate 8 and actin-related protein 3, were mislocalized and down-regulated at the actin-rich anchoring junction between germ and Sertoli cells (apical ectoplasmic specialization) after adjudin treatment. Nonreceptor tyrosine kinase p-FAK-Tyr407, known to regulate F-actin nucleation via actin-related protein 3, was also mislocalized and down-regulated at the apical ectoplasmic specialization, corroborating the observation of actin cytoskeleton disruption. Additionally, spatiotemporal expression of MT regulatory protein end-binding protein 1, shown to be involved in MT-actin cross talk herein, was also disrupted after adjudin treatment. In summary, spermatid/phagosome transport across the epithelium during spermatogenesis requires the coordination between actin- and MT-based cytoskeletons. PMID:26894662

Quantifying the plant actin cytoskeleton response to applied pressure using nanoindentation.

PubMed

Branco, Rémi; Pearsall, Eliza-Jane; Rundle, Chelsea A; White, Rosemary G; Bradby, Jodie E; Hardham, Adrienne R

2017-03-01

Detection of potentially pathogenic microbes through recognition by plants and animals of both physical and chemical signals associated with the pathogens is vital for host well-being. Signal perception leads to the induction of a variety of responses that augment pre-existing, constitutive defences. The plant cell wall is a highly effective preformed barrier which becomes locally reinforced at the infection site through delivery of new wall material by the actin cytoskeleton. Although mechanical stimulation can produce a reaction, there is little understanding of the nature of physical factors capable of triggering plant defence. Neither the magnitude of forces nor the contact time required has been quantified. In the study reported here, mechanical stimulation with a tungsten microneedle has been used to quantify the response of Arabidopsis plants expressing an actin-binding protein tagged with green fluorescent protein (GFP) to reveal the organisation of the actin cytoskeleton. Using confocal microscopy, the response time for actin reorganisation in epidermal cells of Arabidopsis hypocotyls was shown to be 116 ± 49 s. Using nanoindentation and a diamond spherical tip indenter, the magnitude of the forces capable of triggering an actin response has been quantified. We show that Arabidopsis hypocotyl cells can detect a force as small as 4 μN applied for as short a time as 21.6 s to trigger reorganisation of the actin cytoskeleton. This force is an order of magnitude less than the potential invasive force determined for a range of fungal and oomycete plant pathogens. To our knowledge, this is the first quantification of the magnitude and duration of mechanical forces capable of stimulating a structural defence response in a plant cell.

HopW1 from Pseudomonas syringae disrupts the actin cytoskeleton to promote virulence in Arabidopsis.

PubMed

Kang, Yongsung; Jelenska, Joanna; Cecchini, Nicolas M; Li, Yujie; Lee, Min Woo; Kovar, David R; Greenberg, Jean T

2014-06-01

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.

Sirtuin1 Maintains Actin Cytoskeleton by Deacetylation of Cortactin in Injured Podocytes

PubMed Central

Motonishi, Shuta; Wada, Takehiko; Ishimoto, Yu; Ohse, Takamoto; Matsusaka, Taiji; Kubota, Naoto; Shimizu, Akira; Kadowaki, Takashi; Tobe, Kazuyuki

2015-01-01

Recent studies have highlighted the renoprotective effect of sirtuin1 (SIRT1), a deacetylase that contributes to cellular regulation. However, the pathophysiologic role of SIRT1 in podocytes remains unclear. Here, we investigated the function of SIRT1 in podocytes. We first established podocyte-specific Sirt1 knockout (SIRT1pod−/−) mice. We then induced glomerular disease by nephrotoxic serum injection. The increase in urinary albumin excretion and BUN and the severity of glomerular injury were all significantly greater in SIRT1pod−/− mice than in wild-type mice. Western blot analysis and immunofluorescence showed a significant decrease in podocyte-specific proteins in SIRT1pod−/− mice, and electron microscopy showed marked exacerbation of podocyte injury, including actin cytoskeleton derangement in SIRT1pod−/− mice compared with wild-type mice. Protamine sulfate-induced podocyte injury was also exacerbated by podocyte-specific SIRT1 deficiency. In vitro, actin cytoskeleton derangement in H2O2-treated podocytes became prominent when the cells were pretreated with SIRT1 inhibitors. Conversely, this H2O2-induced derangement was ameliorated by SIRT1 activation. Furthermore, SIRT1 activation deacetylated the actin-binding and -polymerizing protein cortactin in the nucleus and facilitated deacetylated cortactin localization in the cytoplasm. Cortactin knockdown or inhibition of the nuclear export of cortactin induced actin cytoskeleton derangement and dissociation of cortactin from F-actin, suggesting the necessity of cytoplasmic cortactin for maintenance of the actin cytoskeleton. Taken together, these findings indicate that SIRT1 protects podocytes and prevents glomerular injury by deacetylating cortactin and thereby, maintaining actin cytoskeleton integrity. PMID:25424328

Requirement for an intact T-cell actin and tubulin cytoskeleton for efficient assembly and spread of human immunodeficiency virus type 1.

PubMed

Jolly, Clare; Mitar, Ivonne; Sattentau, Quentin J

2007-06-01

Human immunodeficiency virus type 1 (HIV-1) infection of CD4(+) T cells leads to the production of new virions that assemble at the plasma membrane. Gag and Env accumulate in the context of lipid rafts at the inner and outer leaflets of the plasma membrane, respectively, forming polarized domains from which HIV-1 buds. HIV-1 budding can result in either release of cell-free virions or direct cell-cell spread via a virological synapse (VS). The recruitment of Gag and Env to these plasma membrane caps in T cells is poorly understood but may require elements of the T-cell secretory apparatus coordinated by the cytoskeleton. Using fixed-cell immunofluorescence labeling and confocal microscopy, we observed a high percentage of HIV-1-infected T cells with polarized Env and Gag in capped, lipid raft-like assembly domains. Treatment of infected T cells with inhibitors of actin or tubulin remodeling disrupted Gag and Env compartmentalization within the polarized raft-like domains. Depolymerization of the actin cytoskeleton reduced Gag release and viral infectivity, and actin and tubulin inhibitors reduced Env incorporation into virions. Live- and fixed-cell confocal imaging and assay of de novo DNA synthesis by real-time PCR allowed quantification of HIV-1 cell-cell transfer. Inhibition of actin and tubulin remodeling in infected cells interfered with cell-cell spread across a VS and reduced new viral DNA synthesis. Based on these data, we propose that HIV-1 requires both actin and tubulin components of the T-cell cytoskeleton to direct its assembly and budding and to elaborate a functional VS.

Function of actin cytoskeleton in gravisensing during spaceflight

NASA Astrophysics Data System (ADS)

Hughes-Fulford, M.

Since astronauts and cosmonauts have significant bone loss in microgravity, we hypothesized that there would be physiological changes in cellular bone growth in the absence of gravity. Our first experiments on STS-56 demonstrated that quiescent osteoblasts activated by sera under microgravity conditions had a significant 60% reduction in growth (p<0.001) and a paradoxical 2 fold increase in release of autocrine PGE2 when compared to ground controls. In addition, there was a significant collapse of the actin cytoskeleton and loss of focal adhesions after 4 days of growth in microgravity. Other investigators have made similar observations of cytoskeletal modifications in microgravity. Later studies in Biorack on STS-76, 81 and 84 confirmed the increased release of PGE2 and collapse of the cytoskeleton in cells grown in microgravity conditions, however flown cells under 1g conditions maintained normal actin cytoskeleton and fibronectin matrix. We do not think that the changes seen in the cytoskeleton are due to alterations in fibronectin message or protein synthesis since no differences were found between microgravity, 1g or ground conditions. The nuclear structure was noticeably different in the flown 0g cells with elongation of the nucleus after 24 hours of microgravity, this alteration in nuclear structure was not seen in the 1g flown or ground control cells. Further examination of total RNA in the cells showed no significant changes between the three gravity conditions suggesting specific not general physiological changes in microgravity. When osteoblast mRNA was analyzed, the immediate early genes, c-myc and cox-2 and the autocrine growth factor FGFb were down-regulated in microgravity. The inability of the 0g grown osteoblast to respond to sera activation suggests that there is a major alteration in anabolic signal transduction under microgravity conditions, most probably through the growth factor receptors and/or the associated kinase pathways. It is still

Dual effect of F-actin targeted carrier combined with antimitotic drug on aggressive colorectal cancer cytoskeleton: Allying dissimilar cell cytoskeleton disrupting mechanisms.

PubMed

Taranejoo, Shahrouz; Janmaleki, Mohsen; Pachenari, Mohammad; Seyedpour, Seyed Morteza; Chandrasekaran, Ramya; Cheng, Wenlong; Hourigan, Kerry

2016-11-20

A recent approach to colon cancer therapy is to employ selective drugs with specific extra/intracellular sites of action. Alteration of cytoskeletal protein reorganization and, subsequently, to cellular biomechanical behaviour during cancer progression highly affects the cancer cell progress. Hence, cytoskeleton targeted drugs are an important class of cancer therapy agents. We have studied viscoelastic alteration of the human colon adenocarcinoma cell line, SW48, after treatment with a drug delivery system comprising chitosan as the carrier and albendazole as the microtubule-targeting agent (MTA). For the first time, we have evaluated the biomechanical characteristics of the cell line, using the micropipette aspiration (MA) method after treatment with drug delivery systems. Surprisingly, employing a chitosan-albendazole pair, in comparison with both neat materials, resulted in more significant change in the viscoelastic parameters of cells, including the elastic constants (K 1 and K 2 ) and the coefficient of viscosity (μ). This difference was more pronounced for cancer cells after 48h of the treatment. Microtubule and actin microfilament (F-actin) contents in the cell line were studied by immunofluorescent staining. Good agreement was observed between the mechanical characteristics results and microtubule/F-actin contents of the treated SW48 cell line, which declined after treatment. The results showed that chitosan affected F-actin more, while MTA was more effective for microtubules. Toxicity studies were performed against two cancer cell lines (SW48 and MCF10CA1h) and compared to normal cells, MCF10A. The results showed cancer selectiveness, safety of formulation, and enhanced anticancer efficacy of the CS/ABZ conjugate. This study suggests that employing such a suitable pair of drug-carriers with dissimilar sites of action, thus allying the different cell cytoskeleton disrupting mechanisms, may provide a more efficient cancer therapy approach. Copyright �

Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in PTEN and p53 status

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

Djuzenova, Cholpon S., E-mail: djuzenova_t@ukw.de; Fiedler, Vanessa; Memmel, Simon

Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut),more » U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion. - Highlights: • We examine 5 glioblastoma lines on the invasion capacity and actin cytoskeleton. • Glioblastoma cell lines mutated in both p53 and PTEN were the most invasive. • Less invasive cells showed much less lamellipodia, but more actin stress fibers. • A mechanism for the differences in tumor cell invasion is proposed.« less

Global architecture of the F-actin cytoskeleton regulates cell shape-dependent endothelial mechanotransduction.

PubMed

Shao, Yue; Mann, Jennifer M; Chen, Weiqiang; Fu, Jianping

2014-03-01

Uniaxial stretch is an important biophysical regulator of cell morphology (or shape) and functions of vascular endothelial cells (ECs). However, it is unclear whether and how cell shape can independently regulate EC mechanotransductive properties under uniaxial stretch. Herein, utilizing a novel uniaxial cell-stretching device integrated with micropost force sensors, we reported the first experimental evidence showing cell shape-dependent EC mechanotransduction via cytoskeleton (CSK) contractile forces in response to uniaxial stretch. Combining experiments and theoretical modeling from first principles, we showed that it was the global architecture of the F-actin CSK that instructed the cell shape-dependent EC mechanotransductive process. Furthermore, a cell shape-dependent nature was relayed in EC mechanotransduction via dynamic focal adhesion (FA) assembly. Our results suggested a novel mechanotransductive process in ECs wherein the global architecture of the F-actin CSK, governed by cell shape, controls mechanotransduction via CSK contractile forces and force-dependent FA assembly under uniaxial stretch.

Cellular chirality arising from the self-organization of the actin cytoskeleton.

PubMed

Tee, Yee Han; Shemesh, Tom; Thiagarajan, Visalatchi; Hariadi, Rizal Fajar; Anderson, Karen L; Page, Christopher; Volkmann, Niels; Hanein, Dorit; Sivaramakrishnan, Sivaraj; Kozlov, Michael M; Bershadsky, Alexander D

2015-04-01

Cellular mechanisms underlying the development of left-right asymmetry in tissues and embryos remain obscure. Here, the development of a chiral pattern of actomyosin was revealed by studying actin cytoskeleton self-organization in cells with isotropic circular shape. A radially symmetrical system of actin bundles consisting of α-actinin-enriched radial fibres (RFs) and myosin-IIA-enriched transverse fibres (TFs) evolved spontaneously into the chiral system as a result of the unidirectional tilting of all RFs, which was accompanied by a tangential shift in the retrograde movement of TFs. We showed that myosin-IIA-dependent contractile stresses within TFs drive their movement along RFs, which grow centripetally in a formin-dependent fashion. The handedness of the chiral pattern was shown to be regulated by α-actinin-1. Computational modelling demonstrated that the dynamics of the RF-TF system can explain the pattern transition from radial to chiral. Thus, actin cytoskeleton self-organization provides built-in machinery that potentially allows cells to develop left-right asymmetry.

NETWORKED 3B: a novel protein in the actin cytoskeleton-endoplasmic reticulum interaction.

PubMed

Wang, Pengwei; Hussey, Patrick J

2017-03-01

In plants movement of the endoplasmic reticulum (ER) is dependent on the actin cytoskeleton. However little is known about proteins that link the ER membrane and the actin cytoskeleton. Here we identified a novel protein, NETWORKED 3B (NET3B), which is associated with the ER and actin cytoskeleton in vivo. NET3B belongs to a superfamily of plant specific actin binding proteins, the NETWORKED family. NET3B associates with the actin cytoskeleton in vivo through an N-terminal NET actin binding (NAB) domain, which has been well-characterized in other members of the NET family. A three amino acid insertion, Val-Glu-Asp, in the NAB domain of NET3B appears to lower its ability to localize to the actin cytoskeleton compared with NET1A, the founding member of the NET family. The C-terminal domain of NET3B links the protein to the ER. Overexpression of NET3B enhanced the association between the ER and the actin cytoskeleton, and the extent of this association was dependent on the amount of NET3B available. Another effect of NET3B overexpression was a reduction in ER membrane diffusion. In conclusion, our results revealed that NET3B modulates ER and actin cytoskeleton interactions in higher plants. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Cell Adhesions: Actin-Based Modules that Mediate Cell-Extracellular Matrix and Cell-Cell Interactions

PubMed Central

Bachir, Alexia; Horwitz, Alan Rick; Nelson, W. James; Bianchini, Julie M.

2018-01-01

Cell adhesions link cells to the extracellular matrix (ECM) and to each other, and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping functional modules. These modules establish physical association with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as sense and translate the mechanical properties of the cellular environment to changes in cell organization and behavior. In this chapter we discuss the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions, and how adhesion molecules mediate crosstalk between cell-ECM and cell-cell adhesion sites. PMID:28679638

Movers and shakers: cell cytoskeleton in cancer metastasis

PubMed Central

Fife, C M; McCarroll, J A; Kavallaris, M

2014-01-01

Metastasis is responsible for the greatest number of cancer deaths. Metastatic disease, or the movement of cancer cells from one site to another, is a complex process requiring dramatic remodelling of the cell cytoskeleton. The various components of the cytoskeleton, actin (microfilaments), microtubules (MTs) and intermediate filaments, are highly integrated and their functions are well orchestrated in normal cells. In contrast, mutations and abnormal expression of cytoskeletal and cytoskeletal-associated proteins play an important role in the ability of cancer cells to resist chemotherapy and metastasize. Studies on the role of actin and its interacting partners have highlighted key signalling pathways, such as the Rho GTPases, and downstream effector proteins that, through the cytoskeleton, mediate tumour cell migration, invasion and metastasis. An emerging role for MTs in tumour cell metastasis is being unravelled and there is increasing interest in the crosstalk between key MT interacting proteins and the actin cytoskeleton, which may provide novel treatment avenues for metastatic disease. Improved understanding of how the cytoskeleton and its interacting partners influence tumour cell migration and metastasis has led to the development of novel therapeutics against aggressive and metastatic disease. Linked Articles This article is part of a themed section on Cytoskeleton, Extracellular Matrix, Cell Migration, Wound Healing and Related Topics. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-24 PMID:24665826

Requirement of the actin cytoskeleton for the association of nectins with other cell adhesion molecules at adherens and tight junctions in MDCK cells.

PubMed

Yamada, Akio; Irie, Kenji; Fukuhara, Atsunori; Ooshio, Takako; Takai, Yoshimi

2004-09-01

Nectins, Ca(2+)-independent immunoglobulin-like cell adhesion molecules (CAMs), first form cell-cell adhesion where cadherins are recruited, forming adherens junctions (AJs) in epithelial cells and fibroblasts. In addition, nectins recruit claudins, occludin, and junctional adhesion molecules (JAMs) to the apical side of AJs, forming tight junctions (TJs) in epithelial cells. Nectins are associated with these CAMs through peripheral membrane proteins (PMPs), many of which are actin filament-binding proteins. We examined here the roles of the actin cytoskeleton in the association of nectins with other CAMs in MDCK cells stably expressing exogenous nectin-1. The nectin-1-based cell-cell adhesion was formed and maintained irrespective of the presence and absence of the actin filament-disrupting agents, such as cytochalasin D and latrunculin A. In the presence of these agents, only afadin remained at the nectin-1-based cell-cell adhesion sites, whereas E-cadherin and other PMPs at AJs, alpha-catenin, beta-catenin, vinculin, alpha-actinin, ADIP, and LMO7, were not concentrated there. The CAMs at TJs, claudin-1, occludin and JAM-1, or the PMPs at TJs, ZO-1 and MAGI-1, were not concentrated there, either. These results indicate that the actin cytoskeleton is required for the association of the nectin-afadin unit with other CAMs and PMPs at AJs and TJs.

Distinct effect of actin cytoskeleton disassembly on exo- and endocytic events in a membrane patch of rat melanotrophs.

PubMed

Chowdhury, Helena H; Kreft, Marko; Zorec, Robert

2002-12-15

We used the cell-attached mode of patch-clamp technique to measure discrete attofarad steps in membrane capacitance (C(m)), reporting area changes in the plasma membrane due to unitary exocytic and endocytic events. To investigate the role of the actin cytoskeleton in elementary exocytic and endocytic events, neuroendocrine rat melanotrophs were treated with Clostridium spiroforme toxin (CST), which specifically depolymerises F-actin. The average amplitude of exocytic events was not significantly different in control and in CST-treated cells. However, the amplitude of endocytic events was significantly smaller in CST-treated cells as compared to controls. The frequency of exocytic events increased by 2-fold in CST-treated cells relative to controls. In control cells the average frequency of exocytic events (upsilon;(exo)) was lower than the frequency of endocytic events (upsilon;(endo)) with a ratio upsilon;(exo)/upsilon;(endo) < 1. In the toxin treated cells, the predominant process was exocytosis with a ratio (upsilon;(exo)/upsilon;(endo) > 1). To study the coupling between the two processes, the slopes of regression lines relating upsilon;(exo) and upsilon;(endo) in a given patch of membrane were studied. The slopes of regression lines were similar, whereas the line intercepts with the y-axis were significantly different. The increased frequency of unitary exocytic events in CST-treated cells is consistent with the view, that the actin cytoskeleton acts as a barrier for exocytosis. While the disassembly of the actin cytoskeleton diminishes the size of unitary endocytic events, suggesting an important role of the actin cytoskeleton in determining the size of endocytic vesicles, the coupling between exocytosis and endocytosis in a given patch of membrane was independent of the state of the actin cytoskeleton.

Distinct effect of actin cytoskeleton disassembly on exo- and endocytic events in a membrane patch of rat melanotrophs

PubMed Central

Chowdhury, Helena H; Kreft, Marko; Zorec, Robert

2002-01-01

We used the cell-attached mode of patch-clamp technique to measure discrete attofarad steps in membrane capacitance (Cm), reporting area changes in the plasma membrane due to unitary exocytic and endocytic events. To investigate the role of the actin cytoskeleton in elementary exocytic and endocytic events, neuroendocrine rat melanotrophs were treated with Clostridium spiroforme toxin (CST), which specifically depolymerises F-actin. The average amplitude of exocytic events was not significantly different in control and in CST-treated cells. However, the amplitude of endocytic events was significantly smaller in CST-treated cells as compared to controls. The frequency of exocytic events increased by 2-fold in CST-treated cells relative to controls. In control cells the average frequency of exocytic events (νexo) was lower than the frequency of endocytic events (νendo) with a ratio νexo/νendo < 1. In the toxin treated cells, the predominant process was exocytosis with a ratio (νexo/νendo > 1). To study the coupling between the two processes, the slopes of regression lines relating νexo and νendo in a given patch of membrane were studied. The slopes of regression lines were similar, whereas the line intercepts with the y-axis were significantly different. The increased frequency of unitary exocytic events in CST-treated cells is consistent with the view, that the actin cytoskeleton acts as a barrier for exocytosis. While the disassembly of the actin cytoskeleton diminishes the size of unitary endocytic events, suggesting an important role of the actin cytoskeleton in determining the size of endocytic vesicles, the coupling between exocytosis and endocytosis in a given patch of membrane was independent of the state of the actin cytoskeleton. PMID:12482893

Design and evaluation of Actichip, a thematic microarray for the study of the actin cytoskeleton

PubMed Central

Muller, Jean; Mehlen, André; Vetter, Guillaume; Yatskou, Mikalai; Muller, Arnaud; Chalmel, Frédéric; Poch, Olivier; Friederich, Evelyne; Vallar, Laurent

2007-01-01

Background The actin cytoskeleton plays a crucial role in supporting and regulating numerous cellular processes. Mutations or alterations in the expression levels affecting the actin cytoskeleton system or related regulatory mechanisms are often associated with complex diseases such as cancer. Understanding how qualitative or quantitative changes in expression of the set of actin cytoskeleton genes are integrated to control actin dynamics and organisation is currently a challenge and should provide insights in identifying potential targets for drug discovery. Here we report the development of a dedicated microarray, the Actichip, containing 60-mer oligonucleotide probes for 327 genes selected for transcriptome analysis of the human actin cytoskeleton. Results Genomic data and sequence analysis features were retrieved from GenBank and stored in an integrative database called Actinome. From these data, probes were designed using a home-made program (CADO4MI) allowing sequence refinement and improved probe specificity by combining the complementary information recovered from the UniGene and RefSeq databases. Actichip performance was analysed by hybridisation with RNAs extracted from epithelial MCF-7 cells and human skeletal muscle. Using thoroughly standardised procedures, we obtained microarray images with excellent quality resulting in high data reproducibility. Actichip displayed a large dynamic range extending over three logs with a limit of sensitivity between one and ten copies of transcript per cell. The array allowed accurate detection of small changes in gene expression and reliable classification of samples based on the expression profiles of tissue-specific genes. When compared to two other oligonucleotide microarray platforms, Actichip showed similar sensitivity and concordant expression ratios. Moreover, Actichip was able to discriminate the highly similar actin isoforms whereas the two other platforms did not. Conclusion Our data demonstrate that

Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy.

PubMed

Engelke, Hanna; Heinrich, Doris; Rädler, Joachim O

2010-12-22

The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.

Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.

PubMed

Lopata, Anna; Hughes, Ruth; Tiede, Christian; Heissler, Sarah M; Sellers, James R; Knight, Peter J; Tomlinson, Darren; Peckham, Michelle

2018-04-26

Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.

Movers and shakers: cell cytoskeleton in cancer metastasis.

PubMed

Fife, C M; McCarroll, J A; Kavallaris, M

2014-12-01

Metastasis is responsible for the greatest number of cancer deaths. Metastatic disease, or the movement of cancer cells from one site to another, is a complex process requiring dramatic remodelling of the cell cytoskeleton. The various components of the cytoskeleton, actin (microfilaments), microtubules (MTs) and intermediate filaments, are highly integrated and their functions are well orchestrated in normal cells. In contrast, mutations and abnormal expression of cytoskeletal and cytoskeletal-associated proteins play an important role in the ability of cancer cells to resist chemotherapy and metastasize. Studies on the role of actin and its interacting partners have highlighted key signalling pathways, such as the Rho GTPases, and downstream effector proteins that, through the cytoskeleton, mediate tumour cell migration, invasion and metastasis. An emerging role for MTs in tumour cell metastasis is being unravelled and there is increasing interest in the crosstalk between key MT interacting proteins and the actin cytoskeleton, which may provide novel treatment avenues for metastatic disease. Improved understanding of how the cytoskeleton and its interacting partners influence tumour cell migration and metastasis has led to the development of novel therapeutics against aggressive and metastatic disease. This article is part of a themed section on Cytoskeleton, Extracellular Matrix, Cell Migration, Wound Healing and Related Topics. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-24. © 2014 The British Pharmacological Society.

Protein Disulfide Isomerase Directly Interacts with β-Actin Cys374 and Regulates Cytoskeleton Reorganization*

PubMed Central

Sobierajska, Katarzyna; Skurzynski, Szymon; Stasiak, Marta; Kryczka, Jakub; Cierniewski, Czeslaw S.; Swiatkowska, Maria

2014-01-01

Recent studies support the role of cysteine oxidation in actin cytoskeleton reorganization during cell adhesion. The aim of this study was to explain whether protein disulfide isomerase (PDI) is responsible for the thiol-disulfide rearrangement in the β-actin molecule of adhering cells. First, we showed that PDI forms a disulfide-bonded complex with β-actin with a molecular mass of 110 kDa. Specific interaction of both proteins was demonstrated by a solid phase binding assay, surface plasmon resonance analysis, and immunoprecipitation experiments. Second, using confocal microscopy, we found that both proteins colocalized when spreading MEG-01 cells on fibronectin. Colocalization of PDI and β-actin could be abolished by the membrane-permeable sulfhydryl blocker, N-ethylmaleimide, by the RGD peptide, and by anti-αIIbβ3 antibodies. Consequently, down-regulation of PDI expression by antisense oligonucleotides impaired the spreading of cells and initiated reorganization of the cytoskeleton. Third, because of transfection experiments followed by immunoprecipitation and confocal analysis, we provided evidence that PDI binds to the β-actin Cys374 thiol. Formation of the β-actin-PDI complex was mediated by integrin-dependent signaling in response to the adhesion of cells to the extracellular matrix. Our data suggest that PDI is released from subcellular compartments to the cytosol and translocated toward the periphery of the cell, where it forms a disulfide bond with β-actin when MEG-01 cells adhere via the αIIbβ3 integrin to fibronectin. Thus, PDI appears to regulate cytoskeletal reorganization by the thiol-disulfide exchange in β-actin via a redox-dependent mechanism. PMID:24415753

The cell wall of Arabidopsis thaliana influences actin network dynamics.

PubMed

Tolmie, Frances; Poulet, Axel; McKenna, Joseph; Sassmann, Stefan; Graumann, Katja; Deeks, Michael; Runions, John

2017-07-20

In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Diadenosine tetraphosphate-gating of cardiac K(ATP) channels requires intact actin cytoskeleton.

PubMed

Jovanović, S; Jovanović, A

2001-09-01

Diadenosine polyphosphates (ApnA) have been recently discovered in the heart, and their levels found to be regulated by ischemia. These signaling molecules are believed to regulate cellular processes that alarm a cell to metabolic stress. In particular, changes in cardiac diadenosine polyphosphates (ApnA) levels may contribute to the regulation of ATP-sensitive K+ (K(ATP)) channel activity, an ion channel that couples the cellular metabolic state with membrane excitability. A feature of myocardial ischemia is the disruption of the actin cytoskeleton which critically regulates the behavior of K(ATP) channels. Whether the integrity of actin microfilaments regulates the interaction of ApnA with K(ATP) channels is not known. The inside-out configuration of the patch-clamp technique was applied to cardiomyocytes isolated from guinea-pig heart. Following patch excision, the prototype dinucleotide, diadenosine tetraphosphate (Ap4A), inhibited K(ATP) channel opening. Treatment of the internal side of membrane patches with either cytochalasin B or DNase I, disrupters of the actin cytoskeleton, prevented Ap4A-induced inhibition of K(ATP) channel opening. Application of purified actin to DNase-treated membrane patches restored the ability of Ap4A to close K(ATP) channels. This study shows that inhibition of cardiac K(ATP) channel by Ap4A, a putative alarmone, requires intact subsarcolemmal actin network. Such interaction between K(ATP) channels, the cardiomyocyte cytoskeleton and intracellular Ap4A could affect different channel-dependent functions.

The actin cytoskeleton may control the polar distribution of an auxin transport protein

NASA Technical Reports Server (NTRS)

Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

2000-01-01

The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

The actin cytoskeleton may control the polar distribution of an auxin transport protein.

PubMed

Muday, G K; Hu, S; Brady, S R

2000-06-01

The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

High level of reactive oxygen species impaired mesenchymal stem cell migration via overpolymerization of F-actin cytoskeleton in systemic lupus erythematosus.

PubMed

Shi, D; Li, X; Chen, H; Che, N; Zhou, S; Lu, Z; Shi, S; Sun, L

2014-12-01

Some lines of evidence have demonstrated abnormalities of bone marrow mesenchymal stem cells (MSCs) in systemic lupus erythematosus (SLE) patients, characterized by defective phenotype of MSCs and slower growth with enhanced apoptosis and senescence. However, whether SLE MSCs demonstrate aberrant migration capacity or abnormalities in cytoskeleton are issues that remain poorly understood. In this study, we found that MSCs from SLE patients did show impairment in migration capacity as well as abnormalities in F-actin cytoskeleton, accompanied by a high level of intracellular reactive oxygen species (ROS). When normal MSCs were treated in vitro with H2O2, which increases intracellular ROS level as an oxidant, both reorganization of F-actin cytoskeleton and impairment of migration capability were observed. On the other hand, treatment with N-acetylcysteine (NAC), as an exogenous antioxidant, made F-actin more orderly and increased migration ratio in SLE MSCs. In addition, oral administration of NAC markedly reduced serum autoantibody levels and ameliorated lupus nephritis (LN) in MRL/lpr mice, partially reversing the abnormalities of MSCs. These results indicate that overpolymerization of F-actin cytoskeleton, which may be associated with high levels of ROS, causes impairment in the migration capacity of SLE MSCs and that oral administration of NAC may have potential therapeutic effects on MRL/lpr mice. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Actin cytoskeleton rearrangements in Arabidopsis roots under stress and during gravitropic response

NASA Astrophysics Data System (ADS)

Pozhvanov, Gregory; Medvedev, Sergei; Suslov, Dmitry; Demidchik, Vadim

Among environmental factors, gravity vector is the only one which is constant in direction and accompanies the whole plant ontogenesis. That said, gravity vector can be considered as an essential factor for correct development of plants. Gravitropism is a plant growth response against changing its position relative to the gravity vector. It is well estableshed that gravitropism is directed by auxin redistribution across the gravistimulated organ. In addition to auxin, actin cytoskeleton was shown to be involved in gravitropism at different stages: gravity perception, signal transduction and gravitropic bending formation. However, the relationship between IAA and actin is still under discussion. In this work we studied rearrangements of actin cytoskeleton during root gravitropic response. Actin microfilaments were visualized in vivo in GFP-fABD2 transgenic Arabidopsis plants, and their angle distribution was acquired from MicroFilament Analyzer software. The curvature of actin microfilaments in root elongation zone was shown to be increased within 30-60 min of gravistimulation, the fraction of axially oriented microfilaments decreased with a concomitant increase in the fraction of oblique and transversally oriented microfilaments. In particular, the fraction of transversally oriented microfilaments (i.e. parallel to the gravity vector) increased 3-5 times. Under 10 min of sub-lethal salt stress impact, actin microfilament orientations widened from an initial axial orientation to a set of peaks at 15(°) , 45(°) and 90(°) . We conclude that the actin cytoskeleton rearrangements observed are associated with the regulation of basic mechanisms of cell extension growth by which the gravitropic bending is formed. Having common stress-related features, gravity-induced actin cytoskeleton rearrangement is slower but results in higher number of g-vector-parallel microfilaments when compared to salt stress-induced rearrangement. Also, differences in gravistimulated root

Regulation of organ straightening and plant posture by an actin-myosin XI cytoskeleton.

PubMed

Okamoto, Keishi; Ueda, Haruko; Shimada, Tomoo; Tamura, Kentaro; Kato, Takehide; Tasaka, Masao; Morita, Miyo Terao; Hara-Nishimura, Ikuko

2015-03-23

Plants are able to bend nearly every organ in response to environmental stimuli such as gravity and light(1,2). After this first phase, the responses to stimuli are restrained by an independent mechanism, or even reversed, so that the organ will stop bending and attain its desired posture. This phenomenon of organ straightening has been called autotropism(3) and autostraightening(4) and modelled as proprioception(5). However, the machinery that drives organ straightening and where it occurs are mostly unknown. Here, we show that the straightening of inflorescence stems is regulated by an actin-myosin XI cytoskeleton in specialized immature fibre cells that are parallel to the stem and encircle it in a thin band. Arabidopsis mutants defective in myosin XI (specifically XIf and XIk) or ACTIN8 exhibit hyperbending of stems in response to gravity, an effect independent of the physical properties of the shoots. The actin-myosin XI cytoskeleton enables organs to attain their new position more rapidly than would an oscillating series of diminishing overshoots in environmental stimuli. We propose that the long actin filaments in elongating fibre cells act as a bending tensile sensor to perceive the organ's posture and trigger the straightening system.

Actin-Based Adhesion Modules Mediate Cell Interactions with the Extracellular Matrix and Neighboring Cells.

PubMed

Bachir, Alexia I; Horwitz, Alan Rick; Nelson, W James; Bianchini, Julie M

2017-07-05

Cell adhesions link cells to the extracellular matrix (ECM) and to each other and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping, functional modules. These modules establish physical associations with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as to sense and translate the mechanical properties of the cellular environment into changes in cell organization and behavior. Here, we review the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions and how adhesion molecules mediate cross talk between cell-ECM and cell-cell adhesion sites. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

Single-cell mechanics--An experimental-computational method for quantifying the membrane-cytoskeleton elasticity of cells.

PubMed

Tartibi, M; Liu, Y X; Liu, G-Y; Komvopoulos, K

2015-11-01

The membrane-cytoskeleton system plays a major role in cell adhesion, growth, migration, and differentiation. F-actin filaments, cross-linkers, binding proteins that bundle F-actin filaments to form the actin cytoskeleton, and integrins that connect the actin cytoskeleton network to the cell plasma membrane and extracellular matrix are major cytoskeleton constituents. Thus, the cell cytoskeleton is a complex composite that can assume different shapes. Atomic force microscopy (AFM)-based techniques have been used to measure cytoskeleton material properties without much attention to cell shape. A recently developed surface chemical patterning method for long-term single-cell culture was used to seed individual cells on circular patterns. A continuum-based cell model, which uses as input the force-displacement response obtained with a modified AFM setup and relates the membrane-cytoskeleton elastic behavior to the cell geometry, while treating all other subcellular components suspended in the cytoplasmic liquid (gel) as an incompressible fluid, is presented and validated by experimental results. The developed analytical-experimental methodology establishes a framework for quantifying the membrane-cytoskeleton elasticity of live cells. This capability may have immense implications in cell biology, particularly in studies seeking to establish correlations between membrane-cytoskeleton elasticity and cell disease, mortality, differentiation, and migration, and provide insight into cell infiltration through nonwoven fibrous scaffolds. The present method can be further extended to analyze membrane-cytoskeleton viscoelasticity, examine the role of other subcellular components (e.g., nucleus envelope) in cell elasticity, and elucidate the effects of mechanical stimuli on cell differentiation and motility. This is the first study to decouple the membrane-cytoskeleton elasticity from cell stiffness and introduce an effective approach for measuring the elastic modulus. The

PKCθ links proximal T cell and Notch signaling through localized regulation of the actin cytoskeleton

PubMed Central

Britton, Graham J; Ambler, Rachel; Clark, Danielle J; Hill, Elaine V; Tunbridge, Helen M; McNally, Kerrie E; Burton, Bronwen R; Butterweck, Philomena; Sabatos-Peyton, Catherine; Hampton-O’Neil, Lea A; Verkade, Paul; Wülfing, Christoph; Wraith, David Cameron

2017-01-01

Notch is a critical regulator of T cell differentiation and is activated through proteolytic cleavage in response to ligand engagement. Using murine myelin-reactive CD4 T cells, we demonstrate that proximal T cell signaling modulates Notch activation by a spatiotemporally constrained mechanism. The protein kinase PKCθ is a critical mediator of signaling by the T cell antigen receptor and the principal costimulatory receptor CD28. PKCθ selectively inactivates the negative regulator of F-actin generation, Coronin 1A, at the center of the T cell interface with the antigen presenting cell (APC). This allows for effective generation of the large actin-based lamellum required for recruitment of the Notch-processing membrane metalloproteinase ADAM10. Such enhancement of Notch activation is critical for efficient T cell proliferation and Th17 differentiation. We reveal a novel mechanism that, through modulation of the cytoskeleton, controls Notch activation at the T cell:APC interface thereby linking T cell receptor and Notch signaling pathways. DOI: http://dx.doi.org/10.7554/eLife.20003.001 PMID:28112644

Sonic hedgehog signaling regulates actin cytoskeleton via Tiam1-Rac1 cascade during spine formation.

PubMed

Sasaki, Nobunari; Kurisu, Junko; Kengaku, Mineko

2010-12-01

The sonic hedgehog (Shh) pathway has essential roles in several processes during development of the vertebrate central nervous system (CNS). Here, we report that Shh regulates dendritic spine formation in hippocampal pyramidal neurons via a novel pathway that directly regulates the actin cytoskeleton. Shh signaling molecules Patched (Ptc) and Smoothened (Smo) are expressed in several types of postmitotic neurons, including cerebellar Purkinje cells and hippocampal pyramidal neurons. Knockdown of Smo induces dendritic spine formation in cultured hippocampal neurons independently of Gli-mediated transcriptional activity. Smo interacts with Tiam1, a guanine nucleotide exchange factor for Rac1, via its cytoplasmic C-terminal region. Inhibition of Tiam1 or Rac1 activity suppresses spine induction by Smo knockdown. Shh induces remodeling of the actin cytoskeleton independently of transcriptional activation in mouse embryonic fibroblasts. These findings demonstrate a novel Shh pathway that regulates the actin cytoskeleton via Tiam1-Rac1 activation. Copyright © 2010 Elsevier Inc. All rights reserved.

Enhanced gravitropism of roots with a disrupted cap actin cytoskeleton

NASA Technical Reports Server (NTRS)

Hou, Guichuan; Mohamalawari, Deepti R.; Blancaflor, Elison B.

2003-01-01

The actin cytoskeleton has been proposed to be a major player in plant gravitropism. However, understanding the role of actin in this process is far from complete. To address this problem, we conducted an analysis of the effect of Latrunculin B (Lat B), a potent actin-disrupting drug, on root gravitropism using various parameters that included detailed curvature kinetics, estimation of gravitropic sensitivity, and monitoring of curvature development after extended clinorotation. Lat B treatment resulted in a promotion of root curvature after a 90 degrees reorientation in three plant species tested. More significantly, the sensitivity of maize (Zea mays) roots to gravity was enhanced after actin disruption, as determined from a comparison of presentation time of Lat B-treated versus untreated roots. A short 10-min gravistimulus followed by extended rotation on a 1-rpm clinostat resulted in extensive gravitropic responses, manifested as curvature that often exceeded 90 degrees. Application of Lat B to the cap or elongation zone of maize roots resulted in the disruption of the actin cytoskeleton, which was confined to the area of localized Lat B application. Only roots with Lat B applied to the cap displayed the strong curvature responses after extended clinorotation. Our study demonstrates that disrupting the actin cytoskeleton in the cap leads to the persistence of a signal established by a previous gravistimulus. Therefore, actin could function in root gravitropism by providing a mechanism to regulate the proliferation of a gravitropic signal originating from the cap to allow the root to attain its correct orientation or set point angle.

Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations

PubMed Central

Westendorf, Christian; Negrete, Jose; Bae, Albert J.; Sandmann, Rabea; Bodenschatz, Eberhard; Beta, Carsten

2013-01-01

The rapid reorganization of the actin cytoskeleton in response to external stimuli is an essential property of many motile eukaryotic cells. Here, we report evidence that the actin machinery of chemotactic Dictyostelium cells operates close to an oscillatory instability. When averaging the actin response of many cells to a short pulse of the chemoattractant cAMP, we observed a transient accumulation of cortical actin reminiscent of a damped oscillation. At the single-cell level, however, the response dynamics ranged from short, strongly damped responses to slowly decaying, weakly damped oscillations. Furthermore, in a small subpopulation, we observed self-sustained oscillations in the cortical F-actin concentration. To substantiate that an oscillatory mechanism governs the actin dynamics in these cells, we systematically exposed a large number of cells to periodic pulse trains of different frequencies. Our results indicate a resonance peak at a stimulation period of around 20 s. We propose a delayed feedback model that explains our experimental findings based on a time-delay in the regulatory network of the actin system. To test the model, we performed stimulation experiments with cells that express GFP-tagged fusion proteins of Coronin and actin-interacting protein 1, as well as knockout mutants that lack Coronin and actin-interacting protein 1. These actin-binding proteins enhance the disassembly of actin filaments and thus allow us to estimate the delay time in the regulatory feedback loop. Based on this independent estimate, our model predicts an intrinsic period of 20 s, which agrees with the resonance observed in our periodic stimulation experiments. PMID:23431176

The actin cytoskeleton in store-mediated calcium entry

PubMed Central

Rosado, Juan A; Sage, Stewart O

2000-01-01

Store-mediated Ca2+ entry is the main pathway for Ca2+ influx in platelets and many other cells. Several hypotheses have considered both direct and indirect coupling mechanisms between the endoplasmic reticulum and the plasma membrane. Here we pay particular attention to new insights into the regulation of store-mediated Ca2+ entry: the role of the cytoskeleton in a secretion-like coupling model. In this model, Ca2+ entry may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, that shows close parallels to the events mediating secretion. As with secretion, the actin cytoskeleton plays an inhibitory role in the activation of Ca2+ entry by preventing the approach and coupling of the endoplasmic reticulum with the plasma membrane, making cytoskeletal remodelling a key event in the activation of Ca2+ entry. We also review recent advances investigating the regulation of store-mediated Ca2+ entry by small GTPases and phosphoinositides, which might be involved in the store-mediated Ca2+ entry pathway through roles in the remodelling of the cytoskeleton. PMID:10896713

Disruption of the actin cytoskeleton results in the promotion of gravitropism in inflorescence stems and hypocotyls of Arabidopsis

NASA Technical Reports Server (NTRS)

Yamamoto, Kazuyoshi; Kiss, John Z.

2002-01-01

The actin cytoskeleton is hypothesized to play a major role in gravity perception and transduction mechanisms in roots of plants. To determine whether actin microfilaments (MFs) are involved in these processes in stem-like organs, we studied gravitropism in Arabidopsis inflorescence stems and hypocotyls. Localization studies using Alexa Fluor-phalloidin in conjugation with confocal microscopy demonstrated a longitudinally and transversely oriented actin MF network in endodermal cells of stems and hypocotyls. Latrunculin B (Lat-B) treatment of hypocotyls caused depolymerization of actin MFs in endodermal cells and a significant reduction of hypocotyl growth rates. Actin MFs in Lat-B-treated inflorescence stems also were disrupted, but growth rates were not affected. Despite disruption of the actin cytoskeleton in these two organs, Lat-B-treated stems and hypocotyls exhibited a promotion of gravitropic curvature in response to reorientation. In contrast, Lat-B reduced gravitropic curvature in roots but also reduced the growth rate. Thus, in contrast to prevailing hypotheses, our results suggest that actin MFs are not a necessary component of gravitropism in inflorescence stems and hypocotyls. Furthermore, this is the first study to demonstrate a prominent actin MF network in endodermal cells in the putative gravity-perceiving cells in stems.

Effects of altered gravity on the cell cycle, actin cytoskeleton and proteome in Physarum polycephalum

NASA Astrophysics Data System (ADS)

He, Jie; Zhang, Xiaoxian; Gao, Yong; Li, Shuijie; Sun, Yeqing

Some researchers suggest that the changes of cell cycle under the effect of microgravity may be associated with many serious adverse physiological changes. In the search for underlying mechanisms and possible new countermeasures, we used the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony to study the effects of altered gravity on the cell cycle, actin cytoskeleton and proteome. In parallel, the cell cycle was analyzed in Physarum incubated (1) in altered gravity for 20 h, (2) in altered gravity for 40 h, (3) in altered gravity for 80 h, and (4) in ground controls. The cell cycle, the actin cytoskeleton, and proteome in the altered gravity and ground controls were examined. The results indicated that the duration of the G2 phase was lengthened 20 min in high aspect ratio vessel (HARV) for 20 h, and prolonged 2 h in altered gravity either for 40 h or for 80 h, whereas the duration of other phases in the cell cycle was unchanged with respect to the control. The microfilaments in G2 phase had a reduced number of fibers and a unique abnormal morphology in altered gravity for 40 h, whereas the microfilaments in other phases of cell cycle were unchanged when compared to controls. Employing classical two-dimensional electrophoresis (2-DE), we examined the effect of the altered gravity on P. polycephalum proteins. The increase in the duration of G2 phase in altered gravity for 40 h was accompanied by changes in the 2-DE protein profiles, over controls. Out of a total of 200 protein spots investigated in G2 phase, which were reproducible in repeated experiments, 72 protein spots were visually identified as specially expressed, and 11 proteins were up-regulated by 2-fold and 28 proteins were down-regulated by 2-fold over controls. Out of a total of three low-expressed proteins in G2 phase in altered gravity for 40 h, two proteins were unknown proteins, and one protein was spherulin 3b by MALDI-TOF mass spectrometry (MS

Radiographic contrast media alterate the localization of actin/band4.9 in the membrane cytoskeleton of human erythrocytes.

PubMed

Franke, R P; Scharnweber, T; Fuhrmann, R; Mrowietz, C; Wenzel, F; Krüger, A; Jung, F

2014-01-01

Different radiographic contrast media (RCM) were shown to induce morphological changes of blood cells (e.g. erythrocytes or thrombocytes) and endothelial cells. The echinocytic shape change of erythrocytes, particularly, affords alterations of the membrane cytoskeleton. The cytoskeleton plays a crucial role for the shape and deformability of the red blood cell. Disruption of the interaction between components of the red blood cell membrane cytoskeleton may cause a loss of structural and functional integrity of the membrane. In this study band4.9 and actin as components of the cytoskeletal junctional complex were examined in human erythrocytes after suspension in autologous plasma or in plasma RCM mixtures (30% v/v Iodixanol-320 or Iopromide-370) followed by a successive double staining with TRITC-/FITC-coupled monoclonal antibodies. After adding Iopromide-370 to the plasma in practically none of the cells the rounded conformation of the membrane cytoskeleton - as it appeared in cells suspended in autologous plasma - was found. In addition, Iopromide-370 induced thin lines and coarse knob-like structures of band4.9 at the cell periphery while most cell centers were devoid of band4.9, and a box-like arrangement of bands of band4.9. A dissociation between colours red (actin) and green (band4.9) occurred as well. In contrast, erythrocytes suspended in a plasma/Iodixanol-320 mixture showed a membrane cytoskeleton comparable to cells suspended in autologous plasma, Similar results were found with respect to the distribution of actin. This study revealed for the first time RCM-dependent differences in band4.9 activities as possible pathophysiological mechanism for the chemotoxicity of radiographic contrast media.

Simulated Microgravity Alters Actin Cytoskeleton and Integrin-Mediated Focal Adhesions of Cultured Human Mesenchymal Stromal Cells

NASA Astrophysics Data System (ADS)

Gershovich, P. M.; Gershovic, J. G.; Buravkova, L. B.

2008-06-01

Cytoskeletal alterations occur in several cell types including lymphocytes, glial cells, and osteoblasts, during spaceflight and under simulated microgravity (SMG) (3, 4). One potential mechanism for cytoskeletal gravisensitivity is disruption of extracellular matrix (ECM) and integrin interactions. Focal adhesions are specialized sites of cell-matrix interaction composed of integrins and the diversity of focal adhesion-associated cytoplasmic proteins including vinculin, talin, α-actinin, and actin filaments (4, 5). Integrins produce signals essential for proper cellular function, survival and differentiation. Therefore, we investigated the effects of SMG on F-actin cytoskeleton structure, vinculin focal adhesions, expression of some integrin subtypes and cellular adhesion molecules (CAMs) in mesenchymal stem cells derived from human bone marrow (hMSCs). Simulated microgravity was produced by 3D-clinostat (Dutch Space, Netherlands). Staining of actin fibers with TRITC-phalloidin showed reorganization even after 30 minutes of simulated microgravity. The increasing of cells number with abnormal F-actin was observed after subsequent terms of 3D-clinorotation (6, 24, 48, 120 hours). Randomization of gravity vector altered dimensional structure of stress fibers and resulted in remodeling of actin fibers inside the cells. In addition, we observed vinculin redistribution inside the cells after 6 hours and prolonged terms of clinorotation. Tubulin fibers in a contrast with F-actin and vinculin didn't show any reorganization even after long 3Dclinorotation (120 hours). The expression of integrin α2 increased 1,5-6-fold in clinorotated hMSCs. Also we observed decrease in number of VCAM-1-positive cells and changes in expression of ICAM-1. Taken together, our findings indicate that SMG leads to microfilament and adhesion alterations of hMSCs most probably associated with involvement of some integrin subtypes.

Organization and dynamics of the actin cytoskeleton during dendritic spine morphological remodeling.

PubMed

Chazeau, Anaël; Giannone, Grégory

2016-08-01

In the central nervous system, most excitatory post-synapses are small subcellular structures called dendritic spines. Their structure and morphological remodeling are tightly coupled to changes in synaptic transmission. The F-actin cytoskeleton is the main driving force of dendritic spine remodeling and sustains synaptic plasticity. It is therefore essential to understand how changes in synaptic transmission can regulate the organization and dynamics of actin binding proteins (ABPs). In this review, we will provide a detailed description of the organization and dynamics of F-actin and ABPs in dendritic spines and will discuss the current models explaining how the actin cytoskeleton sustains both structural and functional synaptic plasticity.

Yeast Rsp5 ubiquitin ligase affects the actin cytoskeleton in vivo and in vitro.

PubMed

Kaminska, Joanna; Spiess, Matthias; Stawiecka-Mirota, Marta; Monkaityte, Rasa; Haguenauer-Tsapis, Rosine; Urban-Grimal, Daniele; Winsor, Barbara; Zoladek, Teresa

2011-12-01

Yeast Rsp5 ubiquitin ligase is involved in several cellular processes, including endocytosis. Actin patches are sites of endocytosis, a process involving actin assembly and disassembly. Here we show Rsp5 localization in cortical patches and demonstrate its involvement in actin cytoskeleton organization and dynamics. We found that the Rsp5-F1-GFP2 N-terminal fragment and full length GFP-Rsp5 were recruited to peripheral patches that temporarily co-localized with Abp1-mCherry, a marker of actin patches. Actin cytoskeleton organization was defective in a strain lacking RSP5 or overexpressing RSP5, and this phenotype was accompanied by morphological abnormalities. Overexpression of RSP5 caused hypersensitivity of cells to Latrunculin A, an actin-depolymerizing drug and was toxic to cells lacking Las17, an activator of actin nucleation. Moreover, Rsp5 was required for efficient actin polymerization in a whole cell extract based in vitro system. Rsp5 interacted with Las17 and Las17-binding proteins, Lsb1 and Lsb2, in a GST-Rsp5-WW2/3 pull down assay. Rsp5 ubiquitinated Lsb1-HA and Lsb2-HA without directing them for degradation. Overexpression of RSP5 increased the cellular level of HA-Las17 in wild type and in lsb1Δ lsb2Δ strains in which the basal level of Las17 was already elevated. This increase was prevented in a strain devoid of Las17-binding protein Sla1 which is also a target of Rsp5 ubiquitination. Thus, Rsp5 together with Lsb1, Lsb2 and Sla1 regulate the level of Las17, an important activator of actin polymerization. Copyright © 2011 Elsevier GmbH. All rights reserved.

Alteration of the Cortical Actin Cytoskeleton Deregulates Ca2+ Signaling, Monospermic Fertilization, and Sperm Entry

PubMed Central

Puppo, A.; Chun, Jong T.; Gragnaniello, Giovanni; Garante, Ezio; Santella, Luigia

2008-01-01

Background When preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca2+) signals during the maturation process. However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear. Methodology/Principal Findings We have measured changes in intracellular Ca2+ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP3 receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton. Conclusions/Significance Our findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca2+ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy. PMID:18974786

Plant actin cytoskeleton re-modeling by plant parasitic nematodes.

PubMed

Engler, Janice de Almeida; Rodiuc, Natalia; Smertenko, Andrei; Abad, Pierre

2010-03-01

The cytoskeleton is an important component of the plant's defense mechanism against the attack of pathogenic organisms. Plants however, are defenseless against parasitic root-knot and cyst nematodes and respond to the invasion by the development of a special feeding site that supplies the parasite with nutrients required for the completion of its life cycle. Recent studies of nematode invasion under treatment with cytoskeletal drugs and in mutant plants where normal functions of the cytoskeleton have been affected, demonstrate the importance of the cytoskeleton in the establishment of a feeding site and successful nematode reproduction. It appears that in the case of microfilaments, nematodes hijack the intracellular machinery that regulates actin dynamics and modulate the organization and properties of the actin filament network. Intervening with this process reduces the nematode infection efficiency and inhibits its life cycle. This discovery uncovers a new pathway that can be exploited for the protection of plants against nematodes.

Disruption of the Actin Cytoskeleton Results in the Promotion of Gravitropism in Inflorescence Stems and Hypocotyls of Arabidopsis1

PubMed Central

Yamamoto, Kazuyoshi; Kiss, John Z.

2002-01-01

The actin cytoskeleton is hypothesized to play a major role in gravity perception and transduction mechanisms in roots of plants. To determine whether actin microfilaments (MFs) are involved in these processes in stem-like organs, we studied gravitropism in Arabidopsis inflorescence stems and hypocotyls. Localization studies using Alexa Fluor-phalloidin in conjugation with confocal microscopy demonstrated a longitudinally and transversely oriented actin MF network in endodermal cells of stems and hypocotyls. Latrunculin B (Lat-B) treatment of hypocotyls caused depolymerization of actin MFs in endodermal cells and a significant reduction of hypocotyl growth rates. Actin MFs in Lat-B-treated inflorescence stems also were disrupted, but growth rates were not affected. Despite disruption of the actin cytoskeleton in these two organs, Lat-B-treated stems and hypocotyls exhibited a promotion of gravitropic curvature in response to reorientation. In contrast, Lat-B reduced gravitropic curvature in roots but also reduced the growth rate. Thus, in contrast to prevailing hypotheses, our results suggest that actin MFs are not a necessary component of gravitropism in inflorescence stems and hypocotyls. Furthermore, this is the first study to demonstrate a prominent actin MF network in endodermal cells in the putative gravity-perceiving cells in stems. PMID:11842170

From filaments to function: The role of the plant actin cytoskeleton in pathogen perception, signaling and immunity.

PubMed

Porter, Katie; Day, Brad

2016-04-01

The eukaryotic actin cytoskeleton is required for numerous cellular processes, including cell shape, development and movement, gene expression and signal transduction, and response to biotic and abiotic stress. In recent years, research in both plants and animal systems have described a function for actin as the ideal surveillance platform, linking the function and activity of primary physiological processes to the immune system. In this review, we will highlight recent advances that have defined the regulation and breadth of function of the actin cytoskeleton as a network required for defense signaling following pathogen infection. Coupled with an overview of recent work demonstrating specific targeting of the plant actin cytoskeleton by a diversity of pathogens, including bacteria, fungi and viruses, we will highlight the importance of actin as a key signaling hub in plants, one that mediates surveillance of cellular homeostasis and the activation of specific signaling responses following pathogen perception. Based on the studies highlighted herein, we propose a working model that posits changes in actin filament organization is in and of itself a highly specific signal, which induces, regulates and physically directs stimulus-specific signaling processes, most importantly, those associated with response to pathogens. © 2015 Institute of Botany, Chinese Academy of Sciences.

The candidate tumor suppressor SASH1 interacts with the actin cytoskeleton and stimulates cell-matrix adhesion.

PubMed

Martini, Melanie; Gnann, Alexandra; Scheikl, Daniela; Holzmann, Bernhard; Janssen, Klaus-Peter

2011-11-01

SASH1, a member of the SLY-family of signal adapter proteins, is a candidate tumor suppressor in breast and colon cancer. Reduced expression of SASH1 is correlated with aggressive tumor growth, metastasis formation, and inferior prognosis. However, the biological role of SASH1 remains largely unknown. To unravel the function of SASH1, we have analyzed the intracellular localization of endogenous SASH1, and have generated structural SASH1 mutants. SASH1 localized to the nucleus as well as to the cytoplasm in epithelial cells. In addition, SASH1 was enriched in lamellipodia and membrane ruffles, where it co-distributed with the actin cytoskeleton. Moreover, we demonstrate a novel interaction of SASH1 with the oncoprotein cortactin, a known regulator of actin polymerization in lamellipodia. Enhanced SASH1 expression significantly increased the content of filamentous actin, leading to the formation of cell protrusions and elongated cell shape. This activity was mapped to the central, evolutionarily conserved domain of SASH1. Furthermore, expression of SASH1 inhibited cell migration and lead to increased cell adhesion to fibronectin and laminin, whereas knock-down of endogenous SASH1 resulted in significantly reduced cell-matrix adhesion. Taken together, our findings unravel for the first time a mechanistic role for SASH1 in tumor formation by regulating the adhesive and migratory behaviour of cancer cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Recruitment Kinetics of Tropomyosin Tpm3.1 to Actin Filament Bundles in the Cytoskeleton Is Independent of Actin Filament Kinetics.

PubMed

Appaduray, Mark A; Masedunskas, Andrius; Bryce, Nicole S; Lucas, Christine A; Warren, Sean C; Timpson, Paul; Stear, Jeffrey H; Gunning, Peter W; Hardeman, Edna C

2016-01-01

The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.

The Nebivolol action on vascular tone is dependent on actin cytoskeleton polymerization and Rho-A activity into ECs and SMCs.

PubMed

Kadi, A; de Isla, N; Moby, V; Lacolley, P; Labrude, P; Stoltz, J F; Menu, P

2014-01-01

Nitric oxide is implicated in the target action of Nebivolol, a selective β1 adrenoceptor blocker used in hypertension treatment. As the Nitric Oxide (NO) production and the actin cytoskeleton are linked, the aim of this work was to study the involvement of actin cytoskeleton on mechanism of action of Nebivolol in cultured endothelial cells. We studied the effect of Nebivolol (200 μM) on actin filaments remodeling and its impact on NO production and eNOS activation. Results showed that Nebivolol perturbs actin filaments polymerization, increases NO production and eNOS activity between 30 minutes and 1 h. Stabilization of actin filaments with phalloïdine (50 μM) abolishes Nebivolol effects on eNOS activation and NO production. Furthermore, Rho-kinase activity decreased during the first hour of Nebivolol treatment, then increased after 3 h, while actin filaments repolymerized, eNOS activation and NO production decreased. In SMCs, Nebivolol induced a decrease in the Rho-kinase activity from 1 h until 24 h of incubation. In conclusion, we suggest that Nebivolol induced NO production in Endothelial Cells (ECs) via complementary actions between actin cytoskeleton remodeling inducing eNOS activation and Rho-kinase implication. The effect of Nebivolol on ECs occurs during the first hour, this effect on SMCs seems to be maintained until 24 h, explaining persisted action of Nebivolol observed in vivo.

The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria.

PubMed

Muñoz-Espín, Daniel; Daniel, Richard; Kawai, Yoshikazu; Carballido-López, Rut; Castilla-Llorente, Virginia; Errington, Jeff; Meijer, Wilfried J J; Salas, Margarita

2009-08-11

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage ϕ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the ϕ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the ϕ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.

X-rays effects on cytoskeleton mechanics of healthy and tumor cells.

PubMed

Panzetta, Valeria; De Menna, Marta; Musella, Ida; Pugliese, Mariagabriella; Quarto, Maria; Netti, Paolo A; Fusco, Sabato

2017-01-01

Alterations in the cytoskeleton structure are frequently found in several diseases and particularly in cancer cells. It is also through the alterations of the cytoskeleton structure that cancer cells acquire most of their common features such as uncontrolled cell proliferation, cell death evasion, and the gaining of migratory and invasive characteristics. Although radiation therapies currently represent one of the most effective treatments for patients, the effects of X-irradiation on the cytoskeleton architecture are still poorly understood. In this case we investigated the effects, over time of two different doses of X-ray irradiation, on cell cytoskeletons of BALB/c3T3 and Sv40-transformed BALB/c 3T3 cells (SVT2). Biophysical parameters - focal adhesion size, actin bundles organization, and cell mechanical properties - were measured before and after irradiations (1 and 2 Gy) at 24 and 72 h, comparing the cytoskeleton properties of normal and transformed cells. The differences, before and after X-irradiation, were revealed in terms of cell morphology and deformability. Finally, such parameters were correlated to the alterations of cytoskeleton dynamics by evaluating cell adhesion at the level of focal adhesion and cytoskeleton mechanics. X-irradiation modifies the structure and the activity of cell cytoskeleton in a dose-dependent manner. For transformed cells, radiation sensitively increased cell adhesion, as indicated by paxillin-rich focal adhesion, flat morphology, a well-organized actin cytoskeleton, and intracellular mechanics. On the other hand, for normal fibroblasts IR had negligible effects on cytoskeletal and adhesive protein organization. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The role of apical cell-cell junctions and associated cytoskeleton in mechanotransduction.

PubMed

Sluysmans, Sophie; Vasileva, Ekaterina; Spadaro, Domenica; Shah, Jimit; Rouaud, Florian; Citi, Sandra

2017-04-01

Tissues of multicellular organisms are characterised by several types of specialised cell-cell junctions. In vertebrate epithelia and endothelia, tight and adherens junctions (AJ) play critical roles in barrier and adhesion functions, and are connected to the actin and microtubule cytoskeletons. The interaction between junctions and the cytoskeleton is crucial for tissue development and physiology, and is involved in the molecular mechanisms governing cell shape, motility, growth and signalling. The machineries which functionally connect tight and AJ to the cytoskeleton comprise proteins which either bind directly to cytoskeletal filaments, or function as adaptors for regulators of the assembly and function of the cytoskeleton. In the last two decades, specific cytoskeleton-associated junctional molecules have been implicated in mechanotransduction, revealing the existence of multimolecular complexes that can sense mechanical cues and translate them into adaptation to tensile forces and biochemical signals. Here, we summarise the current knowledge about the machineries that link tight and AJ to actin filaments and microtubules, and the molecular basis for mechanotransduction at epithelial and endothelial AJ. © 2017 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

The glomerular epithelial cell anti-adhesin podocalyxin associates with the actin cytoskeleton through interactions with ezrin.

PubMed

Orlando, R A; Takeda, T; Zak, B; Schmieder, S; Benoit, V M; McQuistan, T; Furthmayr, H; Farquhar, M G

2001-08-01

During development, renal glomerular epithelial cells (podocytes) undergo extensive morphologic changes necessary for creation of the glomerular filtration apparatus. These changes include formation of interdigitating foot processes, replacement of tight junctions with slit diaphragms, and the concomitant opening of intercellular urinary spaces. It was postulated previously and confirmed recently that podocalyxin, a sialomucin, plays a major role in maintaining the urinary space open by virtue of the physicochemical properties of its highly negatively charged ectodomain. This study examined whether the highly conserved cytoplasmic tail of podocalyxin also contributes to the unique organization of podocytes by interacting with the cytoskeletal network found in their cell bodies and foot processes. By immunocytochemistry, it was shown that podocalyxin and the actin binding protein ezrin are co-expressed in podocytes and co-localize along the apical plasma membrane, where they form a co-immunoprecipitable complex. Selective detergent extraction followed by differential centrifugation revealed that some of the podocalyxin cosediments with actin filaments. Moreover, its sedimentation is dependent on polymerized actin and is mediated by complex formation with ezrin. Once formed, podocalyxin/ezrin complexes are very stable, because they are insensitive to actin depolymerization or inactivation of Rho kinase, which is known to be necessary for regulation of ezrin and to mediate Rho-dependent actin organization. These data indicate that in podocytes, podocalyxin is complexed with ezrin, which mediates its link to the actin cytoskeleton. Thus, in addition to its ectodomain, the cytoplasmic tail of podocalyxin also likely contributes to maintaining the unique podocyte morphology.

Cell-cell interactions mediate cytoskeleton organization and collective endothelial cell chemotaxis.

PubMed

Shamloo, Amir

2014-09-01

This study investigates the role of cell-cell and cell-ligand interactions in cytoskeleton organization of endothelial cells (ECs) and their directional migration within a microfluidic device. The migration of ECs in response to a biochemical factor was studied. Mathematical analysis of the cell migration pathways and cellular cytoskeleton revealed that directional migration, migration persistence length, migration speed, and cytoskeletal stress fiber alignment can be mediated by the level of cell contacts as well as the presence or absence of a biochemical polarizing factor. It was shown that in the presence of a biochemical polarizing factor, higher cell density and more frequent cell contacts has a reinforcing effect on collective cell chemotaxis. In contrast, in the absence of a polarizing factor, high cell density can decrease or suppress the ability of the cells to migrate. Also, the correlation of actin stress fiber organization and alignment with directional migration of ECs was investigated. It was shown that in the presence of a biochemical polarizing factor, stress fibers within the cytoskeleton of ECs can be significantly aligned parallel to the gradient direction when the cells have higher level of contacts. The results also show that the organization and alignment of actin stress fibers is mediated by cell adhesion junctions during collective cell migration and introduce cell-cell interactions as a key factor during collective cell chemotaxis. © 2014 Wiley Periodicals, Inc.

Asb2α-Filamin A Axis Is Essential for Actin Cytoskeleton Remodeling During Heart Development.

PubMed

Métais, Arnaud; Lamsoul, Isabelle; Melet, Armelle; Uttenweiler-Joseph, Sandrine; Poincloux, Renaud; Stefanovic, Sonia; Valière, Amélie; Gonzalez de Peredo, Anne; Stella, Alexandre; Burlet-Schiltz, Odile; Zaffran, Stéphane; Lutz, Pierre G; Moog-Lutz, Christel

2018-03-16

Heart development involves differentiation of cardiac progenitors and assembly of the contractile sarcomere apparatus of cardiomyocytes. However, little is known about the mechanisms that regulate actin cytoskeleton remodeling during cardiac cell differentiation. The Asb2α (Ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2) CRL5 (cullin 5 RING E3 ubiquitin ligase) triggers polyubiquitylation and subsequent degradation by the proteasome of FLNs (filamins). Here, we investigate the role of Asb2α in heart development and its mechanisms of action. Using Asb2 knockout embryos, we show that Asb2 is an essential gene, critical to heart morphogenesis and function, although its loss does not interfere with the overall patterning of the embryonic heart tube. We show that the Asb2α E3 ubiquitin ligase controls Flna stability in immature cardiomyocytes. Importantly, Asb2α-mediated degradation of the actin-binding protein Flna marks a previously unrecognized intermediate step in cardiac cell differentiation characterized by cell shape changes and actin cytoskeleton remodeling. We further establish that in the absence of Asb2α, myofibrils are disorganized and that heartbeats are inefficient, leading to embryonic lethality in mice. These findings identify Asb2α as an unsuspected key regulator of cardiac cell differentiation and shed light on the molecular and cellular mechanisms determining the onset of myocardial cell architecture and its link with early cardiac function. Although Flna is known to play roles in cytoskeleton organization and to be required for heart function, this study now reveals that its degradation mediated by Asb2α ensures essential functions in differentiating cardiac progenitors. © 2018 American Heart Association, Inc.

Actin Out: Regulation of the Synaptic Cytoskeleton

PubMed Central

Spence, Erin F.; Soderling, Scott H.

2015-01-01

The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during spinogenesis and experience-dependent plasticity. Multiple mutations associated with human neurodevelopmental and psychiatric disorders involve genes that encode regulators of the synaptic cytoskeleton. A major, unresolved question is how the disruption of specific actin filament structures leads to the onset and progression of complex synaptic and behavioral phenotypes. This review will cover established and emerging mechanisms of actin cytoskeletal remodeling and how this influences specific aspects of spine biology that are implicated in disease. PMID:26453304

MicroFilament Analyzer identifies actin network organizations in epidermal cells of Arabidopsis thaliana roots

PubMed Central

Jacques, Eveline; Lewandowski, Michal; Buytaert, Jan; Fierens, Yves; Verbelen, Jean-Pierre; Vissenberg, Kris

2013-01-01

The plant cytoskeleton plays a crucial role in the cells’ growth and development during different developmental stages and it undergoes many rearrangements. In order to describe the arrangements of the F-actin cytoskeleton in root epidermal cells of Arabidopsis thaliana, the recently developed software MicroFilament Analyzer (MFA) was exploited. This software enables high-throughput identification and quantification of the orientation of filamentous structures on digital images in a highly standardized and fast way. Using confocal microscopy and transgenic GFP-FABD2-GFP plants the actin cytoskeleton was visualized in the root epidermis. MFA analysis revealed that during the early stages of cell development F-actin is organized in a mainly random pattern. As the cells grow, they preferentially adopt a longitudinal organization, a pattern that is also preserved in the largest cells. In the evolution from young to old cells, an approximately even distribution of transverse, oblique or combined orientations is always present besides the switch from random to a longitudinal oriented actin cytoskeleton. PMID:23656865

Espins and the actin cytoskeleton of hair cell stereocilia and sensory cell microvilli

PubMed Central

Sekerková, Gabriella; Zheng, Lili; Loomis, Patricia A.; Mugnaini, Enrico; Bartles, James R.

2008-01-01

The espins are novel actin-bundling proteins that are produced in multiple isoforms from a single gene. They are present at high concentration in the parallel actin bundle of hair cell stereocilia and are the target of deafness mutations in mice and humans. Espins are also enriched in the microvilli of taste receptor cells, solitary chemoreceptor cells, vomeronasal sensory neurons and Merkel cells, suggesting that espins play important roles in the microvillar projections of vertebrate sensory cells. Espins are potent actin-bundling proteins that are not inhibited by Ca2+. In cells, they efficiently elongate parallel actin bundles and, thereby, help determine the steady-state length of microvilli and stereocilia. Espins bind actin monomer via their WH2 domain and can assemble actin bundles in cells. Certain espin isoforms can also bind phosphatidylinositol 4,5-bisphosphate, profilins or SH3 proteins. These biological activities distinguish espins from other actin-bundling proteins and may make them well-suited to sensory cells. PMID:16909209

Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton

PubMed Central

Takeda, Tetsuro; McQuistan, Tammie; Orlando, Robert A.; Farquhar, Marilyn G.

2001-01-01

Podocalyxin (PC), the major sialoprotein of glomerular epithelial cells (GECs), helps maintain the characteristic architecture of the foot processes and the patency of the filtration slits. PC associates with actin via ezrin, a member of the ERM family of cytoskeletal linker proteins. Here we show that PC is linked to ezrin and the actin cytoskeleton via Na+/H+-exchanger regulatory factor 2 (NHERF2), a scaffold protein containing two PDZ (PSD-95/Dlg/ZO-1) domains and an ERM-binding region. The cytoplasmic tail of PC contains a C-terminal PDZ-binding motif (DTHL) that binds to the second PDZ domain of NHERF2 in yeast two-hybrid and in vitro pull-down assays. By immunocytochemistry NHERF2 colocalizes with PC and ezrin along the apical domain of the GEC plasma membrane. NHERF2 and ezrin form a multimeric complex with PC, as they coimmunoprecipitate with PC. The PC/NHERF2/ezrin complex interacts with the actin cytoskeleton, and this interaction is disrupted in GECs from puromycin aminonucleoside–, protamine sulfate–, or sialidase-treated rats, which show a dramatic loss of foot processes, comparable to that seen in the nephrotic syndrome. Thus NHERF2 appears to function as a scaffold protein linking PC to ezrin and the actin cytoskeleton. PC/NHERF2/ezrin/actin interactions are disrupted in pathologic conditions associated with changes in GEC foot processes, indicating their importance for maintaining the unique organization of this epithelium. J. Clin. Invest. 108:289–301 (2001). DOI:10.1172/JCI200112539. PMID:11457882

2,4-Dichlorophenoxyacetic acid promotes S-nitrosylation and oxidation of actin affecting cytoskeleton and peroxisomal dynamics

PubMed Central

Rodríguez-Serrano, M.; Pazmiño, D. M.; Sparkes, I.; Rochetti, A.; Hawes, C.; Romero-Puertas, M. C.; Sandalio, L. M.

2014-01-01

2,4-Dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin used as a herbicide to control weeds in agriculture. A high concentration of 2,4-D promotes leaf epinasty and cell death. In this work, the molecular mechanisms involved in the toxicity of this herbicide are studied by analysing in Arabidopsis plants the accumulation of reactive oxygen species (ROS) and nitric oxide (NO), and their effect on cytoskeleton structure and peroxisome dynamics. 2,4-D (23mM) promotes leaf epinasty, whereas this process was prevented by EDTA, which can reduce ·OH accumulation. The analysis of ROS accumulation by confocal microscopy showed a 2,4-D-dependent increase in both H2O2 and O2·–, whereas total NO was not affected by the treatment. The herbicide promotes disturbances on the actin cytoskeleton structure as a result of post-translational modification of actin by oxidation and S-nitrosylation, which could disturb actin polymerization, as suggested by the reduction of the F-actin/G-actin ratio. These effects were reduced by EDTA, and the reduction of ROS production in Arabidopsis mutants deficient in xanthine dehydrogenase (Atxdh) gave rise to a reduction in actin oxidation. Also, 2,4-D alters the dynamics of the peroxisome, slowing the speed and shortening the distances by which these organelles are displaced. It is concluded that 2,4-D promotes oxidative and nitrosative stress, causing disturbances in the actin cytoskeleton, thereby affecting the dynamics of peroxisomes and some other organelles such as the mitochondria, with xanthine dehydrogenase being involved in ROS production under these conditions. These structural changes in turn appear to be responsible for the leaf epinasty. PMID:24913628

Fucus as a Model System to Study the Role of Auxin Transport and the Actin Cytoskeleton in Gravity Response

NASA Technical Reports Server (NTRS)

Muday, Gloria K.

2003-01-01

The overarching goal of this proposal was to examine the mechanisms for the cellular asymmetry in auxin transport proteins. As auxin transport polarity changes in response to reorientation of algal and plant cells relative to the gravity vector, it was critical to ask how auxin transport polarity is established and how this transport polarity may change in response to gravity stimulation. The experiments conducted with this NASA grant fell into two categories. The first area of experimentation was to explore the biochemical interactions between an auxin transport protein and the actin cytoskeleton. These experiments used biochemical techniques, including actin affinity chromatography, to demonstrate that one auxin transport protein interacts with the actin cytoskeleton. The second line of experiments examined whether in the initially symmetrical single celled embryos of Fucus distichus, whether auxin regulates development and whether gravity is a cue to control the morphogenesis of these embryos and whether gravi-morphogenesis is auxin dependent. Results in these two areas are summarized separately below. As a result of this funding, in combination with results from other investigators, we have strong evidence for an important role for the actin cytoskeleton in both establishing and change auxin transport polarity. It is also clear that Fucus distichus embryos are auxin responsive and gravity controls their morphogenesis.

Palytoxins and cytoskeleton: An overview.

PubMed

Louzao, M Carmen; Ares, Isabel R; Cagide, Eva; Espiña, Begoña; Vilariño, Natalia; Alfonso, Amparo; Vieytes, Mercedes R; Botana, Luis M

2011-03-01

Cytoskeleton is a dynamic structure essential for a wide variety of normal cellular processes, including the maintenance of cell shape and morphology, volume regulation, membrane dynamics and signal transduction. Cytoskeleton is organized into microtubules, actin meshwork and intermediate filaments. Actin has been identified as a major target for destruction during apoptosis and is also important under pathological conditions such as cancers. Several natural compounds actively modulate actin organization by specific signaling cascades being useful tools to study cytoskeleton dynamics. Palytoxin is a large bioactive compound, first isolated from zoanthids, with a complex structure and different analogs such as ostreocin-D or ovatoxin-a. This toxin has been identified as a potent tumor promoter and cytotoxic molecule, which leads to actin filament distortion and triggers cell death or apoptosis. In this review we report the findings on the involvement of palytoxin and analogues modulating the actin cytoskeleton within different cellular models. Copyright © 2010 Elsevier Ltd. All rights reserved.

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.

Distribution and dynamics of the cytoskeleton in graviresponding protonemata and rhizoids of characean algae: exclusion of microtubules and a convergence of actin filaments in the apex suggest an actin-mediated gravitropism.

PubMed

Braun, M; Wasteneys, G O

1998-05-01

The organization of the microtubule (MT) and actin microfilament (MF) cytoskeleton of tip-growing rhizoids and protonemata of characean green algae was examined by confocal laser scanning microscopy. This analysis included microinjection of fluorescent tubulin and phallotoxins into living cells, as well as immunofluorescence labeling of fixed material and fluorescent phallotoxin labeling of unfixed material. Although the morphologically very similar positively gravitropic (downward growing) rhizoids and negatively gravitropic (upward growing) protonemata show opposite gravitropic responses, no differences were detected in the extensive three-dimensional distribution of actin MFs and MTs in both cell types. Tubulin microinjection revealed that in contrast to internodal cells, fluorescent tubulin incorporated very slowly into the MT arrays of rhizoids, suggesting that MT dynamics are very different in tip-growing and diffusely expanding cells. Microtubules assembled from multiple sites at the plasma membrane in the basal zone, and a dense subapical array emerged from a diffuse nucleation centre on the basal side of the nuclear envelope. Immunofluorescence confirmed these distribution patterns but revealed more extensive MT arrays. In the basal zone, short branching clusters of MTs form two cortical hemicylinders. Subapical, axially oriented MTs are distributed in equal density throughout the peripheral and inner cytoplasm and are closely associated with subapical organelles. Microtubules, however, are completely absent from the apical zones of rhizoids and protonemata. Actin MFs were found in all zones of rhizoids and protonemata including the apex. Two files of axially oriented bundles of subcortical actin MFs and ring-like actin structures in the streaming endoplasm of rhizoids were detected in the basal zones by microinjection or rhodamine-phalloidin labeling. The subapical zone contains a dense array of mainly axially oriented actin MFs that co-distribute with

Actin Bodies in Yeast Quiescent Cells: An Immediately Available Actin Reserve?

