Sample records for actin cytoskeleton functions

  1. The yeast actin cytoskeleton.

    PubMed

    Mishra, Mithilesh; Huang, Junqi; Balasubramanian, Mohan K

    2014-03-01

    The actin cytoskeleton is a complex network of dynamic polymers, which plays an important role in various fundamental cellular processes, including maintenance of cell shape, polarity, cell division, cell migration, endocytosis, vesicular trafficking, and mechanosensation. Precise spatiotemporal assembly and disassembly of actin structures is regulated by the coordinated activity of about 100 highly conserved accessory proteins, which nucleate, elongate, cross-link, and sever actin filaments. Both in vivo studies in a wide range of organisms from yeast to metazoans and in vitro studies of purified proteins have helped shape the current understanding of actin dynamics and function. Molecular genetics, genome-wide functional analysis, sophisticated real-time imaging, and ultrastructural studies in concert with biochemical analysis have made yeast an attractive model to understand the actin cytoskeleton, its molecular dynamics, and physiological function. Studies of the yeast actin cytoskeleton have contributed substantially in defining the universal mechanism regulating actin assembly and disassembly in eukaryotes. Here, we review some of the important insights generated by the study of actin cytoskeleton in two important yeast models the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  2. 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

  3. Actin cytoskeleton and exocytosis in rat melanotrophs.

    PubMed

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

    2000-01-01

    We monitored secretory activity of single rat melanotrophs by the patch-clamp membrane capacitance measurements (C m ). Secretory activity was stimulated by cytosol dialysis with a patch-pipette solution containing 1μM [Ca 2+ ] i . Actin cytoskeleton was disaggregated by pretreating cells with Clostridium spiroforme toxin, which specifically ADP-ribosylates cellular actin. The extent of cytoskeleton disaggregation was monitored by phalloidin immunostaining. The maximal rate of secretion increases two folds in toxin-treated cells in comparison to controls, whereas the extent of calcium-induced secretory response was similar to that obtained in the non-treated cells. The results show that the subcortical actin network attenuates the rate of secretory activity, which we interpret to reflect a barrier function of cytoskeleton for exocytosis.

  4. Actin cytoskeleton and exocytosis in rat melanotrophs.

    PubMed

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

    2000-01-01

    We monitored secretory activity of single rat melanotrophs by the patch-clamp membrane capacitance measurements (Cm). Secretory activity was stimulated by cytosol dialysis with a patch-pipette solution containing 1 microM [Ca2+]i. Actin cytoskeleton was disaggregated by pretreating cells with Clostridium spiroforme toxin, which specifically ADP-ribosylates cellular actin. The extent of cytoskeleton disaggregation was monitored by phalloidin immunostaining. The maximal rate of secretion increases two folds in toxin-treated cells in comparison to controls, whereas the extent of calcium-induced secretory response was similar to that obtained in the non-treated cells. The results show that the subcortical actin network attenuates the rate of secretory activity, which we interpret to reflect a barrier function of cytoskeleton for exocytosis.

  5. Diversification of caldesmon-linked actin cytoskeleton in cell motility

    PubMed Central

    Mayanagi, Taira

    2011-01-01

    The actin cytoskeleton plays a key role in regulating cell motility. Caldesmon (CaD) is an actin-linked regulatory protein found in smooth muscle and non-muscle cells that is conserved among a variety of vertebrates. It binds and stabilizes actin filaments, as well as regulating actin-myosin interaction in a calcium (Ca2+)/calmodulin (CaM)- and/or phosphorylation-dependent manner. CaD function is regulated qualitatively by Ca2+/CaM and by its phosphorylation state and quantitatively at the mRNA level, by three different transcriptional regulation of the CALD1 gene. CaD has numerous functions in cell motility, such as migration, invasion and proliferation, exerted via the reorganization of the actin cytoskeleton. Here we will outline recent findings regarding CaD's structural features and functions. PMID:21350330

  6. Organization and function of the actin cytoskeleton in developing root cells.

    PubMed

    Blancaflor, Elison B; Wang, Yuh-Shuh; Motes, Christy M

    2006-01-01

    The actin cytoskeleton is a highly dynamic structure, which mediates various cellular functions in large part through accessory proteins that tilt the balance between monomeric G-actin and filamentous actin (F-actin) or by facilitating interactions between actin and the plasma membrane, microtubules, and other organelles. Roots have become an attractive model to study actin in plant development because of their simple anatomy and accessibility of some root cell types such as root hairs for microscopic analyses. Roots also exhibit a remarkable developmental plasticity and possess a delicate sensory system that is easily manipulated, so that one can design experiments addressing a range of important biological questions. Many facets of root development can be regulated by the diverse actin network found in the various root developmental regions. Various molecules impinge on this actin scaffold to define how a particular root cell type grows or responds to a specific environmental signal. Although advances in genomics are leading the way toward elucidating actin function in roots, more significant strides will be realized when such tools are combined with improved methodologies for accurately depicting how actin is organized in plant cells.

  7. Course 6: Physics of Composite Cell Membrane and Actin Based Cytoskeleton

    NASA Astrophysics Data System (ADS)

    Sackmann, E.; Bausch, A. R.; Vonna, L.

    1 Architecture of composite cell membranes 1.1 The lipid/protein bilayer is a multicomponent smectic phase with mosaic like architecture 1.2 The spectrin/actin cytoskeleton as hyperelastic cell stabilizer 1.3 The actin cortex: Architecture and function 2 Physics of the actin based cytoskeleton 2.1 Actin is a living semiflexible polymer 2.2 Actin network as viscoelastic body 2.3 Correlation between macroscopic viscoelasticity and molecular 3 Heterogeneous actin gels in cells and biological function 3.1 Manipulation of actin gels 3.2 Control of organization and function of actin cortex by cell signalling 4 Micromechanics and microrheometry of cells 5 Activation of endothelial cells: On the possibility of formation of stress fibers as phase transition of actin-network triggered by cell signalling pathways 6 On cells as adaptive viscoplastic bodies 7 Controll of cellular protrusions controlled by actin/myosin cortex

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Live-Cell Imaging of Mitochondria and the Actin Cytoskeleton in Budding Yeast.

    PubMed

    Higuchi-Sanabria, Ryo; Swayne, Theresa C; Boldogh, Istvan R; Pon, Liza A

    2016-01-01

    Maintenance and regulation of proper mitochondrial dynamics and functions are necessary for cellular homeostasis. Numerous diseases, including neurodegeneration and muscle myopathies, and overall cellular aging are marked by declining mitochondrial function and subsequent loss of multiple other cellular functions. For these reasons, optimized protocols are needed for visualization and quantification of mitochondria and their function and fitness. In budding yeast, mitochondria are intimately associated with the actin cytoskeleton and utilize actin for their movement and inheritance. This chapter describes optimal approaches for labeling mitochondria and the actin cytoskeleton in living budding yeast cells, for imaging the labeled cells, and for analyzing the resulting images.

  13. 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

  14. Multiple roles for the actin cytoskeleton during regulated exocytosis

    PubMed Central

    Porat-Shliom, Natalie; Milberg, Oleg; Masedunskas, Andrius; Weigert, Roberto

    2014-01-01

    Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e. secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules. PMID:22986507

  15. Glutathione depletion triggers actin cytoskeleton changes via actin-binding proteins.

    PubMed

    Zepeta-Flores, Nahum; Valverde, Mahara; Lopez-Saavedra, Alejandro; Rojas, Emilio

    2018-06-04

    The importance of glutathione (GSH) in alternative cellular roles to the canonically proposed, were analyzed in a model unable to synthesize GSH. Gene expression analysis shows that the regulation of the actin cytoskeleton pathway is strongly impacted by the absence of GSH. To test this hypothesis, we evaluate the effect of GSH depletion via buthionine sulfoximine (5 and 12.5 mM) in human neuroblastoma MSN cells. In the present study, 70% of GSH reduction did not induce reactive oxygen species, lipoperoxidation, or cytotoxicity, which enabled us to evaluate the effect of glutathione in the absence of oxidative stress. The cells with decreasing GSH levels acquired morphology changes that depended on the actin cytoskeleton and not on tubulin. We evaluated the expression of three actin-binding proteins: thymosin β4, profilin and gelsolin, showing a reduced expression, both at gene and protein levels at 24 hours of treatment; however, this suppression disappears after 48 hours of treatment. These changes were sufficient to trigger the co-localization of the three proteins towards cytoplasmic projections. Our data confirm that a decrease in GSH in the absence of oxidative stress can transiently inhibit the actin binding proteins and that this stimulus is sufficient to induce changes in cellular morphology via the actin cytoskeleton.

  16. Three’s company: The fission yeast actin cytoskeleton

    PubMed Central

    Kovar, David R.; Sirotkin, Vladimir; Lord, Matthew

    2010-01-01

    How the actin cytoskeleton assembles into different structures to drive diverse cellular processes is a fundamental cell biological question. In addition to orchestrating the appropriate combination of regulators and actin-binding proteins, different actin-based structures must insulate themselves from one another to maintain specificity within a crowded cytoplasm. Actin specification is particularly vexing in complex eukaryotes where a multitude of protein isoforms and actin structures operate within the same cell. Fission yeast Schizosaccharomyces pombe possesses a single actin isoform that functions in three distinct structures throughout the cell cycle. In this review, we explore recent studies in fission yeast that help unravel how different actin structures operate in cells. PMID:21145239

  17. 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.

  18. Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides.

    PubMed

    Saarikangas, Juha; Zhao, Hongxia; Lappalainen, Pekka

    2010-01-01

    The plasma membrane and the underlying cortical actin cytoskeleton undergo continuous dynamic interplay that is responsible for many essential aspects of cell physiology. Polymerization of actin filaments against cellular membranes provides the force for a number of cellular processes such as migration, morphogenesis, and endocytosis. Plasma membrane phosphoinositides (especially phosphatidylinositol bis- and trisphosphates) play a central role in regulating the organization and dynamics of the actin cytoskeleton by acting as platforms for protein recruitment, by triggering signaling cascades, and by directly regulating the activities of actin-binding proteins. Furthermore, a number of actin-associated proteins, such as BAR domain proteins, are capable of directly deforming phosphoinositide-rich membranes to induce plasma membrane protrusions or invaginations. Recent studies have also provided evidence that the actin cytoskeleton-plasma membrane interactions are misregulated in a number of pathological conditions such as cancer and during pathogen invasion. Here, we summarize the wealth of knowledge on how the cortical actin cytoskeleton is regulated by phosphoinositides during various cell biological processes. We also discuss the mechanisms by which interplay between actin dynamics and certain membrane deforming proteins regulate the morphology of the plasma membrane.

  19. 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.

  20. Chronophin activation is necessary in Doxorubicin-induced actin cytoskeleton alteration.

    PubMed

    Lee, Su Jin; Park, Jeen Woo; Kang, Beom Sik; Lee, Dong-Seok; Lee, Hyun-Shik; Choi, Sooyoung; Kwon, Oh-Shin

    2017-06-01

    Although doxorubicin (Dox)-induced oxidative stress is known to be associated with cytotoxicity, the precise mechanism remains unclear. Genotoxic stress not only generates free radicals, but also affects actin cytoskeleton stability. We showed that Dox-induced RhoA signaling stimulated actin cytoskeleton alterations, resulting in central stress fiber disruption at early time points and cell periphery cortical actin formation at a later stage, in HeLa cells. Interestingly, activation of a cofilin phosphatase, chronophin (CIN), was initially evoked by Dox-induced RhoA signaling, resulting in a rapid phosphorylated cofilin turnover leading to actin cytoskeleton remodeling. In addition, a novel interaction between CIN and 14-3-3ζ was detected in the absence of Dox treatment. We demonstrated that CIN activity is quite contrary to 14-3-3ζ binding, and the interaction leads to enhanced phosphorylated cofilin levels. Therefore, initial CIN activation regulation could be critical in Dox-induced actin cytoskeleton remodeling through RhoA/cofilin signaling. [BMB Reports 2017; 50(6): 335-340].

  1. 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

  2. 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.

  3. 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.

  4. The actin cytoskeleton in whole mount preparations and sections.

    PubMed

    Resch, Guenter P; Urban, Edit; Jacob, Sonja

    2010-01-01

    In non-muscle cells, the actin cytoskeleton plays a key role by providing a scaffold contributing to the definition of cell shape, force for driving cell motility, cytokinesis, endocytosis, and propulsion of pathogens, as well as tracks for intracellular transport. A thorough understanding of these processes requires insight into the spatial and temporal organisation of actin filaments into diverse higher-order structures, such as networks, parallel bundles, and contractile arrays. Transmission and scanning electron microscopy can be used to visualise the actin cytoskeleton, but due to the delicate nature of actin filaments, they are easily affected by standard preparation protocols, yielding variable degrees of ultrastructural preservation. In this chapter, we describe different conventional and cryo-approaches to visualise the actin cytoskeleton using transmission electron microscopy and discuss their specific advantages and drawbacks. In the first part, we present three different whole mount techniques, which allow visualisation of actin in the peripheral, thinly spread parts of cells grown in monolayers. In the second part, we describe specific issues concerning the visualisation of actin in thin sections. Techniques for three-dimensional visualisation of actin, protein localisation, and correlative light and electron microscopy are also included. Copyright © 2010 Elsevier Inc. All rights reserved.

  5. Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

    PubMed Central

    Navarro-Garcia, Fernando; Serapio-Palacios, Antonio; Ugalde-Silva, Paul; Tapia-Pastrana, Gabriela; Chavez-Dueñas, Lucia

    2013-01-01

    The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology. PMID:23509714

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Profilin as a regulator of the membrane-actin cytoskeleton interface in plant cells

    PubMed Central

    Sun, Tiantian; Li, Shanwei; Ren, Haiyun

    2013-01-01

    Membrane structures and cytoskeleton dynamics are intimately inter-connected in the eukaryotic cell. Recently, the molecular mechanisms operating at this interface have been progressively addressed. Many experiments have revealed that the actin cytoskeleton can interact with membranes through various discrete membrane domains. The actin-binding protein, profilin has been proven to inhibit actin polymerization and to promote F-actin elongation. This is dependent on many factors, such as the profilin/G-actin ratio and the ionic environment of the cell. Additionally, profilin has specific domains that interact with phosphoinositides and poly-L-proline rich proteins; theoretically, this gives profilin the opportunity to interact with membranes, and a large number of experiments have confirmed this possibility. In this article, we summarize recent findings in plant cells, and discuss the evidence of the connections among actin cytoskeleton, profilin and biomembranes through direct or indirect relationships. PMID:24391654

  16. System-wide organization of actin cytoskeleton determines organelle transport in hypocotyl plant cells

    PubMed Central

    Nowak, Jacqueline; Ivakov, Alexander; Somssich, Marc; Persson, Staffan; Nikoloski, Zoran

    2017-01-01

    The actin cytoskeleton is an essential intracellular filamentous structure that underpins cellular transport and cytoplasmic streaming in plant cells. However, the system-level properties of actin-based cellular trafficking remain tenuous, largely due to the inability to quantify key features of the actin cytoskeleton. Here, we developed an automated image-based, network-driven framework to accurately segment and quantify actin cytoskeletal structures and Golgi transport. We show that the actin cytoskeleton in both growing and elongated hypocotyl cells has structural properties facilitating efficient transport. Our findings suggest that the erratic movement of Golgi is a stable cellular phenomenon that might optimize distribution efficiency of cell material. Moreover, we demonstrate that Golgi transport in hypocotyl cells can be accurately predicted from the actin network topology alone. Thus, our framework provides quantitative evidence for system-wide coordination of cellular transport in plant cells and can be readily applied to investigate cytoskeletal organization and transport in other organisms. PMID:28655850

  17. 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

  18. Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

    PubMed Central

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R.; Bryce, Nicole S.; Whan, Renee M.; Hardeman, Edna C.

    2015-01-01

    The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments. PMID:25978408

  19. 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.

  20. 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).

  1. Characterization of biomechanical properties of cells through dielectrophoresis-based cell stretching and actin cytoskeleton modeling.

    PubMed

    Bai, Guohua; Li, Ying; Chu, Henry K; Wang, Kaiqun; Tan, Qiulin; Xiong, Jijun; Sun, Dong

    2017-04-04

    Cytoskeleton is a highly dynamic network that helps to maintain the rigidity of a cell, and the mechanical properties of a cell are closely related to many cellular functions. This paper presents a new method to probe and characterize cell mechanical properties through dielectrophoresis (DEP)-based cell stretching manipulation and actin cytoskeleton modeling. Leukemia NB4 cells were used as cell line, and changes in their biological properties were examined after chemotherapy treatment with doxorubicin (DOX). DEP-integrated microfluidic chip was utilized as a low-cost and efficient tool to study the deformability of cells. DEP forces used in cell stretching were first evaluated through computer simulation, and the results were compared with modeling equations and with the results of optical stretching (OT) experiments. Structural parameters were then extracted by fitting the experimental data into the actin cytoskeleton model, and the underlying mechanical properties of the cells were subsequently characterized. The DEP forces generated under different voltage inputs were calculated and the results from different approaches demonstrate good approximations to the force estimation. Both DEP and OT stretching experiments confirmed that DOX-treated NB4 cells were stiffer than the untreated cells. The structural parameters extracted from the model and the confocal images indicated significant change in actin network after DOX treatment. The proposed DEP method combined with actin cytoskeleton modeling is a simple engineering tool to characterize the mechanical properties of cells.

  2. 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

  3. Actin cytoskeleton stiffness grades metastatic potential of ovarian carcinoma Hey A8 cells via nanoindentation mapping.

    PubMed

    Zhou, Z L; Sun, X X; Ma, J; Tong, M H; To, S K Y; Wong, A S T; Ngan, A H W

    2017-07-26

    Recent studies have indicated that the nanoindentation measured stiffness of carcinoma adherent cells is in general lower than normal cells, thus suggesting that cell stiffness may serve as a bio-marker for carcinoma. However, the proper establishment of such a conclusion would require biophysical understanding of the underlying mechanism of the cell stiffness. In this work, we compared the elastic moduli of the actin cytoskeletons of Hey A8 ovarian carcinoma cells with and without metastasis (HM and NM), as measured by 2D atomic force microscopy (AFM) with low-depth nanoindentation via a rate-jump method. The results indicate clearly that HM cells showed lower actin cytoskeleton stiffness atop of their nucleus position and higher actin cytoskeleton stiffness at their rims, compared to NM cells, suggesting that the local stiffness on the cytoskeleton can reflect actin filament distribution. Immunofluorescence staining and scanning electron microscopy (SEM) also indicated that the difference in stiffness in Hey A8 cells with different metastasis is associated with their F-actin rearrangement. Finite-element modelling (FEM) shows that a migrating cell would have its actin filaments bundled together to form stress fibers, which would exhibit lower indentation stiffness than the less aligned arrangement of filaments in a non-migrating cell. The results here indicate that the actin cytoskeleton stiffness can serve as a reliable marker for grading the metastasis of adherent carcinoma cells due to their cytoskeleton change and potentially predicting the migration direction of the cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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 subapical MT array. In the apex, actin MFs form thicker bundles that converge into a remarkably distinct actin patch in the apical dome, whose position coincides with the position of the endoplasmic reticulum aggregate in the centre of the Spitzenkörper. Actin MFs radiate from the actin patch towards the apical membrane. Together with results from previous inhibitor studies (Braun and Sievers, 1994, Eur J Cell Biol 63: 289-298), these results suggest that MTs have a stabilizing function in maintaining the polar cytoplasmic and cytoskeletal organization. The motile processes, however, are mediated by actin. In particular, the actin cytoskeleton appears to be involved in the structural and functional organization of the Spitzenkörper and thus is responsible for controlling cell shape and growth direction. Despite the similar structural arrangements of the actin cytoskeleton, major differences in the function of actin MFs have been observed in rhizoids and protonemata. Since actin MFs are more directly involved in the gravitropic response of protonemata than of rhizoids, the opposite gravitropsim in the two cell types seems to be based mainly on different properties and activities of the actin cytoskeleton.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

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

    Vieira da Silva, Claudio; Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sao Paulo, Rua Botucatu, 862, 6o andar, 04023-062 Sao Paulo, SP; Alves da Silva, Erika

    2009-01-16

    Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RHmore » strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP{sub 2} and PIP{sub 3} to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.« less

  19. 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.

  20. 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

  1. 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. ...

  2. 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.

  3. 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

  4. 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.

  5. Cytoskeleton and Cytoskeleton-Bound RNA Visualization in Frog and Insect Oocytes.

    PubMed

    Kloc, Malgorzata; Bilinski, Szczepan; Kubiak, Jacek Z

    2016-01-01

    The majority of oocyte functions involves and depends on the cytoskeletal elements, which include microtubules and actin and cytokeratin filaments. Various structures and molecules are temporarily or permanently bound to the cytoskeletal elements and their functions rely on cytoskeleton integrity and its timely assembly. Thus the accurate visualization of cytoskeleton is often crucial for studies and analyses of oocyte structure and functions. Here we describe several reliable methods for microtubule and/or microfilaments preservation and visualization in Xenopus oocyte extracts, and in situ in live and fixed insect and frog (Xenopus) oocytes. In addition, we describe visualization of cytoskeleton-bound RNAs using molecular beacons in live Xenopus oocytes.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. Microtubule-Actin Cross-Linking Factor 1: Domains, Interaction Partners, and Tissue-Specific Functions.

    PubMed

    Goryunov, Dmitry; Liem, Ronald K H

    2016-01-01

    The cytoskeleton of most eukaryotic cells is composed of three principal filamentous components: actin filaments, microtubules (MTs), and intermediate filaments. It is a highly dynamic system that plays crucial roles in a wide range of cellular processes, including migration, adhesion, cytokinesis, morphogenesis, intracellular traffic and signaling, and structural flexibility. Among the large number of cytoskeleton-associated proteins characterized to date, microtubule-actin cross-linking factor 1 (MACF1) is arguably the most versatile integrator and modulator of cytoskeleton-related processes. MACF1 belongs to the plakin family of proteins, and within it, to the spectraplakin subfamily. These proteins are characterized by the ability to bridge MT and actin cytoskeletal networks in a dynamic fashion, which underlies their involvement in the regulation of cell migration, axonal extension, and vesicular traffic. Studying MACF1 functions has provided insights not only into the regulation of the cytoskeleton but also into molecular mechanisms of both normal cellular physiology and cellular pathology. Multiple MACF1 isoforms exist, composed of a large variety of alternatively spliced domains. Each of these domains mediates a specific set of interactions and functions. These functions are manifested in tissue and cell-specific phenotypes observed in conditional MACF1 knockout mice. The conditional models described to date reveal critical roles of MACF1 in mammalian skin, nervous system, heart muscle, and intestinal epithelia. Complete elimination of MACF1 is early embryonic lethal, indicating an essential role for MACF1 in early development. Further studies of MACF1 domains and their interactions will likely reveal multiple new roles of this protein in various tissues. © 2016 Elsevier Inc. All rights reserved.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. Capillary electrophoretic analysis reveals subcellular binding between individual mitochondria and cytoskeleton

    PubMed Central

    Kostal, Vratislav; Arriaga, Edgar A.

    2011-01-01

    Interactions between the cytoskeleton and mitochondria are essential for normal cellular function. An assessment of such interactions is commonly based on bulk analysis of mitochondrial and cytoskeletal markers present in a given sample, which assumes complete binding between these two organelle types. Such measurements are biased because they rarely account for non-bound ‘free’ subcellular species. Here we report on the use of capillary electrophoresis with dual laser induced fluorescence detection (CE-LIF) to identify, classify, count and quantify properties of individual binding events of mitochondria and cytoskeleton. Mitochondria were fluorescently labeled with DsRed2 while F-actin, a major cytoskeletal component, was fluorescently labeled with Alexa488-phalloidin. In a typical subcellular fraction of L6 myoblasts, 79% of mitochondrial events did not have detectable levels of F-actin, while the rest had on average ~2 zeptomole F-actin, which theoretically represents a ~ 2.5-μm long network of actin filaments per event. Trypsin treatment of L6 subcellular fractions prior to analysis decreased the fraction of mitochondrial events with detectable levels of F-actin, which is expected from digestion of cytoskeletal proteins on the surface of mitochondria. The electrophoretic mobility distributions of the individual events were also used to further distinguish between cytoskeleton-bound from cytoskeleton-free mitochondrial events. The CE-LIF approach described here could be further developed to explore cytoskeleton interactions with other subcellular structures, the effects of cytoskeleton destabilizing drugs, and the progression of viral infections. PMID:21309532

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. RNase L Interacts with Filamin A To Regulate Actin Dynamics and Barrier Function for Viral Entry

    PubMed Central

    Siddiqui, Mohammad Adnan; Dayal, Shubham; Naji, Merna; Ezelle, Heather J.; Zeng, Chun; Zhou, Aimin; Hassel, Bret A.

    2014-01-01

    ABSTRACT The actin cytoskeleton and its network of associated proteins constitute a physical barrier that viruses must circumvent to gain entry into cells for productive infection. The mechanisms by which the physical signals of infection are sensed by the host to activate an innate immune response are not well understood. The antiviral endoribonuclease RNase L is ubiquitously expressed in a latent form and activated upon binding 2-5A, a unique oligoadenylate produced during viral infections. We provide evidence that RNase L in its inactive form interacts with the actin-binding protein Filamin A to modulate the actin cytoskeleton and inhibit virus entry. Cells lacking either RNase L or Filamin A displayed increased virus entry which was exacerbated in cells lacking both proteins. RNase L deletion mutants that reduced Filamin A interaction displayed a compromised ability to restrict virus entry, supporting the idea of an important role for the RNase L-Filamin A complex in barrier function. Remarkably, both the wild type and a catalytically inactive RNase L mutant were competent to reduce virus entry when transfected into RNase L-deficient cells, indicating that this novel function of RNase L is independent of its enzymatic activity. Virus infection and RNase L activation disrupt its association with Filamin A and release RNase L to mediate its canonical nuclease-dependent antiviral activities. The dual functions of RNase L as a constitutive component of the actin cytoskeleton and as an induced mediator of antiviral signaling and effector functions provide insights into its mechanisms of antiviral activity and opportunities for the development of novel antiviral agents. PMID:25352621

  20. F-actin distribution and function during sexual development in Eimeria maxima.

    PubMed

    Frölich, Sonja; Wallach, Michael

    2015-06-01

    To determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling 'beads on a string'. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima.

  1. CRMPs colocalize and interact with cytoskeleton in hippocampal neurons

    PubMed Central

    Yang, Yuhao; Zhao, Bo; Ji, Zhisheng; Zhang, Guowei; Zhang, Jifeng; Li, Sumei; Guo, Guoqing; Lin, Hongsheng

    2015-01-01

    CRMP family proteins (CRMPs) are widely expressed in the developing neurons, mediating a variety of fundamental functions such as growth cone guidance, neuronal polarity and axon elongation. However, whether all the CRMP proteins interact with cytoskeleton remains unknown. In this study, we found that in cultured hippocampal neurons, CRMPs mainly colocalized with tubulin and actin network in neurites. In growth cones, CRMPs colocalized with tubulinmainly in the central (C-) domain and transition zone (T-zone), less in the peripheral (P-) domain and colocalized with actin in all the C-domain, T-zone and P-domain. The correlation efficiency of CRMPs between actin was significantly higher than that between tubulin, especially in growth cones. We successfully constructed GST-CRMPs plasmids, expressed and purified the GST-CRMP proteins. By GST-pulldown assay, all the CRMP family proteins were found to beinteracted with cytoskeleton proteins. Taken together, we revealed that CRMPs were colocalized with cytoskeleton in hippocampal neurons, especially in growth cones. CRMPs can interact with both tubulin and actin, thus mediating neuronal development. PMID:26885211

  2. 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

  3. 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.

  4. Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function

    PubMed Central

    McGough, Amy; Pope, Brian; Chiu, Wah; Weeds, Alan

    1997-01-01

    Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter ‘actin crossovers' (∼75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled. PMID:9265645

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Axon Initial Segment Cytoskeleton: Architecture, Development, and Role in Neuron Polarity

    PubMed Central

    Svitkina, Tatyana M.

    2016-01-01

    The axon initial segment (AIS) is a specialized structure in neurons that resides in between axonal and somatodendritic domains. The localization of the AIS in neurons is ideal for its two major functions: it serves as the site of action potential firing and helps to maintain neuron polarity. It has become increasingly clear that the AIS cytoskeleton is fundamental to AIS functions. In this review, we discuss current understanding of the AIS cytoskeleton with particular interest in its unique architecture and role in maintenance of neuron polarity. The AIS cytoskeleton is divided into two parts, submembrane and cytoplasmic, based on localization, function, and molecular composition. Recent studies using electron and subdiffraction fluorescence microscopy indicate that submembrane cytoskeletal components (ankyrin G, βIV-spectrin, and actin filaments) form a sophisticated network in the AIS that is conceptually similar to the polygonal/triangular network of erythrocytes, with some important differences. Components of the AIS cytoplasmic cytoskeleton (microtubules, actin filaments, and neurofilaments) reside deeper within the AIS shaft and display structural features distinct from other neuronal domains. We discuss how the AIS submembrane and cytoplasmic cytoskeletons contribute to different aspects of AIS polarity function and highlight recent advances in understanding their AIS cytoskeletal assembly and stability. PMID:27493806

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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...

  8. 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.

  9. 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.

  10. [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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. Co-transcriptional nuclear actin dynamics

    PubMed Central

    Percipalle, Piergiorgio

    2013-01-01

    Actin is a key player for nuclear structure and function regulating both chromosome organization and gene activity. In the cell nucleus actin interacts with many different proteins. Among these proteins several studies have identified classical nuclear factors involved in chromatin structure and function, transcription and RNA processing as well as proteins that are normally involved in controlling the actin cytoskeleton. These discoveries have raised the possibility that nuclear actin performs its multi task activities through tight interactions with different sets of proteins. This high degree of promiscuity in the spectrum of protein-to-protein interactions correlates well with the conformational plasticity of actin and the ability to undergo regulated changes in its polymerization states. Several of the factors involved in controlling head-to-tail actin polymerization have been shown to be in the nucleus where they seem to regulate gene activity. By focusing on the multiple tasks performed by actin and actin-binding proteins, possible models of how actin dynamics controls the different phases of the RNA polymerase II transcription cycle are being identified. PMID:23138849

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. Myosin motor function: the ins and outs of actin-based membrane protrusions

    PubMed Central

    Nambiar, Rajalakshmi; McConnell, Russell E.

    2011-01-01

    Cells build plasma membrane protrusions supported by parallel bundles of F-actin to enable a wide variety of biological functions, ranging from motility to host defense. Filopodia, microvilli and stereocilia are three such protrusions that have been the focus of intense biological and biophysical investigation in recent years. While it is evident that actin dynamics play a significant role in the formation of these organelles, members of the myosin superfamily have also been implicated as key players in the maintenance of protrusion architecture and function. Based on a simple analysis of the physical forces that control protrusion formation and morphology, as well as our review of available data, we propose that myosins play two general roles within these structures: (1) as cargo transporters to move critical regulatory components toward distal tips and (2) as mediators of membrane-cytoskeleton adhesion. PMID:20107861

  2. 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

  3. 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.

  4. 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.

  5. The nano-architecture of the axonal cytoskeleton.

    PubMed

    Leterrier, Christophe; Dubey, Pankaj; Roy, Subhojit

    2017-12-01

    The corporeal beauty of the neuronal cytoskeleton has captured the imagination of generations of scientists. One of the easiest cellular structures to visualize by light microscopy, its existence has been known for well over 100 years, yet we have only recently begun to fully appreciate its intricacy and diversity. Recent studies combining new probes with super-resolution microscopy and live imaging have revealed surprising details about the axonal cytoskeleton and, in particular, have discovered previously unknown actin-based structures. Along with traditional electron microscopy, these newer techniques offer a nanoscale view of the axonal cytoskeleton, which is important for our understanding of neuronal form and function, and lay the foundation for future studies. In this Review, we summarize existing concepts in the field and highlight contemporary discoveries that have fundamentally altered our perception of the axonal cytoskeleton.

  6. A functional interplay between the small GTPase Rab11a and mitochondria-shaping proteins regulates mitochondrial positioning and polarization of the actin cytoskeleton downstream of Src family kinases.

    PubMed

    Landry, Marie-Claude; Champagne, Claudia; Boulanger, Marie-Chloé; Jetté, Alexandra; Fuchs, Margit; Dziengelewski, Claire; Lavoie, Josée N

    2014-01-24

    It is believed that mitochondrial dynamics is coordinated with endosomal traffic rates during cytoskeletal remodeling, but the mechanisms involved are largely unknown. The adenovirus early region 4 ORF4 protein (E4orf4) subverts signaling by Src family kinases (SFK) to perturb cellular morphology, membrane traffic, and organellar dynamics and to trigger cell death. Using E4orf4 as a model, we uncovered a functional connection between mitochondria-shaping proteins and the small GTPase Rab11a, a key regulator of polarized transport via recycling endosomes. We found that E4orf4 induced dramatic changes in the morphology of mitochondria along with their mobilization at the vicinity of a polarized actin network typifying E4orf4 action, in a manner controlled by SFK and Rab11a. Mitochondrial remodeling was associated with increased proximity between Rab11a and mitochondrial membranes, changes in fusion-fission dynamics, and mitochondrial relocalization of the fission factor dynamin-related protein 1 (Drp1), which was regulated by the Rab11a effector protein FIP1/RCP. Knockdown of FIP1/RCP or inhibition of Drp1 markedly impaired mitochondrial remodeling and actin assembly, involving Rab11a-mediated mitochondrial dynamics in E4orf4-induced signaling. A similar mobilization of mitochondria near actin-rich structures was mediated by Rab11 and Drp1 in viral Src-transformed cells and contributed to the biogenesis of podosome rosettes. These findings suggest a role for Rab11a in the trafficking of Drp1 to mitochondria upon SFK activation and unravel a novel functional interplay between Rab11a and mitochondria during reshaping of the cell cytoskeleton, which would facilitate mitochondria redistribution near energy-requiring actin-rich structures.

  7. 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

  8. 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.

  9. Remodeling of tick cytoskeleton in response to infection with Anaplasma phagocytophilum.

    PubMed

    Cabezas-Cruz, Alejandro; Alberdi, Pilar; Valdes, James J; Villar, Margarita; de la Fuente, Jose

    2017-06-01

    The obligate intracellular pathogen Anaplasma phagocytophilum infects vertebrate and tick hosts. In this study, a genome-wide search for cytoskeleton components was performed in the tick vector, Ixodes scapularis . The available transcriptomics and proteomics data was then used to characterize the mRNA and protein levels of I. scapularis cytoskeleton components in response to A. phagocytophilum infection. The results showed that cytoskeleton components described in other model organisms were present in the I. scapularis genome. One type of intermediate filaments (lamin), a family of septins that was recently implicated in the cellular response to intracellular pathogens, and several members of motor proteins (kinesins and dyneins) that could be implicated in the cytoplasmic movements of A. phagocytophilum were found. The results showed that levels of tubulin, actin, septin, actin-related proteins and motor proteins were affected by A. phagocytophilum , probably to facilitate infection in I. scapularis . Functional studies demonstrated a role for selected cytoskeleton components in pathogen infection. These results provided a more comprehensive view of the cytoskeletal components involved in the response to A. phagocytophilum infection in ticks.

  10. Actin cable dynamics and Rho/Rock orchestrate a polarized cytoskeletal architecture in the early steps of assembling a stratified epithelium.

    PubMed

    Vaezi, Alec; Bauer, Christoph; Vasioukhin, Valeri; Fuchs, Elaine

    2002-09-01

    To enable stratification and barrier function, the epidermis must permit self-renewal while maintaining adhesive connections. By generating K14-GFP-actin mice to monitor actin dynamics in cultured primary keratinocytes, we uncovered a role for the actin cytoskeleton in establishing cellular organization. During epidermal sheet formation, a polarized network of nascent intercellular junctions and radial actin cables assemble in the apical plane of the monolayer. These actin fibers anchor to a central actin-myosin network, creating a tension-based plane of cytoskeleton across the apical surface of the sheet. Movement of the sheet surface relative to its base expands the zone of intercellular overlap, catalyzing new sites for nascent intercellular junctions. This polarized cytoskeleton is dependent upon alpha-catenin, Rho, and Rock, and its regulation may be important for wound healing and/or stratification, where coordinated tissue movements are involved.

  11. 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

  12. 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.

  13. 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

  14. Gfi1b controls integrin signaling-dependent cytoskeleton dynamics and organization in megakaryocytes.

    PubMed

    Beauchemin, Hugues; Shooshtarizadeh, Peiman; Vadnais, Charles; Vassen, Lothar; Pastore, Yves D; Möröy, Tarik

    2017-03-01

    Mutations in GFI1B are associated with inherited bleeding disorders called GFI1B -related thrombocytopenias. We show here that mice with a megakaryocyte-specific Gfi1b deletion exhibit a macrothrombocytopenic phenotype along a megakaryocytic dysplasia reminiscent of GFI1B -related thrombocytopenia. GFI1B deficiency increases megakaryocyte proliferation and affects their ploidy, but also abrogates their responsiveness towards integrin signaling and their ability to spread and reorganize their cytoskeleton. Gfi1b -null megakaryocytes are also unable to form proplatelets, a process independent of integrin signaling. GFI1B-deficient megakaryocytes exhibit aberrant expression of several components of both the actin and microtubule cytoskeleton, with a dramatic reduction of α-tubulin. Inhibition of FAK or ROCK, both important for actin cytoskeleton organization and integrin signaling, only partially restored their response to integrin ligands, but the inhibition of PAK, a regulator of the actin cytoskeleton, completely rescued the responsiveness of Gfi1b -null megakaryocytes to ligands, but not their ability to form proplatelets. We conclude that Gfi1b controls major functions of megakaryocytes such as integrin-dependent cytoskeleton organization, spreading and migration through the regulation of PAK activity whereas the proplatelet formation defect in GFI1B-deficient megakaryocytes is due, at least partially, to an insufficient α-tubulin content. Copyright© Ferrata Storti Foundation.

  15. 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.

  16. 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

  17. The Role of Actin Cytoskeleton in Dendritic Spines in the Maintenance of Long-Term Memory.

    PubMed

    Basu, Sreetama; Lamprecht, Raphael

    2018-01-01

    Evidence indicates that long-term memory formation involves alterations in synaptic efficacy produced by modifications in neural transmission and morphology. However, it is not clear how such alterations induced by learning, that encode memory, are maintained over long period of time to preserve long-term memory. This is especially intriguing as the half-life of most of the proteins that underlie such changes is usually in the range of hours to days and these proteins may change their location over time. In this review we describe studies that indicate the involvement of dendritic spines in memory formation and its maintenance. These studies show that learning leads to changes in the number and morphology of spines. Disruption in spines morphology or manipulations that lead to alteration in their number after consolidation are associated with impairment in memory maintenance. We further ask how changes in dendritic spines morphology, induced by learning and reputed to encode memory, are maintained to preserve long-term memory. We propose a mechanism, based on studies described in the review, whereby the actin cytoskeleton and its regulatory proteins involved in the initial alteration in spine morphology induced by learning are also essential for spine structural stabilization that maintains long-term memory. In this model glutamate receptors and other synaptic receptors activation during learning leads to the creation of new actin cytoskeletal scaffold leading to changes in spines morphology and memory formation. This new actin cytoskeletal scaffold is preserved beyond actin and its regulatory proteins turnover and dynamics by active stabilization of the level and activity of actin regulatory proteins within these memory spines.