PubMed Central

Pinson, Benoît; Salin, Bénédicte; Daignan-Fornier, Bertrand

2006-01-01

Most eukaryotic cells spend most of their life in a quiescent state, poised to respond to specific signals to proliferate. In Saccharomyces cerevisiae, entry into and exit from quiescence are dependent only on the availability of nutrients in the environment. The transition from quiescence to proliferation requires not only drastic metabolic changes but also a complete remodeling of various cellular structures. Here, we describe an actin cytoskeleton organization specific of the yeast quiescent state. When cells cease to divide, actin is reorganized into structures that we named “actin bodies.” We show that actin bodies contain F-actin and several actin-binding proteins such as fimbrin and capping protein. Furthermore, by contrast to actin patches or cables, actin bodies are mostly immobile, and we could not detect any actin filament turnover. Finally, we show that upon cells refeeding, actin bodies rapidly disappear and actin cables and patches can be assembled in the absence of de novo protein synthesis. This led us to propose that actin bodies are a reserve of actin that can be immediately mobilized for actin cables and patches formation upon reentry into a proliferation cycle. PMID:16914523

Fluid Shear Stress-Induced JNK Activity Leads to Actin Remodeling for Cell Alignment

PubMed Central

Mengistu, Meron; Brotzman, Hannah; Ghadiali, Samir; Lowe-Krentz, Linda

2012-01-01

Fluid shear stress (FSS) exerted on endothelial cell surfaces induces actin cytoskeleton remodeling through mechanotransduction. This study was designed to determine whether FSS activates Jun N-terminal kinase (JNK), to examine the spatial and temporal distribution of active JNK relative to the actin cytoskeleton in endothelial cells exposed to different FSS conditions, and to evaluate the effects of active JNK on actin realignment. Exposure to 15 and 20 dyn/cm2 FSS induced higher activity levels of JNK than the lower 2 and 4 dyn/cm2 flow conditions. At the higher FSS treatments, JNK activity increased with increasing exposure time, peaking 30 minutes after flow onset with an 8-fold activity increase compared to cells in static culture. FSS-induced phospho-JNK co-localized with actin filaments at cell peripheries, as well as with stress fibers. Pharmacologically blocking JNK activity altered FSS-induced actin structure and distribution as a response to FSS. Our results indicate that FSS-induced actin remodeling occurs in three phases, and that JNK plays a role in at least one, suggesting that this kinase activity is involved in mechanotransduction from the apical surface to the actin cytoskeleton in endothelial cells. PMID:20626006

The Yeast Gene, MDM20, Is Necessary for Mitochondrial Inheritance and Organization of the Actin Cytoskeleton

PubMed Central

Hermann, Greg J.; King, Edward J.; Shaw, Janet M.

1997-01-01

In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament–binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20Δ cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton. PMID:9105043

2,4-Dichlorophenoxyacetic acid promotes S-nitrosylation and oxidation of actin affecting cytoskeleton and peroxisomal dynamics.

PubMed

Rodríguez-Serrano, M; Pazmiño, D M; Sparkes, I; Rochetti, A; Hawes, C; Romero-Puertas, M C; Sandalio, L M

2014-09-01

2,4-Dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin used as a herbicide to control weeds in agriculture. A high concentration of 2,4-D promotes leaf epinasty and cell death. In this work, the molecular mechanisms involved in the toxicity of this herbicide are studied by analysing in Arabidopsis plants the accumulation of reactive oxygen species (ROS) and nitric oxide (NO), and their effect on cytoskeleton structure and peroxisome dynamics. 2,4-D (23 mM) promotes leaf epinasty, whereas this process was prevented by EDTA, which can reduce ·OH accumulation. The analysis of ROS accumulation by confocal microscopy showed a 2,4-D-dependent increase in both H2O2 and O2·(-), whereas total NO was not affected by the treatment. The herbicide promotes disturbances on the actin cytoskeleton structure as a result of post-translational modification of actin by oxidation and S-nitrosylation, which could disturb actin polymerization, as suggested by the reduction of the F-actin/G-actin ratio. These effects were reduced by EDTA, and the reduction of ROS production in Arabidopsis mutants deficient in xanthine dehydrogenase (Atxdh) gave rise to a reduction in actin oxidation. Also, 2,4-D alters the dynamics of the peroxisome, slowing the speed and shortening the distances by which these organelles are displaced. It is concluded that 2,4-D promotes oxidative and nitrosative stress, causing disturbances in the actin cytoskeleton, thereby affecting the dynamics of peroxisomes and some other organelles such as the mitochondria, with xanthine dehydrogenase being involved in ROS production under these conditions. These structural changes in turn appear to be responsible for the leaf epinasty. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Hyperpolarization of the plasma membrane potential provokes reorganization of the actin cytoskeleton and increases the stability of adherens junctions in bovine corneal endothelial cells in culture.

PubMed

Nin, Verónica; Hernández, Julio A; Chifflet, Silvia

2009-12-01

In previous works we showed that the depolarization of the plasma membrane potential (PMP) determines a reorganization of the cytoskeleton of diverse epithelia in culture, consisting mainly of a reallocation of peripheral actin toward the cell center, ultimately provoking intercellular disruption. In view of this evidence, we explored in this study the possible effects of membrane potential hyperpolarization on the cytoskeletal organization and adherens junction (AJ) morphology and the stability of confluent bovine corneal endothelial cells in culture. For this purpose, hyperpolarization was achieved by substitution of extracellular sodium by nondiffusible cations or via the incorporation of valinomycin to the control solution. Actin compactness at the cell periphery was assessed by quantitative analysis of fluorescence microscopy images. The stability of the AJ was challenged by calcium deprivation or temperature decrease. Our results showed that plasma membrane hyperpolarization provokes a compaction of AJ-associated actin filaments toward the plasma membrane and an increase in the stability of the AJs. We also observed that the hyperpolarizing procedures determined similar modifications in the actin cytoskeleton of endothelial cells in whole bovine corneas. Together with our previous work, the results of this study contribute to the idea that modifications in the PMP of nonexcitable cells participate in cellular adaptive responses involving reorganization of cytoskeletal components. (c) 2009 Wiley-Liss, Inc.

A new F-actin structure in fungi: actin ring formation around the cell nucleus of Cryptococcus neoformans.

PubMed

Kopecká, Marie; Kawamoto, Susumu; Yamaguchi, Masashi

2013-04-01

The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.

CecropinXJ, a silkworm antimicrobial peptide, induces cytoskeleton disruption in esophageal carcinoma cells.

PubMed

Xia, Lijie; Wu, Yanling; Kang, Su; Ma, Ji; Yang, Jianhua; Zhang, Fuchun

2014-10-01

Antimicrobial peptides exist in the non-specific immune system of organism and participate in the innate host defense of each species. CecropinXJ, a cationic antimicrobial peptide, possesses potent anticancer activity and acts preferentially on cancer cells instead of normal cells, but the mechanism of cancer cell death induced by cecropinXJ remains largely unknown. This study was performed to investigate the cytoskeleton-disrupting effects of cecropinXJ on human esophageal carcinoma cell line Eca109 using scanning electron microscopy observation, fluorescence imaging, cell migration and invasion assays, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. The electronic microscope and fluorescence imaging observation suggested that cecropinXJ could result in morphological changes and induce damage to microtubules and actin of Eca109 cells in a dose-dependent manner. The cell migration and invasion assays demonstrated that cecropinXJ could inhibit migration and invasion of tumor cells. Western blot and qRT-PCR analysis showed that there was obvious correlation between microtubule depolymerization and actin polymerization induced by cecropinXJ. Moreover, cecropinXJ might also cause decreased expression of α-actin, β-actin, γ-actin, α-tubulin, and β-tubulin genes in concentration- and time-dependent manners. In summary, this study indicates that cecropinXJ triggers cytotoxicity in Eca109 cells through inducing the cytoskeleton destruction and regulating the expression of cytoskeleton proteins. This cecropinXJ-mediated cytoskeleton-destruction effect is instrumental in our understanding of the detailed action of antimicrobial peptides in human cancer cells and cecropinXJ might be a potential therapeutic agent for the treatment of cancer in the future. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology

Triptolide disrupts the actin-based Sertoli-germ cells adherens junctions by inhibiting Rho GTPases expression

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

Wang, Xiang; Zhao, Fang

Triptolide (TP), derived from the medicinal plant Triterygium wilfordii Hook. f. (TWHF), is a diterpene triepoxide with variety biological and pharmacological activities. However, TP has been restricted in clinical application due to its narrow therapeutic window especially in reproductive system. During spermatogenesis, Sertoli cell cytoskeleton plays an essential role in facilitating germ cell movement and cell-cell actin-based adherens junctions (AJ). At Sertoli cell-spermatid interface, the anchoring device is a kind of AJ, known as ectoplasmic specializations (ES). In this study, we demonstrate that β-actin, an important component of cytoskeleton, has been significantly down-regulated after TP treatment. TP can inhibit themore » expression of Rho GTPase such as, RhoA, RhoB, Cdc42 and Rac1. Downstream of Rho GTPase, Rho-associated protein kinase (ROCKs) gene expressions were also suppressed by TP. F-actin immunofluorescence proved that TP disrupts Sertoli cells cytoskeleton network. As a result of β-actin down-regulation, TP treatment increased expression of testin, which indicating ES has been disassembled. In summary, this report illustrates that TP induces cytoskeleton dysfunction and disrupts cell-cell adherens junctions via inhibition of Rho GTPases. - Highlights: • Triptolide induced the disruption of Sertoli-germ cell adherens junction. • Rho GTPases expression and actin dynamics have been suppressed by triptolide. • Actin-based adherens junction is a potential antifertility target of triptolide. • Rho-Rock is involved in the regulation of actin dynamics.« less

Prokaryotic cytoskeletons: protein filaments organizing small cells.

PubMed

Wagstaff, James; Löwe, Jan

2018-04-01

Most, if not all, bacterial and archaeal cells contain at least one protein filament system. Although these filament systems in some cases form structures that are very similar to eukaryotic cytoskeletons, the term 'prokaryotic cytoskeletons' is used to refer to many different kinds of protein filaments. Cytoskeletons achieve their functions through polymerization of protein monomers and the resulting ability to access length scales larger than the size of the monomer. Prokaryotic cytoskeletons are involved in many fundamental aspects of prokaryotic cell biology and have important roles in cell shape determination, cell division and nonchromosomal DNA segregation. Some of the filament-forming proteins have been classified into a small number of conserved protein families, for example, the almost ubiquitous tubulin and actin superfamilies. To understand what makes filaments special and how the cytoskeletons they form enable cells to perform essential functions, the structure and function of cytoskeletal molecules and their filaments have been investigated in diverse bacteria and archaea. In this Review, we bring these data together to highlight the diverse ways that linear protein polymers can be used to organize other molecules and structures in bacteria and archaea.

The skeleton in the closet: actin cytoskeletal remodeling in β-cell function.

PubMed

Arous, Caroline; Halban, Philippe A

2015-10-01

Over the last few decades, biomedical research has considered not only the function of single cells but also the importance of the physical environment within a whole tissue, including cell-cell and cell-extracellular matrix interactions. Cytoskeleton organization and focal adhesions are crucial sensors for cells that enable them to rapidly communicate with the physical extracellular environment in response to extracellular stimuli, ensuring proper function and adaptation. The involvement of the microtubular-microfilamentous cytoskeleton in secretion mechanisms was proposed almost 50 years ago, since when the evolution of ever more sensitive and sophisticated methods in microscopy and in cell and molecular biology have led us to become aware of the importance of cytoskeleton remodeling for cell shape regulation and its crucial link with signaling pathways leading to β-cell function. Emerging evidence suggests that dysfunction of cytoskeletal components or extracellular matrix modification influences a number of disorders through potential actin cytoskeleton disruption that could be involved in the initiation of multiple cellular functions. Perturbation of β-cell actin cytoskeleton remodeling could arise secondarily to islet inflammation and fibrosis, possibly accounting in part for impaired β-cell function in type 2 diabetes. This review focuses on the role of actin remodeling in insulin secretion mechanisms and its close relationship with focal adhesions and myosin II. Copyright © 2015 the American Physiological Society.

Jmy regulates oligodendrocyte differentiation via modulation of actin cytoskeleton dynamics.

PubMed

Azevedo, Maria M; Domingues, Helena S; Cordelières, Fabrice P; Sampaio, Paula; Seixas, Ana I; Relvas, João B

2018-05-06

During central nervous system development, oligodendrocytes form structurally and functionally distinct actin-rich protrusions that contact and wrap around axons to assemble myelin sheaths. Establishment of axonal contact is a limiting step in myelination that relies on the oligodendrocyte's ability to locally coordinate cytoskeletal rearrangements with myelin production, under the control of a transcriptional differentiation program. The molecules that provide fine-tuning of actin dynamics during oligodendrocyte differentiation and axon ensheathment remain largely unidentified. We performed transcriptomics analysis of soma and protrusion fractions from rat brain oligodendrocyte progenitors and found a subcellular enrichment of mRNAs in newly-formed protrusions. Approximately 30% of protrusion-enriched transcripts encode proteins related to cytoskeleton dynamics, including the junction mediating and regulatory protein Jmy, a multifunctional regulator of actin polymerization. Here, we show that expression of Jmy is upregulated during myelination and is required for the assembly of actin filaments and protrusion formation during oligodendrocyte differentiation. Quantitative morphodynamics analysis of live oligodendrocytes showed that differentiation is driven by a stereotypical actin network-dependent "cellular shaping" program. Disruption of actin dynamics via knockdown of Jmy leads to a program fail resulting in oligodendrocytes that do not acquire an arborized morphology and are less efficient in contacting neurites and forming myelin wraps in co-cultures with neurons. Our findings provide new mechanistic insight into the relationship between cell shape dynamics and differentiation in development. © 2018 Wiley Periodicals, Inc.

Phosphoinositide 3-Kinase Regulates Glycolysis through Mobilization of Aldolase from the Actin Cytoskeleton.

PubMed

Hu, Hai; Juvekar, Ashish; Lyssiotis, Costas A; Lien, Evan C; Albeck, John G; Oh, Doogie; Varma, Gopal; Hung, Yin Pun; Ullas, Soumya; Lauring, Josh; Seth, Pankaj; Lundquist, Mark R; Tolan, Dean R; Grant, Aaron K; Needleman, Daniel J; Asara, John M; Cantley, Lewis C; Wulf, Gerburg M

2016-01-28

The phosphoinositide 3-kinase (PI3K) pathway regulates multiple steps in glucose metabolism and also cytoskeletal functions, such as cell movement and attachment. Here, we show that PI3K directly coordinates glycolysis with cytoskeletal dynamics in an AKT-independent manner. Growth factors or insulin stimulate the PI3K-dependent activation of Rac, leading to disruption of the actin cytoskeleton, release of filamentous actin-bound aldolase A, and an increase in aldolase activity. Consistently, PI3K inhibitors, but not AKT, SGK, or mTOR inhibitors, cause a significant decrease in glycolysis at the step catalyzed by aldolase, while activating PIK3CA mutations have the opposite effect. These results point toward a master regulatory function of PI3K that integrates an epithelial cell's metabolism and its form, shape, and function, coordinating glycolysis with the energy-intensive dynamics of actin remodeling. Copyright © 2016 Elsevier Inc. All rights reserved.

Imaging Cytoskeleton Components by Electron Microscopy.

PubMed

Svitkina, Tatyana

2016-01-01

The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers-actin filaments, microtubules, and intermediate filaments-are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton. This article describes application of rotary shadowing (or metal replica) EM for visualization of the cytoskeleton. The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction of cells to expose their cytoskeleton, chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved, and individual proteins can be identified by immunogold labeling. More importantly, replica EM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high resolution structural organization of the cytoskeleton in the same cell.

Reorganization of Actin Cytoskeleton by the Phosphoinositide Metabolite Glycerophosphoinositol 4-Phosphate

PubMed Central

Mancini, Raffaella; Piccolo, Enza; Mariggio', Stefania; Filippi, Beatrice Maria; Iurisci, Cristiano; Pertile, Paolo; Berrie, Christopher P.; Corda, Daniela

2003-01-01

Glycerophosphoinositol 4-phosphate (GroPIns-4P) is a biologically active, water-soluble phospholipase A metabolite derived from phosphatidylinositol 4-phosphate, whose cellular concentrations have been reported to increase in Ras-transformed cells. It is therefore important to understand its biological activities. Herein, we have examined whether GroPIns-4P can regulate the organization of the actin cytoskeleton, because this could be a Ras-related function involved in cell motility and metastatic invasion. We find that in serum-starved Swiss 3T3 cells, exogenously added GroPIns-4P rapidly and potently induces the formation of membrane ruffles, and, later, the formation of stress fibers. These actin structures can be regulated by the small GTPases Cdc42, Rac, and Rho. To analyze the mechanism of action of GroPIns-4P, we selectively inactivated each of these GTPases. GroPIns-4P requires active Rac and Rho, but not Cdc42, for ruffle and stress fiber formation, respectively. Moreover, GroPIns-4P induces a rapid translocation of the green fluorescent protein-tagged Rac into ruffles, and increases the fraction of GTP-bound Rac, in intact cells. The activation of Rac by GroPIns-4P was near maximal and long-lasting. Interestingly, this feature seems to be critical in the induction of actin ruffles by GroPIns-4P. PMID:12589050

TGFβ1-Induced Differentiation of Human Bone Marrow-Derived MSCs Is Mediated by Changes to the Actin Cytoskeleton.

PubMed

Elsafadi, Mona; Manikandan, Muthurangan; Almalki, Sami; Mobarak, Mohammad; Atteya, Muhammad; Iqbal, Zafar; Hashmi, Jamil Amjad; Shaheen, Sameerah; Alajez, Nehad; Alfayez, Musaad; Kassem, Moustapha; Dawud, Raed Abu; Mahmood, Amer

2018-01-01

TGF β is a potent regulator of several biological functions in many cell types, but its role in the differentiation of human bone marrow-derived skeletal stem cells (hMSCs) is currently poorly understood. In the present study, we demonstrate that a single dose of TGF β 1 prior to induction of osteogenic or adipogenic differentiation results in increased mineralized matrix or increased numbers of lipid-filled mature adipocytes, respectively. To identify the mechanisms underlying this TGF β -mediated enhancement of lineage commitment, we compared the gene expression profiles of TGF β 1-treated hMSC cultures using DNA microarrays. In total, 1932 genes were upregulated, and 1298 genes were downregulated. Bioinformatics analysis revealed that TGF β l treatment was associated with an enrichment of genes in the skeletal and extracellular matrix categories and the regulation of the actin cytoskeleton. To investigate further, we examined the actin cytoskeleton following treatment with TGF β 1 and/or cytochalasin D. Interestingly, cytochalasin D treatment of hMSCs enhanced adipogenic differentiation but inhibited osteogenic differentiation. Global gene expression profiling revealed a significant enrichment of pathways related to osteogenesis and adipogenesis and of genes regulated by both TGF β 1 and cytochalasin D. Our study demonstrates that TGF β 1 enhances hMSC commitment to either the osteogenic or adipogenic lineages by reorganizing the actin cytoskeleton.

Correlated light and electron microscopy observations of the uterine epithelial cell actin cytoskeleton using fluorescently labeled resin-embedded sections.

PubMed

Moore, Chad L; Cheng, Delfine; Shami, Gerald J; Murphy, Christopher R

2016-05-01

In order to perform correlative light and electron microscopy (CLEM) more precisely, we have modified existing specimen preparation protocols allowing fluorescence retention within embedded and sectioned tissue, facilitating direct observation across length scales. We detail a protocol which provides a precise correlation accuracy using accessible techniques in biological specimen preparation. By combining a pre-embedding uranyl acetate staining step with the progressive lowering of temperature (PLT) technique, a methacrylate embedded tissue specimen is ultrathin sectioned and mounted onto a TEM finder grid for immediate viewing in the confocal and electron microscope. In this study, the protocol is applied to rat uterine epithelial cells in vivo during early pregnancy. Correlative overlay data was used to track changes in filamentous actin that occurs in these cells from fertilization (Day 1) to implantation on Day 6 as part of the plasma membrane transformation, a process essential in the development of uterine receptivity in the rat. CLEM confirmed that the actin cytoskeleton is disrupted as apical microvilli are progressively lost toward implantation, and revealed the thick and continuous terminal web is replaced by a thinner and irregular actin band, with individually distinguishable filaments connecting actin meshworks which correspond with remaining plasma membrane protrusions. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Cytoskeleton-Autophagy Connection.

PubMed

Kast, David J; Dominguez, Roberto

2017-04-24

Actin cytoskeleton dynamics play vital roles in most forms of intracellular trafficking by promoting the biogenesis and transport of vesicular cargoes. Mounting evidence indicates that actin dynamics and membrane-cytoskeleton scaffolds also have essential roles in macroautophagy, the process by which cellular waste is isolated inside specialized vesicles called autophagosomes for recycling and degradation. Branched actin polymerization is necessary for the biogenesis of autophagosomes from the endoplasmic reticulum (ER) membrane. Actomyosin-based transport is then used to feed the growing phagophore with pre-selected cargoes and debris derived from different membranous organelles inside the cell. Finally, mature autophagosomes detach from the ER membrane by an as yet unknown mechanism, undergo intracellular transport and then fuse with lysosomes, endosomes and multivesicular bodies through mechanisms that involve actin- and microtubule-mediated motility, cytoskeleton-membrane scaffolds and signaling proteins. In this review, we highlight the considerable progress made recently towards understanding the diverse roles of the cytoskeleton in autophagy. Published by Elsevier Ltd.

The Cytoskeleton-Autophagy Connection

PubMed Central

Kast, David J.; Dominguez, Roberto

2017-01-01

Summary Actin cytoskeleton dynamics plays vital roles in most forms of intracellular trafficking by promoting the biogenesis and transport of vesicular cargoes. Mounting evidence indicates that actin dynamics and membrane-cytoskeleton scaffolds also play essential roles in macroautophagy, the process by which cellular waste is isolated inside specialized vesicles called autophagosomes for recycling and degradation. Thus, branched-actin polymerization is necessary for the biogenesis of autophagosomes from the endoplasmic reticulum (ER) membrane. Actomyosin-based transport is then used to feed the growing phagophore with pre-selected cargoes and debris derived from different membranous organelles inside the cell. Mature autophagosomes then detach from the ER membrane by an unknown mechanism, and are transported and fused with lysosomes, endosomes and multi-vesicular bodies through mechanisms that involve actin- and microtubule-based motility, cytoskeleton-membrane scaffolds and signaling proteins. In this minireview, we highlight the considerable progress made recently towards understanding the diverse roles of the cytoskeleton in autophagy. PMID:28441569

The Nck family of adapter proteins: regulators of actin cytoskeleton.

PubMed

Buday, László; Wunderlich, Livius; Tamás, Peter

2002-09-01

SH2/SH3 domain-containing adapter proteins, such as the Nck family, play a major role in regulating tyrosine kinase signalling. They serve to recruit proline-rich effector molecules to tyrosine-phosphorylated kinases or their substrates. Initially, it was not clear why cells from nematodes to vertebrates contain redundant and closely related SH2/SH3 adapters, such as Grb2, Crk and Nck. Recent evidence suggests that their biological roles are clearly different, whereas, for example, Grb2 connects activated receptor tyrosine kinases to Sos and Ras, leading to cell proliferation. The proteins of Nck family are implicated in organisation of actin cytoskeleton, cell movement or axon guidance in flies. In this review, the author attempts to summarise signalling pathways in which Nck plays a critical role.

MRP-1/CD9 gene transduction regulates the actin cytoskeleton through the downregulation of WAVE2.

PubMed

Huang, C-L; Ueno, M; Liu, D; Masuya, D; Nakano, J; Yokomise, H; Nakagawa, T; Miyake, M

2006-10-19

Motility-related protein-1 (MRP-1/CD9) is involved in cell motility. We studied the change in the actin cytoskeleton, and the expression of actin-related protein (Arp) 2 and Arp3 and the Wiskott-Aldrich syndrome protein (WASP) family according to MRP-1/CD9 gene transduction into HT1080 cells. The frequency of cells with lamellipodia was significantly lower in MRP-1/CD9-transfected HT1080 cells than in control HT1080 cells (P<0.0001). MRP-1/CD9 gene transduction affected the subcellular localization of Arp2 and Arp3 proteins. Furthermore, MRP-1/CD9 gene transduction induced a downregulation of WAVE2 expression (P<0.0001). However, no difference was observed in the expression of Arp2, Arp3 or other WASPs. A neutralizing anti-MRP-1/CD9 monoclonal antibody inhibited downregulation of WAVE2 in MRP-1/CD9-transfected HT1080 cells (P<0.0001), and reversed the morphological effects of MRP-1/CD9 gene transduction. Furthermore, downregulation of WAVE2 by transfection of WAVE2-specific small interfering RNA (siRNA) mimicked the morphological effects of MRP-1/CD9 gene transduction and suppressed cell motility. However, transfection of each siRNA for Wnt1, Wnt2b1 or Wnt5a did not affect WAVE2 expression. Transfection of WAVE2-specific siRNA also did not affect expressions of these Wnts. These results indicate that MRP-1/CD9 regulates the actin cytoskeleton by downregulating of the WAVE2, through the Wnt-independent signal pathway.

Par-4-mediated recruitment of Amida to the actin cytoskeleton leads to the induction of apoptosis

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

Boosen, Meike; Vetterkind, Susanne; Koplin, Ansgar

Par-4 (prostate apoptosis response-4) sensitizes cells to apoptotic stimuli, but the exact mechanisms are still poorly understood. Using Par-4 as bait in a yeast two-hybrid screen, we identified Amida as a novel interaction partner, a ubiquitously expressed protein which has been suggested to be involved in apoptotic processes. Complex formation of Par-4 and Amida occurs in vitro and in vivo and is mediated via the C-termini of both proteins, involving the leucine zipper of Par-4. Amida resides mainly in the nucleus but displays nucleo-cytoplasmic shuttling in heterokaryons. Upon coexpression with Par-4 in REF52.2 cells, Amida translocates to the cytoplasm andmore » is recruited to actin filaments by Par-4, resulting in enhanced induction of apoptosis. The synergistic effect of Amida/Par-4 complexes on the induction of apoptosis is abrogated when either Amida/Par-4 complex formation or association of these complexes with the actin cytoskeleton is impaired, indicating that the Par-4-mediated relocation of Amida to the actin cytoskeleton is crucial for the pro-apoptotic function of Par-4/Amida complexes in REF52.2 cells. The latter results in enhanced phosphorylation of the regulatory light chain of myosin II (MLC) as has previously been shown for Par-4-mediated recruitment of DAP-like kinase (Dlk), suggesting that the recruitment of nuclear proteins involved in the regulation of apoptotic processes to the actin filament system by Par-4 represents a potent mechanism how Par-4 can trigger apoptosis.« less

p53 regulates cytoskeleton remodeling to suppress tumor progression.

PubMed

Araki, Keigo; Ebata, Takahiro; Guo, Alvin Kunyao; Tobiume, Kei; Wolf, Steven John; Kawauchi, Keiko

2015-11-01

Cancer cells possess unique characteristics such as invasiveness, the ability to undergo epithelial-mesenchymal transition, and an inherent stemness. Cell morphology is altered during these processes and this is highly dependent on actin cytoskeleton remodeling. Regulation of the actin cytoskeleton is, therefore, important for determination of cell fate. Mutations within the TP53 (tumor suppressor p53) gene leading to loss or gain of function (GOF) of the protein are often observed in aggressive cancer cells. Here, we highlight the roles of p53 and its GOF mutants in cancer cell invasion from the perspective of the actin cytoskeleton; in particular its reorganization and regulation by cell adhesion molecules such as integrins and cadherins. We emphasize the multiple functions of p53 in the regulation of actin cytoskeleton remodeling in response to the extracellular microenvironment, and oncogene activation. Such an approach provides a new perspective in the consideration of novel targets for anti-cancer therapy.

Characterization of the Endothelial Cell Cytoskeleton following HLA Class I Ligation

PubMed Central

Ziegler, Mary E.; Souda, Puneet; Jin, Yi-Ping; Whitelegge, Julian P.; Reed, Elaine F.

2012-01-01

Background Vascular endothelial cells (ECs) are a target of antibody-mediated allograft rejection. In vitro, when the HLA class I molecules on the surface of ECs are ligated by anti-HLA class I antibodies, cell proliferation and survival pathways are activated and this is thought to contribute to the development of antibody-mediated rejection. Crosslinking of HLA class I molecules by anti-HLA antibodies also triggers reorganization of the cytoskeleton, which induces the formation of F-actin stress fibers. HLA class I induced stress fiber formation is not well understood. Methodology and Principal Findings The present study examines the protein composition of the cytoskeleton fraction of ECs treated with HLA class I antibodies and compares it to other agonists known to induce alterations of the cytoskeleton in endothelial cells. Analysis by tandem mass spectrometry revealed unique cytoskeleton proteomes for each treatment group. Using annotation tools a candidate list was created that revealed 12 proteins, which were unique to the HLA class I stimulated group. Eleven of the candidate proteins were phosphoproteins and exploration of their predicted kinases provided clues as to how these proteins may contribute to the understanding of HLA class I induced antibody-mediated rejection. Three of the candidates, eukaryotic initiation factor 4A1 (eIF4A1), Tropomyosin alpha 4-chain (TPM4) and DDX3X, were further characterized by Western blot and found to be associated with the cytoskeleton. Confocal microscopy analysis showed that class I ligation stimulated increased eIF4A1 co-localization with F-actin and paxillin. Conclusions/Significance Colocalization of eIF4A1 with F-actin and paxillin following HLA class I ligation suggests that this candidate protein could be a target for understanding the mechanism(s) of class I mediated antibody-mediated rejection. This proteomic approach for analyzing the cytoskeleton of ECs can be applied to other agonists and various cells types

Branched actin networks push against each other at adherens junctions to maintain cell-cell adhesion.

PubMed

Efimova, Nadia; Svitkina, Tatyana M

2018-05-07

Adherens junctions (AJs) are mechanosensitive cadherin-based intercellular adhesions that interact with the actin cytoskeleton and carry most of the mechanical load at cell-cell junctions. Both Arp2/3 complex-dependent actin polymerization generating pushing force and nonmuscle myosin II (NMII)-dependent contraction producing pulling force are necessary for AJ morphogenesis. Which actin system directly interacts with AJs is unknown. Using platinum replica electron microscopy of endothelial cells, we show that vascular endothelial (VE)-cadherin colocalizes with Arp2/3 complex-positive actin networks at different AJ types and is positioned at the interface between two oppositely oriented branched networks from adjacent cells. In contrast, actin-NMII bundles are located more distally from the VE-cadherin-rich zone. After Arp2/3 complex inhibition, linear AJs split, leaving gaps between cells with detergent-insoluble VE-cadherin transiently associated with the gap edges. After NMII inhibition, VE-cadherin is lost from gap edges. We propose that the actin cytoskeleton at AJs acts as a dynamic push-pull system, wherein pushing forces maintain extracellular VE-cadherin transinteraction and pulling forces stabilize intracellular adhesion complexes. © 2018 Efimova and Svitkina.

Actin cytoskeleton depolymerization with clostridium spiroforme toxin enhances the secretory activity of rat melanotrophs.

PubMed

Chowdhury, H H; Popoff, M R; Zorec, R

1999-12-01

1. We measured membrane capacitance (Cm) in cultured rat melanotrophs pretreated with Clostridium spiroforme toxin (CST), which specifically depolymerizes cortical filamentous actin (F-actin). Phalloidin staining confirmed that CST treatment depolymerised the F-actin. 2. In control cells, cytosol dialysis with 1 microM Ca2+i increased Cm by 23 +/- 4 % (n = 11) relative to the resting Cm 400 s after the start of patch rupture. In CST-treated cells the increase in Cm was 32 +/- 5 % (n = 15), not significantly different from controls. The rate of Cm increase was affected transiently by CST treatment, peaking at 1 min after patch rupture. The maximal rate of Cm increase was 4.27 +/- 0.85 fF s-1 (n = 12; measured 200 s after the start of patch rupture) in controls and 8.0 +/- 1.35 fF s-1 (n = 23; measured 75 s after the start of patch rupture) in CST-treated cells (P < 0.01). 3. In control cells cytosol dialysis with 0 microM Ca2+i decreased Cm by 9 +/- 3 % (n = 7), in CST-treated cells Cm increased by 11 +/- 3 % (n = 7) relative to resting Cm 400 s after the start of cytosol dialysis. The rate of change in Cm remained constant (controls: -1 to -2 fF s-1; CST treatment: 1-2 fF s-1). 4. Transient and sustained effects of CST treatment on changes in Cm at high or low [Ca2+]i, respectively, suggest a distinct role of cytoskeleton in Ca2+-dependent and Ca2+-independent changes in Cm. Transient enhancement of the rate of Cm by CST is consistent with a barrier role of cytoskeleton in regulated exocytosis. The sustained effect of CST on Ca2+-independent changes in Cm suggests cytoskeletal involvement in endocytosis.

Actin cytoskeleton depolymerization with Clostridium spiroforme toxin enhances the secretory activity of rat melanotrophs

PubMed Central

Chowdhury, Helena H; Popoff, Michel R; Zorec, Robert

1999-01-01

We measured membrane capacitance (Cm) in cultured rat melanotrophs pretreated with Clostridium spiroforme toxin (CST), which specifically depolymerises cortical filamentous actin (F-actin). Phalloidin staining confirmed that CST treatment depolymerised the F-actin. In control cells, cytosol dialysis with 1 μm Ci2+ increased Cm by 23 ± 4% (n = 11) relative to the resting Cm 400 s after the start of patch rupture. In CST-treated cells the increase in Cm was 32 ± 5% (n = 15), not significantly different from controls. The rate of Cm increase was affected transiently by CST treatment, peaking at 1 min after patch rupture. The maximal rate of Cm increase was 4.27 ± 0.85 fF s−1 (n = 12; measured 200 s after the start of patch rupture) in controls and 8.0 ± 1.35 fF s−1 (n = 23; measured 75 s after the start of patch rupture) in CST-treated cells (P < 0.01). In control cells cytosol dialysis with 0 μm Ci2+ decreased Cm by 9 ± 3% (n = 7), in CST-treated cells Cm increased by 11 ± 3% (n = 7) relative to resting Cm 400 s after the start of cytosol dialysis. The rate of change in Cm remained constant (controls: -1 to -2 fF s−1; CST treatment: 1-2 fF s−1). Transient and sustained effects of CST treatment on changes in Cm at high or low [Ca2+]i, respectively, suggest a distinct role of cytoskeleton in Ca2+-dependent and Ca2+-independent changes in Cm. Transient enhancement of the rate of Cm by CST is consistent with a barrier role of cytoskeleton in regulated exocytosis. The sustained effect of CST on Ca2+-independent changes in Cm suggests cytoskeletal involvement in endocytosis. PMID:10581310

Divergent regulation of the sarcomere and the cytoskeleton.

PubMed

Schevzov, Galina; Fath, Thomas; Vrhovski, Bernadette; Vlahovich, Nicole; Rajan, Sudarsan; Hook, Jeff; Joya, Josephine E; Lemckert, Frances; Puttur, Franz; Lin, Jim J-C; Hardeman, Edna C; Wieczorek, David F; O'Neill, Geraldine M; Gunning, Peter W

2008-01-04

The existence of a feedback mechanism regulating the precise amounts of muscle structural proteins, such as actin and the actin-associated protein tropomyosin (Tm), in the sarcomeres of striated muscles is well established. However, the regulation of nonmuscle or cytoskeletal actin and Tms in nonmuscle cell structures has not been elucidated. Unlike the thin filaments of striated muscles, the actin cytoskeleton in nonmuscle cells is intrinsically dynamic. Given the differing requirements for the structural integrity of the actin thin filaments of the sarcomere compared with the requirement for dynamicity of the actin cytoskeleton in nonmuscle cells, we postulated that different regulatory mechanisms govern the expression of sarcomeric versus cytoskeletal Tms, as key regulators of the properties of the actin cytoskeleton. Comprehensive analyses of tissues from transgenic and knock-out mouse lines that overexpress the cytoskeletal Tms, Tm3 and Tm5NM1, and a comparison with sarcomeric Tms provide evidence for this. Moreover, we show that overexpression of a cytoskeletal Tm drives the amount of filamentous actin.

Micropatterned Azopolymer Surfaces Modulate Cell Mechanics and Cytoskeleton Structure.

PubMed

Rianna, Carmela; Ventre, Maurizio; Cavalli, Silvia; Radmacher, Manfred; Netti, Paolo A

2015-09-30

Physical and chemical characteristics of materials are important regulators of cell behavior. In particular, cell elasticity is a fundamental parameter that reflects the state of a cell. Surface topography finely modulates cell fate and function via adhesion mediated signaling and cytoskeleton generated forces. However, how topographies alter cell mechanics is still unclear. In this work we have analyzed the mechanical properties of peripheral and nuclear regions of NIH-3T3 cells on azopolymer substrates with different topographic patterns. Micrometer scale patterns in the form of parallel ridges or square lattices of surface elevations were encoded on light responsive azopolymer films by means of contactless optical methods. Cell mechanics was investigated by atomic force microscopy (AFM). Cells and consequently the cell cytoskeleton were oriented along the linear patterns affecting cytoskeletal structures, e.g., formation of actin stress fibers. Our data demonstrate that topographic substrate patterns are recognized by cells and mechanical information is transferred by the cytoskeleton. Furthermore, cytoskeleton generated forces deform the nucleus, changing its morphology that appears to be related to different mechanical properties in the nuclear region.

Phosphoinositide 3-Kinase Regulates Glycolysis through Mobilization of Aldolase from the Actin cytoskeleton

PubMed Central

Hu, Hai; Juvekar, Ashish; Lyssiotis, Costas A.; Lien, Evan C.; Albeck, John G.; Oh, Doogie; Varma, Gopal; Hung, Yin Pun; Ullas, Soumya; Lauring, Josh; Seth, Pankaj; Lundquist, Mark R.; Tolan, Dean R.; Grant, Aaron K.; Needleman, Daniel J.; Asara, John M.; Cantley, Lewis C.

2016-01-01

Summary The Phosphoinositide 3-Kinase (PI3K) pathway regulates multiple steps in glucose metabolism but also cytoskeletal functions, such as cell movement and attachment. Here we show that PI3K directly coordinates glycolysis with cytoskeletal dynamics in an AKT-independent manner. Growth factors or insulin stimulate the PI3K-dependent activation of Rac, leading to disruption of the actin cytoskeleton, release of filamentous actin-bound aldolase A and an increase in aldolase activity. Consistently, PI3K-, but not AKT-, SGK- or mTOR-inhibitors, cause a significant decrease in glycolysis at the step catalyzed by aldolase, while activating PIK3CA mutations have the opposite effect. These results point towards a master regulatory function of PI3K that integrates an epithelial cell’s metabolism and its form, shape and function, coordinating glycolysis with the energy-intensive dynamics of actin remodeling. PMID:26824656

The mechanosensor of mesenchymal stem cells: mechanosensitive channel or cytoskeleton?

PubMed

Xiao, E; Chen, Chider; Zhang, Yi

2016-09-20

Mesenchymal stem cells (MSCs) are multipotent adult stem cells. MSCs and their potential for use in regenerative medicine have been investigated extensively. Recently, the mechanisms by which MSCs detect mechanical stimuli have been described in detail. As in other cell types, both mechanosensitive channels, such as transient receptor potential melastatin 7 (TRPM7), and the cytoskeleton, including actin and actomyosin, have been implicated in mechanosensation in MSCs. This review will focus on discussing the precise role of TRPM7 and the cytoskeleton in mechanosensation in MSCs.

Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro

PubMed Central

Lv, Zhimei; Hu, Mengsi; Ren, Xiaoxu; Fan, Minghua; Zhen, Junhui; Chen, Liqun; Lin, Jiangong; Ding, Nannan; Wang, Qun; Wang, Rong

2016-01-01

Fyn, a member of the Src family of tyrosine kinases, is a key regulator in cytoskeletal remodeling in a variety of cell types. Recent studies have demonstrated that Fyn is responsible for nephrin tyrosine phosphorylation, which will result in polymerization of actin filaments and podocyte damage. Thus detailed involvement of Fyn in podocytes is to be elucidated. In this study, we investigated the potential role of Fyn/ROCK signaling and its interactions with paxillin. Our results presented that high glucose led to filamentous actin (F-actin) rearrangement in podocytes, accompanied by paxillin phosphorylation and increased cell motility, during which Fyn and ROCK were markedly activated. Gene knockdown of Fyn by siRNA showed a reversal effect on high glucose-induced podocyte damage and ROCK activation; however, inhibition of ROCK had no significant effects on Fyn phosphorylation. These observations demonstrate that in vitro Fyn mediates high glucose-induced actin cytoskeleton remodeling of podocytes via promoting ROCK activation and paxillin phosphorylation. PMID:26881253

CaMKIIβ Association with the Actin Cytoskeleton Is Regulated by Alternative Splicing

PubMed Central

O'Leary, Heather; Lasda, Erika

2006-01-01

The Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII)β has morphogenic functions in neurons not shared by the α isoform. CaMKIIβ contains three exons (v1, v3, and v4) not present in the CaMKIIα gene, and two of these exons (v1 and v4) are subject to differential alternative splicing. We show here that CaMKIIβ, but not α, mediated bundling of F-actin filaments in vitro. Most importantly, inclusion of exon v1 was required for CaMKIIβ association with the F-actin cytoskeleton within cells. CaMKIIβe, which is the dominant variant around birth and lacks exon v1 sequences, failed to associate with F-actin. By contrast, CaMKIIβ′, which instead lacks exon v4, associated with F-actin as full-length CaMKIIβ. Previous studies with CaMKIIβ mutants have indicated a role of nonstimulated kinase activity in enhancing dendritic arborization. Here, we show that F-actin–targeted CaMKIIβ, but not α, was able to phosphorylate actin in vitro even by nonstimulated basal activity in absence of Ca2+/CaM. In rat pancreatic islets and in skeletal muscle, the actin-associated CaMKIIβ′ and βM were the predominant variants, respectively. Thus, cytoskeletal targeting may mediate functions of CaMKIIβ variants also outside the nervous system. PMID:16928958

HIV compromises integrity of the podocyte actin cytoskeleton through downregulation of the vitamin D receptor

PubMed Central

Chandel, Nirupama; Sharma, Bipin; Husain, Mohammad; Salhan, Divya; Singh, Tejinder; Rai, Partab; Mathieson, Peter W.; Saleem, Moin A.; Malhotra, Ashwani

2013-01-01

Alterations in the podocyte actin cytoskeleton have been implicated in the development of proteinuric kidney diseases. In the present study, we evaluated the effect of HIV on the podocyte actin cytoskeleton and the mechanism involved. We hypothesized that HIV may be compromising the actin cytoskeleton via downregulation of the vitamin D receptor (VDR) of conditionally immortalized differentiated human podocytes (CIDHPs). HIV-transduced podocytes (HIV/CIDHPs) not only displayed downregulation of VDR but also showed activation of the renin-angiotensin system (RAS) in the form of enhanced expression of renin and increased production of ANG II. Moreover, CIDHPs lacking VDR displayed enhanced ANG II production, and treatment of HIV/CIDHPs with EB1089 (vitamin D3; VD) attenuated ANG II production. HIV/CIDHPs as well as ANG II-treated CIDHPs exhibited enhanced expression of cathepsin (CTS) L. Additionally, losartan (an ANG II type I receptor blocker) inhibited both HIV- and ANG II-induced podocyte cathepsin L expression. Furthermore, VD downregulated HIV-induced podocyte CTSL expression. Both losartan and free radical scavengers attenuated HIV- and ANG II-induced podocyte reactive oxygen species (ROS) generation. HIV also led to cytosolic CTSL accumulation through enhancement of podocyte lysosomal membrane permeabilization; on the other hand, VD, losartan, and superoxide dismutase (SOD) attenuated HIV-induced enhanced podocyte cytosolic CTSL accumulation. Morphological evaluation of HIV/CIDHPs revealed sparse actin filaments and attenuated expression of dynamin. Interestingly, podocytes lacking CTSL displayed enhanced dynamin expression, and HIV/CIDHPs expressing CTSL exhibited downregulation of dynamin. These findings indicate that HIV-induced downregulation of podocyte VDR and associated RAS activation and cytosolic CTSL accumulation compromised the actin cytoskeleton. PMID:23467424

Letrozole regulates actin cytoskeleton polymerization dynamics in a SRC-1 dependent manner in the hippocampus of mice.

PubMed

Zhao, Yangang; Yu, Yanlan; Zhang, Yuanyuan; He, Li; Qiu, Linli; Zhao, Jikai; Liu, Mengying; Zhang, Jiqiang

2017-03-01

In the hippocampus, local estrogens (E 2 ) derived from testosterone that is catalyzed by aromatase play important roles in the regulation of hippocampal neural plasticity, but the underlying mechanisms remain unclear. The actin cytoskeleton contributes greatly to hippocampal synaptic plasticity; however, whether it is regulated by local E 2 and the related mechanisms remain to be elucidated. In this study, we first examined the postnatal developmental profiles of hippocampal aromatase and specific proteins responsible for actin cytoskeleton dynamics. Then we used aromatase inhibitor letrozole (LET) to block local E 2 synthesis and examined the changes of these proteins and steroid receptor coactivator-1 (SRC-1), the predominant coactivator for steroid nuclear receptors. Finally, SRC-1 specific RNA interference was used to examine the effects of SRC-1 on the expression of these actin remodeling proteins. The results showed a V-type profile for aromatase and increased profiles for actin cytoskeleton proteins in both male and female hippocampus without obvious sex differences. LET treatment dramatically decreased the F-actin/G-actin ratio, the expression of Rictor, phospho-AKT (ser473), Profilin-1, phospho-Cofilin (Ser3), and SRC-1 in a dose-dependent manner. In vitro studies demonstrated that LET induced downregulation of these proteins could be reversed by E 2 , and E 2 induced increase of these proteins were significantly suppressed by SRC-1 shRNA interference. These results for the first time clearly demonstrated that local E 2 inhibition could induce aberrant actin polymerization; they also showed an important role of SRC-1 in the mediation of local E 2 action on hippocampal synaptic plasticity by regulation of actin cytoskeleton dynamics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differential nuclear shape dynamics of invasive andnon-invasive breast cancer cells are associated with actin cytoskeleton organization and stability.

PubMed

Chiotaki, Rena; Polioudaki, Hara; Theodoropoulos, Panayiotis A

2014-08-01

Cancer cells often exhibit characteristic aberrations in their nuclear architecture, which are indicative of their malignant potential. In this study, we have examined the nuclear and cytoskeletal composition, attachment configuration dynamics, and osmotic or drug treatment response of invasive (Hs578T and MDA-MB-231) and non-invasive (MCF-10A and MCF-7) breast cancer cell lines. Unlike MCF-10A and MCF-7, Hs578T and MDA-MB-231 cells showed extensive nuclear elasticity and deformability and displayed distinct kinetic profiles during substrate attachment. The nuclear shape of MCF-10A and MCF-7 cells remained almost unaffected upon detachment, hyperosmotic shock, or cytoskeleton depolymerization, while Hs578T and MDA-MB-231 revealed dramatic nuclear contour malformations following actin reorganization.

RIC8A is essential for the organisation of actin cytoskeleton and cell-matrix interaction.

PubMed

Ruisu, Katrin; Meier, Riho; Kask, Keiu; Tõnissoo, Tambet; Velling, Teet; Pooga, Margus

2017-08-15

RIC8A functions as a chaperone and guanine nucleotide exchange factor for a subset of G protein α subunits. Multiple G protein subunits mediate various signalling events that regulate cell adhesion and migration and the involvement of RIC8A in some of these processes has been demonstrated. We have previously shown that the deficiency of RIC8A causes a failure in mouse gastrulation and neurogenesis - major events in embryogenesis that rely on proper association of cells with the extracellular matrix (ECM) and involve active cell migration. To elaborate on these findings, we used Ric8a -/- mouse embryonic stem cells and Ric8a-deficient mouse embryonic fibroblasts, and found that RIC8A plays an important role in the organisation and remodelling of actin cytoskeleton and cell-ECM association. Ric8a-deficient cells were able to attach to different ECM components, but were unable to spread correctly, and did not form stress fibres or focal adhesion complexes. We also found that the presence of RIC8A is necessary for the activation of β1 integrins and integrin-mediated cell migration. Copyright © 2017 Elsevier Inc. All rights reserved.

A spatiotemporal characterization method for the dynamic cytoskeleton

PubMed Central

Alhussein, Ghada; Shanti, Aya; Farhat, Ilyas A. H.; Timraz, Sara B. H.; Alwahab, Noaf S. A.; Pearson, Yanthe E.; Martin, Matthew N.; Christoforou, Nicolas

2016-01-01

The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box‐counting is used to infer a fractal dimension (D f) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin‐treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically disrupted actin networks, and quantifies rates of cytoskeletal degradation. Furthermore, GVI distributions were found to be inversely proportional to D f, having several biophysical implications for cytoskeleton formation/degradation. We additionally demonstrated detection sensitivity of differences in D f and GVI for cells seeded on substrates with varying degrees of stiffness, and coated with different attachment proteins. This general approach can be further implemented to gain insights on dynamic growth, disruption, and structure of the cytoskeleton (and other complex biological morphology) due to biological, chemical, or physical stimuli. © 2016 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc. PMID:27015595

Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells.

PubMed

Fujimoto, Tomokazu; Inoue, Toshihiro; Inoue-Mochita, Miyuki; Tanihara, Hidenobu

2016-04-01

The regulation of the actin cytoskeleton in trabecular meshwork (TM) cells is important for controlling outflow of the aqueous humor. In some reports, dexamethasone (DEX) increased the aqueous humor outflow resistance and induced unusual actin structures, such as cross-linked actin networks (CLAN), in TM cells. However, the functions and dynamics of CLAN in TM cells are not completely known, partly because actin stress fibers have been observed only in fixed cells. We conducted live-cell imaging of the actin dynamics in TM cells with or without DEX treatment. An actin-green fluorescent protein (GFP) fusion construct with a modified insect virus was transfected into porcine TM cells. Time-lapse imaging of live TM cells treated with 25 μM Y-27632 and 100 nM DEX was performed using an inverted fluorescence microscope. Fluorescent images were recorded every 15 s for 30 min after Y-27632 treatment or every 30 min for 72 h after DEX treatment. The GFP-actin was expressed in 22.7 ± 10.9% of the transfected TM cells. In live TM cells, many actin stress fibers were observed before the Y-27632 treatment. Y-27632 changed the cell shape and decreased stress fibers in a time-dependent manner. In fixed cells, CLAN-like structures were seen in 26.5 ± 1.7% of the actin-GFP expressed PTM cells treated with DEX for 72 h. In live imaging, there was 28% CLAN-like structure formation at 72 h after DEX treatment, and the lifetime of CLAN-like structures increased after DEX treatment. The DEX-treated cells with CLAN-like structures showed less migration than DEX-treated cells without CLAN-like structures. Furthermore, the control cells (without DEX treatment) with CLAN-like structures also showed less migration than the control cells without CLAN-like structures. These results suggested that CLAN-like structure formation was correlated with cell migration in TM cells. Live cell imaging of the actin cytoskeleton provides valuable information on the actin dynamics in TM

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells.

PubMed

Reifenberger, Matthew S; Yu, Ling; Bao, Hui-Fang; Duke, Billie Jeanne; Liu, Bing-Chen; Ma, He-Ping; Alli, Ahmed A; Eaton, Douglas C; Alli, Abdel A

2014-07-01

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex. Copyright © 2014 the American Physiological Society.

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells

PubMed Central

Reifenberger, Matthew S.; Yu, Ling; Bao, Hui-Fang; Duke, Billie Jeanne; Liu, Bing-Chen; Ma, He-Ping; Eaton, Douglas C.; Alli, Abdel A.

2014-01-01

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na+ channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex. PMID:24829507

TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics

PubMed Central

Bailly, Aurelien; Zwiewka, Marta; Sovero, Valpuri; Ge, Pei; Aryal, Bibek; Hao, Pengchao; Linnert, Miriam; Burgardt, Noelia Inés; Lücke, Christian; Weiwad, Matthias; Michel, Max; Weiergräber, Oliver H.; Pollmann, Stephan; Azzarello, Elisa; Fukao, Yoichiro; Hoffmann, Céline; Wedlich-Söldner, Roland

2016-01-01

Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity. PMID:27053424

The formin DIAPH1 (mDia1) regulates megakaryocyte proplatelet formation by remodeling the actin and microtubule cytoskeletons.

PubMed

Pan, Jiajia; Lordier, Larissa; Meyran, Deborah; Rameau, Philippe; Lecluse, Yann; Kitchen-Goosen, Susan; Badirou, Idinath; Mokrani, Hayat; Narumiya, Shuh; Alberts, Arthur S; Vainchenker, William; Chang, Yunhua

2014-12-18

Megakaryocytes are highly specialized precursor cells that produce platelets via cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) requires profound changes in microtubule and actin organization. In this work, we demonstrated that DIAPH1 (mDia1), a mammalian homolog of Drosophila diaphanous that works as an effector of the small GTPase Rho, negatively regulates PPF by controlling the dynamics of the actin and microtubule cytoskeletons. Moreover, we showed that inhibition of both DIAPH1 and the Rho-associated protein kinase (Rock)/myosin pathway increased PPF via coordination of both cytoskeletons. We provide evidence that 2 major effectors of the Rho GTPase pathway (DIAPH1 and Rock/myosin II) are involved not only in Rho-mediated stress fibers assembly, but also in the regulation of microtubule stability and dynamics during PPF. © 2014 by The American Society of Hematology.

Role of ANC-1 in tethering nuclei to the actin cytoskeleton.

PubMed

Starr, Daniel A; Han, Min

2002-10-11

Mutations in anc-1 (nuclear anchorage defective) disrupt the positioning of nuclei and mitochondria in Caenorhabditis elegans. ANC-1 is shown to consist of mostly coiled regions with a nuclear envelope localization domain (called the KASH domain) and an actin-binding domain; this structure was conserved with the Drosophila protein Msp-300 and the mammalian Syne proteins. Antibodies against ANC-1 localized cytoplasmically and were enriched at the nuclear periphery in an UNC-84-dependent manner. Overexpression of the KASH domain or the actin-binding domain caused a dominant negative anchorage defect. Thus, ANC-1 may connect nuclei to the cytoskeleton by interacting with UNC-84 at the nuclear envelope and with actin in the cytoplasm.

A spatiotemporal characterization method for the dynamic cytoskeleton.

PubMed

Alhussein, Ghada; Shanti, Aya; Farhat, Ilyas A H; Timraz, Sara B H; Alwahab, Noaf S A; Pearson, Yanthe E; Martin, Matthew N; Christoforou, Nicolas; Teo, Jeremy C M

2016-05-01

The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box-counting is used to infer a fractal dimension (Df ) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin-treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically disrupted actin networks, and quantifies rates of cytoskeletal degradation. Furthermore, GVI distributions were found to be inversely proportional to Df , having several biophysical implications for cytoskeleton formation/degradation. We additionally demonstrated detection sensitivity of differences in Df and GVI for cells seeded on substrates with varying degrees of stiffness, and coated with different attachment proteins. This general approach can be further implemented to gain insights on dynamic growth, disruption, and structure of the cytoskeleton (and other complex biological morphology) due to biological, chemical, or physical stimuli. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Cyclin-Dependent Kinase 5 Links Extracellular Cues to Actin Cytoskeleton During Dendritic Spine Development

PubMed Central

Fu, Amy KY

2007-01-01

Emerging evidence has indicated a regulatory role of cyclin-dependent kinase 5 (Cdk5) in synaptic plasticity as well as in higher brain functions, such as learning and memory. However, the molecular and cellular mechanisms underlying the actions of Cdk5 at synapses remain unclear. Recent findings demonstrate that Cdk5 regulates dendritic spine morphogenesis through modulating actin dynamics. Ephexin1 and WAVE-1, two important regulators of the actin cytoskeleton, have both been recently identified as substrates for Cdk5. Importantly, phosphorylation of these proteins by Cdk5 leads to dendritic spine loss, revealing a potential mechanism by which Cdk5 regulates synapse remodeling. Furthermore, Cdk5-dependent phosphorylation of ephexin1 is required for the ephrin-A1 mediated spine retraction, pointing to a critical role of Cdk5 in conveying signals from extracellular cues to actin cytoskeleton at synapses. Taken together, understanding the precise regulation of Cdk5 and its downstream targets at synapses would provide important insights into the multi-regulatory roles of Cdk5 in actin remodeling during dendritic spine development. PMID:19270534

Novel Regulation of Ski Protein Stability and Endosomal Sorting by Actin Cytoskeleton Dynamics in Hepatocytes*

PubMed Central

Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R.; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A.; Macías-Silva, Marina

2015-01-01

TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. PMID:25561741

Actin polymerization drives polar growth in Arabidopsis root hair cells.

PubMed

Vazquez, Luis Alfredo Bañuelos; Sanchez, Rosana; Hernandez-Barrera, Alejandra; Zepeda-Jazo, Isaac; Sánchez, Federico; Quinto, Carmen; Torres, Luis Cárdenas

2014-01-01

In plants, the actin cytoskeleton is a prime regulator of cell polarity, growth, and cytoplasmic streaming. Tip growth, as observed in root hairs, caulonema, and pollen tubes, is governed by many factors, including calcium gradients, exocytosis and endocytosis, reactive oxygen species, and the cytoskeleton. Several studies indicate that the polymerization of G-actin into F-actin also contributes to tip growth. The structure and function of F-actin within the apical dome is variable, ranging from a dense meshwork to sparse single filaments. The presence of multiple F-actin structures in the elongating apices of tip-growing cells suggests that this cytoskeletal array is tightly regulated. We recently reported that sublethal concentrations of fluorescently labeled cytochalasin could be used to visualize the distribution of microfilament plus ends using fluorescence microscopy, and found that the tip region of the growing root hair cells of a legume plant exhibits a clear response to the nodulation factors secreted by Rhizobium. (1) In this current work, we expanded our analysis using confocal microscopy and demonstrated the existence of highly dynamic fluorescent foci along Arabidopsis root hair cells. Furthermore, we show that the strongest fluorescence signal accumulates in the tip dome of the growing root hair and seems to be in close proximity to the apical plasma membrane. Based on these findings, we propose that actin polymerization within the dome of growing root hair cells regulates polar growth.

Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation

PubMed Central

Roa-Espitia, Ana L.; Hernández-Rendón, Eva R.; Baltiérrez-Hoyos, Rafael; Muñoz-Gotera, Rafaela J.; Cote-Vélez, Antonieta; Jiménez, Irma; González-Márquez, Humberto

2016-01-01

ABSTRACT Several focal adhesion proteins are known to cooperate with integrins to link the extracellular matrix to the actin cytoskeleton; as a result, many intracellular signaling pathways are activated and several focal adhesion complexes are formed. However, how these proteins function in mammalian spermatozoa remains unknown. We confirm the presence of focal adhesion proteins in guinea pig spermatozoa, and we explore their role during capacitation and the acrosome reaction, and their relationship with the actin cytoskeleton. Our results suggest the presence of a focal adhesion complex formed by β1-integrin, focal adhesion kinase (FAK), paxillin, vinculin, talin, and α-actinin in the acrosomal region. Inhibition of FAK during capacitation affected the protein tyrosine phosphorylation associated with capacitation that occurs within the first few minutes of capacitation, which caused the acrosome reaction to become increasingly Ca2+ dependent and inhibited the polymerization of actin. The integration of vinculin and talin into the complex, and the activation of FAK and paxillin during capacitation, suggests that the complex assembles at this time. We identify that vinculin and α-actinin increase their interaction with F-actin while it remodels during capacitation, and that during capacitation focal adhesion complexes are structured. FAK contributes to acrosome integrity, likely by regulating the polymerization and the remodeling of the actin cytoskeleton. PMID:27402964

A prestressed cable network model of the adherent cell cytoskeleton.

PubMed

Coughlin, Mark F; Stamenović, Dimitrije

2003-02-01

A prestressed cable network is used to model the deformability of the adherent cell actin cytoskeleton. The overall and microstructural model geometries and cable mechanical properties were assigned values based on observations from living cells and mechanical measurements on isolated actin filaments, respectively. The models were deformed to mimic cell poking (CP), magnetic twisting cytometry (MTC) and magnetic bead microrheometry (MBM) measurements on living adherent cells. The models qualitatively and quantitatively captured the fibroblast cell response to the deformation imposed by CP while exhibiting only some qualitative features of the cell response to MTC and MBM. The model for CP revealed that the tensed peripheral actin filaments provide the key resistance to indentation. The actin filament tension that provides mechanical integrity to the network was estimated at approximately 158 pN, and the nonlinear mechanical response during CP originates from filament kinematics. The MTC and MBM simulations revealed that the model is incomplete, however, these simulations show cable tension as a key determinant of the model response.

F-actin clustering and cell dysmotility induced by the pathological W148R missense mutation of filamin B at the actin-binding domain.

PubMed

Zhao, Yongtong; Shapiro, Sandor S; Eto, Masumi

2016-01-01

Filamin B (FLNB) is a dimeric actin-binding protein that orchestrates the reorganization of the actin cytoskeleton. Congenital mutations of FLNB at the actin-binding domain (ABD) are known to cause abnormalities of skeletal development, such as atelosteogenesis types I and III and Larsen's syndrome, although the underlying mechanisms are poorly understood. Here, using fluorescence microscopy, we characterized the reorganization of the actin cytoskeleton in cells expressing each of six pathological FLNB mutants that have been linked to skeletal abnormalities. The subfractionation assay showed a greater accumulation of the FLNB ABD mutants W148R and E227K than the wild-type protein to the cytoskeleton. Ectopic expression of FLNB-W148R and, to a lesser extent, FLNB-E227K induced prominent F-actin accumulations and the consequent rearrangement of focal adhesions, myosin II, and septin filaments and results in a delayed directional migration of the cells. The W148R protein-induced cytoskeletal rearrangement was partially attenuated by the inhibition of myosin II, p21-activated protein kinase, or Rho-associated protein kinase. The expression of a single-head ABD fragment with the mutations partially mimicked the rearrangement induced by the dimer. The F-actin clustering through the interaction with the mutant FLNB ABD may limit the cytoskeletal reorganization, preventing normal skeletal development. Copyright © 2016 the American Physiological Society.

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

Function of the cytoskeleton in gravisensing during spaceflight

NASA Astrophysics Data System (ADS)

Hughes-Fulford, M.

2003-10-01

Since astronauts and cosmonauts have significant bone loss in microgravity we hypothesized that there would be physiological changes in cellular bone growth and cytoskeleton in the absence of gravity. Investigators from around the world have studied a multitude of bone cells in microgravity including Ros 17/2.8, Mc3T3-E1, MG-63, hFOB and primary chicken calvaria. Changes in cytoskeleton and extracellular matrix (ECM) have been noted in many of these studies. Investigators have noted changes in shape of cells exposed to as little as 20 seconds of microgravity in parabolic flight. Our laboratory reported that quiescent osteoblasts activated by sera under microgravity conditions had a significant 60% reduction in growth (p<0.001) but a paradoxical 2-folf increase in release of the osteoblast autocrine factor PGE 2 when compared to ground controls. In addition, a collapse of the osteoblast actin cytoskeleton and loss of focal adhesions has been noted after 4 days in microgravity. Later studies in Biorack on STS-76, 81 and 84 confirmed the increased release of PGE 2 and collapse of the actin cytoskeleton in cells grown in microgravity conditions, however flown cells under 1g conditions maintained normal actin cytoskeleton and fibronectin matrix. The changes seen in the cytoskeleton are probably not due to alterations in fibronectin message or protein synthesis since no differences have been noted in microgravity. Multiple investigators have observed actin and microtubule cytoskeletal modifications in microgravity, suggesting a common root cause for the change in cell architecture. The inability of the Og grown osteoblast to respond to sera activation suggests that there is a major alteration in anabolic signal transduction under microgravity conditions, most probably through the growth factor receptors and/or the associated kinase pathways that are connected to the cytoskeleton. Cell cycle is dependent on the cytoskeleton. Alterations in cytoskeletal structure can block

OPC-compounds prevent oxidant-induced carbonylation and depolymerization of the F-actin cytoskeleton and intestinal barrier hyperpermeability.

PubMed

Banan, A; Fitzpatrick, L; Zhang, Y; Keshavarzian, A

2001-02-01

Rebamipide (OPC-12759), a quinolone derivative, and OPC-6535, a thiazol-carboxylic acid derivative, are compounds with ability to protect gastrointestinal (GI) mucosal integrity against reactive oxygen metabolites (ROM). The underlying mechanism of OPC-mediated protection remains poorly understood. It is now established that ROM can injure the mucosa by disruption of the cytoskeletal network, a key component of mucosal barrier integrity. We, therefore, investigated whether OPC compounds prevent the oxidation, disassembly, and instability of the cytoskeletal protein actin and, in turn, protect intestinal barrier function against ROM. Human intestinal (Caco-2) cell monolayers were pretreated with OPC (-12759 or -6535) prior to incubation with ROM (H2O2) or HOCl). Effects on cell integrity (ethidium homodimer-1), epithelial barrier function (fluorescein sulfonic acid clearance), and actin cytoskeletal integrity (high-resolution laser confocal) were then determined. Cells were also processed for quantitative immunoblotting of G- and F-actin to measure oxidation (carbonylation) and disassembly of actin. In monolayers exposed to ROM, preincubation with OPC compounds prevented actin oxidation, decreased depolymerized G-actin, and enhanced the stable F-actin. Concomitantly, OPC agents abolished both actin cytoskeletal disruption and monolayer barrier dysfunction. Data suggest for the first time that OPC drugs prevent oxidation of actin and lead to the protection of actin cytoskeleton and intestinal barrier integrity against oxidant insult. Accordingly, these compounds may be used as novel therapeutic agents for the treatment of a variety of oxidative inflammatory intestinal disorders with an abnormal mucosal barrier such as inflammatory bowel disease.

[Effect of different oxygen tension on the cytoskeleton remodeling of goat temporomandibular joint disc cells].

PubMed

Xiaolan, He; Guangjie, Bao; Linglu, Sun; Xue, Zhang; Shanying, Bao; Hong, Kang

2017-08-01

Objective The effect of different oxygen tensions on the cytoskeleton remodeling of goat temporomandibular joint (TMJ) disc cells were investigated. Methods Goat TMJ disc cells were cultured under normoxia (21% O₂) and hypoxia (2%, 4%, and 8% O₂). Toluidine blue, picrosirius red, and type Ⅰ collagen immunocytochemical staining were performed to observe the changes in cell phenotype under different oxygen levels. Immunofluorescent staining and real-time reverse transcription-polymerase chain reaction analysis were then performed to identify actin, tubulin, and vimentin in the cultured disc cells. Results TMJ disc cells still displayed fibroblast characteristics under different oxygen levels and their cytoskeletons had regular arrangement. The fluorescence intensities of actin and vimentin were lowest at 4% O₂(P<0.05), whereas that of tubulin was highest at 2% O₂ (P<0.05). No significant difference among the other groups was observed (P>0.05). Actin mRNA levels were considerably decreased at 2% O₂ and 4% O₂ in hypoxic conditions, while actin mRNA expression was highest in 21% O₂. Tubulin mRNA levels considerably increased at 2% O₂, while tubulin mRNA expression was lowest in 8% O₂ (P<0.05). Vimentin mRNA expression was lowest at 4% O₂ and highest at 21% O₂, and significant differences were observed between vimentin mRNA expression levels among these oxygen levels (P<0.05). Conclusion Cytoskeletons were reconstructed in different oxygen tensions, and 2% O₂ may be the optimal oxygen level required to proliferate TMJ disc cells.

Drebrin coordinates the actin and microtubule cytoskeleton during the initiation of axon collateral branches.

PubMed

Ketschek, Andrea; Spillane, Mirela; Dun, Xin-Peng; Hardy, Holly; Chilton, John; Gallo, Gianluca

2016-10-01

Drebrin is a cytoskeleton-associated protein which can interact with both actin filaments and the tips of microtubules. Its roles have been studied mostly in dendrites, and the functions of drebrin in axons are less well understood. In this study, we analyzed the role of drebrin, through shRNA-mediated depletion and overexpression, in the collateral branching of chicken embryonic sensory axons. We report that drebrin promotes the formation of axonal filopodia and collateral branches in vivo and in vitro. Live imaging of cytoskeletal dynamics revealed that drebrin promotes the formation of filopodia from precursor structures termed axonal actin patches. Endogenous drebrin localizes to actin patches and depletion studies indicate that drebrin contributes to the development of patches. In filopodia, endogenous drebrin localizes to the proximal portion of the filopodium. Drebrin was found to promote the stability of axonal filopodia and the entry of microtubule plus tips into axonal filopodia. The effects of drebrin on the stabilization of filopodia are independent of its effects on promoting microtubule targeting to filopodia. Inhibition of myosin II induces a redistribution of endogenous drebrin distally into filopodia, and further increases branching in drebrin overexpressing neurons. Finally, a 30 min treatment with the branch-inducing signal nerve growth factor increases the levels of axonal drebrin. This study determines the specific roles of drebrin in the regulation of the axonal cytoskeleton, and provides evidence that drebrin contributes to the coordination of the actin and microtubule cytoskeleton during the initial stages of axon branching. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1092-1110, 2016. © 2016 Wiley Periodicals, Inc.

Drebrin Coordinates the Actin and Microtubule Cytoskeleton During the Initiation of Axon Collateral Branches

PubMed Central

Ketschek, Andrea; Spillane, Mirela; Dun, Xin-Peng; Hardy, Holly; Chilton, John; Gallo, Gianluca

2016-01-01

Drebrin is a cytoskeleton-associated protein which can interact with both actin filaments and the tips of microtubules. Its roles have been studied mostly in dendrites, and the functions of drebrin in axons are less well understood. In this work we analyzed the role of drebrin, through shRNA-mediated depletion and over-expression, in the collateral branching of chicken embryonic sensory axons. We report that drebrin promotes the formation of axonal filopodia and collateral branches in vivo and in vitro. Live imaging of cytoskeletal dynamics revealed that drebrin promotes the formation of filopodia from precursor structures termed axonal actin patches. Endogenous drebrin localizes to actin patches and depletion studies indicate that drebrin contributes to the development of patches. In filopodia, endogenous drebrin localizes to the proximal portion of the filopodium. Drebrin was found to promote the stability of axonal filopodia and the entry of microtubule plus tips into axonal filopodia. The effects of drebrin on the stabilization of filopodia are independent of its effects on promoting microtubule targeting to filopodia. Inhibition of myosin II induces a redistribution of endogenous drebrin distally into filopodia, and further increases branching in drebrin overexpressing neurons. Finally, a 30 minute treatment with the branch inducing signal nerve growth factor increases the levels of axonal drebrin. The current study determines the specific roles of drebrin in the regulation of the axonal cytoskeleton, and provides evidence that drebrin contributes to the coordination of the actin and microtubule cytoskeleton during the initial stages of axon branching. PMID:26731339

Role of cortactin in dynamic actin remodeling events in gonadotrope cells.

PubMed

Navratil, Amy M; Dozier, Melissa G; Whitesell, Jennifer D; Clay, Colin M; Roberson, Mark S

2014-02-01

GnRH induces marked activation of the actin cytoskeleton in gonadotropes; however, the physiological consequences and cellular mechanisms responsible have yet to be fully elucidated. The current studies focus on the actin scaffolding protein cortactin. Using the gonadotrope-derived αT3-1 cell line, we found that cortactin is phosphorylated at Y(421), S(405), and S(418) in a time-dependent manner in response to the GnRH agonist buserelin (GnRHa). GnRHa induced translocation of cortactin to the leading edge of the plasma membrane where it colocalizes with actin and actin-related protein 3 (Arp3). Incubation of αT3-1 cells with the c-src inhibitor phosphoprotein phosphatase 1, blocked tyrosine phosphorylation of cortactin, reduced cortactin association with Arp3, and blunted actin reorganization in response to GnRHa. Additionally, we used RNA silencing strategies to knock down cortactin in αT3-1 cells. Knockdown of cortactin blocked the ability of αT3-1 cells to generate filopodia, lamellipodia, and membrane ruffles in response to GnRHa. We show that lamellipodia and filopodia are capable of LHβ mobilization in primary pituitary culture after GnRHa treatment, and disruption of these structures using jasplakinolide reduces LH secretion. Collectively, our findings suggest that after GnRHa activation, src activity leads to tyrosine phosphorylation of cortactin, which facilitates its association with Arp3 to engage the actin cytoskeleton. The reorganization of actin by cortactin potentially underlies GnRHa-induced secretory events within αT3-1 cells.

Effect of cooling (4°C) and cryopreservation on cytoskeleton actin and protein tyrosine phosphorylation in buffalo spermatozoa.

PubMed

Naresh, Sai

2016-02-01

Semen cryopreservation is broadly utilized as a part of the bovine reproducing industry, a large portion of the spermatozoa does not survive and the majority of those that do survive experience various molecular and physiological changes that influence their fertilizing capacity. The main aim of this study is to determine the effect of cooling (4 °C) and cryopreservation on cytoskeleton actin, tyrosine phosphorylation and quality of buffalo spermatozoa, and to determine the similarity between in vitro capacitation and cryopreservation induced capacitation like changes. To achieve this, Western blot was used to examine the changes in actin expression and protein tyrosine phosphorylation, whereas changes in actin polymerization, localization of actin and protein tyrosine phosphorylation during capacitation and cryopreservation were evaluated by indirect immunofluorescence technique. Localization studies revealed that the actin localized to flagella and acrosome membrane regions and following, capacitation it migrated towards the acrosome region of sperm. Time dependent increase in actin polymerization and protein tyrosine phosphorylation was observed during in vitro capacitation. The cooling phase (4 °C) and cryopreservation processes resulted in the loss/damage of cytoskeleton actin. In addition, we performed the actin polymerization and protein tyrosine phosphorylation in cooled and cryopreserved buffalo spermatozoa. Interestingly, cooling and cryopreservation induces actin polymerization and protein tyrosine phosphorylation, which were similar to in vitro capacitation (cryo-capacitation). These changes showed 1.3 folds reduction in the sperm quality parameters which includes motility, viability and plasma membrane integrity. Furthermore, our findings indicate that cooling and cryopreservation damages the cytoskeleton actin and also induces capacitation like changes such as protein tyrosine phosphorylation and actin polymerization. This could be one of the

Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

PubMed Central

Phee, Hyewon; Au-Yeung, Byron B; Pryshchep, Olga; O'Hagan, Kyle Leonard; Fairbairn, Stephanie Grace; Radu, Maria; Kosoff, Rachelle; Mollenauer, Marianne; Cheng, Debra; Chernoff, Jonathan; Weiss, Arthur

2014-01-01

The molecular mechanisms that govern thymocyte development and maturation are incompletely understood. The P21-activated kinase 2 (Pak2) is an effector for the Rho family GTPases Rac and Cdc42 that regulate actin cytoskeletal remodeling, but its role in the immune system remains poorly understood. In this study, we show that T-cell specific deletion of Pak2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development, maturation, and egress of thymocytes. Pak2 was required for pre-TCR β-selection and positive selection. Surprisingly, Pak2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2. Mechanistically, Pak2 is required for actin cytoskeletal remodeling triggered by TCR. Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of Pak2, uncovering the critical function of Pak2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation. DOI: http://dx.doi.org/10.7554/eLife.02270.001 PMID:24843022

T Lymphocyte Migration: An Action Movie Starring the Actin and Associated Actors.

PubMed

Dupré, Loïc; Houmadi, Raïssa; Tang, Catherine; Rey-Barroso, Javier

2015-01-01

The actin cytoskeleton is composed of a dynamic filament meshwork that builds the architecture of the cell to sustain its fundamental properties. This physical structure is characterized by a continuous remodeling, which allows cells to accomplish complex motility steps such as directed migration, crossing of biological barriers, and interaction with other cells. T lymphocytes excel in these motility steps to ensure their immune surveillance duties. In particular, actin cytoskeleton remodeling is a key to facilitate the journey of T lymphocytes through distinct tissue environments and to tune their stop and go behavior during the scanning of antigen-presenting cells. The molecular mechanisms controlling actin cytoskeleton remodeling during T lymphocyte motility have been only partially unraveled, since the function of many actin regulators has not yet been assessed in these cells. Our review aims to integrate the current knowledge into a comprehensive picture of how the actin cytoskeleton drives T lymphocyte migration. We will present the molecular actors that control actin cytoskeleton remodeling, as well as their role in the different T lymphocyte motile steps. We will also highlight which challenges remain to be addressed experimentally and which approaches appear promising to tackle them.

The cytoskeleton in cell-autonomous immunity: structural determinants of host defence

PubMed Central

Mostowy, Serge; Shenoy, Avinash R.

2016-01-01

Host cells use antimicrobial proteins, pathogen-restrictive compartmentalization and cell death in their defence against intracellular pathogens. Recent work has revealed that four components of the cytoskeleton — actin, microtubules, intermediate filaments and septins, which are well known for their roles in cell division, shape and movement — have important functions in innate immunity and cellular self-defence. Investigations using cellular and animal models have shown that these cytoskeletal proteins are crucial for sensing bacteria and for mobilizing effector mechanisms to eliminate them. In this Review, we highlight the emerging roles of the cytoskeleton as a structural determinant of cell-autonomous host defence. PMID:26292640

Calreticulin attenuated microwave radiation-induced human microvascular endothelial cell injury through promoting actin acetylation and polymerization.

PubMed

Xu, Feifei; Wang, You; Tao, Tianqi; Song, Dandan; Liu, Xiuhua

2017-01-01

Recent work reveals that actin acetylation modification has been linked to different normal and disease processes and the effects associated with metabolic and environmental stressors. Herein, we highlight the effects of calreticulin on actin acetylation and cell injury induced by microwave radiation in human microvascular endothelial cell (HMEC). HMEC injury was induced by high-power microwave of different power density (10, 30, 60, 100 mW/cm 2 , for 6 min) with or without exogenous recombinant calreticulin. The cell injury was assessed by lactate dehydrogenase (LDH) activity and Cell Counting Kit-8 in culture medium, migration ability, intercellular junction, and cytoskeleton staining in HMEC. Western blotting analysis was used to detected calreticulin expression in cytosol and nucleus and acetylation of globular actin (G-actin). We found that HMEC injury was induced by microwave radiation in a dose-dependent manner. Pretreatment HMEC with calreticulin suppressed microwave radiation-induced LDH leakage and increased cell viability and improved microwave radiation-induced decrease in migration, intercellular junction, and cytoskeleton. Meanwhile, pretreatment HMEC with exogenous calreticulin upregulated the histone acetyltransferase activity and the acetylation level of G-actin and increased the fibrous actin (F-actin)/G-actin ratio. We conclude that exogenous calreticulin protects HMEC against microwave radiation-induced injury through promoting actin acetylation and polymerization.

The plant cytoskeleton controls regulatory volume increase.