  18. Effects of perinatal asphyxia on rat striatal cytoskeleton.

    PubMed

    Saraceno, G E; Ayala, M V; Badorrey, M S; Holubiec, M; Romero, J I; Galeano, P; Barreto, G; Giraldez-Alvárez, L D; Kölliker-Fres, R; Coirini, H; Capani, F

    2012-01-01

    Perinatal asphyxia (PA) is a medical condition associated with a high short-term morbimortality and different long-term neurological diseases. In previous works, we have shown that neuronal and synaptic changes in rat striatum lead to ubi-protein accumulation in post-synaptic density (PSD) after six months of sub-severe PA. However, very little is known about the synaptic and related structural modifications induced by PA in young rats. In the present work, we studied neuronal cytoskeleton modifications in striatum induced by subsevere PA in 30-day-old rats. We observed a significant decrease in the number of neurons, in particular calbindin immunoreactive neurons after PA. In addition, it was also observed that actin cytoskeleton was highly modified in the PSD as well as an increment of F-actin staining by Phalloidin-alexa(488) in the striatum of PA rats. Using correlative fluorescence-electron microscopy photooxidation, we confirmed and extended confocal observations. F-actin staining augmentation was mostly related with an increment in the number of mushroom-shaped spines. Consistent with microscopic data, Western blot analysis revealed a β-actin increment in PSD in PA rats. On the other hand, MAP-2 immunostaining was decreased after PA, being NF-200 expression unmodified. Although neuronal death was observed, signs of generalized neurodegeneration were absent. Taken together these results showed early post-synaptic F-actin cytoskeleton changes induced by PA with slightly modifications in the other components of the neuronal cytoskeleton, suggesting that F-actin accumulation in the dendritic spines could be involved in the neuronal loss induced by PA. Copyright © 2011 Wiley Periodicals, Inc.

  19. The sarcomeric cytoskeleton: from molecules to motion.

    PubMed

    Gautel, Mathias; Djinović-Carugo, Kristina

    2016-01-01

    Highly ordered organisation of striated muscle is the prerequisite for the fast and unidirectional development of force and motion during heart and skeletal muscle contraction. A group of proteins, summarised as the sarcomeric cytoskeleton, is essential for the ordered assembly of actin and myosin filaments into sarcomeres, by combining architectural, mechanical and signalling functions. This review discusses recent cell biological, biophysical and structural insight into the regulated assembly of sarcomeric cytoskeleton proteins and their roles in dissipating mechanical forces in order to maintain sarcomere integrity during passive extension and active contraction. α-Actinin crosslinks in the Z-disk show a pivot-and-rod structure that anchors both titin and actin filaments. In contrast, the myosin crosslinks formed by myomesin in the M-band are of a ball-and-spring type and may be crucial in providing stable yet elastic connections during active contractions, especially eccentric exercise. © 2016. Published by The Company of Biologists Ltd.

  20. 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

  1. 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

  2. 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).

  3. Liquid behavior of cross-linked actin bundles.

    PubMed

    Weirich, Kimberly L; Banerjee, Shiladitya; Dasbiswas, Kinjal; Witten, Thomas A; Vaikuntanathan, Suriyanarayanan; Gardel, Margaret L

    2017-02-28

    The actin cytoskeleton is a critical regulator of cytoplasmic architecture and mechanics, essential in a myriad of physiological processes. Here we demonstrate a liquid phase of actin filaments in the presence of the physiological cross-linker, filamin. Filamin condenses short actin filaments into spindle-shaped droplets, or tactoids, with shape dynamics consistent with a continuum model of anisotropic liquids. We find that cross-linker density controls the droplet shape and deformation timescales, consistent with a variable interfacial tension and viscosity. Near the liquid-solid transition, cross-linked actin bundles show behaviors reminiscent of fluid threads, including capillary instabilities and contraction. These data reveal a liquid droplet phase of actin, demixed from the surrounding solution and dominated by interfacial tension. These results suggest a mechanism to control organization, morphology, and dynamics of the actin cytoskeleton.

  4. 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.

  5. Nuclear Functions of Actin

    PubMed Central

    Visa, Neus; Percipalle, Piergiorgio

    2010-01-01

    Actin participates in several essential processes in the cell nucleus. Even though the presence of actin in the nucleus was proposed more than 30 years ago, nuclear processes that require actin have been only recently identified. Actin is part of chromatin remodeling complexes; it is associated with the transcription machineries; it becomes incorporated into newly synthesized ribonucleoproteins; and it influences long-range chromatin organization. As in the cytoplasm, nuclear actin works in conjunction with different types of actin-binding proteins that regulate actin function and bridge interactions between actin and other nuclear components. PMID:20452941

  6. 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

  7. 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.

  8. Eukaryotic chaperonin containing T-complex polypeptide 1 interacts with filamentous actin and reduces the initial rate of actin polymerization in vitro

    PubMed Central

    Grantham, Julie; Ruddock, Lloyd W.; Roobol, Anne; Carden, Martin J.

    2002-01-01

    We have previously observed that subunits of the chaperonin required for actin production (type-II chaperonin containing T-complex polypeptide 1 [CCT]) localize at sites of microfilament assembly. In this article we extend this observation by showing that substantially substoichiometric CCT reduces the initial rate of pyrene-labeled actin polymerization in vitro where eubacterial chaperonin GroEL had no such effect. CCT subunits bound selectively to F-actin in cosedimentation assays, and CCT reduced elongation rates from both purified actin filament “seeds” and the short and stabilized, minus-end blocked filaments in erythrocyte membrane cytoskeletons. These observations suggest CCT might remain involved in biogenesis of the actin cytoskeleton, by acting at filament (+) ends, beyond its already well-established role in producing new actin monomers. PMID:12482199

  9. Identification of sucrose synthase as an actin-binding protein

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  10. 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

  11. Septin Ring Assembly Requires Concerted Action of Polarisome Components, a PAK Kinase Cla4p, and the Actin Cytoskeleton in Saccharomyces cerevisiae

    PubMed Central

    Kadota, Jun; Yamamoto, Takaharu; Yoshiuchi, Shiro; Bi, Erfei; Tanaka, Kazuma

    2004-01-01

    Septins are filament-forming proteins that function in cytokinesis in a wide variety of organisms. In budding yeast, the small GTPase Cdc42p triggers the recruitment of septins to the incipient budding site and the assembly of septins into a ring. We herein report that Bni1p and Cla4p, effectors of Cdc42p, are required for the assembly of the septin ring during the initiation of budding but not for its maintenance after the ring converts to a septin collar. In bni1Δ cla4-75-td mutant, septins were recruited to the incipient budding site. However, the septin ring was not assembled, and septins remained at the polarized growing sites. Bni1p, a formin family protein, is a member of the polarisome complex with Spa2p, Bud6p, and Pea2p. All spa2Δ cla4-75-td, bud6Δ cla4-75-td, and pea2Δ cla4-75-td mutants showed defects in septin ring assembly. Bni1p stimulates actin polymerization for the formation of actin cables. Point mutants of BNI1 that are specifically defective in actin cable formation also exhibited septin ring assembly defects in the absence of Cla4p. Consistently, treatment of cla4Δ mutant with the actin inhibitor latrunculin A inhibited septin ring assembly. Our results suggest that polarisome components and Cla4p are required for the initial assembly of the septin ring and that the actin cytoskeleton is involved in this process. PMID:15371547

  12. 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

  13. 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.

  14. miR-181c-BRK1 axis plays a key role in actin cytoskeleton-dependent T cell function.

    PubMed

    Lim, Shok Ping; Ioannou, Nikolaos; Ramsay, Alan G; Darling, David; Gäken, Joop; Mufti, Ghulam J

    2018-05-01

    MicroRNAs are short endogenous noncoding RNAs that play pivotal roles in a diverse range of cellular processes. The miR-181 family is important in T cell development, proliferation, and activation. In this study, we have identified BRK1 as a potential target of miR-181c using a dual selection functional assay and have showed that miR-181c regulates BRK1 by translational inhibition. Given the importance of miR-181 in T cell function and the potential role of BRK1 in the involvement of WAVE2 complex and actin polymerization in T cells, we therefore investigated the influence of miR-181c-BRK1 axis in T cell function. Stimulation of PBMC derived CD3 + T cells resulted in reduced miR-181c expression and up-regulation of BRK1 protein expression, suggesting that miR-181c-BRK1 axis is important in T cell activation. We further showed that overexpression of miR-181c or suppression of BRK1 resulted in inhibition of T cell activation and actin polymerization coupled with defective lamellipodia generation and immunological synapse formation. Additionally, we found that BRK1 silencing led to reduced expressions of other proteins in the WAVE2 complex, suggesting that the impairment of T cell actin dynamics was a result of the instability of the WAVE2 complex following BRK1 depletion. Collectively, we demonstrated that miR-181c reduces BRK1 protein expression level and highlighted the important role of miR-181c-BRK1 axis in T cell activation and actin polymerization-mediated T cell functions. ©2018 Society for Leukocyte Biology.

  15. Environmental toxicants perturb human Sertoli cell adhesive function via changes in F-actin organization mediated by actin regulatory proteins

    PubMed Central

    Xiao, Xiang; Mruk, Dolores D.; Tang, Elizabeth I.; Wong, Chris K.C.; Lee, Will M.; John, Constance M.; Turek, Paul J.; Silvestrini, Bruno; Cheng, C. Yan

    2014-01-01

    STUDY QUESTION Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood–testis barrier (BTB)? SUMMARY ANSWER Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. WHAT IS KNOWN ALREADY Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. STUDY DESIGN, SIZE AND DURATION We examined the effects of two environmental toxicants: cadmium chloride (0.5–20 µM) and bisphenol A (0.4–200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. PARTICIPANTS/MATERIALS, SETTING, METHODS Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). MAIN RESULTS AND THE ROLE OF CHANCE Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by

  16. Orchestrating cytoskeleton and intracellular vesicle traffic to build functional immunological synapses.

    PubMed

    Soares, Helena; Lasserre, Rémi; Alcover, Andrés

    2013-11-01

    Immunological synapses are specialized cell-cell contacts formed between T lymphocytes and antigen-presenting cells. They are induced upon antigen recognition and are crucial for T-cell activation and effector functions. The generation and function of immunological synapses depend on an active T-cell polarization process, which results from a finely orchestrated crosstalk between the antigen receptor signal transduction machinery, the actin and microtubule cytoskeletons, and controlled vesicle traffic. Although we understand how some of these particular events are regulated, we still lack knowledge on how these multiple cellular elements are harmonized to ensure appropriate T-cell responses. We discuss here our view on how T-cell receptor signal transduction initially commands cytoskeletal and vesicle traffic polarization, which in turn sets the immunological synapse molecular design that regulates T-cell activation. We also discuss how the human immunodeficiency virus (HIV-1) hijacks some of these processes impairing immunological synapse generation and function. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. [Regulation of cortical cytoskeleton dynamics during migration of free-living amoebae].

    PubMed

    Kłopocka, Wanda; Redowicz, Maria Jolanta; Wasik, Anna

    2009-01-01

    Amoeba proteus and smaller by an order of magnitude (and evolutionary younger) Acanthamoeba castellanii have been for many years model cells for studies of amoeboidal (crawling) type of movement, characteristic also for some of metazoan cells such as fibroblasts, granulocytes and macrophages. Amoeboidal migration is indispensable of organization and dynamics of actin-based cytoskeleton. While there is a number of data on molecular mechanisms of motility of A. castellanii, there is very little known about bases of migration of A. proteus. Noteworthy, a large A. proteus (length approximately 600 microm) have been from over a century an object for studies on biology and physiology of cellular migration. This review describes the current knowledge on molecular aspects of force generation required for migration of these two amoebae and attempts to compare the functioning and regulation of actin cytoskeleton in these free-living unicellular species.

  3. Marine toxins and the cytoskeleton: a new view of palytoxin toxicity.

    PubMed

    Louzao, M Carmen; Ares, Isabel R; Cagide, Eva

    2008-12-01

    Palytoxin is a marine toxin first isolated from zoanthids (genus Palythoa), even though dinoflagellates of the genus Ostreopsis are the most probable origin of the toxin. Ostreopsis has a wide distribution in tropical and subtropical areas, but recently these dinoflagellates have also started to appear in the Mediterranean Sea. Two of the most remarkable properties of palytoxin are the large and complex structure (with different analogs, such as ostreocin-D or ovatoxin-a) and the extreme acute animal toxicity. The Na(+)/K(+)-ATPase has been proposed as receptor for palytoxin. The marine toxin is known to act on the Na(+) pump and elicit an increase in Na(+) permeability, which leads to depolarization and a secondary Ca(2+) influx, interfering with some functions of cells. Studies on the cellular cytoskeleton have revealed that the signaling cascade triggered by palytoxin leads to actin filament system distortion. The activity of palytoxin on the actin cytoskeleton is only partially associated with the cytosolic Ca(2+) changes; therefore, this ion represents an important factor in altering this structure, but it is not the only cause. The goal of the present minireview is to compile the findings reported to date about: (a) how palytoxin and analogs are able to modify the actin cytoskeleton within different cellular models; and (b) what signaling mechanisms could be involved in the modulation of cytoskeletal dynamics by palytoxin.

  4. 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

  5. Evolution of the cytoskeleton

    PubMed Central

    Erickson, Harold P.

    2009-01-01

    Summary The eukaryotic cytoskeleton appears to have evolved from ancestral precursors related to prokaryotic FtsZ and MreB. FtsZ and MreB show 40−50% sequence identity across different bacterial and archaeal species. Here I suggest that this represents the limit of divergence that is consistent with maintaining their functions for cytokinesis and cell shape. Previous analyses have noted that tubulin and actin are highly conserved across eukaryotic species, but so divergent from their prokaryotic relatives as to be hardly recognizable from sequence comparisons. One suggestion for this extreme divergence of tubulin and actin is that it occurred as they evolved very different functions from FtsZ and MreB. I will present new arguments favoring this suggestion, and speculate on pathways. Moreover, the extreme conservation of tubulin and actin across eukaryotic species is not due to an intrinsic lack of variability, but is attributed to their acquisition of elaborate mechanisms for assembly dynamics and their interactions with multiple motor and binding proteins. A new structure-based sequence alignment identifies amino acids that are conserved from FtsZ to tubulins. The highly conserved amino acids are not those forming the subunit core or protofilament interface, but those involved in binding and hydrolysis of GTP. PMID:17563102

  6. 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

  7. 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

  8. Identifying the dynamics of actin and tubulin polymerization in iPSCs and in iPSC-derived neurons

    PubMed Central

    Magliocca, Valentina; Petrini, Stefania; Franchin, Tiziana; Borghi, Rossella; Niceforo, Alessia; Abbaszadeh, Zeinab; Bertini, Enrico; Compagnucci, Claudia

    2017-01-01

    The development of the nervous system requires cytoskeleton-mediated processes coordinating self-renewal, migration, and differentiation of neurons. It is not surprising that many neurodevelopmental problems and neurodegenerative disorders are caused by deficiencies in cytoskeleton-related genes. For this reason, we focus on the cytoskeletal dynamics in proliferating iPSCs and in iPSC-derived neurons to better characterize the underpinnings of cytoskeletal organization looking at actin and tubulin repolymerization studies using the cell permeable probes SiR-Actin and SiR-Tubulin. During neurogenesis, each neuron extends an axon in a complex and changing environment to reach its final target. The dynamic behavior of the growth cone and its capacity to respond to multiple spatial information allows it to find its correct target. We decided to characterize various parameters of the actin filaments and microtubules. Our results suggest that a rapid re-organization of the cytoskeleton occurs 45 minutes after treatments with de-polymerizing agents in iPSCs and 60 minutes in iPSC-derived neurons in both actin filaments and microtubules. The quantitative data confirm that the actin filaments have a primary role in the re-organization of the cytoskeleton soon after de-polymerization, while microtubules have a major function following cytoskeletal stabilization. In conclusion, we investigate the possibility that de-polymerization of the actin filaments may have an impact on microtubules organization and that de-polymerization of the microtubules may affect the stability of the actin filaments. Our results suggest that a reciprocal influence of the actin filaments occurs over the microtubules and vice versa in both in iPSCs and iPSC-derived neurons. PMID:29340040

  9. 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

  10. 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.

  11. 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

  12. Diverse roles of actin in C. elegans early embryogenesis

    PubMed Central

    Velarde, Nathalie; Gunsalus, Kristin C; Piano, Fabio

    2007-01-01

    Background The actin cytoskeleton plays critical roles in early development in Caenorhabditis elegans. To further understand the complex roles of actin in early embryogenesis we use RNAi and in vivo imaging of filamentous actin (F-actin) dynamics. Results Using RNAi, we found processes that are differentially sensitive to levels of actin during early embryogenesis. Mild actin depletion shows defects in cortical ruffling, pseudocleavage, and establishment of polarity, while more severe depletion shows defects in polar body extrusion, cytokinesis, chromosome segregation, and eventually, egg production. These defects indicate that actin is required for proper oocyte development, fertilization, and a wide range of important events during early embryogenesis, including proper chromosome segregation. In vivo visualization of the cortical actin cytoskeleton shows dynamics that parallel but are distinct from the previously described myosin dynamics. Two distinct types of actin organization are observed at the cortex. During asymmetric polarization to the anterior, or the establishment phase (Phase I), actin forms a meshwork of microfilaments and focal accumulations throughout the cortex, while during the anterior maintenance phase (Phase II) it undergoes a morphological transition to asymmetrically localized puncta. The proper asymmetric redistribution is dependent on the PAR proteins, while both asymmetric redistribution and morphological transitions are dependent upon PFN-1 and NMY-2. Just before cytokinesis, actin disappears from most of the cortex and is only found around the presumptive cytokinetic furrow. Finally, we describe dynamic actin-enriched comets in the early embryo. Conclusion During early C. elegans embryogenesis actin plays more roles and its organization is more dynamic than previously described. Morphological transitions of F-actin, from meshwork to puncta, as well as asymmetric redistribution, are regulated by the PAR proteins. Results from this study

  13. Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner.

    PubMed

    Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

    2016-03-04

    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. 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.

  15. Disruption of Spectrin-Like Cytoskeleton in Differentiating Keratinocytes by PKCδ Activation Is Associated with Phosphorylated Adducin

    PubMed Central

    Zhao, Kong-Nan; Masci, Paul P.; Lavin, Martin F.

    2011-01-01

    Spectrin is a central component of the cytoskeletal protein network in a variety of erythroid and non-erythroid cells. In keratinocytes, this protein has been shown to be pericytoplasmic and plasma membrane associated, but its characteristics and function have not been established in these cells. Here we demonstrate that spectrin increases dramatically in amount and is assembled into the cytoskeleton during differentiation in mouse and human keratinocytes. The spectrin-like cytoskeleton was predominantly organized in the granular and cornified layers of the epidermis and disrupted by actin filament inhibitors, but not by anti-mitotic drugs. When the cytoskeleton was disrupted PKCδ was activated by phosphorylation on Thr505. Specific inhibition of PKCδ(Thr505) activation with rottlerin prevented disruption of the spectrin-like cytoskeleton and the associated morphological changes that accompany differentiation. Rottlerin also inhibited specific phosphorylation of the PKCδ substrate adducin, a cytoskeletal protein. Furthermore, knock-down of endogenous adducin affected not only expression of adducin, but also spectrin and PKCδ, and severely disrupted organization of the spectrin-like cytoskeleton and cytoskeletal distribution of both adducin and PKCδ. These results demonstrate that organization of a spectrin-like cytoskeleton is associated with keratinocytes differentiation, and disruption of this cytoskeleton is mediated by either PKCδ(Thr505) phosphorylation associated with phosphorylated adducin or due to reduction of endogenous adducin, which normally connects and stabilizes the spectrin-actin complex. PMID:22163289

  16. Role of actin in auxin transport and transduction of gravity

    NASA Astrophysics Data System (ADS)

    Hu, S.; Basu, S.; Brady, S.; Muday, G.

    Transport of the plant hormone auxin is polar and the direction of the hormone movement appears to be controlled by asymmetric distribution of auxin transport protein complexes. Changes in the direction of auxin transport are believed to drive asymmetric growth in response to changes in the gravity vector. To test the possibility that asymmetric distribution of the auxin transport protein complex is mediated by attachment to the actin cytoskeleton, a variety of experimental approaches have been used. The most direct demonstration of the role of the actin cytoskeleton in localization of the protein complex is the ability of one protein in this complex to bind to affinity columns containing actin filaments. Additionally, treatments of plant tissues with drugs that fragment the actin c toskeleton reducey polar transport. In order to explore this actin interaction and the affect of gravity on auxin transport and developmental polarity, embryos of the brown alga, Fucus have been examined. Fucus zygotes are initially symmetrical, but develop asymmetry in response to environmental gradients, with light gradients being the best- characterized signal. Gravity will polarize these embryos and gravity-induced polarity is randomized by clinorotation. Auxin transport also appears necessary for environmental controls of polarity, since auxin efflux inhibitors perturb both photo- and gravity-polarization at a very discrete temporal window within six hours after fertilization. The actin cytoskeleton has previously been shown to reorganize after fertilization of Fucus embryos leading to formation of an actin patch at the site of polar outgrowth. These actin patches still form in Fucus embryos treated with auxin efflux inhibitors, yet the position of these patches is randomized. Together, these results suggest that there are connections between the actin cytoskeleton, auxin transport, and gravity oriented growth and development. (Supported by NASA Grant: NAG2-1203)

  17. Oligomerization of coronin: Implication on actin filament length in Leishmania.

    PubMed

    Srivastava, Rashmi; Prasadareddy Kajuluri, Lova; Pathak, Neelam; Gupta, Chhitar M; Sahasrabuddhe, Amogh A

    2015-12-01

    Coronin proteins bind with actin filaments and participate in regulation of actin-dependent processes. These proteins contain a coiled-coil domain at their C-terminus, which is responsible for their dimeric or trimeric forms. However, the functional significance of these oligomeric configurations in organizing the actin cytoskeleton is obscure. Here, we report that the Leishmania coronin exists in a higher oligomeric form through its coiled-coil domain, the truncation of which ablates the ability of Leishmania coronin to assist actin-filament formation. F-actin co-sedimentation assay using purified proteins shows that the coiled-coil domain does not interact with actin-filaments and its absence does not abrogate actin-coronin interaction. Furthermore, it was shown that unlike other coronins, Leishmania coronin interacts with actin-filaments through its unique region. These results provided important insights into the role of coronin oligomerization in modulating actin-network. © 2015 Wiley Periodicals, Inc.

  18. 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.

  19. In vitro studies of actin filament and network dynamics

    PubMed Central

    Mullins, R Dyche; Hansen, Scott D

    2013-01-01

    Now that many genomes have been sequenced, a central concern of cell biology is to understand how the proteins they encode work together to create living matter. In vitro studies form an essential part of this program because understanding cellular functions of biological molecules often requires isolating them and reconstituting their activities. In particular, many elements of the actin cytoskeleton were first discovered by biochemical methods and their cellular functions deduced from in vitro experiments. We highlight recent advances that have come from in vitro studies, beginning with studies of actin filaments, and ending with multi-component reconstitutions of complex actin-based processes, including force-generation and cell spreading. We describe both scientific results and the technical innovations that made them possible. PMID:23267766

  20. 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

  1. CPG2 Recruits Endophilin B2 to the Cytoskeleton for Activity-Dependent Endocytosis of Synaptic Glutamate Receptors.

    PubMed

    Loebrich, Sven; Benoit, Marc Robert; Konopka, Jaclyn Aleksandra; Cottrell, Jeffrey Richard; Gibson, Joanne; Nedivi, Elly

    2016-02-08

    Internalization of glutamate receptors at the postsynaptic membrane via clathrin-mediated endocytosis (CME) is a key mechanism for regulating synaptic strength. A role for the F-actin cytoskeleton in CME is well established, and recently, PKA-dependent association of candidate plasticity gene 2 (CPG2) with the spine-cytoskeleton has been shown to mediate synaptic glutamate receptor internalization. Yet, how the endocytic machinery is physically coupled to the actin cytoskeleton to facilitate glutamate receptor internalization has not been demonstrated. Moreover, there has been no distinction of endocytic-machinery components that are specific to activity-dependent versus constitutive glutamate receptor internalization. Here, we show that CPG2, through a direct physical interaction, recruits endophilin B2 (EndoB2) to F-actin, thus anchoring the endocytic machinery to the spine cytoskeleton and facilitating glutamate receptor internalization. Regulation of CPG2 binding to the actin cytoskeleton by protein kinase A directly impacts recruitment of EndoB2 and clathrin. Specific disruption of EndoB2 or the CPG2-EndoB2 interaction impairs activity-dependent, but not constitutive, internalization of both NMDA- and AMPA-type glutamate receptors. These results demonstrate that, through direct interactions with F-actin and EndoB2, CPG2 physically bridges the spine cytoskeleton and the endocytic machinery, and this tripartite association is critical specifically for activity-dependent CME of synaptic glutamate receptors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. 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

  3. 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.

  4. Analysis of the cytoskeleton organization and its possible functions in male earthworm germ-line cysts equipped with a cytophore.

    PubMed

    Małota, Karol; Świątek, Piotr

    2016-10-01

    We studied the organization of F-actin and the microtubular cytoskeleton in male germ-line cysts in the seminal vesicles of the earthworm Dendrobaena veneta using light, fluorescent and electron microscopy along with both chemically fixed tissue and life cell imaging. Additionally, in order to follow the functioning of the cytoskeleton, we incubated the cysts in colchicine, nocodazole, cytochalasin D and latrunculin A. The male germ-line cells of D. veneta are interconnected via stable intercellular bridges (IB), and form syncytial cysts. Each germ cell has only one IB that connects it to the anuclear central cytoplasmic mass, the cytophore. During the studies, we analyzed the cytoskeleton in spermatogonial, spermatocytic and spermatid cysts. F-actin was detected in the cortical cytoplasm and forms distinct rings in the IBs. The arrangement of the microtubules changed dynamically during spermatogenesis. The microtubules are distributed evenly in whole spermatogonial and spermatocytic cysts; however, they primarily accumulate within the IBs in spermatogonia. In early spermatids, microtubules pass through the IBs and are present in whole cysts. During spermatid elongation, the microtubules form a manchette while they are absent in the cytophore and in the IBs. Use of cytoskeletal drugs did not alter the general morphology of the cysts. Detectable effects-the occurrence of nuclei in the late spermatids and manchette fragments in the cytophore-were observed only after incubation in nocodazole. Our results suggest that the microtubules are responsible for cytoplasmic/organelle transfer between the germ cells and the cytophore during spermatogenesis and for the positioning of the spermatid nuclei.

  5. 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.

  6. Functional correction of dystrophin actin binding domain mutations by genome editing

    PubMed Central

    Kyrychenko, Viktoriia; Kyrychenko, Sergii; Tiburcy, Malte; Shelton, John M.; Long, Chengzu; Schneider, Jay W.; Zimmermann, Wolfram-Hubertus; Bassel-Duby, Rhonda

    2017-01-01

    Dystrophin maintains the integrity of striated muscles by linking the actin cytoskeleton with the cell membrane. Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD) that result in progressive, debilitating muscle weakness, cardiomyopathy, and a shortened lifespan. Mutations of dystrophin that disrupt the amino-terminal actin-binding domain 1 (ABD-1), encoded by exons 2–8, represent the second-most common cause of DMD. In the present study, we compared three different strategies for CRISPR/Cas9 genome editing to correct mutations in the ABD-1 region of the DMD gene by deleting exons 3–9, 6–9, or 7–11 in human induced pluripotent stem cells (iPSCs) and by assessing the function of iPSC-derived cardiomyocytes. All three exon deletion strategies enabled the expression of truncated dystrophin protein and restoration of cardiomyocyte contractility and calcium transients to varying degrees. We show that deletion of exons 3–9 by genomic editing provides an especially effective means of correcting disease-causing ABD-1 mutations. These findings represent an important step toward eventual correction of common DMD mutations and provide a means of rapidly assessing the expression and function of internally truncated forms of dystrophin-lacking portions of ABD-1. PMID:28931764

  7. 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

  8. 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

  9. 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.

  10. Transforming growth factor β-induced superficial zone protein accumulation in the surface zone of articular cartilage is dependent on the cytoskeleton.

    PubMed

    McNary, Sean M; Athanasiou, Kyriacos A; Reddi, A Hari

    2014-03-01

    The phenotype of articular chondrocytes is dependent on the cytoskeleton, specifically the actin microfilament architecture. Articular chondrocytes in monolayer culture undergo dedifferentiation and assume a fibroblastic phenotype. This process can be reversed by altering the actin cytoskeleton by treatment with cytochalasin. Whereas dedifferentiation has been studied on chondrocytes isolated from the whole cartilage, the effects of cytoskeletal alteration on specific zones of cells such as superficial zone chondrocytes are not known. Chondrocytes from the superficial zone secrete superficial zone protein (SZP), a lubricating proteoglycan that reduces the coefficient of friction of articular cartilage. A better understanding of this phenomenon may be useful in elucidating chondrocyte dedifferentiation in monolayer and accumulation of the cartilage lubricant SZP, with an eye toward tissue engineering functional articular cartilage. In this investigation, the effects of cytoskeletal modulation on the ability of superficial zone chondrocytes to secrete SZP were examined. Primary superficial zone chondrocytes were cultured in monolayer and treated with a combination of cytoskeleton modifying reagents and transforming growth factor β (TGFβ) 1, a critical regulator of SZP production. Whereas cytochalasin D maintains the articular chondrocyte phenotype, the hallmark of the superficial zone chondrocyte, SZP, was inhibited in the presence of TGFβ1. A decrease in TGFβ1-induced SZP accumulation was also observed when the microtubule cytoskeleton was modified using paclitaxel. These effects of actin and microtubule alteration were confirmed through the application of jasplakinolide and colchicine, respectively. As Rho GTPases regulate actin organization and microtubule polymerization, we hypothesized that the cytoskeleton is critical for TGFβ-induced SZP accumulation. TGFβ-mediated SZP accumulation was inhibited by small molecule inhibitors ML141 (Cdc42), NSC23766 (Rac1

  11. 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.

  12. Drosophila Spire is an actin nucleation factor.

    PubMed

    Quinlan, Margot E; Heuser, John E; Kerkhoff, Eugen; Mullins, R Dyche

    2005-01-27

    The actin cytoskeleton is essential for many cellular functions including shape determination, intracellular transport and locomotion. Previous work has identified two factors--the Arp2/3 complex and the formin family of proteins--that nucleate new actin filaments via different mechanisms. Here we show that the Drosophila protein Spire represents a third class of actin nucleation factor. In vitro, Spire nucleates new filaments at a rate that is similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex, and it remains associated with the slow-growing pointed end of the new filament. Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer. Maximal nucleation activity requires all four WH2 domains along with an additional actin-binding motif, conserved among Spire proteins. Spire itself is conserved among metazoans and, together with the formin Cappuccino, is required for axis specification in oocytes and embryos, suggesting that multiple actin nucleation factors collaborate to construct essential cytoskeletal structures.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. Actin Turnover-Mediated Gravity Response in Maize Root Apices

    PubMed Central

    Mancuso, Stefano; Barlow, Peter W; Volkmann, Dieter

    2006-01-01

    The dynamic actin cytoskeleton has been proposed to be linked to gravity sensing in plants but the mechanistic understanding of these processes remains unknown. We have performed detailed pharmacological analyses of the role of the dynamic actin cytoskeleton in gravibending of maize (Zea mays) root apices. Depolymerization of actin filaments with two drugs having different mode of their actions, cytochalasin D and latrunculin B, stimulated root gravibending. By contrast, drug-induced stimulation of actin polymerization and inhibition of actin turnover, using two different agents phalloidin and jasplakinolide, compromised the root gravibending. Importantly, all these actin drugs inhibited root growth to similar extents suggesting that high actin turnover is essential for the gravity-related growth responses rather than for the general growth process. Both latrunculin B and cytochalasin D treatments inhibited root growth but restored gravibending of the decapped root apices, indicating that there is a strong potential for effective actin-mediated gravity sensing outside the cap. This elusive gravity sensing outside the root cap is dependent not only on the high rate of actin turnover but also on weakening of myosin activities, as general inhibition of myosin ATPases induced stimulation of gravibending of the decapped root apices. Collectively, these data provide evidence for the actin turnover-mediated gravity sensing outside the root cap. PMID:19521476

  2. 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 cytoskeletonactin, 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

  3. Actin dynamics at focal adhesions: a common endpoint and putative therapeutic target for proteinuric kidney diseases.

    PubMed

    Sever, Sanja; Schiffer, Mario

    2018-06-01

    Proteinuria encompasses diverse causes including both genetic diseases and acquired forms such as diabetic and hypertensive nephropathy. The basis of proteinuria is a disturbance in size selectivity of the glomerular filtration barrier, which largely depends on the podocyte: a terminally differentiated epithelial cell type covering the outer surface of the glomerulus. Compromised podocyte structure is one of the earliest signs of glomerular injury. The phenotype of diverse animal models and podocyte cell culture firmly established the essential role of the actin cytoskeleton in maintaining functional podocyte structure. Podocyte foot processes, actin-based membrane extensions, contain 2 molecularly distinct "hubs" that control actin dynamics: a slit diaphragm and focal adhesions. Although loss of foot processes encompasses disassembly of slit diaphragm multiprotein complexes, as long as cells are attached to the glomerular basement membrane, focal adhesions will be the sites in which stress due to filtration flow is counteracted by forces generated by the actin network in foot processes. Numerous studies within last 20 years have identified actin binding and regulatory proteins as well as integrins as essential components of signaling and actin dynamics at focal adhesions in podocytes, suggesting that some of them may become novel, druggable targets for proteinuric kidney diseases. Here we review evidence supporting the idea that current treatments for chronic kidney diseases beneficially and directly target the podocyte actin cytoskeleton associated with focal adhesions and suggest that therapeutic reagents that target the focal adhesion-regulated actin cytoskeleton in foot processes have potential to modernize treatments for chronic kidney diseases. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  4. Dendrite architecture organized by transcriptional control of the F-actin nucleator Spire.

    PubMed

    Ferreira, Tiago; Ou, Yimiao; Li, Sally; Giniger, Edward; van Meyel, Donald J

    2014-02-01

    The architectures of dendritic trees are crucial for the wiring and function of neuronal circuits because they determine coverage of receptive territories, as well as the nature and strength of sensory or synaptic inputs. Here, we describe a cell-intrinsic pathway sculpting dendritic arborization (da) neurons in Drosophila that requires Longitudinals Lacking (Lola), a BTB/POZ transcription factor, and its control of the F-actin cytoskeleton through Spire (Spir), an actin nucleation protein. Loss of Lola from da neurons reduced the overall length of dendritic arbors, increased the expression of Spir, and produced inappropriate F-actin-rich dendrites at positions too near the cell soma. Selective removal of Lola from only class IV da neurons decreased the evasive responses of larvae to nociception. The increased Spir expression contributed to the abnormal F-actin-rich dendrites and the decreased nocifensive responses because both were suppressed by reduced dose of Spir. Thus, an important role of Lola is to limit expression of Spir to appropriate levels within da neurons. We found Spir to be expressed in dendritic arbors and to be important for their development. Removal of Spir from class IV da neurons reduced F-actin levels and total branch number, shifted the position of greatest branch density away from the cell soma, and compromised nocifensive behavior. We conclude that the Lola-Spir pathway is crucial for the spatial arrangement of branches within dendritic trees and for neural circuit function because it provides balanced control of the F-actin cytoskeleton.

  5. Ultrastructure and cytoskeleton of Chara rhizoids in microgravity.

    PubMed

    Braun, M; Buchen, B; Sievers, A

    1999-01-01

    Gravitropic tip growth of Chara rhizoids is dependent on the presence and functional interaction between statoliths, cytoskeleton and the tip-growth-organizing complex, the Spitzenkorper. Microtubules are essential for the polar cytoplasmic zonation but are excluded from the apex and do not play a crucial role in the primary steps of gravisensing and graviresponse. Actin filaments form a dense meshwork in the subapical zone and converge into a prominent apical actin patch which is associated with the endoplasmic reticulum (ER) aggregate representing the structural center of the Spitzenkorper. The position of the statoliths is regulated by gravity and a counteracting force mediated by actomyosin. Reducing the acceleration forces in microgravity experiments causes a basipetal displacement of the statoliths. Rhizoids grow randomly in all directions. However, they express the same cell shape and cytoplasmic zonation as ground controls. The ultrastructure of the Spitzenkorper, including the aggregation of ER, the assembly of vesicles in the apex, the polar distribution of proplastids, mitochondria, dictyosomes and ER cisternae in the subapical zone is maintained. The unaltered cytoskeletal organization, growth rates and gravitropic responsiveness indicate that microgravity has no major effect on gravitropic tip-growing Chara rhizoids. However, the threshold value of gravisensitivity might be different from ground controls due to the altered position of statoliths, a possibly reduced amount of BaSO4 in statoliths and a possible adaptation of the actin cytoskeleton to microgravity conditions.

  6. Ultrastructure and cytoskeleton of Chara rhizoids in microgravity

    NASA Astrophysics Data System (ADS)

    Braun, M.; Buchen, B.; Sievers, A.

    1999-01-01

    Gravitropic tip growth of Chara rhizoids is dependent on the presence and functional interaction between statoliths, cytoskeleton and the tip-growth-organizing complex, the Spitzenkörper. Microtubules are essential for the polar cytoplasmic zonation but are excluded from the apex and do not play a crucial role in the primary steps of gravisensing and graviresponse. Actin filaments form a dense meshwork in the subapical zone and converge into a prominent apical actin patch which is associated with the endoplasmic reticulum (ER) aggregate representing the structural center of the Spitzenkörper. The position of the statoliths is regulated by gravity and a counteracting force mediated by actomyosin. Reducing the acceleration forces in microgravity experiments causes a basipetal displacement of the statoliths. Rhizoids grow randomly in all directions. However, they express the same cell shape and cytoplasmic zonation as ground controls. The ultrastructure of the Spitzenkörper, including the aggregation of ER, the assembly of vesicles in the apex, the polar distribution of proplastids, mitochondria, dictyosomes and ER cisternae in the subapical zone is maintained. The unaltered cytoskeletal organization, growth rates and gravitropic responsiveness indicate that microgravity has no major effect on gravitropic tip-growing Chara rhizoids. However, the threshold value of gravisensitivity might be different from ground controls due to the altered position of statoliths, a possibly reduced amount of BaSO4 in statoliths and a possible adaptation of the actin cytoskeleton to microgravity conditions.