PubMed

Liu, Qiong; Qiao, Fei; Ismail, Ahmed; Chang, Xiaoli; Nick, Peter

2013-09-01

The ability to adjust cell volume is required for the adaptation to osmotic stress. Plant protoplasts can swell within seconds in response to hypoosmotic shock suggesting that membrane material is released from internal stores. Since the stability of plant membranes depends on submembraneous actin, we asked, whether this regulatory volume control depends on the cytoskeleton. As system we used two cell lines from grapevine which differ in their osmotic tolerance and observed that the cytoskeleton responded differently in these two cell lines. To quantify the ability for regulatory volume control, we used hydraulic conductivity (Lp) as readout and demonstrated a role of the cytoskeleton in protoplast swelling. Chelation of calcium, inhibition of calcium channels, or manipulation of membrane fluidity, did not significantly alter Lp, whereas direct manipulation of the cytoskeleton via specific chemical reagents, or indirectly, through the bacterial elicitor Harpin or activation of phospholipase D, was effective. By optochemical engineering of actin using a caged form of the phytohormone auxin we can break the symmetry of actin organisation resulting in a localised deformation of cell shape indicative of a locally increased Lp. We interpret our findings in terms of a model, where the submembraneous cytoskeleton controls the release of intracellular membrane stores during regulatory volume change. Copyright © 2013 Elsevier B.V. All rights reserved.

Large gradient high magnetic field affects the association of MACF1 with actin and microtubule cytoskeleton.

PubMed

Qian, Ai-Rong; Hu, Li-Fang; Gao, Xiang; Zhang, Wei; Di, Sheng-Meng; Tian, Zong-Cheng; Yang, Peng-Fei; Yin, Da-Chuan; Weng, Yuan-Yuan; Shang, Peng

2009-10-01

The intense inhomogeneous magnetic fields acting on the diamagnetic materials naturally present in cells can generate strong magnetic forces. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can produce three magnetic force fields of -1360, 0, and 1312 T(2)/m, and three corresponding apparent gravity levels, namely 0, 1, and 2-g for diamagnetic materials. In this study, the effects of different magnetic force fields on osteoblast-like cells (MG-63 and MC3T3-E1) viability, microtubule actin crosslinking factor 1 (MACF1) expression and its association with cytoskeleton were investigated. Results showed that cell viability increased to different degrees after exposure to 0 or 1-g conditions for 24 h, but it decreased by about 30% under 2-g conditions compared with control conditions. An increase in MACF1 expression at the RNA or protein level was observed in osteoblast-like cells under the magnetic force field of -1360 T(2)/m (0-g) relative to 1312 T(2)/m (2-g). Under control conditions, anti-MACF1 staining was scattered in the cytoplasm and partially colocalized with actin filaments (AFs) or microtubules (MTs) in the majority of osteoblast-like cells. Under 0-g conditions, MACF1 labeling was concentrated at perinuclear region and colocalization was not apparent. The patterns of anti-MACF1 labeling on MTs varied with MTs' changing under LG-HMF environment. In conclusion, LG-HMF affects osteoblast-like cell viability, MACF1 distribution, expression, and its association with cytoskeleton to some extent.

Disassembly of actin structures by nanosecond pulsed electric field is a downstream effect of cell swelling.

PubMed

Pakhomov, Andrei G; Xiao, Shu; Pakhomova, Olga N; Semenov, Iurii; Kuipers, Marjorie A; Ibey, Bennett L

2014-12-01

Disruption of the actin cytoskeleton structures was reported as one of the characteristic effects of nanosecond-duration pulsed electric field (nsPEF) in both mammalian and plant cells. We utilized CHO cells that expressed the monomeric fluorescent protein (mApple) tagged to actin to test if nsPEF modifies the cell actin directly or as a consequence of cell membrane permeabilization. A train of four 600-ns pulses at 19.2 kV/cm (2 Hz) caused immediate cell membrane poration manifested by YO-PRO-1 dye uptake, gradual cell rounding and swelling. Concurrently, bright actin features were replaced by dimmer and uniform fluorescence of diffuse actin. To block the nsPEF-induced swelling, the bath buffer was isoosmotically supplemented with an electropore-impermeable solute (sucrose). A similar addition of a smaller, electropore-permeable solute (adonitol) served as a control. We demonstrated that sucrose efficiently blocked disassembly of actin features by nsPEF, whereas adonitol did not. Sucrose also attenuated bleaching of mApple-tagged actin in nsPEF-treated cells (as integrated over the cell volume), although did not fully prevent it. We conclude that disintegration of the actin cytoskeleton was a result of cell swelling, which, in turn, was caused by cell permeabilization by nsPEF and transmembrane diffusion of solutes which led to the osmotic imbalance. Copyright © 2014 Elsevier B.V. All rights reserved.

A Prestressed Cable Network Model of the Adherent Cell Cytoskeleton

PubMed Central

Coughlin, Mark F.; Stamenović, Dimitrije

2003-01-01

A prestressed cable network is used to model the deformability of the adherent cell actin cytoskeleton. The overall and microstructural model geometries and cable mechanical properties were assigned values based on observations from living cells and mechanical measurements on isolated actin filaments, respectively. The models were deformed to mimic cell poking (CP), magnetic twisting cytometry (MTC) and magnetic bead microrheometry (MBM) measurements on living adherent cells. The models qualitatively and quantitatively captured the fibroblast cell response to the deformation imposed by CP while exhibiting only some qualitative features of the cell response to MTC and MBM. The model for CP revealed that the tensed peripheral actin filaments provide the key resistance to indentation. The actin filament tension that provides mechanical integrity to the network was estimated at ∼158 pN, and the nonlinear mechanical response during CP originates from filament kinematics. The MTC and MBM simulations revealed that the model is incomplete, however, these simulations show cable tension as a key determinant of the model response. PMID:12547813

The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions

PubMed Central

Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M.; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel

2015-01-01

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate–bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA–MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein–cytoskeleton interactions are a universally conserved feature. PMID:26169353

Cytoskeleton in Mast Cell Signaling

PubMed Central

Dráber, Pavel; Sulimenko, Vadym; Dráberová, Eduarda

2012-01-01

Mast cell activation mediated by the high affinity receptor for IgE (FcεRI) is a key event in allergic response and inflammation. Other receptors on mast cells, as c-Kit for stem cell factor and G protein-coupled receptors (GPCRs) synergistically enhance the FcεRI-mediated release of inflammatory mediators. Activation of various signaling pathways in mast cells results in changes in cell morphology, adhesion to substrate, exocytosis, and migration. Reorganization of cytoskeleton is pivotal in all these processes. Cytoskeletal proteins also play an important role in initial stages of FcεRI and other surface receptors induced triggering. Highly dynamic microtubules formed by αβ-tubulin dimers as well as microfilaments build up from polymerized actin are affected in activated cells by kinases/phosphatases, Rho GTPases and changes in concentration of cytosolic Ca2+. Also important are nucleation proteins; the γ-tubulin complexes in case of microtubules or Arp 2/3 complex with its nucleation promoting factors and formins in case of microfilaments. The dynamic nature of microtubules and microfilaments in activated cells depends on many associated/regulatory proteins. Changes in rigidity of activated mast cells reflect changes in intermediate filaments build up from vimentin. This review offers a critical appraisal of current knowledge on the role of cytoskeleton in mast cells signaling. PMID:22654883

A Gly65Val substitution in an actin, GhACT_LI1, disrupts cell polarity and membrane anchoring of F-actin resulting in dwarf, lintless Li1 cotton plants

USDA-ARS?s Scientific Manuscript database

• Actin polymerizes to form the cytoskeleton and organize polar growth in all eukaryotic cells. Species with numerous actin genes are especially useful for the dissection of actin molecular function due to redundancy and neofunctionalization. Here, we investigated the role of a cotton (Gossypium hi...

Bacterial actin MreB assembles in complex with cell shape protein RodZ.

PubMed

van den Ent, Fusinita; Johnson, Christopher M; Persons, Logan; de Boer, Piet; Löwe, Jan

2010-03-17

Bacterial actin homologue MreB is required for cell shape maintenance in most non-spherical bacteria, where it assembles into helical structures just underneath the cytoplasmic membrane. Proper assembly of the actin cytoskeleton requires RodZ, a conserved, bitopic membrane protein that colocalises to MreB and is essential for cell shape determination. Here, we present the first crystal structure of bacterial actin engaged with a natural partner and provide a clear functional significance of the interaction. We show that the cytoplasmic helix-turn-helix motif of Thermotoga maritima RodZ directly interacts with monomeric as well as filamentous MreB and present the crystal structure of the complex. In vitro and in vivo analyses of mutant T. maritima and Escherichia coli RodZ validate the structure and reveal the importance of the MreB-RodZ interaction in the ability of cells to propagate as rods. Furthermore, the results elucidate how the bacterial actin cytoskeleton might be anchored to the membrane to help constrain peptidoglycan synthesis in the periplasm.

Comparison of pre-processing techniques for fluorescence microscopy images of cells labeled for actin.

PubMed

Muralidhar, Gautam S; Channappayya, Sumohana S; Slater, John H; Blinka, Ellen M; Bovik, Alan C; Frey, Wolfgang; Markey, Mia K

2008-11-06

Automated analysis of fluorescence microscopy images of endothelial cells labeled for actin is important for quantifying changes in the actin cytoskeleton. The current manual approach is laborious and inefficient. The goal of our work is to develop automated image analysis methods, thereby increasing cell analysis throughput. In this study, we present preliminary results on comparing different algorithms for cell segmentation and image denoising.

Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin.

PubMed

Gupton, Stephanie L; Anderson, Karen L; Kole, Thomas P; Fischer, Robert S; Ponti, Aaron; Hitchcock-DeGregori, Sarah E; Danuser, Gaudenz; Fowler, Velia M; Wirtz, Denis; Hanein, Dorit; Waterman-Storer, Clare M

2005-02-14

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin-binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle alphaTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.

Tyrosine kinases activate store-mediated Ca2+ entry in human platelets through the reorganization of the actin cytoskeleton.

PubMed Central

Rosado, J A; Graves, D; Sage, S O

2000-01-01

We have recently reported that store-mediated Ca(2+) entry in platelets is likely to be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, a model termed 'secretion-like coupling'. In this model the actin cytoskeleton plays a key regulatory role. Since tyrosine kinases have been shown to be important for Ca(2+) entry in platelets and other cells, we have now investigated the possible involvement of tyrosine kinases in the secretion-like-coupling model. Treatment of platelets with thrombin or thapsigargin induced actin polymerization by a calcium-independent pathway. Methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor, prevented thrombin- or thapsigargin-induced actin polymerization. The effects of tyrosine kinases in store-mediated Ca(2+) entry were found to be entirely dependent on the actin cytoskeleton. PP1, an inhibitor of the Src family of proteins, partially inhibited store-mediated Ca(2+) entry. In addition, depletion of intracellular Ca(2+) stores stimulated cytoskeletal association of the cytoplasmic tyrosine kinase pp60(src), a process that was sensitive to treatment with cytochalasin D and PP1, but not to inhibition of Ras proteins using prenylcysteine analogues. Finally, combined inhibition of both Ras proteins and tyrosine kinases resulted in complete inhibition of Ca(2+) entry, suggesting that these two families of proteins have independent effects in the activation of store-mediated Ca(2+) entry in human platelets. PMID:11023829

Human somatic cells acquire the plasticity to generate embryoid-like metamorphosis via the actin cytoskeleton in injured tissues.

PubMed

Diaz, Jairo A; Murillo, Mauricio F; Mendoza, Jhonan A; Barreto, Ana M; Poveda, Lina S; Sanchez, Lina K; Poveda, Laura C; Mora, Katherine T

2016-01-01

Emergent biological responses develop via unknown processes dependent on physical collision. In hypoxia, when the tissue architecture collapses but the geometric core is stable, actin cytoskeleton filament components emerge, revealing a hidden internal order that identifies how each molecule is reassembled into the original mold, using one common connection, i.e., a fractal self-similarity that guides the system from the beginning in reverse metamorphosis, with spontaneous self-assembly of past forms that mimics an embryoid phenotype. We captured this hidden collective filamentous assemblage in progress: Hypoxic deformed cells enter into intercellular collisions, generate migratory ejected filaments, and produce self-assembly of triangular chiral hexagon complexes; this dynamic geometry guides the microenvironment scaffold in which this biological process is incubated, recapitulating embryonic morphogenesis. In all injured tissues, especially in damaged skeletal (striated) muscle cells, visibly hypertrophic intercalated actin-myosin filaments are organized in zebra stripe pattern along the anterior-posterior axis in the interior of the cell, generating cephalic-caudal polarity segmentation, with a high selective level of immunopositivity for Actin, Alpha Skeletal Muscle antibody and for Neuron-Specific Enolase expression of ectodermal differentiation. The function of actin filaments in emergent responses to tissue injury is to reconstitute, reactivate and orchestrate cellular metamorphosis, involving the re-expression of fetal genes, providing evidence of the reverse flow of genetic information within a biological system. The resultant embryoid phenotype emerges as a microscopic fractal template copy of the organization of the whole body, likely allowing the modification and reprogramming of the phenotype of the tumor in which these structures develop, as well as establishing a reverse primordial microscopic mold to collectively re-form cellular building blocks to

Human somatic cells acquire the plasticity to generate embryoid-like metamorphosis via the actin cytoskeleton in injured tissues

PubMed Central

Diaz, Jairo A; Murillo, Mauricio F; Mendoza, Jhonan A; Barreto, Ana M; Poveda, Lina S; Sanchez, Lina K; Poveda, Laura C; Mora, Katherine T

2016-01-01

Emergent biological responses develop via unknown processes dependent on physical collision. In hypoxia, when the tissue architecture collapses but the geometric core is stable, actin cytoskeleton filament components emerge, revealing a hidden internal order that identifies how each molecule is reassembled into the original mold, using one common connection, i.e., a fractal self-similarity that guides the system from the beginning in reverse metamorphosis, with spontaneous self-assembly of past forms that mimics an embryoid phenotype. We captured this hidden collective filamentous assemblage in progress: Hypoxic deformed cells enter into intercellular collisions, generate migratory ejected filaments, and produce self-assembly of triangular chiral hexagon complexes; this dynamic geometry guides the microenvironment scaffold in which this biological process is incubated, recapitulating embryonic morphogenesis. In all injured tissues, especially in damaged skeletal (striated) muscle cells, visibly hypertrophic intercalated actin-myosin filaments are organized in zebra stripe pattern along the anterior-posterior axis in the interior of the cell, generating cephalic-caudal polarity segmentation, with a high selective level of immunopositivity for Actin, Alpha Skeletal Muscle antibody and for Neuron-Specific Enolase expression of ectodermal differentiation. The function of actin filaments in emergent responses to tissue injury is to reconstitute, reactivate and orchestrate cellular metamorphosis, involving the re-expression of fetal genes, providing evidence of the reverse flow of genetic information within a biological system. The resultant embryoid phenotype emerges as a microscopic fractal template copy of the organization of the whole body, likely allowing the modification and reprogramming of the phenotype of the tumor in which these structures develop, as well as establishing a reverse primordial microscopic mold to collectively re-form cellular building blocks to

Physically-Induced Cytoskeleton Remodeling of Cells in Three-Dimensional Culture

PubMed Central

Lee, Sheng-Lin; Nekouzadeh, Ali; Butler, Boyd; Pryse, Kenneth M.; McConnaughey, William B.; Nathan, Adam C.; Legant, Wesley R.; Schaefer, Pascal M.; Pless, Robert B.

2012-01-01

Characterizing how cells in three-dimensional (3D) environments or natural tissues respond to biophysical stimuli is a longstanding challenge in biology and tissue engineering. We demonstrate a strategy to monitor morphological and mechanical responses of contractile fibroblasts in a 3D environment. Cells responded to stretch through specific, cell-wide mechanisms involving staged retraction and reinforcement. Retraction responses occurred for all orientations of stress fibers and cellular protrusions relative to the stretch direction, while reinforcement responses, including extension of cellular processes and stress fiber formation, occurred predominantly in the stretch direction. A previously unreported role of F-actin clumps was observed, with clumps possibly acting as F-actin reservoirs for retraction and reinforcement responses during stretch. Responses were consistent with a model of cellular sensitivity to local physical cues. These findings suggest mechanisms for global actin cytoskeleton remodeling in non-muscle cells and provide insight into cellular responses important in pathologies such as fibrosis and hypertension. PMID:23300512

A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a low-affinity state in MreB.

PubMed

Bean, G J; Flickinger, S T; Westler, W M; McCully, M E; Sept, D; Weibel, D B; Amann, K J

2009-06-09

S-(3,4-Dichlorobenzyl)isothiourea (A22) disrupts the actin cytoskeleton of bacteria, causing defects of morphology and chromosome segregation. Previous studies have suggested that the actin homologue MreB itself is the target of A22, but there has been no direct observation of A22 binding to MreB and no mechanistic explanation of its mode of action. We show that A22 binds MreB with at least micromolar affinity in its nucleotide-binding pocket in a manner that is sterically incompatible with simultaneous ATP binding. A22 negatively affects both the time course and extent of MreB polymerization in vitro in the presence of ATP. A22 prevents assembly of MreB into long, rigid polymers, as determined by both fluorescence microscopy and sedimentation assays. A22 increases the critical concentration of ATP-bound MreB assembly from 500 nM to approximately 2000 nM. We therefore conclude that A22 is a competitive inhibitor of ATP binding to MreB. A22-bound MreB is capable of polymerization, but with assembly properties that more closely resemble those of the ADP-bound state. Because the cellular concentration of MreB is in the low micromolar range, this mechanism explains the ability of A22 to largely disassemble the actin cytoskeleton in bacterial cells. It also represents a novel mode of action for a cytoskeletal drug and the first biochemical characterization of the interaction between a small molecule inhibitor of the bacterial cytoskeleton and its target.

Spiral actin-polymerization waves can generate amoeboidal cell crawling

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

Dreher, A.; Aranson, I. S.; Kruse, K.

2014-05-01

Amoeboidal cell crawling on solid substrates is characterized by protrusions that seemingly appear randomly along the cell periphery and drive the cell forward. For many cell types, it is known that the protrusions result from polymerization of the actin cytoskeleton. However, little is known about how the formation of protrusions is triggered and whether the appearance of subsequent protrusions is coordinated. Recently, the spontaneous formation of actin-polymerization waves was observed. These waves have been proposed to orchestrate the cytoskeletal dynamics during cell crawling. Here, we study the impact of cytoskeletal polymerization waves on cell migration using a phase-field approach. Inmore » addition to directionally moving cells, we find states reminiscent of amoeboidal cell crawling. In this framework, new protrusions are seen to emerge from a nucleation process, generating spiral actin waves in the cell interior. Nucleation of new spirals does not require noise, but occurs in a state that is apparently displaying spatio-temporal chaos.« less

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 metapneumovirus Induces Reorganization of the Actin Cytoskeleton for Direct Cell-to-Cell Spread

PubMed Central

El Najjar, Farah; Cifuentes-Muñoz, Nicolás; Zhu, Haining; Buchholz, Ursula J.; Moncman, Carole L.; Dutch, Rebecca Ellis

2016-01-01

Paramyxovirus spread generally involves assembly of individual viral particles which then infect target cells. We show that infection of human bronchial airway cells with human metapneumovirus (HMPV), a recently identified paramyxovirus which causes significant respiratory disease, results in formation of intercellular extensions and extensive networks of branched cell-associated filaments. Formation of these structures is dependent on actin, but not microtubule, polymerization. Interestingly, using a co-culture assay we show that conditions which block regular infection by HMPV particles, including addition of neutralizing antibodies or removal of cell surface heparan sulfate, did not prevent viral spread from infected to new target cells. In contrast, inhibition of actin polymerization or alterations to Rho GTPase signaling pathways significantly decreased cell-to-cell spread. Furthermore, viral proteins and viral RNA were detected in intercellular extensions, suggesting direct transfer of viral genetic material to new target cells. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated, we show that the HMPV phosphoprotein extensively co-localized with actin and induced formation of cellular extensions when transiently expressed, supporting a new model in which a paramyxovirus phosphoprotein is a key player in assembly and spread. Our results reveal a novel mechanism for HMPV direct cell-to-cell spread and provide insights into dissemination of respiratory viruses. PMID:27683250

The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions.

PubMed

Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel; Søgaard-Andersen, Lotte; Mignot, Tâm

2015-07-20

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate-bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA-MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein-cytoskeleton interactions are a universally conserved feature. © 2015 Treuner-Lange et al.

Spatial Cytoskeleton Organization Supports Targeted Intracellular Transport

NASA Astrophysics Data System (ADS)

Hafner, Anne E.; Rieger, Heiko

2018-03-01

The efficiency of intracellular cargo transport from specific source to target locations is strongly dependent upon molecular motor-assisted motion along the cytoskeleton. Radial transport along microtubules and lateral transport along the filaments of the actin cortex underneath the cell membrane are characteristic for cells with a centrosome. The interplay between the specific cytoskeleton organization and the motor performance realizes a spatially inhomogeneous intermittent search strategy. In order to analyze the efficiency of such intracellular search strategies we formulate a random velocity model with intermittent arrest states. We evaluate efficiency in terms of mean first passage times for three different, frequently encountered intracellular transport tasks: i) the narrow escape problem, which emerges during cargo transport to a synapse or other specific region of the cell membrane, ii) the reaction problem, which considers the binding time of two particles within the cell, and iii) the reaction-escape problem, which arises when cargo must be released at a synapse only after pairing with another particle. Our results indicate that cells are able to realize efficient search strategies for various intracellular transport tasks economically through a spatial cytoskeleton organization that involves only a narrow actin cortex rather than a cell body filled with randomly oriented actin filaments.

Actin depolymerization enhances adipogenic differentiation in human stromal stem cells.

PubMed

Chen, Li; Hu, Huimin; Qiu, Weimin; Shi, Kaikai; Kassem, Moustapha

2018-05-01

Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Copyright © 2018 Elsevier B.V. All rights reserved.

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

NASA Technical Reports Server (NTRS)

Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

2000-01-01

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography.

PubMed

Hu, S; Brady, S R; Kovar, D R; Staiger, C J; Clark, G B; Roux, S J; Muday, G K

2000-10-01

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Vinculin promotes cell spreading by mechanically coupling integrins to the cytoskeleton

NASA Technical Reports Server (NTRS)

Ezzell, R. M.; Goldmann, W. H.; Wang, N.; Parasharama, N.; Ingber, D. E.

1997-01-01

Mouse F9 embryonic carcinoma 5.51 cells that lack the cytoskeletal protein vinculin spread poorly on extracellular matrix compared with wild-type F9 cells or two vinculin-transfected clones (5.51Vin3 and Vin4; Samuels et al., 1993, J. Cell Biol. 121, 909-921). In the present study, we used this model system to determine how the presence of vinculin promotes cytoskeletal alterations and associated changes in cell shape. Microscopic analysis of cell spreading at early times, revealed that 5.51 cells retained the ability to form filopodia; however, they could not form lamellipodia, assemble stress fibers, or efficiently spread over the culture substrate. Detergent (Triton X-100) studies revealed that these major differences in cell morphology and cytoskeletal organization did not result from differences in levels of total polymerized or cross-linked actin. Biochemical studies showed that 5.51 cells, in addition to lacking vinculin, exhibited slightly reduced levels of alpha-actinin and paxillin in their detergent-insoluble cytoskeleton. The absence of vinculin correlated with a decrease in the mechanical stiffness of the integrin-cytoskeleton linkage, as measured using cell magnetometry. Furthermore, when vinculin was replaced by transfection in 5.51Vin3 and 5.51Vin4 cells, the levels of cytoskeletal-associated alpha-actinin and paxillin, the efficiency of transmembrane mechanical coupling, and the formation of actin stress fibers were all restored to near wild-type levels. These findings suggest that vinculin may promote cell spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, rather than by altering the total level of actin polymerization or cross-linking.

The distinct roles of the nucleus and nucleus-cytoskeleton connections in three-dimensional cell migration

PubMed Central

Khatau, Shyam B.; Bloom, Ryan J.; Bajpai, Saumendra; Razafsky, David; Zang, Shu; Giri, Anjil; Wu, Pei-Hsun; Marchand, Jorge; Celedon, Alfredo; Hale, Christopher M.; Sun, Sean X.; Hodzic, Didier; Wirtz, Denis

2012-01-01

Cells often migrate in vivo in an extracellular matrix that is intrinsically three-dimensional (3D) and the role of actin filament architecture in 3D cell migration is less well understood. Here we show that, while recently identified linkers of nucleoskeleton to cytoskeleton (LINC) complexes play a minimal role in conventional 2D migration, they play a critical role in regulating the organization of a subset of actin filament bundles – the perinuclear actin cap - connected to the nucleus through Nesprin2giant and Nesprin3 in cells in 3D collagen I matrix. Actin cap fibers prolong the nucleus and mediate the formation of pseudopodial protrusions, which drive matrix traction and 3D cell migration. Disruption of LINC complexes disorganizes the actin cap, which impairs 3D cell migration. A simple mechanical model explains why LINC complexes and the perinuclear actin cap are essential in 3D migration by providing mechanical support to the formation of pseudopodial protrusions. PMID:22761994

Actin cytoskeletal rearrangement and dysfunction due to activation of the receptor for advanced glycation end products is inhibited by thymosin beta 4

PubMed Central

Kim, Sokho; Kwon, Jungkee

2015-01-01

The receptor of advanced glycation end products (RAGE) is a cell-surface receptor that is a key factor in the pathogenesis of diabetic complications, including vascular disorders. Dysfunction of the actin cytoskeleton contributes to disruption of cell membrane repair in response to various type of endothelial cell damage. However, mechanism underlying RAGE remodelling of the actin cytoskeleton, by which globular actin (G-actin) forms to filamentous actin (F-actin), remains unclear. In this study we examined the role of thymosin beta 4 (Tβ4) – which binds to actin, blocks actin polymerization, and maintains the dynamic equilibrium between G-actin and F-actin in human umbilical vein endothelial cells (HUVECs) – in the response to RAGE. Tβ4 increased cell viability and decreased levels of reactive oxygen species in HUVECs incubated with AGEs. Tβ4 reduced the expression of RAGE, consistent with a down-regulation of the F-actin to G-actin ratio. The effect of remodelling of the actin cytoskeleton on RAGE expression was clarified by adding Phalloidin, which stabilizes F-actin. Moreover, small interfering RNA was used to determine whether intrinsic Tβ4 regulates RAGE expression in the actin cytoskeleton. The absence of intrinsic Tβ4 in HUVECs evoked actin cytoskeleton disorder and increased RAGE expression. These findings suggest that regulation of the actin cytoskeleton by Tβ4 plays a pivotal role in the RAGE response to AGEs. PMID:25640761

Exploitation of host cell cytoskeleton and signalling during Listeria monocytogenes entry into mammalian cells.

PubMed

Pizarro-Cerdá, Javier; Sousa, Sandra; Cossart, Pascale

2004-02-01

Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells during infection is a key issue for the understanding how this food-borne pathogen causes a pleiotropic disease ranging from gastro-enteritis to meningitis and abortions. Using multidisciplinary approaches, essentially combining bacterial genetics and cell biology, we have identified two bacterial proteins critical for entry into target cells, InlA and InlB. Their cellular ligands have been also identified: InlA interacts with the adhesion molecule E-cadherin, while InlB interacts with the receptor for the globular head of the complement factor C1q (gC1q-R), with the hepatocyte growth factor receptor (c-Met) and with glycosaminoglycans (including heparan sulphate). The dynamic interaction between these cellular receptors and the actin cytoskeleton is currently under investigation. Several intracellular molecules have been recognized as key effectors for Listeria entry into target cells, including catenins (implicated in the connection of E-cadherin to actin) and the actin depolymerising factor/cofilin (involved in the rearrangement of the cytoskeleton in the InlB-dependent internalisation pathway). At the organism level, species specificity has been discovered concerning the interaction between InlA and E-cadherin, leading to the generation of transgenic mice expressing the human E-cadherin, in which the critical role of InlA in the crossing of the intestinal barrier has been clearly determined. Listeria appears as an instrumental model for addressing critical questions concerning both the complex process of bacterial pathogenesis and also fundamental molecular processes, such as phagocytosis.

Exploitation of host cell cytoskeleton and signalling during Listeria monocytogenes entry into mammalian cells.

PubMed

Pizarro-Cerdá, Javier; Sousa, Sandra; Cossart, Pascale

2004-06-01

Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells during infection isa key issue for the understanding how this food-borne pathogen causes a pleiotropic disease ranging from gastro-enteritis to meningitis and abortions. Using multidisciplinary approaches, essentially combining bacterial genetics and cell biology, we have identified two bacterial proteins critical for entry into target cells, InlA and InlB. Their cellular ligands have been also identified: InlA interacts with the adhesion molecule E-cadherin, while InlB interacts with the receptor for the globular head of the complement factor Clq (gClq-R), with the hepatocyte growth factor receptor (c-Met) and with glycosaminoglycans(including heparan sulphate). The dynamic interaction between these cellular receptors and the actin cytoskeleton is currently under investigation. Several intracellular molecules have been recognized as key effectors for Listeria entry into target cells,including catenins (implicated in the connection of E-cadherin to actin) and the actin depolymerising factor/cofilin (involved in the rearrangement of the cytoskeleton in the InlB-dependent internalisation pathway). At the organism level, species specificity has been discovered concerning the interaction between InlA and E-cadherin, leading to the generation of transgenic mice expressing the human E-cadherin, in which the critical role of InlA in the crossing of the intestinal barrier has been clearly determined. Listeria appears as an instrumental model for addressing critical questions concerning both the complex process of bacterial pathogenesis and also fundamental molecular processes, such as phagocytosis.

The calcium feedback loop and T cell activation: how cytoskeleton networks control intracellular calcium flux.

PubMed

Joseph, Noah; Reicher, Barak; Barda-Saad, Mira

2014-02-01

During T cell activation, the engagement of a T cell with an antigen-presenting cell (APC) results in rapid cytoskeletal rearrangements and a dramatic increase of intracellular calcium (Ca(2+)) concentration, downstream to T cell antigen receptor (TCR) ligation. These events facilitate the organization of an immunological synapse (IS), which supports the redistribution of receptors, signaling molecules and organelles towards the T cell-APC interface to induce downstream signaling events, ultimately supporting T cell effector functions. Thus, Ca(2+) signaling and cytoskeleton rearrangements are essential for T cell activation and T cell-dependent immune response. Rapid release of Ca(2+) from intracellular stores, e.g. the endoplasmic reticulum (ER), triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels, residing in the plasma membrane. These channels facilitate a sustained influx of extracellular Ca(2+) across the plasma membrane in a process termed store-operated Ca(2+) entry (SOCE). Because CRAC channels are themselves inhibited by Ca(2+) ions, additional factors are suggested to enable the sustained Ca(2+) influx required for T cell function. Among these factors, we focus here on the contribution of the actin and microtubule cytoskeleton. The TCR-mediated increase in intracellular Ca(2+) evokes a rapid cytoskeleton-dependent polarization, which involves actin cytoskeleton rearrangements and microtubule-organizing center (MTOC) reorientation. Here, we review the molecular mechanisms of Ca(2+) flux and cytoskeletal rearrangements, and further describe the way by which the cytoskeletal networks feedback to Ca(2+) signaling by controlling the spatial and temporal distribution of Ca(2+) sources and sinks, modulating TCR-dependent Ca(2+) signals, which are required for an appropriate T cell response. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters

Rac-WAVE-mediated actin reorganization is required for organization and maintenance of cell-cell adhesion.

PubMed

Yamazaki, Daisuke; Oikawa, Tsukasa; Takenawa, Tadaomi

2007-01-01

During cadherin-dependent cell-cell adhesion, the actin cytoskeleton undergoes dynamic reorganization in epithelial cells. Rho-family small GTPases, which regulate actin dynamics, play pivotal roles in cadherin-dependent cell-cell adhesion; however, the precise molecular mechanisms that underlie cell-cell adhesion formation remain unclear. Here we show that Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE)-mediated reorganization of actin, downstream of Rac plays an important role in normal development of cadherin-dependent cell-cell adhesions in MDCK cells. Rac-induced development of cadherin-dependent adhesions required WAVE2-dependent actin reorganization. The process of cell-cell adhesion is divided into three steps: formation of new cell-cell contacts, stabilization of these new contacts and junction maturation. WAVE1 and WAVE2 were expressed in MDCK cells. The functions of WAVE1 and WAVE2 were redundant in this system but WAVE2 appeared to play a more significant role. During the first step, WAVE2-dependent lamellipodial protrusions facilitated formation of cell-cell contacts. During the second step, WAVE2 recruited actin filaments to new cell-cell contacts and stabilized newly formed cadherin clusters. During the third step, WAVE2-dependent actin reorganization was required for organization and maintenance of mature cell-cell adhesions. Thus, Rac-WAVE-dependent actin reorganization is not only involved in formation of cell-cell adhesions but is also required for their maintenance.

A QUICK Screen for Lrrk2 Interaction Partners – Leucine-rich Repeat Kinase 2 is Involved in Actin Cytoskeleton Dynamics*

PubMed Central

Meixner, Andrea; Boldt, Karsten; Van Troys, Marleen; Askenazi, Manor; Gloeckner, Christian J.; Bauer, Matthias; Marto, Jarrod A.; Ampe, Christophe; Kinkl, Norbert; Ueffing, Marius

2011-01-01

Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinson's disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement, and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function. PMID:20876399

Cell polarity, cell adhesion, and spermatogenesis: role of cytoskeletons

PubMed Central

Li, Linxi; Gao, Ying; Chen, Haiqi; Jesus, Tito; Tang, Elizabeth; Li, Nan; Lian, Qingquan; Ge, Ren-shan; Cheng, C. Yan

2017-01-01

In the rat testis, studies have shown that cell polarity, in particular spermatid polarity, to support spermatogenesis is conferred by the coordinated efforts of the Par-, Crumbs-, and Scribble-based polarity complexes in the seminiferous epithelium. Furthermore, planar cell polarity (PCP) is conferred by PCP proteins such as Van Gogh-like 2 (Vangl2) in the testis. On the other hand, cell junctions at the Sertoli cell–spermatid (steps 8–19) interface are exclusively supported by adhesion protein complexes (for example, α6β1-integrin-laminin-α3,β3,γ3 and nectin-3-afadin) at the actin-rich apical ectoplasmic specialization (ES) since the apical ES is the only anchoring device in step 8–19 spermatids. For cell junctions at the Sertoli cell–cell interface, they are supported by adhesion complexes at the actin-based basal ES (for example, N-cadherin-β-catenin and nectin-2-afadin), tight junction (occludin-ZO-1 and claudin 11-ZO-1), and gap junction (connexin 43-plakophilin-2) and also intermediate filament-based desmosome (for example, desmoglein-2-desmocollin-2). In short, the testis-specific actin-rich anchoring device known as ES is crucial to support spermatid and Sertoli cell adhesion. Accumulating evidence has shown that the Par-, Crumbs-, and Scribble-based polarity complexes and the PCP Vangl2 are working in concert with actin- or microtubule-based cytoskeletons (or both) and these polarity (or PCP) protein complexes exert their effects through changes in the organization of the cytoskeletal elements across the seminiferous epithelium of adult rat testes. As such, there is an intimate relationship between cell polarity, cell adhesion, and cytoskeletal function in the testis. Herein, we critically evaluate these recent findings based on studies on different animal models. We also suggest some crucial future studies to be performed. PMID:28928959

Alterations of the cytoskeleton in human cells in space proved by life-cell imaging.

PubMed

Corydon, Thomas J; Kopp, Sascha; Wehland, Markus; Braun, Markus; Schütte, Andreas; Mayer, Tobias; Hülsing, Thomas; Oltmann, Hergen; Schmitz, Burkhard; Hemmersbach, Ruth; Grimm, Daniela

2016-01-28

Microgravity induces changes in the cytoskeleton. This might have an impact on cells and organs of humans in space. Unfortunately, studies of cytoskeletal changes in microgravity reported so far are obligatorily based on the analysis of fixed cells exposed to microgravity during a parabolic flight campaign (PFC). This study focuses on the development of a compact fluorescence microscope (FLUMIAS) for fast live-cell imaging under real microgravity. It demonstrates the application of the instrument for on-board analysis of cytoskeletal changes in FTC-133 cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin during the 24(th) DLR PFC and TEXUS 52 rocket mission. Although vibration is an inevitable part of parabolic flight maneuvers, we successfully for the first time report life-cell cytoskeleton imaging during microgravity, and gene expression analysis after the 31(st) parabola showing a clear up-regulation of cytoskeletal genes. Notably, during the rocket flight the FLUMIAS microscope reveals significant alterations of the cytoskeleton related to microgravity. Our findings clearly demonstrate the applicability of the FLUMIAS microscope for life-cell imaging during microgravity, rendering it an important technological advance in live-cell imaging when dissecting protein localization.

Alterations of the cytoskeleton in human cells in space proved by life-cell imaging

PubMed Central

Corydon, Thomas J.; Kopp, Sascha; Wehland, Markus; Braun, Markus; Schütte, Andreas; Mayer, Tobias; Hülsing, Thomas; Oltmann, Hergen; Schmitz, Burkhard; Hemmersbach, Ruth; Grimm, Daniela

2016-01-01

Microgravity induces changes in the cytoskeleton. This might have an impact on cells and organs of humans in space. Unfortunately, studies of cytoskeletal changes in microgravity reported so far are obligatorily based on the analysis of fixed cells exposed to microgravity during a parabolic flight campaign (PFC). This study focuses on the development of a compact fluorescence microscope (FLUMIAS) for fast live-cell imaging under real microgravity. It demonstrates the application of the instrument for on-board analysis of cytoskeletal changes in FTC-133 cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin during the 24th DLR PFC and TEXUS 52 rocket mission. Although vibration is an inevitable part of parabolic flight maneuvers, we successfully for the first time report life-cell cytoskeleton imaging during microgravity, and gene expression analysis after the 31st parabola showing a clear up-regulation of cytoskeletal genes. Notably, during the rocket flight the FLUMIAS microscope reveals significant alterations of the cytoskeleton related to microgravity. Our findings clearly demonstrate the applicability of the FLUMIAS microscope for life-cell imaging during microgravity, rendering it an important technological advance in live-cell imaging when dissecting protein localization. PMID:26818711

Spatial organization of the cytoskeleton enhances cargo delivery to specific target areas on the plasma membrane of spherical cells.

PubMed

Hafner, Anne E; Rieger, Heiko

2016-11-15

Intracellular transport is vital for the proper functioning and survival of a cell. Cargo (proteins, vesicles, organelles, etc) is transferred from its place of creation to its target locations via molecular motor assisted transport along cytoskeletal filaments. The transport efficiency is strongly affected by the spatial organization of the cytoskeleton, which constitutes an inhomogeneous, complex network. In cells with a centrosome microtubules grow radially from the central microtubule organizing center towards the cell periphery whereas actin filaments form a dense meshwork, the actin cortex, underneath the cell membrane with a broad range of orientations. The emerging ballistic motion along filaments is frequently interrupted due to constricting intersection nodes or cycles of detachment and reattachment processes in the crowded cytoplasm. In order to investigate the efficiency of search strategies established by the cell's specific spatial organization of the cytoskeleton we formulate a random velocity model with intermittent arrest states. With extensive computer simulations we analyze the dependence of the mean first passage times for narrow escape problems on the structural characteristics of the cytoskeleton, the motor properties and the fraction of time spent in each state. We find that an inhomogeneous architecture with a small width of the actin cortex constitutes an efficient intracellular search strategy.

Characterization of mRNA-Cytoskeleton Interactions In Situ Using FMTRIP and Proximity Ligation

PubMed Central

Jung, Jeenah; Lifland, Aaron W.; Alonas, Eric J.; Zurla, Chiara; Santangelo, Philip J.

2013-01-01

Many studies have demonstrated an association between the cytoskeleton and mRNA, as well as the asymmetric distribution of mRNA granules within the cell in response to various signaling events. It is likely that the extensive cytoskeletal network directs mRNA transport and localization, with different cytoskeletal elements having their own specific roles. In order to understand the spatiotemporal changes in the interactions between the mRNA and the cytoskeleton as a response to a stimulus, a technique that can visualize and quantify these changes across a population of cells while capturing cell-to-cell variations is required. Here, we demonstrate a method for imaging and quantifying mRNA-cytoskeleton interactions on a per cell basis with single-interaction sensitivity. Using a proximity ligation assay with flag-tagged multiply-labeled tetravalent RNA imaging probes (FMTRIP), we quantified interactions between mRNAs and β-tubulin, vimentin, or filamentous actin (F-actin) for two different mRNAs, poly(A) + and β-actin mRNA, in two different cell types, A549 cells and human dermal fibroblasts (HDF). We found that the mRNAs interacted predominantly with F-actin (>50% in HDF, >20% in A549 cells), compared to β-tubulin (<5%) and vimentin (11-13%). This likely reflects differences in mRNA management by the two cell types. We then quantified changes in these interactions in response to two perturbations, F-actin depolymerization and arsenite-induced oxidative stress, both of which alter either the cytoskeleton itself and mRNA localization. Both perturbations led to a decrease in poly(A) + mRNA interactions with F-actin and an increase in the interactions with microtubules, in a time dependent manner. PMID:24040294

The Bcr-Abl kinase regulates the actin cytoskeleton via a GADS/Slp-76/Nck1 adaptor protein pathway.

PubMed

Preisinger, Christian; Kolch, Walter

2010-05-01

Bcr-Abl is the transforming principle underlying chronic myelogenous leukaemia (CML). Here, we use a functional interaction proteomics approach to map pathways by which Bcr-Abl regulates defined cellular processes. The results show that Bcr-Abl regulates the actin cytoskeleton and non-apoptotic membrane blebbing via a GADS/Slp-76/Nck1 adaptor protein pathway. The binding of GADS to Bcr-Abl requires Bcr-Abl tyrosine kinase activity and is sensitive to the Bcr-Abl inhibitor imatinib, while the GADS/Slp-76 and Slp-76/Nck interactions are tyrosine phosphorylation independent. All three adaptor proteins co-localize with cortical actin in membrane blebs. Downregulation of each adaptor protein disrupts the actin cytoskeleton and membrane blebbing in a similar fashion and similar to imatinib. These findings highlight the importance of protein interaction dependent adaptor protein pathways in oncogenic kinase signaling. 2010 Elsevier Inc. All rights reserved.

Altered Actin Dynamics and Functions of Osteoblast-Like Cells in Parabolic Flight may Involve ERK1/2

NASA Astrophysics Data System (ADS)

Dai, Zhongquan; Tan, Yingjun; Yang, Fen; Qu, Lina; Zhang, Hongyu; Wan, Yumin; Li, Yinghui

2011-01-01

Osteoblasts are sensitive to mechanical stressors such as gravity and alter their cytoskeletons and functions to adapt; however, the contribution of gravity to this phenomenon is not well understood. In this study, we investigated the effects of acute gravitational changes on the structure and function of osteoblast ROS17/2.8 as generated by parabolic flight. The changes in microfilament cytoskeleton was observed by immunofluorescence stain of Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I for F-actin and G-actin, respectively. To examine osteoblast function, ALP (alkaline phosphatase) activity, osteocalcin secretions and the expression of ALP, COL1A1 (collagen type I alpha 1 chain) and osteocalcin were detected by modified Gomori methods, radioimmunity and RT-PCR, respectively. Double fluorescence staining of phosphorylated p44/42 and F-actin were performed to observe their colocalization relationship. The established semi-quantitative analysis method of fluorescence intensity of EGFP was used to detect the activity changes of COL1A1 promoter in EGFP-ROS cells with MAPK inhibitor PD98059 or F-actin inhibitor cytochalasin B. Results indicate that the altered gravity induced the reorganization of microfilament cytoskeletons of osteoblasts. After 3 h parabolic flight, F-actin of osteoblast cytoskeleton became thicker and directivity, whereas G-actin shrunk and became more concentrated at the edge of nucleus. The excretion of osteocalcin, the activity of ALP and the expression of mRNA decreased. Colocalization analysis indicated that phosphorylated p44/42 MAPK was coupled with F-actin. Inhibitor PD98059 and cytochalasin B decreased the fluorescence intensity of EGFP-ROS cells. Above results suggest that short time gravity variations induce the adjustment of osteoblast structure and functional and ERK1/2 signaling maybe involve these responses. We believe that it is an adaptive method of the osteoblasts to gravity alteration that structure

Poisons, ruffles and rockets: bacterial pathogens and the host cell cytoskeleton.

PubMed

Steele-Mortimer, O; Knodler, L A; Finlay, B B

2000-02-01

The cytoskeleton of eukaryotic cells is affected by a number of bacterial and viral pathogens. In this review we consider three recurring themes of cytoskeletal involvement in bacterial pathogenesis: 1) the effect of bacterial toxins on actin-regulating small GTP-binding proteins; 2) the invasion of non-phagocytic cells by the bacterial induction of ruffles at the plasma membrane; 3) the formation of actin tails and pedestals by intracellular and extracellular bacteria, respectively. Considerable progress has been made recently in the characterization of these processes. It is becoming clear that bacterial pathogens have developed a variety of sophisticated mechanisms for utilizing the complex cytoskeletal system of host cells. These bacterially-induced processes are now providing unique insights into the regulation of fundamental eukaryotic mechanisms.

25 Years of Tension over Actin Binding to the Cadherin Cell Adhesion Complex: The Devil is in the Details.

PubMed

Nelson, W James; Weis, William I

2016-07-01

Over the past 25 years, there has been a conceptual (re)evolution in understanding how the cadherin cell adhesion complex, which contains F-actin-binding proteins, binds to the actin cytoskeleton. There is now good synergy between structural, biochemical, and cell biological results that the cadherin-catenin complex binds to F-actin under force. Copyright © 2016 Elsevier Ltd. All rights reserved.

Apicomplexans pulling the strings: manipulation of the host cell cytoskeleton dynamics.

PubMed

Cardoso, Rita; Soares, Helena; Hemphill, Andrew; Leitão, Alexandre

2016-07-01

Invasive stages of apicomplexan parasites require a host cell to survive, proliferate and advance to the next life cycle stage. Once invasion is achieved, apicomplexans interact closely with the host cell cytoskeleton, but in many cases the different species have evolved distinct mechanisms and pathways to modulate the structural organization of cytoskeletal filaments. The host cell cytoskeleton is a complex network, largely, but not exclusively, composed of microtubules, actin microfilaments and intermediate filaments, all of which are modulated by associated proteins, and it is involved in diverse functions including maintenance of cell morphology and mechanical support, migration, signal transduction, nutrient uptake, membrane and organelle trafficking and cell division. The ability of apicomplexans to modulate the cytoskeleton to their own advantage is clearly beneficial. We here review different aspects of the interactions of apicomplexans with the three main cytoskeletal filament types, provide information on the currently known parasite effector proteins and respective host cell targets involved, and how these interactions modulate the host cell physiology. Some of these findings could provide novel targets that could be exploited for the development of preventive and/or therapeutic strategies.

F5-peptide induces aspermatogenesis by disrupting organization of actin- and microtubule-based cytoskeletons in the testis

PubMed Central

Gao, Ying; Mruk, Dolores D.; Lui, Wing-yee; Lee, Will M.; Cheng, C. Yan

2016-01-01

During the release of sperm at spermiation, a biologically active F5-peptide, which can disrupt the Sertoli cell tight junction (TJ) permeability barrier, is produced at the site of the degenerating apical ES (ectoplasmic specialization). This peptide coordinates the events of spermiation and blood-testis barrier (BTB) remodeling at stage VIII of the epithelial cycle, creating a local apical ES-BTB axis to coordinate cellular events across the epithelium. The mechanism(s) by which F5-peptide perturbs BTB restructuring, and its involvement in apical ES dynamics remain unknown. F5-peptide, besides perturbing BTB integrity, was shown to induce germ cell release from the epithelium following its efficient in vivo overexpression in the testis. Overexpression of F5-peptide caused disorganization of actin- and microtubule (MT)-based cytoskeletons, mediated by altering the spatiotemporal expression of actin binding/regulatory proteins in the seminiferous epithelium. F5-peptide perturbed the ability of actin microfilaments and/or MTs from converting between their bundled and unbundled/defragmented configuration, thereby perturbing adhesion between spermatids and Sertoli cells. Since apical ES and basal ES/BTB are interconnected through the underlying cytoskeletal networks, this thus provides an efficient and novel mechanism to coordinate different cellular events across the epithelium during spermatogenesis through changes in the organization of actin microfilaments and MTs. These findings also illustrate the potential of F5-peptide being a male contraceptive peptide for men. PMID:27611949

Image-based model of the spectrin cytoskeleton for red blood cell simulation.

PubMed

Fai, Thomas G; Leo-Macias, Alejandra; Stokes, David L; Peskin, Charles S

2017-10-01

We simulate deformable red blood cells in the microcirculation using the immersed boundary method with a cytoskeletal model that incorporates structural details revealed by tomographic images. The elasticity of red blood cells is known to be supplied by both their lipid bilayer membranes, which resist bending and local changes in area, and their cytoskeletons, which resist in-plane shear. The cytoskeleton consists of spectrin tetramers that are tethered to the lipid bilayer by ankyrin and by actin-based junctional complexes. We model the cytoskeleton as a random geometric graph, with nodes corresponding to junctional complexes and with edges corresponding to spectrin tetramers such that the edge lengths are given by the end-to-end distances between nodes. The statistical properties of this graph are based on distributions gathered from three-dimensional tomographic images of the cytoskeleton by a segmentation algorithm. We show that the elastic response of our model cytoskeleton, in which the spectrin polymers are treated as entropic springs, is in good agreement with the experimentally measured shear modulus. By simulating red blood cells in flow with the immersed boundary method, we compare this discrete cytoskeletal model to an existing continuum model and predict the extent to which dynamic spectrin network connectivity can protect against failure in the case of a red cell subjected to an applied strain. The methods presented here could form the basis of disease- and patient-specific computational studies of hereditary diseases affecting the red cell cytoskeleton.

Image-based model of the spectrin cytoskeleton for red blood cell simulation

PubMed Central

Stokes, David L.; Peskin, Charles S.

2017-01-01

We simulate deformable red blood cells in the microcirculation using the immersed boundary method with a cytoskeletal model that incorporates structural details revealed by tomographic images. The elasticity of red blood cells is known to be supplied by both their lipid bilayer membranes, which resist bending and local changes in area, and their cytoskeletons, which resist in-plane shear. The cytoskeleton consists of spectrin tetramers that are tethered to the lipid bilayer by ankyrin and by actin-based junctional complexes. We model the cytoskeleton as a random geometric graph, with nodes corresponding to junctional complexes and with edges corresponding to spectrin tetramers such that the edge lengths are given by the end-to-end distances between nodes. The statistical properties of this graph are based on distributions gathered from three-dimensional tomographic images of the cytoskeleton by a segmentation algorithm. We show that the elastic response of our model cytoskeleton, in which the spectrin polymers are treated as entropic springs, is in good agreement with the experimentally measured shear modulus. By simulating red blood cells in flow with the immersed boundary method, we compare this discrete cytoskeletal model to an existing continuum model and predict the extent to which dynamic spectrin network connectivity can protect against failure in the case of a red cell subjected to an applied strain. The methods presented here could form the basis of disease- and patient-specific computational studies of hereditary diseases affecting the red cell cytoskeleton. PMID:28991926

A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin

DOE PAGES

Liu, Yao; Zhu, Wenhan; Tan, Yunhao; ...

2017-01-27

Legionella pneumophila, the etiological agent of Legionnaires' disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H 95E XXH 99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cellmore » rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Furthermore, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.« less

Spatial organization of the cytoskeleton enhances cargo delivery to specific target areas on the plasma membrane of spherical cells

NASA Astrophysics Data System (ADS)

Hafner, Anne E.; Rieger, Heiko

2016-12-01

Intracellular transport is vital for the proper functioning and survival of a cell. Cargo (proteins, vesicles, organelles, etc) is transferred from its place of creation to its target locations via molecular motor assisted transport along cytoskeletal filaments. The transport efficiency is strongly affected by the spatial organization of the cytoskeleton, which constitutes an inhomogeneous, complex network. In cells with a centrosome microtubules grow radially from the central microtubule organizing center towards the cell periphery whereas actin filaments form a dense meshwork, the actin cortex, underneath the cell membrane with a broad range of orientations. The emerging ballistic motion along filaments is frequently interrupted due to constricting intersection nodes or cycles of detachment and reattachment processes in the crowded cytoplasm. In order to investigate the efficiency of search strategies established by the cell’s specific spatial organization of the cytoskeleton we formulate a random velocity model with intermittent arrest states. With extensive computer simulations we analyze the dependence of the mean first passage times for narrow escape problems on the structural characteristics of the cytoskeleton, the motor properties and the fraction of time spent in each state. We find that an inhomogeneous architecture with a small width of the actin cortex constitutes an efficient intracellular search strategy.

Clarin-1, encoded by the Usher Syndrome III causative gene, forms a membranous microdomain: possible role of clarin-1 in organizing the actin cytoskeleton.

PubMed

Tian, Guilian; Zhou, Yun; Hajkova, Dagmar; Miyagi, Masaru; Dinculescu, Astra; Hauswirth, William W; Palczewski, Krzysztof; Geng, Ruishuang; Alagramam, Kumar N; Isosomppi, Juha; Sankila, Eeva-Marja; Flannery, John G; Imanishi, Yoshikazu

2009-07-10

Clarin-1 is the protein product encoded by the gene mutated in Usher syndrome III. Although the molecular function of clarin-1 is unknown, its primary structure predicts four transmembrane domains similar to a large family of membrane proteins that include tetraspanins. Here we investigated the role of clarin-1 by using heterologous expression and in vivo model systems. When expressed in HEK293 cells, clarin-1 localized to the plasma membrane and concentrated in low density compartments distinct from lipid rafts. Clarin-1 reorganized actin filament structures and induced lamellipodia. This actin-reorganizing function was absent in the modified protein encoded by the most prevalent North American Usher syndrome III mutation, the N48K form of clarin-1 deficient in N-linked glycosylation. Proteomics analyses revealed a number of clarin-1-interacting proteins involved in cell-cell adhesion, focal adhesions, cell migration, tight junctions, and regulation of the actin cytoskeleton. Consistent with the hypothesized role of clarin-1 in actin organization, F-actin-enriched stereocilia of auditory hair cells evidenced structural disorganization in Clrn1(-/-) mice. These observations suggest a possible role for clarin-1 in the regulation and homeostasis of actin filaments, and link clarin-1 to the interactive network of Usher syndrome gene products.

Formaldehyde fixation is detrimental to actin cables in glucose-depleted S. cerevisiae cells

PubMed Central

Vasicova, Pavla; Rinnerthaler, Mark; Haskova, Danusa; Novakova, Lenka; Malcova, Ivana; Breitenbach, Michael; Hasek, Jiri

2016-01-01

Actin filaments form cortical patches and emanating cables in fermenting cells of Saccharomyces cerevisiae. This pattern has been shown to be depolarized in glucose-depleted cells after formaldehyde fixation and staining with rhodamine-tagged phalloidin. Loss of actin cables in mother cells was remarkable. Here we extend our knowledge on actin in live glucose-depleted cells co-expressing the marker of actin patches (Abp1-RFP) with the marker of actin cables (Abp140-GFP). Glucose depletion resulted in appearance of actin patches also in mother cells. However, even after 80 min of glucose deprivation these cells showed a clear network of actin cables labeled with Abp140-GFP in contrast to previously published data. In live cells with a mitochondrial dysfunction (rho0 cells), glucose depletion resulted in almost immediate appearance of Abp140-GFP foci partially overlapping with Abp1-RFP patches in mother cells. Residual actin cables were clustered in patch-associated bundles. A similar overlapping “patchy” pattern of both actin markers was observed upon treatment of glucose-deprived rho+ cells with FCCP (the inhibitor of oxidative phosphorylation) and upon treatment with formaldehyde. While the formaldehyde-targeted process stays unknown, our results indicate that published data on yeast actin cytoskeleton obtained from glucose-depleted cells after fixation should be considered with caution. PMID:28357356

Interaction between Flavivirus and Cytoskeleton during Virus Replication

PubMed Central

Foo, Kar Yue; Chee, Hui-Yee

2015-01-01

Flaviviruses are potentially human pathogens that cause major epidemics worldwide. Flavivirus interacts with host cell factors to form a favourable virus replication site. Cell cytoskeletons have been observed to have close contact with flaviviruses, which expands the understanding of cytoskeleton functions during virus replication, although many detailed mechanisms are still unclear. The interactions between the virus and host cytoskeletons such as actin filaments, microtubules, and intermediate filaments have provided insight into molecular alterations during the virus infection, such as viral entry, in-cell transport, scaffold assembly, and egress. This review article focuses on the utilization of cytoskeleton by Flavivirus and the respective functions during virus replication. PMID:26347881

Unique ζ-chain motifs mediate a direct TCR-actin linkage critical for immunological synapse formation and T-cell activation.

PubMed

Klieger, Yair; Almogi-Hazan, Osnat; Ish-Shalom, Eliran; Pato, Aviad; Pauker, Maor H; Barda-Saad, Mira; Wang, Lynn; Baniyash, Michal

2014-01-01

TCR-mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ-chain motifs, mediating the TCR's direct interaction with actin and inducing actin bundling. While T cells expressing the ζ-chain mutated in these motifs lack cytoskeleton (actin) associated (cska)-TCRs, they express normal levels of non-cska and surface TCRs as cells expressing wild-type ζ-chain. However, such mutant cells are unable to display activation-dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR-actin linkage, providing the missing gap linking between TCR-mediated Ag recognition, specific cytoskeleton orientation toward the T-cell-APC interacting pole and long-lived IS maintenance. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells

PubMed Central

Lechuga, Susana; Baranwal, Somesh; Li, Chao; Naydenov, Nayden G.; Kuemmerle, John F.; Dugina, Vera; Chaponnier, Christine; Ivanov, Andrei I.

2014-01-01

Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA–depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis. PMID:25143399

Nuclear positioning by actin cables and perinuclear actin

PubMed Central

Huelsmann, Sven; Brown, Nicholas H

2014-01-01

Nuclear positioning is an important process during development and homeostasis. Depending on the affected tissue, mislocalized nuclei can alter cellular processes such as polarization, differentiation, or migration and lead ultimately to diseases. Many cells actively control the position of their nucleus using their cytoskeleton and motor proteins. We have recently shown that during Drosophila oogenesis, nurse cells employ cytoplasmic actin cables in association with perinuclear actin to position their nucleus. Here, we briefly summarize our work and discuss why nuclear positioning in nurse cells is specialized but the molecular mechanisms are likely to be more generally used. PMID:24905988

Connectingthe puzzle pieces between cytoskeleton andsecretory pathway

PubMed Central

Gurel, Pinar S.; Hatch, Anna L.; Higgs, Henry N.

2014-01-01

A tendency in cell biology is to divide and conquer. For example, decades of painstaking work have led to an understanding of endoplasmic reticulum (ER) and Golgi structure, dynamics, and transport. In parallel, cytoskeletal researchers have revealed a fantastic diversity of structure and cellular function in both actin and microtubules. Increasingly, these areas overlap, necessitating an understanding of both organelle and cytoskeletal biology. This review addressesconnections between the actin/microtubule cytoskeletons and organelles in animal cells, focusing on threetopics: ER structure/function, ER-to-Golgi transport; and Golgi structure/function. Making these connections has been challenging, due to 1) the small sizes and dynamic characteristics of some components, 2) the fact that organelle-specific cytoskeleton can easily be obscured by more abundant cytoskeletal structures, and 3) the difficulties in imaging membranes and cytoskeleton simultaneously, especially at the ultra-structural level. One major concept is that the cytoskeleton is frequently used to generate force for membrane movement, with two potential consequences: translocation of the organelle, or deformation of the organelle membrane. While initially discussing issues common to metazoan cells in general, we subsequently highlight specific features of neurons, since these highly polarized cells present unique challenges for organellar distribution and dynamics. PMID:25050967

[Cytoskeletal actin and its associated proteins. Some examples in Protista].

PubMed

Guillén, N; Carlier, M F; Brugerolle, G; Tardieux, I; Ausseil, J

1998-06-01

Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin

Regulation of microtubule (MT)-based cytoskeleton in the seminiferous epithelium during spermatogenesis

PubMed Central

Tang, Elizabeth I.; Mruk, Dolores D.; Cheng, C. Yan

2016-01-01

In rodents and humans, testicular cells, similar to other mammalian cells, are supported by actin-, microtubule (MT)- and intermediate filament-based cytoskeletons to regulate spermatogenesis during the epithelial cycle. However, most of the published findings in the literature are limited to studies that visualize these cytoskeletons in the seminiferous epithelium during spermatogenesis. Few are focus on the underlying molecular mechanism that regulates their organization in the epithelium in response to changes in the stages of the epithelial cycle remains largely explored. Functional studies in the last decade have begun to focus on the role of binding proteins that regulate these cytoskeletons, and some interesting data have been rapidly emerging in the field. Since the actin- and intermediate-based cytoskeletons have been recently reviewed, herein we focus on the MT-based cytoskeleton for two reasons. First, besides serving as a structural support cytoskeleton, MT is known to serve as the track to support and facilitate the transport of germ cells, such as preleptotene spermatocytes connected in clones and elongating/elongated spermatids during spermiogenesis across the blood-testis barrier (BTB) and the adluminal compartment, respectively, during spermatogenesis. While these cellular events are crucial to the completion of spermatogenesis, they have been largely ignored in the past. Second, MT-based cytoskeleton is working in concert with the actin-based cytoskeleton to provide structural support to the transport of intracellular organelles across the cell cytosol, such as endosome-based vesicles, and residual bodies, phagosomes in Sertoli cells, to maintain the cellular homeostasis in the seminiferous epithelium. We critically evaluate some recent published findings herein to support a hypothesis regarding the role of MT in conferring germ cell transport in the seminiferous epithelium. PMID:26791048

Triggering signaling pathways using F-actin self-organization.

PubMed

Colin, A; Bonnemay, L; Gayrard, C; Gautier, J; Gueroui, Z

2016-10-04

The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity.

Triggering signaling pathways using F-actin self-organization

PubMed Central

Colin, A.; Bonnemay, L.; Gayrard, C.; Gautier, J.; Gueroui, Z.

2016-01-01

The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity. PMID:27698406

Role of G protein signaling in the formation of the fibrin(ogen)-integrin αIIbβ3-actin cytoskeleton complex in platelets.

PubMed

Budnik, Ivan; Shenkman, Boris; Savion, Naphtali

2016-09-01

Effective platelet function requires formation of a physical link between fibrin(ogen), integrin αIIbβ3, and cytoplasmic actin filaments. We investigated the role of the Gαq, Gαi, and Gα12/13 families of heterotrimeric GTP-binding proteins (G proteins) in the assembly of a ligand-αIIbβ3-actin cytoskeleton complex. Selective and combined activation of the G proteins was achieved by using combinations of various platelet agonists and inhibitors. Formation and stability of fibrinogen-αIIbβ3 interaction were evaluated by the extent of platelet aggregation and the rate of eptifibatide-induced platelet disaggregation; association of αIIbβ3 with the cytoskeleton was analyzed by western blot. Formation of the fibrin-αIIbβ3-actin cytoskeleton complex was evaluated by rotational thromboelastometry assay in which clot formation was induced by the mixture of reptilase and factor XIIIa. We demonstrated that involvement of heterotrimeric G proteins in the formation of the ligand-αIIbβ3-cytoskeleton complex depends on whether fibrinogen or fibrin serves as the integrin ligand. Formation of the fibrinogen-αIIbβ3-cytoskeleton complex requires combined activation of at least two G protein pathways while the maximal αIIbβ3-cytoskeleton association and the strongest αIIbβ3-fibrinogen binding supporting irreversible platelet aggregation require combined activation of all three-Gαq, Gαi, and Gα12/13-G protein families. In contrast, formation of the fibrin-αIIbβ3-cytoskeleton complex mediating clot retraction is critically dependent on the activation of the Gαi family, especially on the activation of Gαz.

The role of the actin cytoskeleton in calcium signaling in starfish oocytes.

PubMed

Santella, Luigia; Puppo, Agostina; Chun, Jong Tai

2008-01-01

Ca2+ is the most universal second messenger in cells from the very first moment of fertilization. In all animal species, fertilized eggs exhibit massive mobilization of intracellular Ca2+ to orchestrate the initial events of development. Echinoderm eggs have been an excellent model system for studying fertilization and the cell cycle due to their large size and abundance. In preparation for fertilization, the cell cycle-arrested oocytes must undergo meiotic maturation. Studies of starfish oocytes have shown that Ca2+ signaling is intimately involved in this process. Our knowledge of the molecular mechanism of meiotic maturation and fertilization has expanded greatly in the past two decades due to the discovery of cell cycle-related kinases and Ca2+-mobilizing second messengers. However, the molecular details of their actions await elucidation of other cellular elements that assist in the creation and transduction of Ca2+ signals. In this regard, the actin cytoskeleton, the receptors for second messengers and the Ca2+-binding proteins also require more attention. This article reviews the physiological significance and the mechanism of intracellular Ca2+ mobilization in starfish oocytes during maturation and fertilization.

Synergistic effect of signaling from receptors of soluble platelet agonists and outside-in signaling in formation of a stable fibrinogen-integrin αIIbβ3-actin cytoskeleton complex.

PubMed

Budnik, Ivan; Shenkman, Boris; Savion, Naphtali

2015-01-01

Thrombus formation in the injured vessel wall is a highly complex process involving various blood-born components that go through specific temporal and spatial changes as observed by intravital videomicroscopy. Platelets bind transiently to the developing thrombus and may either become stably incorporated into or disengage from the thrombus. The aim of the present study was to reveal the processes involved in the formation of a stable thrombus. Platelet-rich plasma and washed platelets were studied by the aggregometer. The aggregate stability was challenged by eptifibatide. Platelet Triton-insoluble fraction was prepared and the actin and αIIb content in the cytoskeleton was analyzed by western blot. Maximal actin polymerization is achieved 1min after platelet activation while maximal αIIbβ3-actin cytoskeleton association requires 5 to 10min of activation and fibrinogen-mediated platelet-to-platelet bridging. Thus, actin polymerization is dependent on platelet activation and requires neither αIIbβ3 integrin occupation nor platelet aggregation. Formation of a stable aggregate requires platelet activation for more than 1min, complete increase in actin cytoskeleton fraction and partial association of αIIbβ3 with the actin cytoskeleton. However, direct αIIbβ3 activation is not sufficient for cytoskeleton complex formation. Thus, stable αIIbβ3-fibrinogen interaction, representing stable aggregate, is achieved after more than 1min agonist activation, involving inside-out and outside-in signaling but not after direct integrin activation, involving only outside-in signaling. Formation of a stable fibrinogen-αIIbβ3-actin cytoskeleton complex is the result of the combined effect of platelet stimulation by soluble agonists, activation of αIIbβ3, fibrinogen binding and platelet-to-platelet bridging. Copyright © 2014 Elsevier Ltd. All rights reserved.

Actin Cytoskeleton-Based Plant Synapse as Gravitransducer in the Transition Zone of the Root Apex

NASA Astrophysics Data System (ADS)

Baluska, Frantisek; Barlow, Peter; Volkmann, Dieter; Mancuso, Stefano

The actin cytoskeleton was originally proposed to act as the signal transducer in the plant gravity sensory-motoric circuit. Surprisingly, however, several studies have documented that roots perfom gravisensing and gravitropism more effectively if exposed to diverse anti-F-actin drugs. Our study, using decapped maize root apices, has revealed that depolymerization of F-actin stimulates gravity perception in cells of the transition zone where root gravitropism is initiated (Mancuso et al. 2006). It has been proposed (Balǔka et al. 2005, 2009a) that s the non-growing adhesive end-poles, enriched with F-actin and myosin VIII, and active in endocytic recycling of both PIN transporters and cell wall pectins cross-linked with calcium and boron, act as the gravisensing domains, and that these impinge directly upon the root motoric responses via control of polar auxin transport. This model suggests that mechanical asymmetry at these plant synapses determines vectorial gravity-controlled auxin transport. Due to the gravity-imposed mechanical load upon the protoplast, a tensional stress is also imposed upon the plasma membrane of the physically lower synaptic cell pole. This stress is then relieved by shifting the endocytosis-exocytosis balance towards exocytosis (Balǔka et al. s 2005, 2009a,b). This `Synaptic Auxin Secretion' hypothesis does not conflict with the `Starch Statolith' hypothesis, which is based on amyloplast sedimentation. In fact, the `Synaptic Auxin Secretion' hypothesis has many elements which allow its unification with the Starch-Statolith model (Balǔka et al. 2005, 2009a,b). s References Balǔka F, Volkmann D, Menzel D (2005) Plant synapses: actin-based adhesion s domains for cell-to-cell communication. Trends Plant Sci 10: 106-111 Balǔka F, Schlicht M, s Wan Y-L, Burbach C, Volkmann D (2009a) Intracellular domains and polarity in root apices: from synaptic domains to plant neurobiology. Nova Acta Leopoldina 96: 103-122 Balǔka s F, Mancuso S

Cytoskeleton Molecular Motors: Structures and Their Functions in Neuron.

PubMed

Xiao, Qingpin; Hu, Xiaohui; Wei, Zhiyi; Tam, Kin Yip

2016-01-01

Cells make use of molecular motors to transport small molecules, macromolecules and cellular organelles to target region to execute biological functions, which is utmost important for polarized cells, such as neurons. In particular, cytoskeleton motors play fundamental roles in neuron polarization, extension, shape and neurotransmission. Cytoskeleton motors comprise of myosin, kinesin and cytoplasmic dynein. F-actin filaments act as myosin track, while kinesin and cytoplasmic dynein move on microtubules. Cytoskeleton motors work together to build a highly polarized and regulated system in neuronal cells via different molecular mechanisms and functional regulations. This review discusses the structures and working mechanisms of the cytoskeleton motors in neurons.

The transcriptional repressor Sum1p counteracts Sir2p in regulation of the actin cytoskeleton, mitochondrial quality control and replicative lifespan in Saccharomyces cerevisiae.

PubMed

Higuchi-Sanabria, Ryo; Vevea, Jason D; Charalel, Joseph K; Sapar, Maria L; Pon, Liza A

2016-01-18

Increasing the stability or dynamics of the actin cytoskeleton can extend lifespan in C. elegans and S. cerevisiae . Actin cables of budding yeast, bundles of actin filaments that mediate cargo transport, affect lifespan control through effects on mitochondrial quality control. Sir2p, the founding member of the Sirtuin family of lifespan regulators, also affects actin cable dynamics, assembly, and function in mitochondrial quality control. Here, we obtained evidence for novel interactions between Sir2p and Sum1p, a transcriptional repressor that was originally identified through mutations that genetically suppress sir2 ∆ phenotypes unrelated to lifespan. We find that deletion of SUM1 in wild-type cells results in increased mitochondrial function and actin cable abundance. Furthermore, deletion of SUM1 suppresses defects in actin cables and mitochondria of sir2 ∆ yeast, and extends the replicative lifespan and cellular health span of sir2 ∆ cells. Thus, Sum1p suppresses Sir2p function in control of specific aging determinants and lifespan in budding yeast.

Actin dynamics in Amoeba proteus motility.

PubMed

Pomorski, P; Krzemiński, P; Wasik, A; Wierzbicka, K; Barańska, J; Kłopocka, W

2007-01-01

We studied the distribution of the endogenous Arp2/3 complex in Amoeba proteus and visualised the ratio of filamentous (F-actin) to total actin in living cells. The presented results show that in the highly motile Amoeba proteus, Arp2/3 complex-dependent actin polymerisation is involved in the formation of the branching network of the contractile layer, adhesive structures, and perinuclear cytoskeleton. The aggregation of the Arp2/3 complex in the cortical network, with the exception of the uroid and advancing fronts, and the spatial orientation of microfilaments at the leading edge suggest that actin polymerisation in this area is not sufficient to provide the driving force for membrane displacement. The examined proteins were enriched in the pinocytotic pseudopodia and the perinuclear cytoskeleton in pinocytotic amoebae. In migrating amoebae, the course of changes in F-actin concentration corresponded with the distribution of tension in the cell cortex. The maximum level of F-actin in migrating amoebae was observed in the middle-posterior region and in the front of retracting pseudopodia. Arp2/3 complex-dependent actin polymerisation did not seem to influence F-actin concentration. The strongly condensed state of the microfilament system could be attributed to strong isometric contraction of the cortical layer accompanied by its retraction from distal cell regions. Isotonic contraction was limited to the uroid.

Adhesion structures and their cytoskeleton-membrane interactions at podosomes of osteoclasts in culture.

PubMed

Akisaka, Toshitaka; Yoshida, Hisaho; Suzuki, Reiko; Takama, Keiko

2008-03-01

The organization of the cytoskeleton in the podosomes of osteoclasts was studied by use of cell shearing, rotary replication, and fluorescence cytochemical techniques. After shearing, clathrin plaques and particles associated with the cytoskeleton were left behind on the exposed cytoplasmic side of the membrane. The cytoskeleton of the podosomes was characterized by two types of actin filaments: relatively long filaments in the portion surrounding the podosome core, and highly branched short filaments in the core. Individual actin filaments radiating from the podosomes interacted with several membrane particles along the length of the filaments. Many lateral contacts with the membrane surface by the particles were made along the length of individual actin filaments. The polarity of actin filaments in podosomes became oriented such that their barbed ends were directed toward the core of podosomes. The actin cytoskeletons terminated or branched at the podosomes, where the membrane tightly adhered to the substratum. Microtubules were not usually present in the podosome structures; however, certain microtubules appeared to be morphologically in direct contact with the podosome core. Most of the larger clathrin plaques consisted of flat sheets of clathrin lattices that interconnected neighboring clathrin lattices to form an extensive clathrin area. However, the small deeply invaginated clathrin plaques and the podosomal cytoskeleton were located close together. Thus, the clathrin plaques on the ventral membrane of osteoclasts might be involved in both cell adhesion and the formation of receptor-ligand complexes, i.e., endocytosis.

Liquid droplets of cross-linked actin filaments

NASA Astrophysics Data System (ADS)

Weirich, Kimberly; Banerjee, Shiladitya; Dasbiswas, Kinjal; Vaikuntanathan, Suriyanarayan; Gardel, Margaret

Soft materials constructed from biomolecules self-assemble into a myriad of structures that work in concert to support cell physiology. One critical soft material is the actin cytoskeleton, a viscoelastic gel composed of cross-linked actin filaments. Although actin networks are primarily known for their elastic properties, which are crucial to regulating cell mechanics, the viscous behavior has been theorized to enable shape changes and flows. We experimentally demonstrate a fluid phase of cross-linked actin, where cross-linker condenses dilute short actin filaments into spindle-shaped droplets, or tactoids. Tactoids have shape dynamics consistent with a continuum model of liquid crystal droplets. The cross-linker, which acts as a long range attractive interaction, analogous to molecular cohesion, controls the tactoid shape and dynamics, which reports on the liquid's interfacial tension and viscosity. We investigate how the cross-linker properties and filament length influence the liquid properties. These results demonstrate a novel mechanism to control organization of the actin cytoskeleton and provide insight into design principles for complex, macromolecular liquid phases.

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

PubMed Central

Swaminathan, Vinay; Kalappurakkal, Joseph Mathew; Moore, Travis I.; Koga, Nobuyasu; Baker, David A.; Oldenbourg, Rudolf; Tani, Tomomi; Springer, Timothy A.; Waterman, Clare M.

2017-01-01

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven “retrograde flow” of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues. PMID:29073038

The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

PubMed Central

Spracklen, Andrew J.; Fagan, Tiffany N.; Lovander, Kaylee E.; Tootle, Tina L.

2015-01-01

Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling

Arp2/3 complex inhibition radically alters lamellipodial actin architecture, suspended cell shape, and the cell spreading process

PubMed Central

Henson, John H.; Yeterian, Mesrob; Weeks, Richard M.; Medrano, Angela E.; Brown, Briana L.; Geist, Heather L.; Pais, Mollyann D.; Oldenbourg, Rudolf; Shuster, Charles B.

2015-01-01

Recent studies have investigated the dendritic actin cytoskeleton of the cell edge's lamellipodial (LP) region by experimentally decreasing the activity of the actin filament nucleator and branch former, the Arp2/3 complex. Here we extend these studies via pharmacological inhibition of the Arp2/3 complex in sea urchin coelomocytes, cells that possess an unusually broad LP region and display correspondingly exaggerated centripetal flow. Using light and electron microscopy, we demonstrate that Arp2/3 complex inhibition via the drug CK666 dramatically altered LP actin architecture, slowed centripetal flow, drove a lamellipodial-to-filopodial shape change in suspended cells, and induced a novel actin structural organization during cell spreading. A general feature of the CK666 phenotype in coelomocytes was transverse actin arcs, and arc generation was arrested by a formin inhibitor. We also demonstrate that CK666 treatment produces actin arcs in other cells with broad LP regions, namely fish keratocytes and Drosophila S2 cells. We hypothesize that the actin arcs made visible by Arp2/3 complex inhibition in coelomocytes may represent an exaggerated manifestation of the elongate mother filaments that could possibly serve as the scaffold for the production of the dendritic actin network. PMID:25568343

Actin filaments as tension sensors.

PubMed

Galkin, Vitold E; Orlova, Albina; Egelman, Edward H

2012-02-07

The field of mechanobiology has witnessed an explosive growth over the past several years as interest has greatly increased in understanding how mechanical forces are transduced by cells and how cells migrate, adhere and generate traction. Actin, a highly abundant and anomalously conserved protein, plays a large role in forming the dynamic cytoskeleton that is so essential for cell form, motility and mechanosensitivity. While the actin filament (F-actin) has been viewed as dynamic in terms of polymerization and depolymerization, new results suggest that F-actin itself may function as a highly dynamic tension sensor. This property may help explain the unusual conservation of actin's sequence, as well as shed further light on actin's essential role in structures from sarcomeres to stress fibers. Copyright © 2012 Elsevier Ltd. All rights reserved.