  7. Disrupting the cortical actin cytoskeleton points to two distinct mechanisms of yeast [PSI+] prion formation

    PubMed Central

    Speldewinde, Shaun H.; Tuite, Mick F.

    2017-01-01

    Mammalian and fungal prions arise de novo; however, the mechanism is poorly understood in molecular terms. One strong possibility is that oxidative damage to the non-prion form of a protein may be an important trigger influencing the formation of its heritable prion conformation. We have examined the oxidative stress-induced formation of the yeast [PSI+] prion, which is the altered conformation of the Sup35 translation termination factor. We used tandem affinity purification (TAP) and mass spectrometry to identify the proteins which associate with Sup35 in a tsa1 tsa2 antioxidant mutant to address the mechanism by which Sup35 forms the [PSI+] prion during oxidative stress conditions. This analysis identified several components of the cortical actin cytoskeleton including the Abp1 actin nucleation promoting factor, and we show that deletion of the ABP1 gene abrogates oxidant-induced [PSI+] prion formation. The frequency of spontaneous [PSI+] prion formation can be increased by overexpression of Sup35 since the excess Sup35 increases the probability of forming prion seeds. In contrast to oxidant-induced [PSI+] prion formation, overexpression-induced [PSI+] prion formation was only modestly affected in an abp1 mutant. Furthermore, treating yeast cells with latrunculin A to disrupt the formation of actin cables and patches abrogated oxidant-induced, but not overexpression-induced [PSI+] prion formation, suggesting a mechanistic difference in prion formation. [PIN+], the prion form of Rnq1, localizes to the IPOD (insoluble protein deposit) and is thought to influence the aggregation of other proteins. We show Sup35 becomes oxidized and aggregates during oxidative stress conditions, but does not co-localize with Rnq1 in an abp1 mutant which may account for the reduced frequency of [PSI+] prion formation. PMID:28369054

  8. 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

  9. Myopodin is an F-actin bundling protein with multiple independent actin-binding regions.

    PubMed

    Linnemann, Anja; Vakeel, Padmanabhan; Bezerra, Eduardo; Orfanos, Zacharias; Djinović-Carugo, Kristina; van der Ven, Peter F M; Kirfel, Gregor; Fürst, Dieter O

    2013-02-01

    The assembly of striated muscle myofibrils is a multistep process in which a variety of proteins is involved. One of the first and most important steps in myofibrillogenesis is the arrangement of thin myofilaments into ordered I-Z-I brushes, requiring the coordinated activity of numerous actin binding proteins. The early expression of myopodin prior to sarcomeric α-actinin, as well as its binding to actin, α-actinin and filamin indicate an important role for this protein in actin cytoskeleton remodelling with the precise function of myopodin in this process yet remaining to be resolved. While myopodin was previously described as a protein capable of cross-linking actin filaments into thick bundles upon transient transfections, it has remained unclear whether myopodin alone is capable of bundling actin, or if additional proteins are involved. We have therefore investigated the in vitro actin binding properties of myopodin. High speed cosedimentation assays with skeletal muscle actin confirmed direct binding of myopodin to F-actin and showed that this interaction is mediated by at least two independent actin binding sites, found in all myopodin isoforms identified to date. Furthermore, low-speed cosedimentation assays revealed that not only full length myopodin, but also the fragment containing only the second binding site, bundles microfilaments in the absence of accessory proteins. Ultrastructural analysis demonstrated that this bundling activity resembled that of α-actinin. Biochemical experiments revealed that bundling was not achieved by myopodin's ability to dimerize, indicating the presence of two individual F-actin binding sites within the second binding segment. Thus full length myopodin contains at least three F-actin binding sites. These data provide further understanding of the mechanisms by which myopodin contributes to actin reorganization during myofibril assembly.

  10. 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.

  11. Involvement of the sieve element cytoskeleton in electrical responses to cold shocks.

    PubMed

    Hafke, Jens B; Ehlers, Katrin; Föller, Jens; Höll, Sabina-Roxana; Becker, Stefanie; van Bel, Aart J E

    2013-06-01

    This study dealt with the visualization of the sieve element (SE) cytoskeleton and its involvement in electrical responses to local cold shocks, exemplifying the role of the cytoskeleton in Ca(2+)-triggered signal cascades in SEs. High-affinity fluorescent phalloidin as well as immunocytochemistry using anti-actin antibodies demonstrated a fully developed parietal actin meshwork in SEs. The involvement of the cytoskeleton in electrical responses and forisome conformation changes as indicators of Ca(2+) influx was investigated by the application of cold shocks in the presence of diverse actin disruptors (latrunculin A and cytochalasin D). Under control conditions, cold shocks elicited a graded initial voltage transient, ΔV1, reduced by external La(3+) in keeping with the involvement of Ca(2+) channels, and a second voltage transient, ΔV2. Cytochalasin D had no effect on ΔV1, while ΔV1 was significantly reduced with 500 nm latrunculin A. Forisome dispersion was triggered by cold shocks of 4°C or greater, which was indicative of an all-or-none behavior. Forisome dispersion was suppressed by incubation with latrunculin A. In conclusion, the cytoskeleton controls cold shock-induced Ca(2+) influx into SEs, leading to forisome dispersion and sieve plate occlusion in fava bean (Vicia faba).

  12. 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.

  13. 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

  14. Inhibition of invasion by glycogen synthase kinase-3 beta inhibitors through dysregulation of actin re-organisation via down-regulation of WAVE2.

    PubMed

    Yoshino, Yuki; Suzuki, Manami; Takahashi, Hidekazu; Ishioka, Chikashi

    2015-08-14

    Cancer cell invasion is a critical phenomenon in cancer pathogenesis. Glycogen synthase kinase-3β (GSK-3β) has been reported to regulate cancer cell invasion both negatively and positively. Thus, the net effect of GSK-3β on invasion is unclear. In this report, we showed that GSK-3β inhibitors induced dysregulation of the actin cytoskeleton and functional insufficiency of focal adhesion, which resulted in suppressed invasion. In addition, WAVE2, an essential molecule for actin fibre branching, was down-regulated after GSK-3β inhibition. Collectively, we propose that the WAVE2-actin cytoskeleton axis is an important target of GSK-3β inhibitors in cancer cell invasion. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. 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

  16. 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

  17. 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

  18. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

    NASA Technical Reports Server (NTRS)

    Butler, J. H.; Hu, S.; Brady, S. R.; Dixon, M. W.; Muday, G. K.

    1998-01-01

    The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.

  19. An epidermal plakin that integrates actin and microtubule networks at cellular junctions.

    PubMed

    Karakesisoglou, I; Yang, Y; Fuchs, E

    2000-04-03

    Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.

  20. Allele-specific Effects of Human Deafness γ-Actin Mutations (DFNA20/26) on the Actin/Cofilin Interaction*

    PubMed Central

    Bryan, Keith E.; Rubenstein, Peter A.

    2009-01-01

    Auditory hair cell function requires proper assembly and regulation of the nonmuscle gamma isoactin-rich cytoskeleton, and six point mutations in this isoactin cause a type of delayed onset autosomal dominant nonsyndromic progressive hearing loss, DFNA20/26. The molecular basis underlying this actin-dependent hearing loss is unknown. To address this problem, the mutations have been introduced into yeast actin, and their effects on actin function were assessed in vivo and in vitro. Because we previously showed that polymerization was unaffected in five of the six mutants, we have focused on proteins that regulate actin, in particular cofilin, which severs F-actin and sequesters actin monomers. The mutations do not affect the interaction of cofilin with G-actin. However, T89I and V370A mutant F-actins are much more susceptible to cofilin disassembly than WT filaments in vitro. Conversely, P332A filaments demonstrate enhanced resistance. Wild type actin solutions containing T89I, K118M, or P332A mutant actins at mole fractions similar to those found in the hair cell respond in vitro toward cofilin in a manner proportional to the level of the mutant present. Finally, depression of cofilin action in vivo by elimination of the cofilin-activating protein, Aip1p, rescues the inability to grow on glycerol caused by K118M, T278I, P332A, and V370A. These results suggest that a filament instability caused by these mutations can be balanced by decreasing a system in vivo that promotes increased filament turnover. Such mutant-dependent filament destabilization could easily result in hair cell malfunction leading to the late-onset hearing loss observed in these patients. PMID:19419963

  1. 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

  2. eNOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1

    PubMed Central

    García-Ortiz, Almudena; Martín-Cofreces, Noa B.; Ibiza, Sales; Ortega, Ángel; Izquierdo-Álvarez, Alicia; Trullo, Antonio; Victor, Víctor M.; Calvo, Enrique; Sot, Begoña; Martínez-Ruiz, Antonio; Vázquez, Jesús; Sánchez-Madrid, Francisco

    2017-01-01

    The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of β-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS. PMID:28394935

  3. Cytoskeleton in motion: the dynamics of keratin intermediate filaments in epithelia.

    PubMed

    Windoffer, Reinhard; Beil, Michael; Magin, Thomas M; Leube, Rudolf E

    2011-09-05

    Epithelia are exposed to multiple forms of stress. Keratin intermediate filaments are abundant in epithelia and form cytoskeletal networks that contribute to cell type-specific functions, such as adhesion, migration, and metabolism. A perpetual keratin filament turnover cycle supports these functions. This multistep process keeps the cytoskeleton in motion, facilitating rapid and protein biosynthesis-independent network remodeling while maintaining an intact network. The current challenge is to unravel the molecular mechanisms underlying the regulation of the keratin cycle in relation to actin and microtubule networks and in the context of epithelial tissue function.

  4. Cytoskeleton in motion: the dynamics of keratin intermediate filaments in epithelia

    PubMed Central

    Windoffer, Reinhard; Beil, Michael; Magin, Thomas M.

    2011-01-01

    Epithelia are exposed to multiple forms of stress. Keratin intermediate filaments are abundant in epithelia and form cytoskeletal networks that contribute to cell type–specific functions, such as adhesion, migration, and metabolism. A perpetual keratin filament turnover cycle supports these functions. This multistep process keeps the cytoskeleton in motion, facilitating rapid and protein biosynthesis–independent network remodeling while maintaining an intact network. The current challenge is to unravel the molecular mechanisms underlying the regulation of the keratin cycle in relation to actin and microtubule networks and in the context of epithelial tissue function. PMID:21893596

  5. Single-Molecule Studies of Actin Assembly and Disassembly Factors

    PubMed Central

    Smith, Benjamin A.; Gelles, Jeff; Goode, Bruce L.

    2014-01-01

    The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks. PMID:24630103

  6. 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.

  7. 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.

  8. Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton

    NASA Technical Reports Server (NTRS)

    Muday, G. K.

    2000-01-01

    In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.

  9. RNA-binding proteins of the NXF (nuclear export factor) family and their connection with the cytoskeleton.

    PubMed

    Mamon, L A; Ginanova, V R; Kliver, S F; Yakimova, A O; Atsapkina, A A; Golubkova, E V

    2017-04-01

    The mutual relationship between mRNA and the cytoskeleton can be seen from two points of view. On the one hand, the cytoskeleton is necessary for mRNA trafficking and anchoring to subcellular domains. On the other hand, cytoskeletal growth and rearrangement require the translation of mRNAs that are connected to the cytoskeleton. β-actin mRNA localization may influence dynamic changes in the actin cytoskeleton. In the cytoplasm, long-lived mRNAs exist in the form of RNP (ribonucleoprotein) complexes, where they interact with RNA-binding proteins, including NXF (Nuclear eXport Factor). Dm NXF1 is an evolutionarily conserved protein in Drosophila melanogaster that has orthologs in different animals. The universal function of nxf1 genes is the nuclear export of different mRNAs in various organisms. In this mini-review, we briefly discuss the evidence demonstrating that Dm NXF1 fulfils not only universal but also specialized cytoplasmic functions. This protein is detected not only in the nucleus but also in the cytoplasm. It is a component of neuronal granules. Dm NXF1 marks nuclear division spindles during early embryogenesis and the dense body on one side of the elongated spermatid nuclei. The characteristic features of sbr mutants (sbr 10 and sbr 5 ) are impairment of chromosome segregation and spindle formation anomalies during female meiosis. sbr 12 mutant sterile males with immobile spermatozoa exhibit disturbances in the axoneme, mitochondrial derivatives and cytokinesis. These data allow us to propose that the Dm NXF1 proteins transport certain mRNAs in neurites and interact with localized mRNAs that are necessary for dynamic changes of the cytoskeleton. © 2017 Wiley Periodicals, Inc.

  10. Changes in actin dynamics are involved in salicylic acid signaling pathway.

    PubMed

    Matoušková, Jindřiška; Janda, Martin; Fišer, Radovan; Sašek, Vladimír; Kocourková, Daniela; Burketová, Lenka; Dušková, Jiřina; Martinec, Jan; Valentová, Olga

    2014-06-01

    Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1 mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200 nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  11. F-Actin Dynamics in Neurospora crassa ▿ †

    PubMed Central

    Berepiki, Adokiye; Lichius, Alexander; Shoji, Jun-Ya; Tilsner, Jens; Read, Nick D.

    2010-01-01

    This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth. PMID:20139238

  12. 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

  13. Biophysical model of the role of actin remodeling on dendritic spine morphology

    PubMed Central

    Miermans, C. A.; Kusters, R. P. T.; Hoogenraad, C. C.; Storm, C.

    2017-01-01

    Dendritic spines are small membranous structures that protrude from the neuronal dendrite. Each spine contains a synaptic contact site that may connect its parent dendrite to the axons of neighboring neurons. Dendritic spines are markedly distinct in shape and size, and certain types of stimulation prompt spines to evolve, in fairly predictable fashion, from thin nascent morphologies to the mushroom-like shapes associated with mature spines. It is well established that the remodeling of spines is strongly dependent upon the actin cytoskeleton inside the spine. A general framework that details the precise role of actin in directing the transitions between the various spine shapes is lacking. We address this issue, and present a quantitative, model-based scenario for spine plasticity validated using realistic and physiologically relevant parameters. Our model points to a crucial role for the actin cytoskeleton. In the early stages of spine formation, the interplay between the elastic properties of the spine membrane and the protrusive forces generated in the actin cytoskeleton propels the incipient spine. In the maturation stage, actin remodeling in the form of the combined dynamics of branched and bundled actin is required to form mature, mushroom-like spines. Importantly, our model shows that constricting the spine-neck aids in the stabilization of mature spines, thus pointing to a role in stabilization and maintenance for additional factors such as ring-like F-actin structures. Taken together, our model provides unique insights into the fundamental role of actin remodeling and polymerization forces during spine formation and maturation. PMID:28158194

  14. 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.

  15. 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.

  16. Dynamics of Actin Cables in Polarized Growth of the Filamentous Fungus Aspergillus nidulans

    PubMed Central

    Bergs, Anna; Ishitsuka, Yuji; Evangelinos, Minoas; Nienhaus, G. U.; Takeshita, Norio

    2016-01-01

    Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although, specific marker proteins have been developed to visualize actin cables in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here, we observed actin cables using tropomyosin (TpmA) and Lifeact fused to fluorescent proteins in living Aspergillus nidulans hyphae and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 μm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules. PMID:27242709

  17. The HDAC complex and cytoskeleton.

    PubMed

    Kovacs, Jeffery J; Hubbert, Charlotte; Yao, Tso-Pang

    2004-01-01

    HDAC6 is a cytoplasmic deacetylase that dynamically associates with the microtubule and actin cytoskeletons. HDAC6 regulates growth factor-induced chemotaxis by its unique deacetylase activity towards microtubules or other substrates. Here we describe a non-catalytic structural domain that is essential for HDAC6 function and places HDAC6 as a critical mediator linking the acetylation and ubiquitination network. This evolutionarily conserved motif, termed the BUZ domain, has features of a zinc finger and binds both mono- and polyubiquitinated proteins. Furthermore, the BUZ domain promotes HDAC6 mono-ubiquitination. These results establish the BUZ domain, in addition to the UIM and CUE domains, as a novel motif that both binds ubiquitin and mediates mono-ubiquitination. Importantly, the BUZ domain is essential for HDAC6 to promote chemotaxis, indicating that communication with the ubiquitin network is critical for proper HDAC6 function. The unique presence of the UIM and CUE domains in proteins involved in endocytic trafficking suggests that HDAC6 might also regulate vesicle transport and protein degradation. Indeed, we have found that HDAC6 is actively transported and concentrated in vesicular compartments. We propose that an integration of reversible acetylation and ubiquitination by HDAC6 may be a novel component in regulating the cytoskeleton, vesicle transport and protein degradation.

  18. 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.

  19. Actin-filament disassembly: it takes two to shrink them fast.

    PubMed

    Winterhoff, Moritz; Faix, Jan

    2015-06-01

    Actin-filament disassembly is indispensable for replenishing the pool of polymerizable actin and allows continuous dynamic remodelling of the actin cytoskeleton. A new study now reveals that ADF/cofilin preferentially dismantles branched networks and provides new insights into the collaborative work of ADF/cofilin and Aip1 on filament disassembly at the molecular level. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. 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

  1. Continuum mechanical model for cross-linked actin networks with contractile bundles

    NASA Astrophysics Data System (ADS)

    Ferreira, J. P. S.; Parente, M. P. L.; Natal Jorge, R. M.

    2018-01-01

    In the context of a mechanical approach to cell biology, there is a close relationship between cellular function and mechanical properties. In recent years, an increasing amount of attention has been given to the coupling between biochemical and mechanical signals by means of constitutive models. In particular, on the active contractility of the actin cytoskeleton. Given the importance of the actin contraction on the physiological functions, this study propose a constitutive model to describe how the filamentous network controls its mechanics actively. Embedded in a soft isotropic ground substance, the network behaves as a viscous mechanical continuum, comprised of isotropically distributed cross-linked actin filaments and actomyosin bundles. Trough virtual rheometry experiments, the present model relates the dynamics of the myosin motors with the network stiffness, which is to a large extent governed by the time-scale of the applied deformations/forces.

  2. Involvement of the Sieve Element Cytoskeleton in Electrical Responses to Cold Shocks1[W

    PubMed Central

    Hafke, Jens B.; Ehlers, Katrin; Föller, Jens; Höll, Sabina-Roxana; Becker, Stefanie; van Bel, Aart J.E.

    2013-01-01

    This study dealt with the visualization of the sieve element (SE) cytoskeleton and its involvement in electrical responses to local cold shocks, exemplifying the role of the cytoskeleton in Ca2+-triggered signal cascades in SEs. High-affinity fluorescent phalloidin as well as immunocytochemistry using anti-actin antibodies demonstrated a fully developed parietal actin meshwork in SEs. The involvement of the cytoskeleton in electrical responses and forisome conformation changes as indicators of Ca2+ influx was investigated by the application of cold shocks in the presence of diverse actin disruptors (latrunculin A and cytochalasin D). Under control conditions, cold shocks elicited a graded initial voltage transient, ΔV1, reduced by external La3+ in keeping with the involvement of Ca2+ channels, and a second voltage transient, ΔV2. Cytochalasin D had no effect on ΔV1, while ΔV1 was significantly reduced with 500 nm latrunculin A. Forisome dispersion was triggered by cold shocks of 4°C or greater, which was indicative of an all-or-none behavior. Forisome dispersion was suppressed by incubation with latrunculin A. In conclusion, the cytoskeleton controls cold shock-induced Ca2+ influx into SEs, leading to forisome dispersion and sieve plate occlusion in fava bean (Vicia faba). PMID:23624858

  3. Rheology of Membrane-Attached Minimal Actin Cortices.

    PubMed

    Nöding, Helen; Schön, Markus; Reinermann, Corinna; Dörrer, Nils; Kürschner, Aileen; Geil, Burkhard; Mey, Ingo; Heussinger, Claus; Janshoff, Andreas; Steinem, Claudia

    2018-04-26

    The actin cortex is a thin cross-linked network attached to the plasma membrane, which is responsible for the cell's shape during migration, division, and growth. In a reductionist approach, we created a minimal actin cortex (MAC) attached to a lipid membrane to correlate the filamentous actin architecture with its viscoelastic properties. The system is composed of a supported 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine bilayer doped with the receptor lipid phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P 2 ) to which a constitutively active mutant of ezrin, which is a direct membrane-cytoskeleton linker, is bound. The formation of the MAC on the supported lipid bilayer is analyzed as a function of increasing PtdIns(4,5)P 2 /ezrin pinning points, revealing an increase in the intersections between actin filaments, that is, the node density of the MAC. Bead tracking microrheology on the membrane-attached actin network provides information about its viscoelastic properties. The results show that ezrin serves as a dynamic cross-linker for the actin cortex attached to the lipid bilayer and that the stiffness of the network is influenced by the pinning point density, relating the plateau storage modulus G 0 to the node density of the MAC.

  4. 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

  5. Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation.

    PubMed

    Barrès, Romain; Grémeaux, Thierry; Gual, Philippe; Gonzalez, Teresa; Gugenheim, Jean; Tran, Albert; Le Marchand-Brustel, Yannick; Tanti, Jean-François

    2006-11-01

    APS (adaptor protein with PH and SH2 domains) initiates a phosphatidylinositol 3-kinase-independent pathway involved in insulin-stimulated glucose transport. We recently identified Enigma, a PDZ and LIM domain-containing protein, as a partner of APS and showed that APS-Enigma complex plays a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma mRNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin-induced actin rearrangements, whereas the expression of Enigma without its LIM domains was without effect. A physiological link between increased expression of Enigma and an alteration in insulin-induced glucose uptake was suggested by the increase in Enigma mRNA expression in adipose tissue of diabetic obese patients. Taken together, these data strongly suggest that the interaction between APS and Enigma is involved in insulin-induced Glut 4 translocation by regulating cortical actin remodeling and raise the possibility that modification of APS/Enigma ratio could participate in the alteration of insulin-induced glucose uptake in adipose tissue.

  6. Scd5p and Clathrin Function Are Important for Cortical Actin Organization, Endocytosis, and Localization of Sla2p in Yeast

    PubMed Central

    Henry, Kenneth R.; D'Hondt, Kathleen; Chang, JiSuk; Newpher, Thomas; Huang, Kristen; Hudson, R. Tod; Riezman, Howard; Lemmon, Sandra K.

    2002-01-01

    SCD5 was identified as a multicopy suppressor of clathrin HC-deficient yeast. SCD5 is essential, but an scd5-Δ338 mutant, expressing Scd5p with a C-terminal truncation of 338 amino acids, is temperature sensitive for growth. Further studies here demonstrate that scd5-Δ338 affects receptor-mediated and fluid-phase endocytosis and normal actin organization. The scd5-Δ338 mutant contains larger and depolarized cortical actin patches and a prevalence of G-actin bars. scd5-Δ338 also displays synthetic negative genetic interactions with mutations in several other proteins important for cortical actin organization and endocytosis. Moreover, Scd5p colocalizes with cortical actin. Analysis has revealed that clathrin-deficient yeast also have a major defect in cortical actin organization and accumulate G-actin. Overexpression of SCD5 partially suppresses the actin defect of clathrin mutants, whereas combining scd5-Δ338 with a clathrin mutation exacerbates the actin and endocytic phenotypes. Both Scd5p and yeast clathrin physically associate with Sla2p, a homologue of the mammalian huntingtin interacting protein HIP1 and the related HIP1R. Furthermore, Sla2p localization at the cell cortex is dependent on Scd5p and clathrin function. Therefore, Scd5p and clathrin are important for actin organization and endocytosis, and Sla2p may provide a critical link between clathrin and the actin cytoskeleton in yeast, similar to HIP1(R) in animal cells. PMID:12181333

  7. Regulation of glutamate receptor internalization by the spine cytoskeleton is mediated by its PKA-dependent association with CPG2

    PubMed Central

    Loebrich, Sven; Djukic, Biljana; Tong, Zachary J.; Cottrell, Jeffrey R.; Turrigiano, Gina G.; Nedivi, Elly

    2013-01-01

    A key neuronal mechanism for adjusting excitatory synaptic strength is clathrin-mediated endocytosis of postsynaptic glutamate receptors (GluRs). The actin cytoskeleton is critical for clathrin-mediated endocytosis, yet we lack a mechanistic understanding of its interaction with the endocytic process and how it may be regulated. Here we show that F-actin in dendritic spines physically binds the synaptic nuclear envelope 1 gene product candidate plasticity gene 2 (CPG2) in a PKA-dependent manner, and that this association is required for synaptic GluR internalization. Mutating two PKA sites on CPG2 disrupts its cytoskeletal association, attenuating GluR endocytosis and affecting the efficacy of synaptic transmission in vivo. These results identify CPG2 as an F-actin binding partner that functionally mediates interaction of the spine cytoskeleton with postsynaptic endocytosis. Further, the regulation of CPG2/F-actin association by PKA provides a gateway for cellular control of synaptic receptor internalization through second messenger signaling pathways. Recent identification of human synaptic nuclear envelope 1 as a risk locus for bipolar disorder suggests that CPG2 could play a role in synaptic dysfunction underlying neuropsychiatric disease. PMID:24191017

  8. Actin assembly factors regulate the gelation kinetics and architecture of F-actin networks.

    PubMed

    Falzone, Tobias T; Oakes, Patrick W; Sees, Jennifer; Kovar, David R; Gardel, Margaret L

    2013-04-16

    Dynamic regulation of the actin cytoskeleton is required for diverse cellular processes. Proteins regulating the assembly kinetics of the cytoskeletal biopolymer F-actin are known to impact the architecture of actin cytoskeletal networks in vivo, but the underlying mechanisms are not well understood. Here, we demonstrate that changes to actin assembly kinetics with physiologically relevant proteins profilin and formin (mDia1 and Cdc12) have dramatic consequences on the architecture and gelation kinetics of otherwise biochemically identical cross-linked F-actin networks. Reduced F-actin nucleation rates promote the formation of a sparse network of thick bundles, whereas increased nucleation rates result in a denser network of thinner bundles. Changes to F-actin elongation rates also have marked consequences. At low elongation rates, gelation ceases and a solution of rigid bundles is formed. By contrast, rapid filament elongation accelerates dynamic arrest and promotes gelation with minimal F-actin density. These results are consistent with a recently developed model of how kinetic constraints regulate network architecture and underscore how molecular control of polymer assembly is exploited to modulate cytoskeletal architecture and material properties. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  9. Oscillatory Increases in Alkalinity Anticipate Growth and May Regulate Actin Dynamics in Pollen Tubes of Lily[W][OA

    PubMed Central

    Lovy-Wheeler, Alenka; Kunkel, Joseph G.; Allwood, Ellen G.; Hussey, Patrick J.; Hepler, Peter K.

    2006-01-01

    Lily (Lilium formosanum or Lilium longiflorum) pollen tubes, microinjected with a low concentration of the pH-sensitive dye bis-carboxyethyl carboxyfluorescein dextran, show oscillating pH changes in their apical domain relative to growth. An increase in pH in the apex precedes the fastest growth velocities, whereas a decline follows growth, suggesting a possible relationship between alkalinity and cell extension. A target for pH may be the actin cytoskeleton, because the apical cortical actin fringe resides in the same region as the alkaline band in lily pollen tubes and elongation requires actin polymerization. A pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain. Acidification causes actin filament destabilization and inhibits growth by 80%. Upon complete growth inhibition, the actin fringe is the first actin cytoskeleton component to disappear. We propose that during normal growth, the pH increase in the alkaline band stimulates the fragmenting activity of ADF/AIP, which in turn generates more sites for actin polymerization. Increased actin polymerization supports faster growth rates and a proton influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH stabilizes and increases, the activity of ADF/AIP again increases, repeating the cycle of events. PMID:16920777

  10. 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.

  11. The cytoskeleton and gravitropism in higher plants

    NASA Technical Reports Server (NTRS)

    Blancaflor, Elison B.

    2002-01-01

    The cellular and molecular mechanisms underlying the gravitropic response of plants have continued to elude plant biologists despite more than a century of research. Lately there has been increased attention on the role of the cytoskeleton in plant gravitropism, but several controversies and major gaps in our understanding of cytoskeletal involvement in gravitropism remain. A major question in the study of plant gravitropism is how the cytoskeleton mediates early sensing and signal transduction events in plants. Much has been made of the actin cytoskeleton as the cellular structure that sedimenting amyloplasts impinge upon to trigger the downstream signaling events leading to the bending response. There is also strong molecular and biochemical evidence that the transport of auxin, an important player in gravitropism, is regulated by actin. Organizational changes in microtubules during the growth response phase of gravitropism have also been well documented, but the significance of such reorientations in controlling differential cellular growth is unclear. Studies employing pharmacological approaches to dissect cytoskeletal involvement in gravitropism have led to conflicting results and therefore need to be interpreted with caution. Despite the current controversies, the revolutionary advances in molecular, biochemical, and cell biological techniques have opened up several possibilities for further research into this difficult area. The myriad proteins associated with the plant cytoskeleton that are being rapidly characterized provide a rich assortment of candidate regulators that could be targets of the gravity signal transduction chain. Cytoskeletal and ion imaging in real time combined with mutant analysis promises to provide a fresh start into this controversial area of research.

  12. Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots.

    PubMed

    Wang, Yuh-Shuh; Motes, Christy M; Mohamalawari, Deepti R; Blancaflor, Elison B

    2004-10-01

    The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins. Copyright 2004 Wiley-Liss, Inc.

  13. 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

  14. 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

  15. Zinc and Copper Effects on Stability of Tubulin and Actin Networks in Dendrites and Spines of Hippocampal Neurons.

    PubMed

    Perrin, Laura; Roudeau, Stéphane; Carmona, Asuncion; Domart, Florelle; Petersen, Jennifer D; Bohic, Sylvain; Yang, Yang; Cloetens, Peter; Ortega, Richard

    2017-07-19

    Zinc and copper ions can modulate the activity of glutamate receptors. However, labile zinc and copper ions likely represent only the tip of the iceberg and other neuronal functions are suspected for these metals in their bound state. We performed synchrotron X-ray fluorescence imaging with 30 nm resolution to image total biometals in dendrites and spines from hippocampal neurons. We found that zinc is distributed all along the dendrites while copper is mainly pinpointed within the spines. In spines, zinc content is higher within the spine head while copper is higher within the spine neck. Such specific distributions suggested metal interactions with cytoskeleton proteins. Zinc supplementation induced the increase of β-tubulin content in dendrites. Copper supplementation impaired the β-tubulin and F-actin networks. Copper chelation resulted in the decrease of F-actin content in dendrites, drastically reducing the number of F-actin protrusions. These results indicate that zinc is involved in microtubule stability whereas copper is essential for actin-dependent stability of dendritic spines, although copper excess can impair the dendritic cytoskeleton.

  16. Actin Age Orchestrates Myosin-5 and Myosin-6 Runlengths

    PubMed Central

    Zimmermann, Dennis; Santos, Alicja; Kovar, David R.; Rock, Ronald S.

    2015-01-01

    Summary Unlike a static and immobile skeleton, the actin cytoskeleton is a highly dynamic network of filamentous actin (F-actin) polymers that continuously turn over. In addition to generating mechanical forces and sensing mechanical deformation, dynamic F-actin networks serve as cellular tracks for myosin motor traffic. However, much of our mechanistic understanding of processive myosins comes from in vitro studies where motility was studied on pre-assembled and artificially stabilized, static F-actin tracks. In this work, we examine the role of actin dynamics in single-molecule myosin motility using assembling F-actin and the two highly processive motors, myosin-5 and myosin-6. These two myosins have distinct functions in the cell and travel in opposite directions along actin filaments [1–3]. Myosin-5 walks towards the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks towards the pointed end of F-actin [5], traveling towards the cell center along older segments of the actin filament. We find that myosin-5 takes 1.3 to 1.5-fold longer runs on ADP•Pi (young) F-actin, while myosin-6 takes 1.7 to 3.6-fold longer runs along ADP (old) F-actin. These results suggest that conformational differences between ADP•Pi and ADP F-actin tailor these myosins to walk farther toward their preferred actin filament end. Taken together, these experiments define a new mechanism by which myosin traffic may sort to different F-actin networks depending on filament age. PMID:26190073

  17. 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.

  18. A structural study of F-actin - filamin networks

    NASA Astrophysics Data System (ADS)

    Ahrens-Braunstein, Ashley; Nguyen, Lam; Hirst, Linda

    2010-03-01

    The cell's ability to move and contract is attributed to the semi-flexible filamentous protein, F -actin, one of the three filaments in the cytoskeleton. Actin bundling can be formed by a cross-linking actin binding protein (ABP) filamin. By examining filamin's cross-linking abilities at different concentrations and molar ratios, we can study the flexibility, structure and multiple network formations created when cross-linking F-actin with this protein. We have studied the phase diagram of this protein system using fluorescence microscopy, analyzing the network structures observed in the context of a coarse grained molecular dynamics simulation carried out by our group.

  19. Actin Assembly Factors Regulate the Gelation Kinetics and Architecture of F-actin Networks

    PubMed Central

    Falzone, Tobias T.; Oakes, Patrick W.; Sees, Jennifer; Kovar, David R.; Gardel, Margaret L.

    2013-01-01

    Dynamic regulation of the actin cytoskeleton is required for diverse cellular processes. Proteins regulating the assembly kinetics of the cytoskeletal biopolymer F-actin are known to impact the architecture of actin cytoskeletal networks in vivo, but the underlying mechanisms are not well understood. Here, we demonstrate that changes to actin assembly kinetics with physiologically relevant proteins profilin and formin (mDia1 and Cdc12) have dramatic consequences on the architecture and gelation kinetics of otherwise biochemically identical cross-linked F-actin networks. Reduced F-actin nucleation rates promote the formation of a sparse network of thick bundles, whereas increased nucleation rates result in a denser network of thinner bundles. Changes to F-actin elongation rates also have marked consequences. At low elongation rates, gelation ceases and a solution of rigid bundles is formed. By contrast, rapid filament elongation accelerates dynamic arrest and promotes gelation with minimal F-actin density. These results are consistent with a recently developed model of how kinetic constraints regulate network architecture and underscore how molecular control of polymer assembly is exploited to modulate cytoskeletal architecture and material properties. PMID:23601318

  20. 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

  1. Role of the microtubule cytoskeleton in gravisensing Chara rhizoids.

    PubMed

    Braun, M; Sievers, A

    1994-04-01

    The arrangement of the microtubule cytoskeleton in tip-growing and gravisensing Chara rhizoids has been documented by immunofluorescence microscopy. Predominantly axially oriented undulating bundles of cortical microtubules were found in the basal zone of the rhizoids and colocalized with the microfilament bundles underlying the cytoplasmic streaming. Microtubules penetrate the subapical zone, forming a three-dimensional network that envelops the nucleus and organelles. Microtubules are present up to 5 to 10 microns basal from the apical cytoplasmic region containing the statoliths. No microtubules were found in the apical zone of the rhizoid which is the site of tip growth and gravitropism. Depolymerization of microtubules by application of oryzalin does not affect cytoplasmic streaming and gravitropic growth until the relatively stationary and polarly organized apical and subapical cytoplasm is converted into streaming cytoplasm. When the statoliths and the apical cytoplasm are included in the cytoplasmic streaming, tip growth and gravitropism are stopped. Oryzalin-induced disruption of the microtubule cytoskeleton also results in a rearrangement of the dense network of apical and subapical microfilaments into thicker bundles, whereas disruption of the microfilament cytoskeleton by cytochalasin D had no effect on the organization of the microtubule cytoskeleton. It is, therefore, concluded that the arrangement of microtubules is essential for the polar cytoplasmic zonation and the functionally polar organization of the actin cytoskeleton which is responsible for the motile processes in rhizoids. Microtubules are not involved in the primary events of gravitropism in Chara rhizoids.

  2. Surfing pathogens and the lessons learned for actin polymerization.

    PubMed

    Frischknecht, F; Way, M

    2001-01-01

    A number of unrelated bacterial species as well as vaccinia virus (ab)use the process of actin polymerization to facilitate and enhance their infection cycle. Studies into the mechanism by which these pathogens hijack and control the actin cytoskeleton have provided many interesting insights into the regulation of actin polymerization in migrating cells. This review focuses on what we have learnt from the actin-based motilities of Listeria, Shigella and vaccinia and discusses what we would still like to learn from our nasty friends, including enteropathogenic Escherichia coli and Rickettsia

  3. Circadian actin dynamics drive rhythmic fibroblast mobilisation during wound healing

    PubMed Central

    Hoyle, Nathaniel P.; Seinkmane, Estere; Putker, Marrit; Feeney, Kevin A.; Krogager, Toke P.; Chesham, Johanna E.; Bray, Liam K.; Thomas, Justyn M.; Dunn, Ken; Blaikley, John; O’Neill, John S.

    2017-01-01

    Fibroblasts are primary cellular protagonists of wound healing. They also exhibit circadian timekeeping which imparts a ~24-hour rhythm to their biological function. We interrogated the functional consequences of the cell-autonomous clockwork in fibroblasts using a proteome-wide screen for rhythmically expressed proteins. We observed temporal coordination of actin regulators that drives cell-intrinsic rhythms in actin dynamics. In consequence the cellular clock modulates the efficiency of actin-dependent processes such as cell migration and adhesion, which ultimately impact the efficacy of wound healing. Accordingly, skin wounds incurred during a mouse’s active phase exhibited increased fibroblast invasion in vivo and ex vivo, as well as in cultured fibroblasts and keratinocytes. Our experimental results correlate with the observation that the time of injury significantly affects healing after burns in humans, with daytime wounds healing ~60% faster than night-time wounds. We suggest that circadian regulation of the cytoskeleton influences wound healing efficacy from the cellular to the organismal scale. PMID:29118260

  4. The cytoskeleton as a novel therapeutic target for old neurodegenerative disorders.

    PubMed

    Eira, Jessica; Silva, Catarina Santos; Sousa, Mónica Mendes; Liz, Márcia Almeida

    2016-06-01

    Cytoskeleton defects, including alterations in microtubule stability, in axonal transport as well as in actin dynamics, have been characterized in several unrelated neurodegenerative conditions. These observations suggest that defects of cytoskeleton organization may be a common feature contributing to neurodegeneration. In line with this hypothesis, drugs targeting the cytoskeleton are currently being tested in animal models and in human clinical trials, showing promising effects. Drugs that modulate microtubule stability, inhibitors of posttranslational modifications of cytoskeletal components, specifically compounds affecting the levels of tubulin acetylation, and compounds targeting signaling molecules which regulate cytoskeleton dynamics, constitute the mostly addressed therapeutic interventions aiming at preventing cytoskeleton damage in neurodegenerative disorders. In this review, we will discuss in a critical perspective the current knowledge on cytoskeleton damage pathways as well as therapeutic strategies designed to revert cytoskeleton-related defects mainly focusing on the following neurodegenerative disorders: Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Amyotrophic Lateral Sclerosis and Charcot-Marie-Tooth Disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. A growing family: the expanding universe of the bacterial cytoskeleton

    PubMed Central

    Ingerson-Mahar, Michael; Gitai, Zemer

    2014-01-01

    Cytoskeletal proteins are important mediators of cellular organization in both eukaryotes and bacteria. In the past, cytoskeletal studies have largely focused on three major cytoskeletal families, namely the eukaryotic actin, tubulin, and intermediate filament (IF) proteins and their bacterial homologs MreB, FtsZ, and crescentin. However, mounting evidence suggests that these proteins represent only the tip of the iceberg, as the cellular cytoskeletal network is far more complex. In bacteria, each of MreB, FtsZ, and crescentin represents only one member of large families of diverse homologs. There are also newly identified bacterial cytoskeletal proteins with no eukaryotic homologs, such as WACA proteins and bactofilins. Furthermore, there are universally conserved proteins, such as the metabolic enzyme CtpS, that assemble into filamentous structures that can be repurposed for structural cytoskeletal functions. Recent studies have also identified an increasing number of eukaryotic cytoskeletal proteins that are unrelated to actin, tubulin, and IFs, such that expanding our understanding of cytoskeletal proteins is advancing the understanding of the cell biology of all organisms. Here, we summarize the recent explosion in the identification of new members of the bacterial cytoskeleton and describe a hypothesis for the evolution of the cytoskeleton from self-assembling enzymes. PMID:22092065

  6. Glutaredoxins Grx4 and Grx3 of Saccharomyces cerevisiae play a role in actin dynamics through their Trx domains, which contributes to oxidative stress resistance.