Real-Time Dynamics of Emerging Actin Networks in Cell-Mimicking Compartments

PubMed Central

Deshpande, Siddharth; Pfohl, Thomas

2015-01-01

Understanding the cytoskeletal functionality and its relation to other cellular components and properties is a prominent question in biophysics. The dynamics of actin cytoskeleton and its polymorphic nature are indispensable for the proper functioning of living cells. Actin bundles are involved in cell motility, environmental exploration, intracellular transport and mechanical stability. Though the viscoelastic properties of actin-based structures have been extensively probed, the underlying microstructure dynamics, especially their disassembly, is not fully understood. In this article, we explore the rich dynamics and emergent properties exhibited by actin bundles within flow-free confinements using a microfluidic set-up and epifluorescence microscopy. After forming entangled actin filaments within cell-sized quasi two-dimensional confinements, we induce their bundling using three different fundamental mechanisms: counterion condensation, depletion interactions and specific protein-protein interactions. Intriguingly, long actin filaments form emerging networks of actin bundles via percolation leading to remarkable properties such as stress generation and spindle-like intermediate structures. Simultaneous sharing of filaments in different links of the network is an important parameter, as short filaments do not form networks but segregated clusters of bundles instead. We encounter a hierarchical process of bundling and its subsequent disassembly. Additionally, our study suggests that such percolated networks are likely to exist within living cells in a dynamic fashion. These observations render a perspective about differential cytoskeletal responses towards numerous stimuli. PMID:25785606

The Cytoskeleton and Force Response Mechanisms

NASA Technical Reports Server (NTRS)

Allen, Philip Goodwin

2003-01-01

The long term aim of this project was to define the mechanisms by which cells sense and respond to the physical forces experienced at 1g and missing in microgravity. Identification and characterization of the elements of the cells force response mechanism could provide pathways and molecules to serve as targets for pharmacological intervention to mitigate the pathologic effects of microgravity. Mechanical forces experienced by the organism can be transmitted to cells through molecules that allow cells to bind to the extracellular matrix and through other types of molecules which bind cells to each other. These molecules are coupled in large complexes of proteins to structural elements such as the actin cytoskeleton that give the cell the ability to sense, resist and respond to force. Application of small forces to tissue culture cells causes local elevation of intracellular calcium through stretch activated ion channels, increased tyrosine phosphorylation and a restructuring of the actin cytoskeleton. Using collagen coated iron oxide beads and strong magnets, we can apply different levels of force to cells in culture. We have found that force application causes the cells to polymerize actin at the site of mechanical deformation and unexpectedly, to depolymerize actin across the rest of the cell. Observations of GFP- actin expressing cells demonstrate that actin accumulates at the site of deformation within the first five minutes of force application and is maintained for many tens of minutes after force is removed. Consistent with the reinforcement of the cytoskeletal structures underlying the integrin-bead interaction, force also alters the motion of bound magnetic beads. This effect is seen following the removal of the magnetic field, and is only partially ablated by actin disruption with cytochalsin B. While actin is polymerizing locally at the site of force application, force also stimulates a global reduction in actin filament content within the cells. We have

The role of actin networks in cellular mechanosensing

NASA Astrophysics Data System (ADS)

Azatov, Mikheil

Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant

The actin filament cross-linker L-plastin confers resistance to TNF-α in MCF-7 breast cancer cells in a phosphorylation-dependent manner

PubMed Central

Janji, Bassam; Vallar, Laurent; Tanoury, Ziad Al; Bernardin, François; Vetter, Guillaume; Schaffner-Reckinger, Elisabeth; Berchem, Guy; Friederich, Evelyne; Chouaib, Salem

2010-01-01

Abstract We used a tumour necrosis factor (TNF)-α resistant breast adenocarcinoma MCF-7 cell line to investigate the involvement of the actin cytoskeleton in the mechanism of cell resistance to this cytokine. We found that TNF resistance correlates with the loss of cell epithelial properties and the gain of a mesenchymal phenotype, reminiscent of an epithelial-to-mesenchymal transition (EMT). Morphological changes were associated with a profound reorganization of the actin cytoskeleton and with a change in the repertoire of expressed actin cytoskeleton genes and EMT markers, as revealed by DNA microarray-based expression profiling. L-plastin, an F-actin cross-linking and stabilizing protein, was identified as one of the most significantly up-regulated genes in TNF-resistant cells. Knockdown of L-plastin in these cells revealed its crucial role in conferring TNF resistance. Importantly, overexpression of wild-type L-plastin in TNF-sensitive MCF-7 cells was sufficient to protect them against TNF-mediated cell death. Furthermore, we found that this effect is dependent on serine-5 phosphorylation of L-plastin and that non-conventional protein kinase C isoforms and the ceramide pathway may regulate its phosphorylation state. The protective role of L-plastin was not restricted to TNF-α resistant MCF-7 cells because a correlation between the expression of L-plastin and the resistance to TNF-α was observed in other breast cancer cell lines. Together, our study discloses a novel unexpected role of the actin bundling protein L-plastin as a cell protective protein against TNF-cytotoxicity. PMID:19799649

Leveraging the membrane-cytoskeleton interface with myosin-1

PubMed Central

McConnell, Russell E.; Tyska, Matthew J.

2010-01-01

Class 1 myosins are small motor proteins with the ability to simultaneously bind to actin filaments and cellular membranes. Given their ability to generate mechanical force, and their high prevalence in many cell types, these molecules are well positioned to carry out a number of important biological functions at the interface of membrane and the actin cytoskeleton. Indeed, recent studies implicate these motors in endocytosis, exocytosis, release of extracellular vesicles, and the regulation of tension between membrane and the cytoskeleton. Many class 1 myosins also exhibit a load-dependent mechano-chemical cycle that enables them to maintain tension for long periods of time without hydrolyzing ATP. These properties put myosins-1 in a unique position to regulate dynamic membrane-cytoskeleton interactions and respond to physical forces during these events. PMID:20471271

Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line.

PubMed

Oh, J-E; Karlmark Raja, K; Shin, J-H; Pollak, A; Hengstschläger, M; Lubec, G

2006-10-01

No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.

Inositol induces mesenchymal-epithelial reversion in breast cancer cells through cytoskeleton rearrangement.

PubMed

Dinicola, Simona; Fabrizi, Gianmarco; Masiello, Maria Grazia; Proietti, Sara; Palombo, Alessandro; Minini, Mirko; Harrath, Abdel Halim; Alwasel, Saleh H; Ricci, Giulia; Catizone, Angela; Cucina, Alessandra; Bizzarri, Mariano

2016-07-01

Inositol displays multi-targeted effects on many biochemical pathways involved in epithelial-mesenchymal transition (EMT). As Akt activation is inhibited by inositol, we investigated if such effect could hamper EMT in MDA-MB-231 breast cancer cells. In cancer cells treated with pharmacological doses of inositol E-cadherin was increased, β-catenin was redistributed behind cell membrane, and metalloproteinase-9 was significantly reduced, while motility and invading capacity were severely inhibited. Those changes were associated with a significant down-regulation of PI3K/Akt activity, leading to a decrease in downstream signaling effectors: NF-kB, COX-2, and SNAI1. Inositol-mediated inhibition of PS1 leads to lowered Notch 1 release, thus contributing in decreasing SNAI1 levels. Overall, these data indicated that inositol inhibits the principal molecular pathway supporting EMT. Similar results were obtained in ZR-75, a highly metastatic breast cancer line. These findings are coupled with significant changes on cytoskeleton. Inositol slowed-down vimentin expression in cells placed behind the wound-healing edge and stabilized cortical F-actin. Moreover, lamellipodia and filopodia, two specific membrane extensions enabling cell migration and invasiveness, were no longer detectable after inositol addiction. Additionally, fascin and cofilin, two mandatory required components for F-actin assembling within cell protrusions, were highly reduced. These data suggest that inositol may induce an EMT reversion in breast cancer cells, suppressing motility and invasiveness through cytoskeleton modifications. Copyright © 2016 Elsevier Inc. All rights reserved.

Conditional N-WASP knockout in mouse brain implicates actin cytoskeleton regulation in hydrocephalus pathology.

PubMed

Jain, Neeraj; Lim, Lee Wei; Tan, Wei Ting; George, Bhawana; Makeyev, Eugene; Thanabalu, Thirumaran

2014-04-01

Cerebrospinal fluid (CSF) is produced by the choroid plexus and moved by multi-ciliated ependymal cells through the ventricular system of the vertebrate brain. Defects in the ependymal layer functionality are a common cause of hydrocephalus. N-WASP (Neural-Wiskott Aldrich Syndrome Protein) is a brain-enriched regulator of actin cytoskeleton and N-WASP knockout caused embryonic lethality in mice with neural tube and cardiac abnormalities. To shed light on the role of N-WASP in mouse brain development, we generated N-WASP conditional knockout mouse model N-WASP(fl/fl); Nestin-Cre (NKO-Nes). NKO-Nes mice were born with Mendelian ratios but exhibited reduced growth characteristics compared to their littermates containing functional N-WASP alleles. Importantly, all NKO-Nes mice developed cranial deformities due to excessive CSF accumulation and did not survive past weaning. Coronal brain sections of these animals revealed dilated lateral ventricles, defects in ciliogenesis, loss of ependymal layer integrity, reduced thickness of cerebral cortex and aqueductal stenosis. Immunostaining for N-cadherin suggests that ependymal integrity in NKO-Nes mice is lost as compared to normal morphology in the wild-type controls. Moreover, scanning electron microscopy and immunofluorescence analyses of coronal brain sections with anti-acetylated tubulin antibodies revealed the absence of cilia in ventricular walls of NKO-Nes mice indicative of ciliogenesis defects. N-WASP deficiency does not lead to altered expression of N-WASP regulatory proteins, Fyn and Cdc42, which have been previously implicated in hydrocephalus pathology. Taken together, our results suggest that N-WASP plays a critical role in normal brain development and implicate actin cytoskeleton regulation as a vulnerable axis frequently deregulated in hydrocephalus. Copyright © 2014 Elsevier Inc. All rights reserved.

Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopy

NASA Astrophysics Data System (ADS)

Clausen, M. P.; Colin-York, H.; Schneider, F.; Eggeling, C.; Fritzsche, M.

2017-02-01

Nanoscale spacing between the plasma membrane and the underlying cortical actin cytoskeleton profoundly modulates cellular morphology, mechanics, and function. Measuring this distance has been a key challenge in cell biology. Current methods for dissecting the nanoscale spacing either limit themselves to complex survey design using fixed samples or rely on diffraction-limited fluorescence imaging whose spatial resolution is insufficient to quantify distances on the nanoscale. Using dual-color super-resolution STED (stimulated-emission-depletion) microscopy, we here overcome this challenge and accurately measure the density distribution of the cortical actin cytoskeleton and the distance between the actin cortex and the membrane in live Jurkat T-cells. We found an asymmetric cortical actin density distribution with a mean width of 230 (+105/-125) nm. The spatial distances measured between the maximum density peaks of the cortex and the membrane were bi-modally distributed with mean values of 50  ±  15 nm and 120  ±  40 nm, respectively. Taken together with the finite width of the cortex, our results suggest that in some regions the cortical actin is closer than 10 nm to the membrane and a maximum of 20 nm in others.

Chirality of the cytoskeleton in the origins of cellular asymmetry

PubMed Central

2016-01-01

Self-assembly of two important components of the cytoskeleton of eukaryotic cells, actin microfilaments and microtubules (MTs) results in polar filaments of one chirality. As is true for bacterial flagella, in actin microfilaments, screw direction is important for assembly processes and motility. For MTs, polar orientation within the cell is paramount. The alignment of these elements in the cell cytoplasm gives rise to emergent properties, including the potential for cell differentiation and specialization. Complex MTs with a characteristic chirality are found in basal bodies and centrioles; this chirality is preserved in cilia. In motile cilia, it is reflected in the direction of the effective stroke. The positioning of the basal body or cilia on the cell surface depends on polarity proteins. In evolution, survival depends on global polarity information relayed to the cell in part by orientation of the MT and actin filament cytoskeletons and the chirality of the basal body to determine left and right coordinates within a defined anterior–posterior cell and tissue axis. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’. PMID:27821520

Nuclear positioning by actin cables and perinuclear actin: Special and general?

PubMed

Huelsmann, Sven; Brown, Nicholas H

2014-01-01

Nuclear positioning is an important process during development and homeostasis. Depending on the affected tissue, mislocalized nuclei can alter cellular processes such as polarization, differentiation, or migration and lead ultimately to diseases. Many cells actively control the position of their nucleus using their cytoskeleton and motor proteins. We have recently shown that during Drosophila oogenesis, nurse cells employ cytoplasmic actin cables in association with perinuclear actin to position their nucleus. Here, we briefly summarize our work and discuss why nuclear positioning in nurse cells is specialized but the molecular mechanisms are likely to be more generally used.

Initial stem cell adhesion on porous silicon surface: molecular architecture of actin cytoskeleton and filopodial growth

PubMed Central

2014-01-01

The way cells explore their surrounding extracellular matrix (ECM) during development and migration is mediated by lamellipodia at their leading edge, acting as an actual motor pulling the cell forward. Lamellipodia are the primary area within the cell of actin microfilaments (filopodia) formation. In this work, we report on the use of porous silicon (pSi) scaffolds to mimic the ECM of mesenchymal stem cells from the dental pulp (DPSC) and breast cancer (MCF-7) cells. Our atomic force microscopy (AFM), fluorescence microscopy, and scanning electron microscopy (SEM) results show that pSi promoted the appearance of lateral filopodia protruding from the DPSC cell body and not only in the lamellipodia area. The formation of elongated lateral actin filaments suggests that pores provided the necessary anchorage points for protrusion growth. Although MCF-7 cells displayed a lower presence of organized actin network on both pSi and nonporous silicon, pSi stimulated the formation of extended cell protrusions. PMID:25386101

Adenomatous polyposis coli nucleates actin assembly to drive cell migration and microtubule-induced focal adhesion turnover

PubMed Central

Eskin, Julian A.; Jaiswal, Richa

2017-01-01

Cell motility depends on tight coordination between the microtubule (MT) and actin cytoskeletons, but the mechanisms underlying this MT–actin cross talk have remained poorly understood. Here, we show that the tumor suppressor protein adenomatous polyposis coli (APC), which is a known MT-associated protein, directly nucleates actin assembly to promote directed cell migration. By changing only two residues in APC, we generated a separation-of-function mutant, APC (m4), that abolishes actin nucleation activity without affecting MT interactions. Expression of full-length APC carrying the m4 mutation (APC (m4)) rescued cellular defects in MT organization, MT dynamics, and mitochondrial distribution caused by depletion of endogenous APC but failed to restore cell migration. Wild-type APC and APC (m4) localized to focal adhesions (FAs), and APC (m4) was defective in promoting actin assembly at FAs to facilitate MT-induced FA turnover. These results provide the first direct evidence for APC-mediated actin assembly in vivo and establish a role for APC in coordinating MTs and actin at FAs to direct cell migration. PMID:28663347

Tubulin and Actin Interplay at the T Cell and Antigen-Presenting Cell Interface

PubMed Central

Martín-Cófreces, Noa Beatriz; Alarcón, Balbino; Sánchez-Madrid, Francisco

2011-01-01

T cells reorganize their actin and tubulin-based cytoskeletons to provide a physical basis to the immune synapse. However, growing evidence shows that their roles on T cell activation are more dynamic than merely serving as tracks or scaffold for different molecules. The crosstalk between both skeletons may be important for the formation and movement of the lamella at the immunological synapse by increasing the adhesion of the T cell to the antigen-presenting cells (APC), thus favoring the transport of components toward the plasma membrane and in turn regulating the T-APC intercellular communication. Microtubules and F-actin appear to be essential for the transport of the different signaling microclusters along the membrane, therefore facilitating the propagation of the signal. Finally, they can also be important for regulating the endocytosis, recycling, and degradation of the T cell receptor signaling machinery, thus helping both to sustain the activated state and to switch it off. PMID:22566814

Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases

PubMed Central

1996-01-01

GPI-anchored surface proteins mediate many important functions, including transport, signal transduction, adhesion, and protection against complement. They cluster into glycolipid-based membrane domains and caveolae, plasmalemmal vesicles involved in the transcytosis and endocytosis of these surface proteins. However, in lymphocytes, neither the characteristic flask shaped caveolae nor caveolin, a transmembrane protein typical of caveolae, have been observed. Here, we show that the GPI-anchored CD59 molecule on Jurkat T cells is internalized after cross-linking, a process inhibited by nystatin, a sterol chelating agent. Clustered CD59 molecules mostly accumulate in non-coated invaginations of the lymphocyte membrane before endocytosis, in marked contrast with the pattern of CD3-TCR internalization. Cytochalasin H blocked CD59 internalization in lymphocytes, but neither CD3 internalization nor transferrin uptake. Confocal microscopy analysis of F-actin distribution within lymphocytes showed that CD59 clusters were associated with patches of polymerized actin. Also, we found that internalization of CD59 was prevented by the protein kinase C inhibitor staurosporine and by the protein kinase A activator forskolin. Thus, in lymphocytes, as in other cell types, glycolipid-based domains provide sites of integration of signaling pathways involved in GPI-anchored protein endocytosis. This process, which is regulated by both protein kinase C and A activity, is tightly controlled by the dynamic organization of actin cytoskeleton, and may be critical for polarized contacts of circulating cells. PMID:8666664

An antifungal protein from Ginkgo biloba binds actin and can trigger cell death.

PubMed

Gao, Ningning; Wadhwani, Parvesh; Mühlhäuser, Philipp; Liu, Qiong; Riemann, Michael; Ulrich, Anne S; Nick, Peter

2016-07-01

Ginkbilobin is a short antifungal protein that had been purified and cloned from the seeds of the living fossil Ginkgo biloba. Homologues of this protein can be detected in all seed plants and the heterosporic fern Selaginella and are conserved with respect to domain structures, peptide motifs, and specific cysteine signatures. To get insight into the cellular functions of these conserved motifs, we expressed green fluorescent protein fusions of full-length and truncated ginkbilobin in tobacco BY-2 cells. We show that the signal peptide confers efficient secretion of ginkbilobin. When this signal peptide is either cleaved or masked, ginkbilobin binds and visualizes the actin cytoskeleton. This actin-binding activity of ginkbilobin is mediated by a specific subdomain just downstream of the signal peptide, and this subdomain can also coassemble with actin in vitro. Upon stable overexpression of this domain, we observe a specific delay in premitotic nuclear positioning indicative of a reduced dynamicity of actin. To elucidate the cellular response to the binding of this subdomain to actin, we use chemical engineering based on synthetic peptides comprising different parts of the actin-binding subdomain conjugated with the cell-penetrating peptide BP100 and with rhodamine B as a fluorescent reporter. Binding of this synthetic construct to actin efficiently induces programmed cell death. We discuss these findings in terms of a working model, where ginkbilobin can activate actin-dependent cell death.

Sensor potency of the moonlighting enzyme-decorated cytoskeleton: the cytoskeleton as a metabolic sensor

PubMed Central

2013-01-01

Background There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear. Presentation of the hypothesis In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels. Testing the hypothesis Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding. Implications of the hypothesis The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined. PMID:23398642

Impact of Simulated Microgravity on Cytoskeleton and Viscoelastic Properties of Endothelial Cell

NASA Astrophysics Data System (ADS)

Janmaleki, M.; Pachenari, M.; Seyedpour, S. M.; Shahghadami, R.; Sanati-Nezhad, A.

2016-09-01

This study focused on the effects of simulated microgravity (s-μg) on mechanical properties, major cytoskeleton biopolymers, and morphology of endothelial cells (ECs). The structural and functional integrity of ECs are vital to regulate vascular homeostasis and prevent atherosclerosis. Furthermore, these highly gravity sensitive cells play a key role in pathogenesis of many diseases. In this research, impacts of s-μg on mechanical behavior of human umbilical vein endothelial cells were investigated by utilizing a three-dimensional random positioning machine (3D-RPM). Results revealed a considerable drop in cell stiffness and viscosity after 24 hrs of being subjected to weightlessness. Cortical rigidity experienced relatively immediate and significant decline comparing to the stiffness of whole cell body. The cells became rounded in morphology while western blot analysis showed reduction of the main cytoskeletal components. Moreover, fluorescence staining confirmed disorganization of both actin filaments and microtubules (MTs). The results were compared statistically among test and control groups and it was concluded that s-μg led to a significant alteration in mechanical behavior of ECs due to remodeling of cell cytoskeleton.

Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina

PubMed Central

Lahne, Manuela; Li, Jingling; Marton, Rebecca M.

2015-01-01

that regulate retinal regeneration in these organisms will help to elucidate approaches to stimulate a similar response in humans. In the damaged zebrafish retina, Müller glia dedifferentiate and proliferate to generate neuronal progenitor cells (NPCs) that differentiate into the lost neurons. We show that the nuclei of Müller glia and NPCs migrate apically and basally in phase with the cell cycle. This migration is facilitated by the actin cytoskeleton and Rho-associated coiled-coil kinases (Rocks). We demonstrate that Rock function is required for sufficient proliferation and the regeneration of photoreceptors, likely via regulating nuclear migration. PMID:26609156

Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina.

PubMed

Lahne, Manuela; Li, Jingling; Marton, Rebecca M; Hyde, David R

2015-11-25

regeneration in these organisms will help to elucidate approaches to stimulate a similar response in humans. In the damaged zebrafish retina, Müller glia dedifferentiate and proliferate to generate neuronal progenitor cells (NPCs) that differentiate into the lost neurons. We show that the nuclei of Müller glia and NPCs migrate apically and basally in phase with the cell cycle. This migration is facilitated by the actin cytoskeleton and Rho-associated coiled-coil kinases (Rocks). We demonstrate that Rock function is required for sufficient proliferation and the regeneration of photoreceptors, likely via regulating nuclear migration. Copyright © 2015 the authors 0270-6474/15/3515612-23$15.00/0.

Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions

PubMed Central

Baranwal, Somesh

2015-01-01

Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis. PMID:25792565

Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions.

PubMed

Lechuga, Susana; Baranwal, Somesh; Ivanov, Andrei I

2015-05-01

Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis. Copyright © 2015 the American Physiological Society.

Actin filaments-A target for redox regulation.

PubMed

Wilson, Carlos; Terman, Jonathan R; González-Billault, Christian; Ahmed, Giasuddin

2016-10-01

Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through noncovalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates-the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL-and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The Stationary-Phase Cells of Saccharomyces cerevisiae Display Dynamic Actin Filaments Required for Processes Extending Chronological Life Span.

PubMed

Vasicova, Pavla; Lejskova, Renata; Malcova, Ivana; Hasek, Jiri

2015-11-01

Stationary-growth-phase Saccharomyces cerevisiae yeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. In S. cerevisiae stationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Our in vivo analyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficient cox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Stationary-Phase Cells of Saccharomyces cerevisiae Display Dynamic Actin Filaments Required for Processes Extending Chronological Life Span

PubMed Central

Lejskova, Renata; Malcova, Ivana

2015-01-01

Stationary-growth-phase Saccharomyces cerevisiae yeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. In S. cerevisiae stationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Our in vivo analyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficient cox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth. PMID:26351139

Interaction between two adapter proteins, PAG and EBP50: a possible link between membrane rafts and actin cytoskeleton.

PubMed

Brdicková, N; Brdicka, T; Andera, L; Spicka, J; Angelisová, P; Milgram, S L; Horejsí, V

2001-10-26

Phosphoprotein associated with GEMs (PAG), also known as Csk-binding protein (Cbp), is a broadly expressed palmitoylated transmembrane adapter protein found in membrane rafts, also called GEMs (glycosphingolipid-enriched membrane microdomains). PAG is known to bind and activate the essential regulator of Src-family kinases, cytoplasmic protein tyrosine kinase Csk. In the present study we used the yeast 2-hybrid system to search for additional proteins which might bind to PAG. We have identified the abundant cytoplasmic adapter protein EBP50 (ezrin/radixin/moesin (ERM)-binding phosphoprotein of 50 kDa), also known as NHERF (Na(+)/H(+) exchanger regulatory factor), as a specific PAG-binding partner. The interaction involves the C-terminal sequence (TRL) of PAG and N-terminal PDZ domain(s) of EBP50. As EBP50 is known to interact via its C-terminal domain with the ERM-family proteins, which in turn bind to actin cytoskeleton, the PAG-EBP50 interaction may be important for connecting membrane rafts to the actin cytoskeleton.

Resinless section electron microscopy reveals the yeast cytoskeleton.

PubMed

Penman, J; Penman, S

1997-04-15

The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective detergent extraction. In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow detergent extraction of the plasma membrane lipids. Gel electropherograms show that the extracted or "soluble" proteins are distinct from the retained or "structural" proteins that presumably comprise the cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters-5 nm and 15-20 nm-which may correspond to actin and intermediate filaments, respectively. A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300-500 nm in diameter, is likely the nucleus. The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture.

Actin retrograde flow controls natural killer cell response by regulating the conformation state of SHP-1.

PubMed

Matalon, Omri; Ben-Shmuel, Aviad; Kivelevitz, Jessica; Sabag, Batel; Fried, Sophia; Joseph, Noah; Noy, Elad; Biber, Guy; Barda-Saad, Mira

2018-03-01

Natural killer (NK) cells are a powerful weapon against viral infections and tumor growth. Although the actin-myosin (actomyosin) cytoskeleton is crucial for a variety of cellular processes, the role of mechanotransduction, the conversion of actomyosin mechanical forces into signaling cascades, was never explored in NK cells. Here, we demonstrate that actomyosin retrograde flow (ARF) controls the immune response of primary human NK cells through a novel interaction between β-actin and the SH2-domain-containing protein tyrosine phosphatase-1 (SHP-1), converting its conformation state, and thereby regulating NK cell cytotoxicity. Our results identify ARF as a master regulator of the NK cell immune response. Since actin dynamics occur in multiple cellular processes, this mechanism might also regulate the activity of SHP-1 in additional cellular systems. © 2018 The Authors.

Hyperosmotically induced volume change and calcium signaling in intervertebral disk cells: the role of the actin cytoskeleton.

PubMed

Pritchard, Scott; Erickson, Geoffrey R; Guilak, Farshid

2002-11-01

Loading of the spine alters the osmotic environment in the intervertebral disk (IVD) as interstitial water is expressed from the tissue. Cells from the three zones of the IVD, the anulus fibrosus (AF), transition zone (TZ), and nucleus pulposus (NP), respond to osmotic stress with altered biosynthesis through a pathway that may involve calcium (Ca(2+)) as a second messenger. We examined the hypothesis that IVD cells respond to hyperosmotic stress by increasing the concentration of intracellular calcium ([Ca(2+)](i)) through a mechanism involving F-actin. In response to hyperosmotic stress, control cells from all zones decreased in volume and cells from the AF and TZ exhibited [Ca(2+)](i) transients, while cells from the NP did not. Extracellular Ca(2+) was necessary to initiate [Ca(2+)](i) transients. Stabilization of F-actin with phalloidin prevented the Ca(2+) response in AF and TZ cells and decreased the rate of volume change in cells from all zones, coupled with an increase in the elastic moduli and apparent viscosity. Conversely, actin breakdown with cytochalasin D facilitated Ca(2+) signaling while decreasing the elastic moduli and apparent viscosity for NP cells. These results suggest that hyperosmotic stress induces volume change in IVD cells and may initiate [Ca(2+)](i) transients through an actin-dependent mechanism.

F-actin distribution at nodes of Ranvier and Schmidt-Lanterman incisures in mammalian sciatic nerves.

PubMed

Kun, Alejandra; Canclini, Lucía; Rosso, Gonzalo; Bresque, Mariana; Romeo, Carlos; Hanusz, Alicia; Cal, Karina; Calliari, Aldo; Sotelo Silveira, José; Sotelo, José R

2012-07-01

Very little is known about the function of the F-actin cytoskeleton in the regeneration and pathology of peripheral nerve fibers. The actin cytoskeleton has been associated with maintenance of tissue structure, transmission of traction and contraction forces, and an involvement in cell motility. Therefore, the state of the actin cytoskeleton strongly influences the mechanical properties of cells and intracellular transport therein. In this work, we analyze the distribution of F-actin at Schmidt-Lanterman Incisures (SLI) and nodes of Ranvier (NR) domains in normal, regenerating and pathologic Trembler J (TrJ/+) sciatic nerve fibers, of rats and mice. F-actin was quantified and it was found increased in TrJ/+, both in SLI and NR. However, SLI and NR of regenerating rat sciatic nerve did not show significant differences in F-actin, as compared with normal nerves. Cytochalasin-D and Latrunculin-A were used to disrupt the F-actin network in normal and regenerating rat sciatic nerve fibers. Both drugs disrupt F-actin, but in different ways. Cytochalasin-D did not disrupt Schwann cell (SC) F-actin at the NR. Latrunculin-A did not disrupt F-actin at the boundary region between SC and axon at the NR domain. We surmise that the rearrangement of F-actin in neurological disorders, as presented here, is an important feature of TrJ/+ pathology as a Charcot-Marie-Tooth (CMT) model. Copyright © 2012 Wiley Periodicals, Inc.

Mip1 associates with both the Mps1 kinase and actin and is required for cell cortex stability and anaphase spindle positioning

USDA-ARS?s Scientific Manuscript database

The Mps1 family of protein kinases contributes to cell cycle control by regulating multiple microtubule cytoskeleton activities. We have uncovered a new Mps1 substrate that provides a novel link between Mps1 and the actin cytoskeleton. We have identified a conserved human Mps1 (hMps1) interacting pr...

The functions of the cytoskeleton and associated proteins during mitosis and cytokinesis in plant cells

PubMed Central

Li, Shanwei; Sun, Tiantian; Ren, Haiyun

2015-01-01

In higher plants, microtubule (MT)-based, and actin filament (AF)-based structures play important roles in mitosis and cytokinesis. Besides the mitotic spindle, the evolution of a band comprising cortical MTs and AFs, namely, the preprophase band (PPB), is evident in plant cells. This band forecasts a specific division plane before the initiation of mitosis. During cytokinesis, another plant-specific cytoskeletal structure called the phragmoplast guides vesicles in the creation of a new cell wall. In addition, a number of cytoskeleton-associated proteins are reportedly involved in the formation and function of the PPB, mitotic spindle, and phragmoplast. This review summarizes current knowledge on the cytoskeleton-associated proteins that mediate the cytoskeletal arrays during mitosis and cytokinesis in plant cells and discusses the interaction between MTs and AFs involved in mitosis and cytokinesis. PMID:25964792

[INFLUENCE OF INHIBITION OF ACTIN POLYMERIZATION ON ADIPOGENIC DIFFERENTIATION OF RAT Achilles-DERIVED TENDON STEM CELLS IN VITRO].

PubMed

Chen, Bo; Tang, Kanglai; Zhang, Jiqiang; Guo, Yupeng; Liu, Xiangzhou; Shi, Youxin

2015-02-01

To investigate the effect of cytoskeleton modification on the adipogenic differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. TSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. After the 3rd passage cells were cultured with DMEM medium containing 15% fetal bovine serum and cytochalasin D (CYD) at the concentrations of 0, 50, 100, 500, and 1 000 ng/mL, the cell survival condition and morphology changes were observed by inverted phase contrast microscope, the cytoskeleton was observed through fibrous actin (F-actin) staining, and the ratio of F-actin/ soluble globular actin (G-actin) was detected and calculated through Western blot. According to the above results, the effective concentration of CYD was selected and used for next experiments. After TSCs were cultured for 3 and 7 days respectively with adipogenic induction media (induction group), adipogenic induction media containing CYD (CYD+induction group), ordinary medium (ordinary group), and ordinary medium containing CYD (CYD+ordinary group), the real-time quantitative PCR (qRT-PCR) and Western blot were carried out to measure the mRNA and protein expressions of adipogenic differentiation-related markers, including peroxisome proliferator-activated receptor y (PPARγ), lipoprotein lipase (LPL), and fatty acid binding protein (aP2). The final CYD concentration of 100 ng/mL can inhibit effectively G-actin polymerization into F-actin, but could not affect TSCs survival, which was used for next experiments. qRT-PCR and Western blot suggested that the mRNA expressions of PPARγ, LPL, and aP2 and the protein expressions of PPARγ and aP2 were increased significantly in the CYD+induction group at 3 and 7 days when compared with the induction group (P < 0.05). In the CYD+ordinary group, there still was a significant increase in the mRNA expressions of PPARγ, LPL, and aP2 when compared with the ordinary

Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances

PubMed Central

Schudt, Gordian; Kolesnikova, Larissa; Dolnik, Olga; Sodeik, Beate; Becker, Stephan

2013-01-01

Transport of large viral nucleocapsids from replication centers to assembly sites requires contributions from the host cytoskeleton via cellular adaptor and motor proteins. For the Marburg and Ebola viruses, related viruses that cause severe hemorrhagic fevers, the mechanism of nucleocapsid transport remains poorly understood. Here we developed and used live-cell imaging of fluorescently labeled viral and host proteins to characterize the dynamics and molecular requirements of nucleocapsid transport in Marburg virus-infected cells under biosafety level 4 conditions. The study showed a complex actin-based transport of nucleocapsids over long distances from the viral replication centers to the budding sites. Only after the nucleocapsids had associated with the matrix viral protein VP40 at the plasma membrane were they recruited into filopodia and cotransported with host motor myosin 10 toward the budding sites at the tip or side of the long cellular protrusions. Three different transport modes and velocities were identified: (i) Along actin filaments in the cytosol, nucleocapsids were transported at ∼200 nm/s; (ii) nucleocapsids migrated from one actin filament to another at ∼400 nm/s; and (iii) VP40-associated nucleocapsids moved inside filopodia at 100 nm/s. Unique insights into the spatiotemporal dynamics of nucleocapsids and their interaction with the cytoskeleton and motor proteins can lead to novel classes of antivirals that interfere with the trafficking and subsequent release of the Marburg virus from infected cells. PMID:23940347

Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances.

PubMed

Schudt, Gordian; Kolesnikova, Larissa; Dolnik, Olga; Sodeik, Beate; Becker, Stephan

2013-08-27

Transport of large viral nucleocapsids from replication centers to assembly sites requires contributions from the host cytoskeleton via cellular adaptor and motor proteins. For the Marburg and Ebola viruses, related viruses that cause severe hemorrhagic fevers, the mechanism of nucleocapsid transport remains poorly understood. Here we developed and used live-cell imaging of fluorescently labeled viral and host proteins to characterize the dynamics and molecular requirements of nucleocapsid transport in Marburg virus-infected cells under biosafety level 4 conditions. The study showed a complex actin-based transport of nucleocapsids over long distances from the viral replication centers to the budding sites. Only after the nucleocapsids had associated with the matrix viral protein VP40 at the plasma membrane were they recruited into filopodia and cotransported with host motor myosin 10 toward the budding sites at the tip or side of the long cellular protrusions. Three different transport modes and velocities were identified: (i) Along actin filaments in the cytosol, nucleocapsids were transported at ∼200 nm/s; (ii) nucleocapsids migrated from one actin filament to another at ∼400 nm/s; and (iii) VP40-associated nucleocapsids moved inside filopodia at 100 nm/s. Unique insights into the spatiotemporal dynamics of nucleocapsids and their interaction with the cytoskeleton and motor proteins can lead to novel classes of antivirals that interfere with the trafficking and subsequent release of the Marburg virus from infected cells.

The Nance-Horan syndrome protein encodes a functional WAVE homology domain (WHD) and is important for co-ordinating actin remodelling and maintaining cell morphology.

PubMed

Brooks, Simon P; Coccia, Margherita; Tang, Hao R; Kanuga, Naheed; Machesky, Laura M; Bailly, Maryse; Cheetham, Michael E; Hardcastle, Alison J

2010-06-15

Nance-Horan syndrome (NHS) is an X-linked developmental disorder, characterized by bilateral congenital cataracts, dental anomalies, facial dysmorphism and mental retardation. Null mutations in a novel gene, NHS, cause the syndrome. The NHS gene appears to have multiple isoforms as a result of alternative transcription, but a cellular function for the NHS protein has yet to be defined. We describe NHS as a founder member of a new protein family (NHS, NHSL1 and NHSL2). Here, we demonstrate that NHS is a novel regulator of actin remodelling and cell morphology. NHS localizes to sites of cell-cell contact, the leading edge of lamellipodia and focal adhesions. The N-terminus of isoforms NHS-A and NHS-1A, implicated in the pathogenesis of NHS, have a functional WAVE homology domain that interacts with the Abi protein family, haematopoietic stem/progenitor cell protein 300 (HSPC300), Nap1 and Sra1. NHS knockdown resulted in the disruption of the actin cytoskeleton. We show that NHS controls cell morphology by maintaining the integrity of the circumferential actin ring and controlling lamellipod formation. NHS knockdown led to a striking increase in cell spreading. Conversely, ectopic overexpression of NHS inhibited lamellipod formation. Remodelling of the actin cytoskeleton and localized actin polymerization into branched actin filaments at the plasma membrane are essential for mediating changes in cell shape, migration and cell contact. Our data identify NHS as a new regulator of actin remodelling. We suggest that NHS orchestrates actin regulatory protein function in response to signalling events during development.

Actin filaments – a target for redox regulation

PubMed Central

Wilson, Carlos; Terman, Jonathan R.; González-Billault, Christian; Ahmed, Giasuddin

2016-01-01

Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through non-covalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates – the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL – and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. PMID:27309342

The Nance–Horan syndrome protein encodes a functional WAVE homology domain (WHD) and is important for co-ordinating actin remodelling and maintaining cell morphology

PubMed Central

Brooks, Simon P.; Coccia, Margherita; Tang, Hao R.; Kanuga, Naheed; Machesky, Laura M.; Bailly, Maryse; Cheetham, Michael E.; Hardcastle, Alison J.

2010-01-01

Nance–Horan syndrome (NHS) is an X-linked developmental disorder, characterized by bilateral congenital cataracts, dental anomalies, facial dysmorphism and mental retardation. Null mutations in a novel gene, NHS, cause the syndrome. The NHS gene appears to have multiple isoforms as a result of alternative transcription, but a cellular function for the NHS protein has yet to be defined. We describe NHS as a founder member of a new protein family (NHS, NHSL1 and NHSL2). Here, we demonstrate that NHS is a novel regulator of actin remodelling and cell morphology. NHS localizes to sites of cell–cell contact, the leading edge of lamellipodia and focal adhesions. The N-terminus of isoforms NHS-A and NHS-1A, implicated in the pathogenesis of NHS, have a functional WAVE homology domain that interacts with the Abi protein family, haematopoietic stem/progenitor cell protein 300 (HSPC300), Nap1 and Sra1. NHS knockdown resulted in the disruption of the actin cytoskeleton. We show that NHS controls cell morphology by maintaining the integrity of the circumferential actin ring and controlling lamellipod formation. NHS knockdown led to a striking increase in cell spreading. Conversely, ectopic overexpression of NHS inhibited lamellipod formation. Remodelling of the actin cytoskeleton and localized actin polymerization into branched actin filaments at the plasma membrane are essential for mediating changes in cell shape, migration and cell contact. Our data identify NHS as a new regulator of actin remodelling. We suggest that NHS orchestrates actin regulatory protein function in response to signalling events during development. PMID:20332100

Gβ Regulates Coupling between Actin Oscillators for Cell Polarity and Directional Migration

PubMed Central

Cai, Huaqing; Sun, Yaohui; Huang, Chuan-Hsiang; Freyre, Mariel; Zhao, Min; Devreotes, Peter N.; Weiner, Orion D.

2016-01-01

For directional movement, eukaryotic cells depend on the proper organization of their actin cytoskeleton. This engine of motility is made up of highly dynamic nonequilibrium actin structures such as flashes, oscillations, and traveling waves. In Dictyostelium, oscillatory actin foci interact with signals such as Ras and phosphatidylinositol 3,4,5-trisphosphate (PIP3) to form protrusions. However, how signaling cues tame actin dynamics to produce a pseudopod and guide cellular motility is a critical open question in eukaryotic chemotaxis. Here, we demonstrate that the strength of coupling between individual actin oscillators controls cell polarization and directional movement. We implement an inducible sequestration system to inactivate the heterotrimeric G protein subunit Gβ and find that this acute perturbation triggers persistent, high-amplitude cortical oscillations of F-actin. Actin oscillators that are normally weakly coupled to one another in wild-type cells become strongly synchronized following acute inactivation of Gβ. This global coupling impairs sensing of internal cues during spontaneous polarization and sensing of external cues during directional motility. A simple mathematical model of coupled actin oscillators reveals the importance of appropriate coupling strength for chemotaxis: moderate coupling can increase sensitivity to noisy inputs. Taken together, our data suggest that Gβ regulates the strength of coupling between actin oscillators for efficient polarity and directional migration. As these observations are only possible following acute inhibition of Gβ and are masked by slow compensation in genetic knockouts, our work also shows that acute loss-of-function approaches can complement and extend the reach of classical genetics in Dictyostelium and likely other systems as well. PMID:26890004

Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.

PubMed

Lechuga, Susana; Baranwal, Somesh; Li, Chao; Naydenov, Nayden G; Kuemmerle, John F; Dugina, Vera; Chaponnier, Christine; Ivanov, Andrei I

2014-10-15

Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis. © 2014 Lechuga, Baranwal, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Bacterial cytoskeleton and implications for new antibiotic targets.

PubMed

Wang, Huan; Xie, Longxiang; Luo, Hongping; Xie, Jianping

2016-01-01

Traditionally eukaryotes exclusive cytoskeleton has been found in bacteria and other prokaryotes. FtsZ, MreB and CreS are bacterial counterpart of eukaryotic tubulin, actin filaments and intermediate filaments, respectively. FtsZ can assemble to a Z-ring at the cell division site, regulate bacterial cell division; MreB can form helical structure, and involve in maintaining cell shape, regulating chromosome segregation; CreS, found in Caulobacter crescentus (C. crescentus), can form curve or helical filaments in intracellular membrane. CreS is crucial for cell morphology maintenance. There are also some prokaryotic unique cytoskeleton components playing crucial roles in cell division, chromosome segregation and cell morphology. The cytoskeleton components of Mycobacterium tuberculosis (M. tuberculosis), together with their dynamics during exposure to antibiotics are summarized in this article to provide insights into the unique organization of this formidable pathogen and druggable targets for new antibiotics.

Microscale force response and morphology of tunable co-polymerized cytoskeleton networks

NASA Astrophysics Data System (ADS)

Ricketts, Shea; Yadav, Vikrant; Ross, Jennifer L.; Robertson-Anderson, Rae M.

The cytoskeleton is largely comprised of actin and microtubules that entangle and crosslink to form complex networks and structures, giving rise to nonlinear multifunctional mechanics in cells. The relative concentrations of semiflexible actin filaments and rigid microtubules tune cytoskeleton function, allowing cells to move and divide while maintaining rigidity and resilience. To elucidate this complex tunability, we create in vitro composites of co-polymerized actin and microtubules with actin:microtubule molar ratios of 0:1-1:0. We use optical tweezers and confocal microscopy to characterize the nonlinear microscale force response and morphology of the composites. We optically drag a microsphere 30 μm through varying actin-microtubule networks at 10 μm/s and 20 μm/s, and measure the force the networks exerts to resist the strain and the force relaxation following strain. We use dual-color confocal microscopy to image distinctly-labeled filaments in the networks, and characterize the integration of actin and microtubules, network connectivity, and filament rigidity. We find that increasing the fraction of microtubules in networks non-monotonically increases elasticity and stiffness, and hinders force relaxation by suppressing network mobility and fluctuations. NSF CAREER Award (DMR-1255446), Scialog Collaborative Innovation Award funded by Research Corporation for Scientific Advancement (Grant No. 24192).

Visualization of highly dynamic F-actin plus ends in growing phaseolus vulgaris root hair cells and their responses to Rhizobium etli nod factors.

PubMed

Zepeda, Isaac; Sánchez-López, Rosana; Kunkel, Joseph G; Bañuelos, Luis A; Hernández-Barrera, Alejandra; Sánchez, Federico; Quinto, Carmen; Cárdenas, Luis

2014-03-01

Legume plants secrete signaling molecules called flavonoids into the rhizosphere. These molecules activate the transcription of rhizobial nod genes, which encode proteins involved in the synthesis of signaling compounds named Nod factors (NFs). NFs, in turn, trigger changes in plant gene expression, cortical cell dedifferentiation and mitosis, depolarization of the root hair cell membrane potential and rearrangement of the actin cytoskeleton. Actin polymerization plays an important role in apical growth in hyphae and pollen tubes. Using sublethal concentrations of fluorescently labeled cytochalasin D (Cyt-Fl), we visualized the distribution of filamentous actin (F-actin) plus ends in living Phaseolus vulgaris and Arabidopsis root hairs during apical growth. We demonstrated that Cyt-Fl specifically labeled the newly available plus ends of actin microfilaments, which probably represent sites of polymerization. The addition of unlabeled competing cytochalasin reduced the signal, suggesting that the labeled and unlabeled forms of the drug bind to the same site on F-actin. Exposure to Rhizobium etli NFs resulted in a rapid increase in the number of F-actin plus ends in P. vulgaris root hairs and in the re-localization of F-actin plus ends to infection thread initiation sites. These data suggest that NFs promote the formation of F-actin plus ends, which results in actin cytoskeleton rearrangements that facilitate infection thread formation.

Prestressed F-actin networks cross-linked by hinged filamins replicate mechanical properties of cells

NASA Astrophysics Data System (ADS)

Gardel, M. L.; Nakamura, F.; Hartwig, J. H.; Crocker, J. C.; Stossel, T. P.; Weitz, D. A.

2006-02-01

We show that actin filaments, shortened to physiological lengths by gelsolin and cross-linked with recombinant human filamins (FLNs), exhibit dynamic elastic properties similar to those reported for live cells. To achieve elasticity values of comparable magnitude to those of cells, the in vitro network must be subjected to external prestress, which directly controls network elasticity. A molecular requirement for the strain-related behavior at physiological conditionsis a flexible hinge found in FLNa and some FLNb molecules. Basic physical properties of the in vitro filamin-F-actin network replicate the essential mechanical properties of living cells. This physical behavior could accommodate passive deformation and internal organelle trafficking at low strains yet resist externally or internally generated high shear forces. cytoskeleton | cell mechanics | nonlinear rheology

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells

PubMed Central

Hu, Lifang; Su, Peihong; Li, Runzhi; Yan, Kun; Chen, Zhihao; Shang, Peng; Qian, Airong

2015-01-01

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function. [BMB Reports 2015; 48(10): 583-588] PMID:26277981

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells.

PubMed

Hu, Lifang; Su, Peihong; Li, Runzhi; Yan, Kun; Chen, Zhihao; Shang, Peng; Qian, Airong

2015-10-01

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function.

Ostreocin-D impact on globular actin of intact cells.

PubMed

Ares, Isabel R; Cagide, Eva; Louzao, M Carmen; Espiña, Begoña; Vieytes, Mercedes R; Yasumoto, Takeshi; Botana, Luis M

2009-02-01

Ostreocin-D, discovered in the past decade, is a marine toxin produced by dinoflagellates. It shares structure with palytoxin, a toxic compound responsible for the seafood intoxication named clupeotoxism. At the cellular level, the action sites and pharmacological effects for ostreocin-D are still almost unknown. Previously, we demonstrated that these toxins change the filamentous actin cytoskeleton, which is essential for multiple cellular functions. However, nothing has yet been reported about what happens with the unpolymerized actin pool. Here (i) the effects induced by ostreocin-D on unpolymerized actin, (ii) the Ca2+ role in such a process, and (iii) the cytotoxic activity of ostreocin-D on the human neuroblastoma BE(2)-M17 cell line are shown for the first time. Fluorescently labeled DNase I was used for staining of monomeric actin prior to detection with both laser-scanning cytometry and confocal microscopy techniques. Cellular viability was tested through a microplate metabolic activity assay. Ostreocin-D elicited a rearrangement of monomeric actin toward the nuclear region. This event was not accompanied by changes in its content. In addition, the presence or absence of external Ca2+ did not change these results. This toxin was also found to cause a decrease in the viability of neuroblastoma cells, which was inhibited by the specific blocker of Na+/K+-ATPase, ouabain. All these responses were comparable to those obtained with palytoxin under identical conditions. The data suggest that ostreocin-D modulates the unassembled actin pool, activating signal transduction pathways not related to Ca2+ influx in the same way as palytoxin.

Boolean gates on actin filaments

NASA Astrophysics Data System (ADS)

Siccardi, Stefano; Tuszynski, Jack A.; Adamatzky, Andrew

2016-01-01

Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications.

Resinless section electron microscopy reveals the yeast cytoskeleton

PubMed Central

Penman, Joshua; Penman, Sheldon

1997-01-01

The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective detergent extraction. In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow detergent extraction of the plasma membrane lipids. Gel electropherograms show that the extracted or “soluble” proteins are distinct from the retained or “structural” proteins that presumably comprise the cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters—5 nm and 15–20 nm—which may correspond to actin and intermediate filaments, respectively. A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300–500 nm in diameter, is likely the nucleus. The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture. PMID:9108046

Actin and microtubule networks contribute differently to cell response for small and large strains

NASA Astrophysics Data System (ADS)

Kubitschke, H.; Schnauss, J.; Nnetu, K. D.; Warmt, E.; Stange, R.; Kaes, J.

2017-09-01

Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (≤5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.

Contractile actin cables induced by Bacillus anthracis lethal toxin depend on the histone acetylation machinery.

PubMed

Rolando, Monica; Stefani, Caroline; Doye, Anne; Acosta, Maria I; Visvikis, Orane; Yevick, Hannah G; Buchrieser, Carmen; Mettouchi, Amel; Bassereau, Patricia; Lemichez, Emmanuel

2015-10-01

It remains a challenge to decode the molecular basis of the long-term actin cytoskeleton rearrangements that are governed by the reprogramming of gene expression. Bacillus anthracis lethal toxin (LT) inhibits mitogen-activated protein kinase (MAPK) signaling, thereby modulating gene expression, with major consequences for actin cytoskeleton organization and the loss of endothelial barrier function. Using a laser ablation approach, we characterized the contractile and tensile mechanical properties of LT-induced stress fibers. These actin cables resist pulling forces that are transmitted at cell-matrix interfaces and at cell-cell discontinuous adherens junctions. We report that treating the cells with trichostatin A (TSA), a broad range inhibitor of histone deacetylases (HDACs), or with MS-275, which targets HDAC1, 2 and 3, induces stress fibers. LT decreased the cellular levels of HDAC1, 2 and 3 and reduced the global HDAC activity in the nucleus. Both the LT and TSA treatments induced Rnd3 expression, which is required for the LT-mediated induction of actin stress fibers. Furthermore, we reveal that treating the LT-intoxicated cells with garcinol, an inhibitor of histone acetyl-transferases (HATs), disrupts the stress fibers and limits the monolayer barrier dysfunctions. These data demonstrate the importance of modulating the flux of protein acetylation in order to control actin cytoskeleton organization and the endothelial cell monolayer barrier. © 2015 Wiley Periodicals, Inc.

Are non-muscle actin isoforms functionally equivalent?

PubMed

Simiczyjew, Aleksandra; Pietraszek-Gremplewicz, Katarzyna; Mazur, Antonina Joanna; Nowak, Dorota

2017-11-01

Actin is highly conserved and it is the most widespread protein in eukaryotic cells. One of the most important features of actin, which allows it to have many different functions, is its ability to polymerize and interact with many other proteins. Actins are the major constituent of the actin cytoskeleton, which is an important system that is involved in various aspects of cell function, including cell motility, structure, integrity, regulation of signal transduction and transcription. Six mammal actin isoforms are highly conserved and share common functions. Two of them, β and γ non-muscle actin isoforms, which differ only by four amino acids located at the N-terminus of the polypeptide chain, are required for survival and proper cell functioning. We also summarized data about actbl2, which is suggested to be a newly discovered isoactin. Here, we review the current knowledge about tissue-specific expression of the non-muscle actin isoforms and possible functional differences between them. We also discuss molecular tools, which in recent years have allowed for a better understanding of the role of these proteins in cell functioning.

Nucleus-associated actin in Amoeba proteus.

PubMed

Berdieva, Mariia; Bogolyubov, Dmitry; Podlipaeva, Yuliya; Goodkov, Andrew

2016-10-01

The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms. Copyright © 2016 Elsevier GmbH. All rights reserved.

Bacterial Actins? An Evolutionary Perspective

NASA Technical Reports Server (NTRS)

Doolittle, Russell F.; York, Amanda L.

2003-01-01

According to the conventional wisdom, the existence of a cytoskeleton in eukaryotes and its absence in prokaryotes constitute a fundamental divide between the two domains of life. An integral part of the dogma is that a cytoskeleton enabled an early eukaryote to feed upon prokaryotes, a consequence of which was the occasional endosymbiosis and the eventual evolution of organelles. Two recent papers present compelling evidence that actin, one of the principal components of a cytoskeleton, has a homolog in Bacteria that behaves in many ways like eukaryotic actin. Sequence comparisons reveml that eukaryotic actin and the bacterial homolog (mreB protein), unlike many other proteins common to eukaryotes and Bacteria, have very different and more highly extended evolutionary histories.

Cell surface dynamics - how Rho GTPases orchestrate the interplay between the plasma membrane and the cortical cytoskeleton.

PubMed

de Curtis, Ivan; Meldolesi, Jacopo

2012-10-01

Small GTPases are known to regulate hundreds of cell functions. In particular, Rho family GTPases are master regulators of the cytoskeleton. By regulating actin nucleation complexes, Rho GTPases control changes in cell shape, including the extension and/or retraction of surface protrusions and invaginations. Protrusion and invagination of the plasma membrane also involves the interaction between the plasma membrane and the cortical cytoskeleton. This interplay between membranes and the cytoskeleton can lead to an increase or decrease in the plasma membrane surface area and its tension as a result of the fusion (exocytosis) or internalization (endocytosis) of membranous compartments, respectively. For a long time, the cytoskeleton and plasma membrane dynamics were investigated separately. However, studies from many laboratories have now revealed that Rho GTPases, their modulation of the cytoskeleton, and membrane traffic are closely connected during the dynamic remodeling of the cell surface. Arf- and Rab-dependent exocytosis of specific vesicles contributes to the targeting of Rho GTPases and their regulatory factors to discrete sites of the plasma membrane. Rho GTPases regulate the tethering of exocytic vesicles and modulate their subsequent fusion. They also have crucial roles in the different forms of endocytosis, where they participate in the sorting of membrane domains as well as the sculpting and sealing of membrane flasks and cups. Here, we discuss how cell surface dynamics depend on the orchestration of the cytoskeleton and the plasma membrane by Rho GTPases.

Effect of omega-3 polyunsaturated fatty acids on the cytoskeleton: an open-label intervention study.

PubMed

Schmidt, Simone; Willers, Janina; Riecker, Sabine; Möller, Katharina; Schuchardt, Jan Philipp; Hahn, Andreas

2015-02-14

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) show beneficial effects on cardiovascular health and cognitive functions, but the underlying molecular mechanisms are not completely understood. Because of the fact that cytoskeleton dynamics affect almost every cellular process, the regulation of cytoskeletal dynamics could be a new pathway by which n-3 PUFAs exert their effects on cellular level. A 12-week open-label intervention study with 12 healthy men was conducted to determine the effects of 2.7 g/d n-3 PUFA on changes in mRNA expression of cytoskeleton-associated genes by quantitative real-time PCR in whole blood. Furthermore, the actin content in red blood cells was analyzed by immunofluorescence imaging. N-3 PUFA supplementation resulted in a significant down-regulation of cytoskeleton-associated genes, in particular three GTPases (RAC1, RHOA, CDC42), three kinases (ROCK1, PAK2, LIMK), two Wiskott-Aldrich syndrome proteins (WASL, WASF2) as well as actin related protein 2/3 complex (ARPC2, ARPC3) and cofilin (CFL1). Variability in F-actin content between subjects was high; reduced actin content was only reduced within group evaluation. Reduced cytoskeleton-associated gene expression after n-3 PUFA supplementation suggests that regulation of cytoskeleton dynamics might be an additional way by which n-3 PUFAs exert their cellular effects. Concerning F-actin, this analysis did not reveal unmistakable results impeding a generalized conclusion.

Differential effects of LifeAct-GFP and actin-GFP on cell mechanics assessed using micropipette aspiration.

PubMed

Sliogeryte, Kristina; Thorpe, Stephen D; Wang, Zhao; Thompson, Clare L; Gavara, Nuria; Knight, Martin M

2016-01-25

The actin cytoskeleton forms a dynamic structure involved in many fundamental cellular processes including the control of cell morphology, migration and biomechanics. Recently LifeAct-GFP (green fluorescent protein) has been proposed for visualising actin structure and dynamics in live cells as an alternative to actin-GFP which has been shown to affect cell mechanics. Here we compare the two approaches in terms of their effect on cellular mechanical behaviour. Human mesenchymal stem cells (hMSCs) were analysed using micropipette aspiration and the effective cellular equilibrium and instantaneous moduli calculated using the standard linear solid model. We show that LifeAct-GFP provides clearer visualisation of F-actin organisation and dynamics. Furthermore, LifeAct-GFP does not alter effective cellular mechanical properties whereas actin-GFP expression causes an increase in the cell modulus. Interestingly, LifeAct-GFP expression did produce a small (~10%) increase in the percentage of cells exhibiting aspiration-induced membrane bleb formation, whilst actin-GFP expression reduced blebbing. Further studies examined the influence of LifeAct-GFP in other cell types, namely chondrogenically differentiated hMSCs and murine chondrocytes. LifeAct-GFP also had no effect on the moduli of these non-blebbing cells for which mechanical properties are largely dependent on the actin cortex. In conclusion we show that LifeAct-GFP enables clearer visualisation of actin organisation and dynamics without disruption of the biomechanical properties of either the whole cell or the actin cortex. Thus the study provides new evidence supporting the use of LifeAct-GFP rather than actin-GFP for live cell microscopy and the study of cellular mechanobiology. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

There is more than one way to model an elephant. Experiment-driven modeling of the actin cytoskeleton.

PubMed

Ditlev, Jonathon A; Mayer, Bruce J; Loew, Leslie M

2013-02-05

Mathematical modeling has established its value for investigating the interplay of biochemical and mechanical mechanisms underlying actin-based motility. Because of the complex nature of actin dynamics and its regulation, many of these models are phenomenological or conceptual, providing a general understanding of the physics at play. But the wealth of carefully measured kinetic data on the interactions of many of the players in actin biochemistry cries out for the creation of more detailed and accurate models that could permit investigators to dissect interdependent roles of individual molecular components. Moreover, no human mind can assimilate all of the mechanisms underlying complex protein networks; so an additional benefit of a detailed kinetic model is that the numerous binding proteins, signaling mechanisms, and biochemical reactions can be computationally organized in a fully explicit, accessible, visualizable, and reusable structure. In this review, we will focus on how comprehensive and adaptable modeling allows investigators to explain experimental observations and develop testable hypotheses on the intracellular dynamics of the actin cytoskeleton. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

There is More Than One Way to Model an Elephant. Experiment-Driven Modeling of the Actin Cytoskeleton

PubMed Central

Ditlev, Jonathon A.; Mayer, Bruce J.; Loew, Leslie M.

2013-01-01

Mathematical modeling has established its value for investigating the interplay of biochemical and mechanical mechanisms underlying actin-based motility. Because of the complex nature of actin dynamics and its regulation, many of these models are phenomenological or conceptual, providing a general understanding of the physics at play. But the wealth of carefully measured kinetic data on the interactions of many of the players in actin biochemistry cries out for the creation of more detailed and accurate models that could permit investigators to dissect interdependent roles of individual molecular components. Moreover, no human mind can assimilate all of the mechanisms underlying complex protein networks; so an additional benefit of a detailed kinetic model is that the numerous binding proteins, signaling mechanisms, and biochemical reactions can be computationally organized in a fully explicit, accessible, visualizable, and reusable structure. In this review, we will focus on how comprehensive and adaptable modeling allows investigators to explain experimental observations and develop testable hypotheses on the intracellular dynamics of the actin cytoskeleton. PMID:23442903

PIP2: choreographer of actin-adaptor proteins in the HIV-1 dance

PubMed Central

Rocha-Perugini, Vera; Gordon-Alonso, Mónica; Sánchez-Madrid, Francisco

2014-01-01

The actin cytoskeleton plays a key role during the replication cycle of human immunodeficiency virus-1 (HIV-1). HIV-1 infection is affected by cellular proteins that influence the clustering of viral receptors or the subcortical actin cytoskeleton. Several of these actin-adaptor proteins are controlled by the second messenger phosphatidylinositol 4,5-biphosphate (PIP2), an important regulator of actin organization. PIP2 production is induced by HIV-1 attachment and facilitates viral infection. However, the importance of PIP2 in regulating cytoskeletal proteins and thus HIV-1 infection has been overlooked. This review examines recent reports describing the roles played by actin-adaptor proteins during HIV-1 infection of CD4+ T cells, highlighting the influence of the signaling lipid PIP2 in this process. PMID:24768560

Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation*

PubMed Central

Lalo, Ulyana; Roberts, Jonathan A.; Evans, Richard J.

2011-01-01

P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation. PMID:21757694

A novel function of twins, B subunit of protein phosphatase 2A, in regulating actin polymerization.

PubMed

Yeh, Po-An; Chang, Ching-Jin

2017-01-01

Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A) is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation.

A novel function of twins, B subunit of protein phosphatase 2A, in regulating actin polymerization

PubMed Central

Chang, Ching-Jin

2017-01-01

Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A) is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation. PMID:28977036

Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia

PubMed Central

Goldman, Robert D.; Louvard, Daniel

2010-01-01

Invasive cancer cells are believed to breach the basement membrane (BM) using specialized protrusions called invadopodia. We found that the crossing of a native BM is a three-stage process: invadopodia indeed form and perforate the BM, elongate into mature invadopodia, and then guide the cell toward the stromal compartment. We studied the remodeling of cytoskeleton networks during invadopodia formation and elongation using ultrastructural analysis, spatial distribution of molecular markers, and RNA interference silencing of protein expression. We show that formation of invadopodia requires only the actin cytoskeleton and filopodia- and lamellipodia-associated proteins. In contrast, elongation of invadopodia is mostly dependent on filopodial actin machinery. Moreover, intact microtubules and vimentin intermediate filament networks are required for further growth. We propose that invadopodia form by assembly of dendritic/diagonal and bundled actin networks and then mature by elongation of actin bundles, followed by the entry of microtubules and vimentin filaments. These findings provide a link between the epithelial to mesenchymal transition and BM transmigration. PMID:20421424

Coupled excitable Ras and F-actin activation mediates spontaneous pseudopod formation and directed cell movement

PubMed Central

van Haastert, Peter J. M.; Keizer-Gunnink, Ineke; Kortholt, Arjan

2017-01-01

Many eukaryotic cells regulate their mobility by external cues. Genetic studies have identified >100 components that participate in chemotaxis, which hinders the identification of the conceptual framework of how cells sense and respond to shallow chemical gradients. The activation of Ras occurs during basal locomotion and is an essential connector between receptor and cytoskeleton during chemotaxis. Using a sensitive assay for activated Ras, we show here that activation of Ras and F-actin forms two excitable systems that are coupled through mutual positive feedback and memory. This coupled excitable system leads to short-lived patches of activated Ras and associated F-actin that precede the extension of protrusions. In buffer, excitability starts frequently with Ras activation in the back/side of the cell or with F-actin in the front of the cell. In a shallow gradient of chemoattractant, local Ras activation triggers full excitation of Ras and subsequently F-actin at the side of the cell facing the chemoattractant, leading to directed pseudopod extension and chemotaxis. A computational model shows that the coupled excitable Ras/F-actin system forms the driving heart for the ordered-stochastic extension of pseudopods in buffer and for efficient directional extension of pseudopods in chemotactic gradients. PMID:28148648

Critical role of actin-associated proteins in smooth muscle contraction, cell proliferation, airway hyperresponsiveness and airway remodeling.

PubMed

Tang, Dale D

2015-10-30

Asthma is characterized by airway hyperresponsiveness and airway remodeling, which are largely attributed to increased airway smooth muscle contractility and cell proliferation. It is known that both chemical and mechanical stimulation regulates smooth muscle contraction. Recent studies suggest that contractile activation and mechanical stretch induce actin cytoskeletal remodeling in smooth muscle. However, the mechanisms that control actin cytoskeletal reorganization are not completely elucidated. This review summarizes our current understanding regarding how actin-associated proteins may regulate remodeling of the actin cytoskeleton in airway smooth muscle. In particular, there is accumulating evidence to suggest that Abelson tyrosine kinase (Abl) plays a critical role in regulating airway smooth muscle contraction and cell proliferation in vitro, and airway hyperresponsiveness and remodeling in vivo. These studies indicate that Abl may be a novel target for the development of new therapy to treat asthma.

Actin Engine in Immunological Synapse

PubMed Central

Piragyte, Indre

2012-01-01

T cell activation and function require physical contact with antigen presenting cells at a specialized junctional structure known as the immunological synapse. Once formed, the immunological synapse leads to sustained T cell receptor-mediated signalling and stabilized adhesion. High resolution microscopy indeed had a great impact in understanding the function and dynamic structure of immunological synapse. Trends of recent research are now moving towards understanding the mechanical part of immune system, expanding our knowledge in mechanosensitivity, force generation, and biophysics of cell-cell interaction. Actin cytoskeleton plays inevitable role in adaptive immune system, allowing it to bear dynamic and precise characteristics at the same time. The regulation of mechanical engine seems very complicated and overlapping, but it enables cells to be very sensitive to external signals such as surface rigidity. In this review, we focus on actin regulators and how immune cells regulate dynamic actin rearrangement process to drive the formation of immunological synapse. PMID:22916042

The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton.

PubMed

Hohaus, Annette; Person, Veronika; Behlke, Joachim; Schaper, Jutta; Morano, Ingo; Haase, Hannelore

2002-08-01

Ahnak is a ubiquitously expressed giant protein of 5643 amino acids implicated in cell differentiation and signal transduction. In a recent study, we demonstrated the association of ahnak with the regulatory beta2 subunit of the cardiac L-type Ca2+ channel. Here we identify the most carboxyl-terminal ahnak region (aa 5262-5643) to interact with recombinant beta2a as well as with beta2 and beta1a isoforms of native muscle Ca2+ channels using a panel of GST fusion proteins. Equilibrium sedimentation analysis revealed Kd values of 55 +/- 11 nM and 328 +/- 24 nM for carboxyl-terminal (aa 195-606) and amino-terminal (aa 1-200) truncates of the beta2a subunit, respectively. The same carboxyl-terminal ahnak region (aa 5262-5643) bound to G-actin and cosedimented with F-actin. Confocal microscopy of human left ventricular tissue localized the carboxyl-terminal ahnak portion to the sarcolemma including the T-tubular system and the intercalated disks of cardiomyocytes. These results suggest that ahnak provides a structural basis for the subsarcolemmal cytoarchitecture and confers the regulatory role of the actin-based cytoskeleton to the L-type Ca2+ channel.

Bcr-Abl induces abnormal cytoskeleton remodeling, beta1 integrin clustering and increased cell adhesion to fibronectin through the Abl interactor 1 pathway.

PubMed

Li, Yingzhu; Clough, Nancy; Sun, Xiaolin; Yu, Weidong; Abbott, Brian L; Hogan, Christopher J; Dai, Zonghan

2007-04-15

Hematopoietic cells isolated from patients with Bcr-Abl-positive leukemia exhibit multiple abnormalities of cytoskeletal and integrin function. These abnormalities are thought to play a role in the pathogenesis of leukemia; however, the molecular events leading to these abnormalities are not fully understood. We show here that the Abi1 pathway is required for Bcr-Abl to stimulate actin cytoskeleton remodeling, integrin clustering and cell adhesion. Expression of Bcr-Abl induces tyrosine phosphorylation of Abi1. This is accompanied by a subcellular translocation of Abi1/WAVE2 to a site adjacent to membrane, where an F-actin-enriched structure containing the adhesion molecules such as beta1-integrin, paxillin and vinculin is assembled. Bcr-Abl-induced membrane translocation of Abi1/WAVE2 requires direct interaction between Abi1 and Bcr-Abl, but is independent of the phosphoinositide 3-kinase pathway. Formation of the F-actin-rich complex correlates with an increased cell adhesion to fibronectin. More importantly, disruption of the interaction between Bcr-Abl and Abi1 by mutations either in Bcr-Abl or Abi1 not only abolished tyrosine phosphorylation of Abi1 and membrane translocation of Abi1/WAVE2, but also inhibited Bcr-Abl-stimulated actin cytoskeleton remodeling, integrin clustering and cell adhesion to fibronectin. Together, these data define Abi1/WAVE2 as a downstream pathway that contributes to Bcr-Abl-induced abnormalities of cytoskeletal and integrin function.

S-Nitrosylation of Cofilin-1 Mediates Estradiol-17β-Stimulated Endothelial Cytoskeleton Remodeling

PubMed Central

Zhang, Hong-hai; Lechuga, Thomas J.; Tith, Tevy; Wang, Wen; Wing, Deborah A.

2015-01-01

Rapid nitric oxide (NO) production via endothelial NO synthase (eNOS) activation represents a major signaling pathway for the cardiovascular protective effects of estrogens; however, the pathways after NO biosynthesis that estrogens use to function remain largely unknown. Covalent adduction of a NO moiety to cysteines, termed S-nitrosylation (SNO), has emerged as a key route for NO to directly regulate protein function. Cofilin-1 (CFL1) is a small actin-binding protein essential for actin dynamics and cytoskeleton remodeling. Despite being identified as a major SNO protein in endothelial cells, whether SNO regulates CFL-1 function is unknown. We hypothesized that estradiol-17β (E2β) stimulates SNO of CFL1 via eNOS-derived NO and that E2β-induced SNO-CFL1 mediates cytoskeleton remodeling in endothelial cells. Point mutation studies determined Cys80 as the primary SNO site among the 4 cysteines (Cys39/80/139/147) in CFL1. Substitutions of Cys80 with Ala or Ser were used to prepare the SNO-mimetic/deficient (C80A/S) CFL1 mutants. Recombinant wild-type (wt) and mutant CFL1 proteins were prepared; their actin-severing activity was determined by real-time fluorescence imaging analysis. The activity of C80A CFL1 was enhanced to that of the constitutively active S3/A CFL1, whereas the other mutants had no effects. C80A/S mutations lowered Ser3 phosphorylation. Treatment with E2β increased filamentous (F)-actin and filopodium formation in endothelial cells, which were significantly reduced in cells overexpressing wt-CFL. Overexpression of C80A, but not C80S, CFL1 decreased basal F-actin and further suppressed E2β-induced F-actin and filopodium formation compared with wt-CFL1 overexpression. Thus, SNOCys80 of cofilin-1 via eNOS-derived NO provides a novel pathway for mediating estrogen-induced endothelial cell cytoskeleton remodeling. PMID:25635941

Intrinsic, Functional, and Structural Properties of β-Thymosins and β-Thymosin/WH2 Domains in the Regulation and Coordination of Actin Self-Assembly Dynamics and Cytoskeleton Remodeling.

PubMed

Renault, L

2016-01-01

β-Thymosins are a family of heat-stable multifunctional polypeptides that are expressed as small proteins of about 5kDa (~45 amino acids) almost exclusively in multicellular animals. They were first isolated from the thymus. As full-length or truncated polypeptides, they appear to stimulate a broad range of extracellular activities in various signaling pathways, including tissue repair and regeneration, inflammation, cell migration, and immune defense. However, their cell surface receptors and structural mechanisms of regulations in these multiple pathways remain still poorly understood. Besides their extracellular activities, they belong to a larger family of small, intrinsically disordered actin-binding domains called WH2/β-thymosin domains that have been identified in more than 1800 multidomain proteins found in different taxonomic domains of life and involved in various actin-based motile processes including cell morphogenesis, motility, adhesions, tissue development, intracellular trafficking, or pathogen infections. This review briefly surveys the main recent findings to understand how these small, intrinsically disordered but functional domains can interact with many unrelated partners and can thus integrate and coordinate various intracellular activities in actin self-assembly dynamics and cell signaling pathways linked to their cytoskeleton remodeling. © 2016 Elsevier Inc. All rights reserved.

Actin dynamics, architecture, and mechanics in cell motility.

PubMed

Blanchoin, Laurent; Boujemaa-Paterski, Rajaa; Sykes, Cécile; Plastino, Julie

2014-01-01

Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or "dashpots" (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles.

Tropomodulins: pointed-end capping proteins that regulate actin filament architecture in diverse cell types

PubMed Central

Yamashiro, Sawako; Gokhin, David S.; Kimura, Sumiko; Nowak, Roberta B.; Fowler, Velia M.

2012-01-01

Tropomodulins are a family of four proteins (Tmods 1–4) that cap the pointed ends of actin filaments in actin cytoskeletal structures in a developmentally regulated and tissue-specific manner. Unique among capping proteins, Tmods also bind tropomyosins (TMs), which greatly enhance the actin filament pointed-end capping activity of Tmods. Tmods are defined by a tropomyosin (TM)-regulated/Pointed-End Actin Capping (TM-Cap) domain in their unstructured N-terminal portion, followed by a compact, folded Leucine-Rich Repeat/Pointed-End Actin Capping (LRR-Cap) domain. By inhibiting actin monomer association and dissociation from pointed ends, Tmods regulate regulate actin dynamics and turnover, stabilizing actin filament lengths and cytoskeletal architecture. In this review, we summarize the genes, structural features, molecular and biochemical properties, actin regulatory mechanisms, expression patterns, and cell and tissue functions of Tmods. By understanding Tmods’ functions in the context of their molecular structure, actin regulation, binding partners, and related variants (leiomodins 1–3), we can draw broad conclusions that can explain the diverse morphological and functional phenotypes that arise from Tmod perturbation experiments in vitro and in vivo. Tmod-based stabilization and organization of intracellular actin filament networks provide key insights into how the emergent properties of the actin cytoskeleton drive tissue morphogenesis and physiology. PMID:22488942

Nuclear Import of β-Dystroglycan Is Facilitated by Ezrin-Mediated Cytoskeleton Reorganization

PubMed Central

Vásquez-Limeta, Alejandra; Wagstaff, Kylie M.; Ortega, Arturo; Crouch, Dorothy H.; Jans, David A.; Cisneros, Bulmaro

2014-01-01

The β-dystroglycan (β-DG) protein has the ability to target to multiple sites in eukaryotic cells, being a member of diverse protein assemblies including the transmembranal dystrophin-associated complex, and a nuclear envelope-localised complex that contains emerin and lamins A/C and B1. We noted that the importin α2/β1-recognised nuclear localization signal (NLS) of β-DG is also a binding site for the cytoskeletal-interacting protein ezrin, and set out to determine whether ezrin binding might modulate β-DG nuclear translocation for the first time. Unexpectedly, we found that ezrin enhances rather than inhibits β-DG nuclear translocation in C2C12 myoblasts. Both overexpression of a phosphomimetic activated ezrin variant (Ez-T567D) and activation of endogenous ezrin through stimulation of the Rho pathway resulted in both formation of actin-rich surface protrusions and significantly increased nuclear translocation of β-DG as shown by quantitative microscopy and subcellular fractionation/Western analysis. In contrast, overexpression of a nonphosphorylatable inactive ezrin variant (Ez-T567A) or inhibition of Rho signaling, decreased nuclear translocation of β-DG concomitant with a lack of cell surface protrusions. Further, a role for the actin cytoskeleton in ezrin enhancement of β-DG nuclear translocation was implicated by the observation that an ezrin variant lacking its actin-binding domain failed to enhance nuclear translocation of β-DG, while disruption of the actin cytoskeleton led to a reduction in β-DG nuclear localization. Finally, we show that ezrin-mediated cytoskeletal reorganization enhances nuclear translocation of the cytoplasmic but not the transmembranal fraction of β-DG. This is the first study showing that cytoskeleton reorganization can modulate nuclear translocation of β-DG, with the implication that β-DG can respond to cytoskeleton-driven changes in cell morphology by translocating from the cytoplasm to the nucleus to orchestrate

Actin capping protein CAPZB regulates cell morphology, differentiation, and neural crest migration in craniofacial morphogenesis†

PubMed Central

Mukherjee, Kusumika; Ishii, Kana; Pillalamarri, Vamsee; Kammin, Tammy; Atkin, Joan F.; Hickey, Scott E.; Xi, Qiongchao J.; Zepeda, Cinthya J.; Gusella, James F.; Talkowski, Michael E.; Morton, Cynthia C.; Maas, Richard L.; Liao, Eric C.

2016-01-01

CAPZB is an actin-capping protein that caps the growing end of F-actin and modulates the cytoskeleton and tethers actin filaments to the Z-line of the sarcomere in muscles. Whole-genome sequencing was performed on a subject with micrognathia, cleft palate and hypotonia that harbored a de novo, balanced chromosomal translocation that disrupts the CAPZB gene. The function of capzb was analyzed in the zebrafish model. capzb−/− mutants exhibit both craniofacial and muscle defects that recapitulate the phenotypes observed in the human subject. Loss of capzb affects cell morphology, differentiation and neural crest migration. Differentiation of both myogenic stem cells and neural crest cells requires capzb. During palate morphogenesis, defective cranial neural crest cell migration in capzb−/− mutants results in loss of the median cell population, creating a cleft phenotype. capzb is also required for trunk neural crest migration, as evident from melanophores disorganization in capzb−/− mutants. In addition, capzb over-expression results in embryonic lethality. Therefore, proper capzb dosage is important during embryogenesis, and regulates both cell behavior and tissue morphogenesis. PMID:26758871

Influence of Pulsed Electric Fields and Mitochondria-Cytoskeleton Interactions on Cell Respiration.

PubMed

Goswami, Ishan; Perry, Justin B; Allen, Mitchell E; Brown, David A; von Spakovsky, Michael R; Verbridge, Scott S

2018-06-19

Pulsed electric fields with microsecond pulse width (μsPEFs) are used clinically; namely, irreversible electroporation/Nanoknife is used for soft tissue tumor ablation. The μsPEF pulse parameters used in irreversible electroporation (0.5-1 kV/cm, 80-100 pulses, ∼100 μs each, 1 Hz frequency) may cause an internal field to develop within the cell because of the disruption of the outer cell membrane and subsequent penetration of the electric field. An internal field may disrupt voltage-sensitive mitochondria, although the research literature has been relatively unclear regarding whether such disruptions occur with μsPEFs. This investigation reports the influence of clinically used μsPEF parameters on mitochondrial respiration in live cells. Using a high-throughput Agilent Seahorse machine, it was observed that μsPEF exposure comprising 80 pulses with amplitudes of 600 or 700 V/cm did not alter mitochondrial respiration in 4T1 cells measured after overnight postexposure recovery. To record alterations in mitochondrial function immediately after μsPEF exposure, high-resolution respirometry was used to measure the electron transport chain state via responses to glutamate-malate and ADP and mitochondrial membrane potential via response to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In addition to measuring immediate mitochondrial responses to μsPEF exposure, measurements were also made on cells permeabilized using digitonin and those with compromised cytoskeleton due to actin depolymerization via treatment with the drug latrunculin B. The former treatment was used as a control to tease out the effects of plasma membrane permeabilization, whereas the latter was used to investigate indirect effects on the mitochondria that may occur if μsPEFs impact the cytoskeleton on which the mitochondria are anchored. Based on the results, it was concluded that within the pulse parameters tested, μsPEFs alone do not hinder mitochondrial physiology but can be used

Mechanical compression and hydrostatic pressure induce reversible changes in actin cytoskeletal organisation in chondrocytes in agarose.

PubMed

Knight, M M; Toyoda, T; Lee, D A; Bader, D L

2006-01-01

In numerous cell types, the cytoskeleton has been widely implicated in mechanotransduction pathways involving stretch-activated ion channels, integrins and deformation of intracellular organelles. Studies have also demonstrated that the cytoskeleton can undergo remodelling in response to mechanical stimuli such as tensile strain or fluid flow. In articular chondrocytes, the mechanotransduction pathways are complex, inter-related and as yet, poorly understood. Furthermore, little is known of how the chondrocyte cytoskeleton responds to physiological mechanical loading. This study utilises the well-characterised chondrocyte-agarose model and an established confocal image-analysis technique to demonstrate that both static and cyclic, compressive strain and hydrostatic pressure all induce remodelling of actin microfilaments. This remodelling was characterised by a change from a uniform to a more punctate distribution of cortical actin around the cell periphery. For some loading regimes, this remodelling was reversed over a subsequent 1h unloaded period. This reversible remodelling of actin cytoskeleton may therefore represent a mechanism through which the chondrocyte alters its mechanical properties and mechanosensitivity in response to physiological mechanical loading.