    PubMed

    Pujol-Carrion, Nuria; de la Torre-Ruiz, Maria Angeles

    2010-12-01

    Grx3 and Grx4 are two monothiol glutaredoxins of Saccharomyces cerevisiae that have previously been characterized as regulators of Aft1 localization and therefore of iron homeostasis. In this study, we present data showing that both Grx3 and Grx4 have new roles in actin cytoskeleton remodeling and in cellular defenses against oxidative stress caused by reactive oxygen species (ROS) accumulation. The Grx4 protein plays a unique role in the maintenance of actin cable integrity, which is independent of its role in the transcriptional regulation of Aft1. Grx3 plays an additive and redundant role, in combination with Grx4, in the organization of the actin cytoskeleton, both under normal conditions and in response to external oxidative stress. Each Grx3 and Grx4 protein contains a thioredoxin domain sequence (Trx), followed by a glutaredoxin domain (Grx). We performed functional analyses of each of the two domains and characterized different functions for them. Each of the two Grx domains plays a role in ROS detoxification and cell viability. However, the Trx domain of each Grx4 and Grx3 protein acts independently of its respective Grx domain in a novel function that involves the polarization of the actin cytoskeleton, which also determines cell resistance against oxidative conditions. Finally, we present experimental evidence demonstrating that Grx4 behaves as an antioxidant protein increasing cell survival under conditions of oxidative stress.

  7. Analysis of Actin-Based Intracellular Trafficking in Pollen Tubes.

    PubMed

    Jiang, Yuxiang; Zhang, Meng; Huang, Shanjin

    2017-01-01

    Underlying rapid and directional pollen tube growth is the active intracellular trafficking system that carries materials necessary for cell wall synthesis and membrane expansion to the expanding point of the pollen tube. The actin cytoskeleton has been shown to control various intracellular trafficking events in the pollen tube, but the underlying cellular and molecular mechanisms remain poorly understood. To better understand how the actin cytoskeleton is involved in the regulation of intracellular trafficking events, we need to establish assays to visualize and quantify the distribution and dynamics of organelles, vesicles, or secreted proteins. In this chapter, we introduce methods regarding the visualization and quantification of the distribution and dynamics of organelles or vesicles in pollen tubes.

  8. A Robust Actin Filaments Image Analysis Framework

    PubMed Central

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-01-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a ‘cartoon’ part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the ‘cartoon’ image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts

  9. 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

  10. Hippocampal Dendritic Spines Are Segregated Depending on Their Actin Polymerization

    PubMed Central

    Domínguez-Iturza, Nuria; Calvo, María; Benoist, Marion; Esteban, José Antonio; Morales, Miguel

    2016-01-01

    Dendritic spines are mushroom-shaped protrusions of the postsynaptic membrane. Spines receive the majority of glutamatergic synaptic inputs. Their morphology, dynamics, and density have been related to synaptic plasticity and learning. The main determinant of spine shape is filamentous actin. Using FRAP, we have reexamined the actin dynamics of individual spines from pyramidal hippocampal neurons, both in cultures and in hippocampal organotypic slices. Our results indicate that, in cultures, the actin mobile fraction is independently regulated at the individual spine level, and mobile fraction values do not correlate with either age or distance from the soma. The most significant factor regulating actin mobile fraction was the presence of astrocytes in the culture substrate. Spines from neurons growing in the virtual absence of astrocytes have a more stable actin cytoskeleton, while spines from neurons growing in close contact with astrocytes show a more dynamic cytoskeleton. According to their recovery time, spines were distributed into two populations with slower and faster recovery times, while spines from slice cultures were grouped into one population. Finally, employing fast lineal acquisition protocols, we confirmed the existence of loci with high polymerization rates within the spine. PMID:26881098

  11. Interactions between Auxin Transport and the Actin Cytoskeleton in Developmental Polarity of Fucus distichus Embryos in Response to Light and Gravity1

    PubMed Central

    Sun, Haiguo; Basu, Swati; Brady, Shari R.; Luciano, Randy L.; Muday, Gloria K.

    2004-01-01

    Land plants orient their growth relative to light and gravity through complex mechanisms that require auxin redistribution. Embryos of brown algae use similar environmental stimuli to orient their developmental polarity. These studies of the brown algae Fucus distichus examined whether auxin and auxin transport are also required during polarization in early embryos and to orient growth in already developed tissues. These embryos polarize with the gravity vector in the absence of a light cue. The auxin, indole-3-acetic acid (IAA), and auxin efflux inhibitors, such as naphthylphthalamic acid (NPA), reduced environmental polarization in response to gravity and light vectors. Young rhizoids are negatively phototropic, and NPA also inhibits rhizoid phototropism. The effect of IAA and NPA on gravity and photopolarization is maximal within 2.5 to 4.5 h after fertilization (AF). Over the first 6 h AF, auxin transport is relatively constant, suggesting that developmentally controlled sensitivity to auxin determines the narrow window during which NPA and IAA reduce environmental polarization. Actin patches were formed during the first hour AF and began to photolocalize within 3 h, coinciding with the time of NPA and IAA action. Treatment with NPA reduced the polar localization of actin patches but not patch formation. Latrunculin B prevented environmental polarization in a time frame that overlaps the formation of actin patches and IAA and NPA action. Latrunculin B also altered auxin transport. Together, these results indicate a role for auxin in the orientation of developmental polarity and suggest interactions between the actin cytoskeleton and auxin transport in F. distichus embryos. PMID:15122028

  12. 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.

  13. Infection and Transport of Herpes Simplex Virus Type 1 in Neurons: Role of the Cytoskeleton

    PubMed Central

    2018-01-01

    Herpes simplex virus type 1 (HSV-1) is a neuroinvasive human pathogen that has the ability to infect and replicate within epithelial cells and neurons and establish a life-long latent infection in sensory neurons. HSV-1 depends on the host cellular cytoskeleton for entry, replication, and exit. Therefore, HSV-1 has adapted mechanisms to promote its survival by exploiting the microtubule and actin cytoskeletons to direct its active transport, infection, and spread between neurons and epithelial cells during primary and recurrent infections. This review will focus on the currently known mechanisms utilized by HSV-1 to harness the neuronal cytoskeleton, molecular motors, and the secretory and exocytic pathways for efficient virus entry, axonal transport, replication, assembly, and exit from the distinct functional compartments (cell body and axon) of the highly polarized sensory neurons. PMID:29473915

  14. Bradykinin increases blood-tumor barrier permeability by down-regulating the expression levels of ZO-1, occludin, and claudin-5 and rearranging actin cytoskeleton.

    PubMed

    Liu, Li-Bo; Xue, Yi-Xue; Liu, Yun-Hui; Wang, Yi-Bao

    2008-04-01

    Bradykinin (BK) has been shown to open blood-tumor barrier (BTB) selectively and to increase permeability of the BTB transiently, but the mechanism is unclear. This study was performed to determine whether BK opens the BTB by affecting the tight junction (TJ)-associated proteins zonula occluden-1 (ZO-1), occludin, and caludin-5 and cytoskeleton protein filamentous actin (F-actin). In rat brain glioma model and BTB model in vitro, we find that the protein expression levels of ZO-1, occludin, and claudin-5 are attenuated by BK induction. Immunohistochemistry and immunofluorescence assays show that the attenuated expression of ZO-1, occludin, and claudin-5 and F-actin is most obvious in the smaller tumor capillaries (<20 microm) after BK infusion, and there is no change in the larger tumor capillaries (>20 microm). The redistribution of ZO-1, occludin, and claudin-5 and rearrangement of F-actin in brain microvascular endothelial cells are observed at the same time. Meanwhile, Evans blue assay shows that the permeability of BTB increases after BK infusion. Transmission electron microscopy indicates that TJ is opened and that pinocytotic vesicular density is increased. Transendothelial electrical resistance (TEER) and horseradish peroxidase flux assays also reveal that TJ is opened by BK induction. In addition, radioimmunity and Western blot assay reveal a significant decrease in expression levels of cAMP and catalytic subunit of protien kinase A (PKAcs) of tumor tissue. This study demonstrates that the increase of BK-mediated BTB permeability is associated with the down-regulation of ZO-1, occludin, and claudin-5 and the rearrangement of F-actin and that cAMP/PKA signal transduction system might be involved in the modulating process.

  15. 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.

  16. 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

  17. 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

  18. Arabidopsis ACTIN-DEPOLYMERIZING FACTOR3 Is Required for Controlling Aphid Feeding from the Phloem1[OPEN

    PubMed Central

    Mondal, Hossain A.; Louis, Joe; Archer, Lani; Patel, Monika; Nalam, Vamsi J.; Sarowar, Sujon; Sivapalan, Vishala

    2018-01-01

    The actin cytoskeleton network has an important role in plant cell growth, division, and stress response. Actin-depolymerizing factors (ADFs) are a group of actin-binding proteins that contribute to reorganization of the actin network. Here, we show that the Arabidopsis (Arabidopsis thaliana) ADF3 is required in the phloem for controlling infestation by Myzus persicae Sülzer, commonly known as the green peach aphid (GPA), which is an important phloem sap-consuming pest of more than fifty plant families. In agreement with a role for the actin-depolymerizing function of ADF3 in defense against the GPA, we show that resistance in adf3 was restored by overexpression of the related ADF4 and the actin cytoskeleton destabilizers, cytochalasin D and latrunculin B. Electrical monitoring of the GPA feeding behavior indicates that the GPA stylets found sieve elements faster when feeding on the adf3 mutant compared to the wild-type plant. In addition, once they found the sieve elements, the GPA fed for a more prolonged period from sieve elements of adf3 compared to the wild-type plant. The longer feeding period correlated with an increase in fecundity and population size of the GPA and a parallel reduction in callose deposition in the adf3 mutant. The adf3-conferred susceptibility to GPA was overcome by expression of the ADF3 coding sequence from the phloem-specific SUC2 promoter, thus confirming the importance of ADF3 function in the phloem. We further demonstrate that the ADF3-dependent defense mechanism is linked to the transcriptional up-regulation of PHYTOALEXIN-DEFICIENT4, which is an important regulator of defenses against the GPA. PMID:29133373

  19. Functional adaptation between yeast actin and its cognate myosin motors.

    PubMed

    Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S; Rubenstein, Peter A; Lord, Matthew

    2011-09-02

    We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins.

  20. 14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote Giardia lamblia

    PubMed Central

    Xu, Jennifer; Steele-Ogus, Melissa; Alas, Germain C. M.

    2017-01-01

    ABSTRACT The phosphoserine/phosphothreonine-binding protein 14-3-3 is known to regulate actin; this function has been previously attributed to sequestration of phosphorylated cofilin. 14-3-3 was identified as an actin-associated protein in the deep-branching eukaryote Giardia lamblia; however, Giardia lacks cofilin and all other canonical actin-binding proteins (ABPs). Thus, the role of G. lamblia 14-3-3 (Gl-14-3-3) in actin regulation was unknown. Gl-14-3-3 depletion resulted in an overall disruption of actin organization characterized by ectopically distributed short actin filaments. Using phosphatase and kinase inhibitors, we demonstrated that actin phosphorylation correlated with destabilization of the actin network and increased complex formation with 14-3-3, while blocking actin phosphorylation stabilized actin filaments and attenuated complex formation. Giardia’s sole Rho family GTPase, Gl-Rac, modulates Gl-14-3-3’s association with actin, providing the first connection between Gl-Rac and the actin cytoskeleton in Giardia. Giardia actin (Gl-actin) contains two putative 14-3-3 binding motifs, one of which (S330) is conserved in mammalian actin. Mutation of these sites reduced, but did not completely disrupt, the association with 14-3-3. Native gels and overlay assays indicate that intermediate proteins are required to support complex formation between 14-3-3 and actin. Overall, our results support a role for 14-3-3 as a regulator of actin; however, the presence of multiple 14-3-3–actin complexes suggests a more complex regulatory relationship than might be expected for a minimalistic parasite. IMPORTANCE Giardia lacks canonical actin-binding proteins. Gl-14-3-3 was identified as an actin interactor, but the significance of this interaction was unknown. Loss of Gl-14-3-3 results in ectopic short actin filaments, indicating that Gl-14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that Gl-14-3-3 complex

  1. 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.

  2. Genome-wide identification, phylogenetic classification, and exon-intron structure characterisation of the tubulin and actin genes in flax (Linum usitatissimum).

    PubMed

    Pydiura, Nikolay; Pirko, Yaroslav; Galinousky, Dmitry; Postovoitova, Anastasiia; Yemets, Alla; Kilchevsky, Aleksandr; Blume, Yaroslav

    2018-06-08

    Flax (Linum usitatissimum L.) is a valuable food and fiber crop cultivated for its quality fiber and seed oil. α-, β-, γ-tubulins and actins are the main structural proteins of the cytoskeleton. α- and γ-tubulin and actin genes have not been characterized yet in the flax genome. In this study, we have identified 6 α-tubulin genes, 13 β-tubulin genes, 2 γ-tubulin genes, and 15 actin genes in the flax genome and analysed the phylogenetic relationships between flax and A. thaliana tubulin and actin genes. Six α-tubulin genes are represented by 3 paralogous pairs, among 13 β-tubulin genes 7 different isotypes can be distinguished, 6 of which are encoded by two paralogous genes each. γ-tubulin is represented by a paralogous pair of genes one of which may be not functional. Fifteen actin genes represent 7 paralogous pairs - 7 actin isotypes and a sequentially duplicated copy of one of the genes of one of the isotypes. Exon-intron structure analysis has shown intron length polymorphism within the β-tubulin genes and intron number variation among the α-tubulin gene: 3 or 4 introns are found in two or four genes, respectively. Intron positioning occurs at conservative sites, as observed in numerous other plant species. Flax actin genes show both intron length polymorphisms and variation in the number of intron that may be 2 or 3. These data will be useful to support further studies on the specificity, functioning, regulation and evolution of the flax cytoskeleton proteins. This article is protected by copyright. All rights reserved.

  3. Structure of the Rigor Actin-Tropomyosin-Myosin Complex

    PubMed Central

    Behrmann, Elmar; Müller, Mirco; Penczek, Pawel A.; Mannherz, Hans Georg; Manstein, Dietmar J.; Raunser, Stefan

    2014-01-01

    The interaction of myosin with actin filaments is the central feature of muscle contraction and cargo movement along actin filaments of the cytoskeleton. Myosin converts the chemical energy stored in ATP into force and movement along actin filaments. Myosin binding to actin induces conformational changes that are coupled to the nucleotide-binding pocket and amplified by a specialized region of the motor domain for efficient force generation. Tropomyosin plays a key role in regulating the productive interaction between myosins and actin. Here, we report the 8 Å resolution structure of the actin-tropomyosin-myosin complex determined by cryo electron microscopy. The pseudo-atomic model of the complex obtained from fitting crystal structures into the map defines the large actin-myosin-tropomyosin interface and the molecular interactions between the proteins in detail and allows us to propose a structural model for tropomyosin dependent myosin binding to actin and actin-induced nucleotide release from myosin. PMID:22817895

  4. The evolution of compositionally and functionally distinct actin filaments.

    PubMed

    Gunning, Peter W; Ghoshdastider, Umesh; Whitaker, Shane; Popp, David; Robinson, Robert C

    2015-06-01

    The actin filament is astonishingly well conserved across a diverse set of eukaryotic species. It has essentially remained unchanged in the billion years that separate yeast, Arabidopsis and man. In contrast, bacterial actin-like proteins have diverged to the extreme, and many of them are not readily identified from sequence-based homology searches. Here, we present phylogenetic analyses that point to an evolutionary drive to diversify actin filament composition across kingdoms. Bacteria use a one-filament-one-function system to create distinct filament systems within a single cell. In contrast, eukaryotic actin is a universal force provider in a wide range of processes. In plants, there has been an expansion of the number of closely related actin genes, whereas in fungi and metazoa diversification in tropomyosins has increased the compositional variety in actin filament systems. Both mechanisms dictate the subset of actin-binding proteins that interact with each filament type, leading to specialization in function. In this Hypothesis, we thus propose that different mechanisms were selected in bacteria, plants and metazoa, which achieved actin filament compositional variation leading to the expansion of their functional diversity. © 2015. Published by The Company of Biologists Ltd.

  5. Formation of Hirano Bodies Induced by Expression of an Actin Cross-Linking Protein with a Gain-of-Function Mutation

    PubMed Central

    Maselli, Andrew; Furukawa, Ruth; Thomson, Susanne A. M.; Davis, Richard C.; Fechheimer, Marcus

    2003-01-01

    Hirano bodies are paracrystalline actin filament-containing structures reported to be associated with a variety of neurodegenerative diseases. However, the biological function of Hirano bodies remains poorly understood, since nearly all prior studies of these structures were done with postmortem samples of tissue. In the present study, we generated a full-length form of a Dictyostelium 34-kDa actin cross-linking protein with point mutations in the first putative EF hand, termed 34-kDa ΔEF1. The 34-kDa ΔEF1 protein binds calcium normally but has activated actin binding that is unregulated by calcium. The expression of the 34-kDa ΔEF1 protein in Dictyostelium induces the formation of Hirano bodies, as assessed by both fluorescence microscopy and transmission electron microscopy. Dictyostelium cells bearing Hirano bodies grow normally, indicating that Hirano bodies are not associated with cell death and are not deleterious to cell growth. Moreover, the expression of the 34-kDa ΔEF1 protein rescues the phenotypes of cells lacking the 34-kDa protein and cells lacking both the 34-kDa protein and α-actinin. Finally, the expression of the 34-kDa ΔEF1 protein also initiates the formation of Hirano bodies in cultured mouse fibroblasts. These results show that the failure to regulate the activity and/or affinity of an actin cross-linking protein can provide a signal for the formation of Hirano bodies. More generally, the formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton. PMID:12912897

  6. Neuroprotective effects of hypothermia on synaptic actin cytoskeletal changes induced by perinatal asphyxia.

    PubMed

    Muñiz, Javier; Romero, Juan; Holubiec, Mariana; Barreto, George; González, Janneth; Saint-Martin, Madeleine; Blanco, Eduardo; Carlos Cavicchia, Juan; Castilla, Rocío; Capani, Francisco

    2014-05-14

    Cerebral hypoxia-ischemia damages synaptic proteins, resulting in cytoskeletal alterations, protein aggregation and neuronal death. In the previous works, we have shown neuronal and synaptic changes in rat neostriatum subjected to hypoxia that leads to ubi-protein accumulation. Recently, we also showed that, changes in F-actin organization could be related to early alterations induced by hypoxia in the Central Nervous System. However, little is known about effective treatment to diminish the damage. The main aim of this work is to study the effects of birth hypothermia on the actin cytoskeleton of neostriatal post-synaptic densities (PSD) in 60 days olds rats by immunohistochemistry, photooxidation and western blot. We used 2 different protocols of hypothermia: (a) intrahypoxic hypothermia at 15°C and (b) post-hypoxia hypothermia at 32°C. Consistent with previous data at 30 days, staining with phalloidin-Alexa(488) followed by confocal microscopy analysis showed an increase of F-actin fluorescent staining in the neostriatum of hypoxic animals. Correlative photooxidation electron microscopy confirmed these observations showing an increment in the number of mushroom-shaped F-actin staining spines in neostriatal excitatory synapses in rats subjected to hypoxia. In addition, western blot revealed β-actin increase in PSDs in hypoxic animals. The optic relative density measurement showed a significant difference between controls and hypoxic animals. When hypoxia was induced under hypothermic conditions, the changes observed in actin cytoskeleton were blocked. Post-hypoxic hypothermia showed similar answer but actin cytoskeleton modifications were not totally reverted as we observed at 15°C. These data suggest that the decrease of the body temperature decreases the actin modifications in dendritic spines preventing the neuronal death. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Regulation of Endothelial Permeability by Glutathione S-Transferase Pi Against Actin Polymerization.

    PubMed

    Yang, Yang; Yin, Fangyuan; Hang, Qiyun; Dong, Xiaoliang; Chen, Jiao; Li, Ling; Cao, Peng; Yin, Zhimin; Luo, Lan

    2018-01-01

    Inflammation-induced injury of the endothelial barrier occurs in several pathological conditions, including atherosclerosis, ischemia, and sepsis. Endothelial cytoskeleton rearrangement is an important pathological mechanism by which inflammatory stimulation triggers an increase of vascular endothelial permeability. However, the mechanism maintaining endothelial cell barrier function against inflammatory stress is not fully understood. Glutathione S-transferase pi (GSTpi) exists in various types of cells and protects them against different stresses. In our previous study, GSTpi was found to act as a negative regulator of inflammatory responses. We used a Transwell permeability assay to test the influence of GSTpi and its transferase activity on the increase of endothelial permeability induced by tumor necrosis factor alpha (TNF-α). TNF-α-induced actin remodeling and the influence of GSTpi were observed by using laser confocal microscopy. Western blotting was used to test the influence of GSTpi on TNF-α-activated p38 mitogen-activated protein kinase (MAPK)/MK2/heat shock protein 27 (HSP27). GSTpi reduced TNF-α-induced stress fiber formation and endothelial permeability increase by restraining actin cytoskeleton rearrangement, and this reduction was unrelated to its transferase activity. We found that GSTpi inhibited p38MAPK phosphorylation by directly binding p38 and influenced downstream substrate HSP27-induced actin remodeling. GSTpi inhibited TNF-α-induced actin remodeling, stress fiber formation and endothelial permeability increase by inhibiting the p38MAPK/HSP27 signaling pathway. © 2018 The Author(s). Published by S. Karger AG, Basel.

  8. Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro

    NASA Technical Reports Server (NTRS)

    Kieffer, J. D.; Plopper, G.; Ingber, D. E.; Hartwig, J. H.; Kupper, T. S.

    1995-01-01

    The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

  9. Regulation of blood-testis barrier by actin binding proteins and protein kinases

    PubMed Central

    Li, Nan; Tang, Elizabeth I.; Cheng, C. Yan

    2016-01-01

    The blood-testis barrier (BTB) is an important ultrastructure in the testis since the onset of spermatogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule (MT)-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases. PMID:26628556

  10. The cytoskeletal scaffold Shank3 is recruited to pathogen-induced actin rearrangements

    PubMed Central

    Huett, Alan; Leong, John M; Podolsky, Daniel K.; Xavier, Ramnik J.

    2009-01-01

    Summary The common gastrointestinal pathogens enteropathogenic Escherichia coli (EPEC) and Salmonella Typhimurium both reorganize the gut epithelial cell actin cytoskeleton to mediate pathogenesis, utilizing mimicry of the host signaling apparatus. The PDZ domain-containing protein Shank3, is a large cytoskeletal scaffold protein with known functions in neuronal morphology and synaptic signaling, and is also capable of acting as a scaffolding adaptor during Ret tyrosine kinase signaling in epithelial cells. Using immunofluorescent and functional RNA-interference approaches we show that Shank3 is present in both EPEC- and S. Typhimurium-induced actin rearrangements and is required for optimal EPEC pedestal formation. We propose that Shank3 is one of a number of host synaptic proteins likely to play key roles in bacteria-host interactions. PMID:19371741

  11. 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.

  12. Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons.

    PubMed

    Leung, C L; Sun, D; Zheng, M; Knowles, D R; Liem, R K

    1999-12-13

    We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends-PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH(2) terminus. However, unlike dystonin, mACF7 does not contain a coiled-coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest-specific protein, Gas2. In this paper, we demonstrate that the NH(2)-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

  13. An αII Spectrin-Based Cytoskeleton Protects Large-Diameter Myelinated Axons from Degeneration.

    PubMed

    Huang, Claire Yu-Mei; Zhang, Chuansheng; Zollinger, Daniel R; Leterrier, Christophe; Rasband, Matthew N

    2017-11-22

    Axons must withstand mechanical forces, including tension, torsion, and compression. Spectrins and actin form a periodic cytoskeleton proposed to protect axons against these forces. However, because spectrins also participate in assembly of axon initial segments (AISs) and nodes of Ranvier, it is difficult to uncouple their roles in maintaining axon integrity from their functions at AIS and nodes. To overcome this problem and to determine the importance of spectrin cytoskeletons for axon integrity, we generated mice with αII spectrin-deficient peripheral sensory neurons. The axons of these neurons are very long and exposed to the mechanical forces associated with limb movement; most lack an AIS, and some are unmyelinated and have no nodes. We analyzed αII spectrin-deficient mice of both sexes and found that, in myelinated axons, αII spectrin forms a periodic cytoskeleton with βIV and βII spectrin at nodes of Ranvier and paranodes, respectively, but that loss of αII spectrin disrupts this organization. Avil-cre;Sptan1 f/f mice have reduced numbers of nodes, disrupted paranodal junctions, and mislocalized Kv1 K + channels. We show that the density of nodal βIV spectrin is constant among axons, but the density of nodal αII spectrin increases with axon diameter. Remarkably, Avil-cre;Sptan1 f/f mice have intact nociception and small-diameter axons, but severe ataxia due to preferential degeneration of large-diameter myelinated axons. Our results suggest that nodal αII spectrin helps resist the mechanical forces experienced by large-diameter axons, and that αII spectrin-dependent cytoskeletons are also required for assembly of nodes of Ranvier. SIGNIFICANCE STATEMENT A periodic axonal cytoskeleton consisting of actin and spectrin has been proposed to help axons resist the mechanical forces to which they are exposed (e.g., compression, torsion, and stretch). However, until now, no vertebrate animal model has tested the requirement of the spectrin cytoskeleton in

  14. Cross-Linking Molecules Modify Composite Actin Networks Independently

    NASA Astrophysics Data System (ADS)

    Schmoller, K. M.; Lieleg, O.; Bausch, A. R.

    2008-09-01

    While cells make use of many actin binding proteins (ABPs) simultaneously to tailor the mechanical properties of the cytoskeleton, the detailed interplay of different ABPs is not understood. By a combination of macrorheological measurements and confocal microscopy, we show that the ABPs fascin and filamin modify the structural and viscoelastic properties of composite in vitro actin networks independently. The outnumbering ABP dictates the local network structure and therefore also dominates the macromechanical network response.

  15. A growing family: the expanding universe of the bacterial cytoskeleton.

    PubMed

    Ingerson-Mahar, Michael; Gitai, Zemer

    2012-01-01

    Cytoskeletal proteins are important mediators of cellular organization in both eukaryotes and bacteria. In the past, cytoskeletal studies have largely focused on three major cytoskeletal families, namely the eukaryotic actin, tubulin, and intermediate filament (IF) proteins and their bacterial homologs MreB, FtsZ, and crescentin. However, mounting evidence suggests that these proteins represent only the tip of the iceberg, as the cellular cytoskeletal network is far more complex. In bacteria, each of MreB, FtsZ, and crescentin represents only one member of large families of diverse homologs. There are also newly identified bacterial cytoskeletal proteins with no eukaryotic homologs, such as WACA proteins and bactofilins. Furthermore, there are universally conserved proteins, such as the metabolic enzyme CtpS, that assemble into filamentous structures that can be repurposed for structural cytoskeletal functions. Recent studies have also identified an increasing number of eukaryotic cytoskeletal proteins that are unrelated to actin, tubulin, and IFs, such that expanding our understanding of cytoskeletal proteins is advancing the understanding of the cell biology of all organisms. Here, we summarize the recent explosion in the identification of new members of the bacterial cytoskeleton and describe a hypothesis for the evolution of the cytoskeleton from self-assembling enzymes. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  16. 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

  17. Microscale Mechanics of Actin Networks During Dynamic Assembly and Dissociation

    NASA Astrophysics Data System (ADS)

    Gurmessa, Bekele; Robertson-Anderson, Rae; Ross, Jennifer; Nguyen, Dan; Saleh, Omar

    Actin is one of the key components of the cytoskeleton, enabling cells to move and divide while maintaining shape by dynamic polymerization, dissociation and crosslinking. Actin polymerization and network formation is driven by ATP hydrolysis and varies depending on the concentrations of actin monomers and crosslinking proteins. The viscoelastic properties of steady-state actin networks have been well-characterized, yet the mechanical properties of these non-equilibrium systems during dynamic assembly and disassembly remain to be understood. We use semipermeable microfluidic devices to induce in situ dissolution and re-polymerization of entangled and crosslinked actin networks, by varying ATP concentrations in real-time, while measuring the mechanical properties during disassembly and re-assembly. We use optical tweezers to sinusoidally oscillate embedded microspheres and measure the resulting force at set time-intervals and in different regions of the network during cyclic assembly/disassembly. We determine the time-dependent viscoelastic properties of non-equilibrium network intermediates and the reproducibility and homogeneity of network formation and dissolution. Results inform the role that cytoskeleton reorganization plays in the dynamic multifunctional mechanics of cells. NSF CAREER Award (DMR-1255446) and a Scialog Collaborative Innovation Award funded by Research Corporation for Scientific Advancement (Grant No. 24192).

  18. Filament organization revealed in platinum replicas of freeze-dried cytoskeletons

    PubMed Central

    1980-01-01

    This report presents the appearance of rapidly frozen, freeze-dried cytoskeletons that have been rotary replicated with platinum and viewed in the transmission electron microscope. The resolution of this method is sufficient to visualize individual filaments in the cytoskeleton and to discriminate among actin, microtubules, and intermediate filaments solely by their surface substructure. This identification has been confirmed by specific decoration with antibodies and selective extraction of individual filament types, and correlated with light microscope immunocytochemistry and gel electrophoresis patterns. The freeze-drying preserves a remarkable degree of three-dimensionality in the organization of these cytoskeletons. They look strikingly similar to the meshwork of strands or "microtrabeculae" seen in the cytoplasm of whole cells by high voltage electron microscopy, in that the filaments form a lattice of the same configutation and with the same proportions of open area as the microtrabeculae seen in whole cells. The major differences between these two views of the structural elements of the cytoplasmic matrix can be attributed to the effects of aldehyde fixation and dehydration. Freeze-dried cytoskeletons thus provide an opportunity to study--at high resolution and in the absence of problems caused by chemical fixation--the detailed organization of filaments in different regions of the cytoplasm and at different stages of cell development. In this report the pattern of actin and intermediate filament organization in various regions of fully spread mouse fibroblasts is described. PMID:6893451

  19. Palmitoylation of LIM Kinase-1 ensures spine-specific actin polymerization and morphological plasticity

    PubMed Central

    George, Joju; Soares, Cary; Montersino, Audrey; Beique, Jean-Claude; Thomas, Gareth M

    2015-01-01

    Precise regulation of the dendritic spine actin cytoskeleton is critical for neurodevelopment and neuronal plasticity, but how neurons spatially control actin dynamics is not well defined. Here, we identify direct palmitoylation of the actin regulator LIM kinase-1 (LIMK1) as a novel mechanism to control spine-specific actin dynamics. A conserved palmitoyl-motif is necessary and sufficient to target LIMK1 to spines and to anchor LIMK1 in spines. ShRNA knockdown/rescue experiments reveal that LIMK1 palmitoylation is essential for normal spine actin polymerization, for spine-specific structural plasticity and for long-term spine stability. Palmitoylation is critical for LIMK1 function because this modification not only controls LIMK1 targeting, but is also essential for LIMK1 activation by its membrane-localized upstream activator PAK. These novel roles for palmitoylation in the spatial control of actin dynamics and kinase signaling provide new insights into structural plasticity mechanisms and strengthen links between dendritic spine impairments and neuropathological conditions. DOI: http://dx.doi.org/10.7554/eLife.06327.001 PMID:25884247

  20. Astrocyte-neuron interaction in diphenyl ditelluride toxicity directed to the cytoskeleton.

    PubMed

    Heimfarth, Luana; da Silva Ferreira, Fernanda; Pierozan, Paula; Mingori, Moara Rodrigues; Moreira, José Cláudio Fonseca; da Rocha, João Batista Teixeira; Pessoa-Pureur, Regina

    2017-03-15

    Diphenylditelluride (PhTe) 2 is a neurotoxin that disrupts cytoskeletal homeostasis. We are showing that different concentrations of (PhTe) 2 caused hypophosphorylation of glial fibrillary acidic protein (GFAP), vimentin and neurofilament subunits (NFL, NFM and NFH) and altered actin organization in co-cultured astrocytes and neurons from cerebral cortex of rats. These mechanisms were mediated by N-methyl-d-aspartate (NMDA) receptors without participation of either L-type voltage-dependent calcium channels (L-VDCC) or metabotropic glutamate receptors. Upregulated Ca 2+ influx downstream of NMDA receptors activated Ca 2+ -dependent protein phosphatase 2B (PP2B) causing hypophosphorylation of astrocyte and neuron IFs. Immunocytochemistry showed that hypophosphorylated intermediate filaments (IF) failed to disrupt their organization into the cytoskeleton. However, phalloidin-actin-FITC stained cytoskeleton evidenced misregulation of actin distribution, cell spreading and increased stress fibers in astrocytes. βIII tubulin staining showed that neurite meshworks are not altered by (PhTe) 2 , suggesting greater susceptibility of astrocytes than neurons to (PheTe) 2 toxicity. These findings indicate that signals leading to IF hypophosphorylation fail to disrupt the cytoskeletal IF meshwork of interacting astrocytes and neurons in vitro however astrocyte actin network seems more susceptible. Our findings support that intracellular Ca 2+ is one of the crucial signals that modulate the action of (PhTe) 2 in co-cultured astrocytes and neurons and highlights the cytoskeleton as an end-point of the neurotoxicity of this compound. Cytoskeletal misregulation is associated with cell dysfunction, therefore, the understanding of the molecular mechanisms mediating the neurotoxicity of this compound is a matter of increasing interest since tellurium compounds are increasingly released in the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Direct Transmembrane Interaction between Actin and the Pore-Competent, Cholesterol-Dependent Cytolysin Pneumolysin

    PubMed Central

    Hupp, Sabrina; Förtsch, Christina; Wippel, Carolin; Ma, Jiangtao; Mitchell, Timothy J.; Iliev, Asparouh I.

    2013-01-01

    The eukaryotic actin cytoskeleton is an evolutionarily well-established pathogen target, as a large number of bacterial factors disturb its dynamics to alter the function of the host cells. These pathogenic factors modulate or mimic actin effector proteins or they modify actin directly, leading to an imbalance of the precisely regulated actin turnover. Here, we show that the pore-forming, cholesterol-dependent cytolysin pneumolysin (PLY), a major neurotoxin of Streptococcus pneumoniae, has the capacity to bind actin directly and to enhance actin polymerisation in vitro. In cells, the toxin co-localised with F-actin shortly after exposure, and this direct interaction was verified by Förster resonance energy transfer. PLY was capable of exerting its effect on actin through the lipid bilayer of giant unilamellar vesicles, but only when its pore competence was preserved. The dissociation constant of G-actin binding to PLY in a biochemical environment was 170–190 nM, which is indicative of a high-affinity interaction, comparable to the affinity of other intracellular actin-binding factors. Our results demonstrate the first example of a direct interaction of a pore-forming toxin with cytoskeletal components, suggesting that the cross talk between pore-forming cytolysins and cells is more complex than previously thought. PMID:23219469

  2. Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines

    PubMed Central

    Chazeau, Anaël; Garcia, Mikael; Czöndör, Katalin; Perrais, David; Tessier, Béatrice; Giannone, Grégory; Thoumine, Olivier

    2015-01-01

    The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin–coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain. PMID:25568337

  3. Cations Modulate Actin Bundle Mechanics, Assembly Dynamics, and Structure.

    PubMed

    Castaneda, Nicholas; Zheng, Tianyu; Rivera-Jacquez, Hector J; Lee, Hyun-Ju; Hyun, Jaekyung; Balaeff, Alexander; Huo, Qun; Kang, Hyeran

    2018-04-12

    Actin bundles are key factors in the mechanical support and dynamic reorganization of the cytoskeleton. High concentrations of multivalent counterions promote bundle formation through electrostatic attraction between actin filaments that are negatively charged polyelectrolytes. In this study, we evaluate how physiologically relevant divalent cations affect the mechanical, dynamic, and structural properties of actin bundles. Using a combination of total internal reflection fluorescence microscopy, transmission electron microscopy, and dynamic light scattering, we demonstrate that divalent cations modulate bundle stiffness, length distribution, and lateral growth. Molecular dynamics simulations of an all-atom model of the actin bundle reveal specific actin residues coordinate cation-binding sites that promote the bundle formation. Our work suggests that specific cation interactions may play a fundamental role in the assembly, structure, and mechanical properties of actin bundles.

  4. The assembly of MreB, a prokaryotic homolog of actin.

    PubMed

    Esue, Osigwe; Cordero, Maria; Wirtz, Denis; Tseng, Yiider

    2005-01-28

    MreB, a major component of the bacterial cytoskeleton, exhibits high structural homology to its eukaryotic counterpart actin. Live cell microscopy studies suggest that MreB molecules organize into large filamentous spirals that support the cell membrane and play a key shape-determining function. However, the basic properties of MreB filament assembly remain unknown. Here, we studied the assembly of Thermotoga maritima MreB triggered by ATP in vitro and compared it to the well-studied assembly of actin. These studies show that MreB filament ultrastructure and polymerization depend crucially on temperature as well as the ions present on solution. At the optimal growth temperature of T. maritima, MreB assembly proceeded much faster than that of actin, without nucleation (or nucleation is highly favorable and fast) and with little or no contribution from filament end-to-end annealing. MreB exhibited rates of ATP hydrolysis and phosphate release similar to that of F-actin, however, with a critical concentration of approximately 3 nm, which is approximately 100-fold lower than that of actin. Furthermore, MreB assembled into filamentous bundles that have the ability to spontaneously form ring-like structures without auxiliary proteins. These findings suggest that despite high structural homology, MreB and actin display significantly different assembly properties.

  5. Thematic Minireview Series: The State of the Cytoskeleton in 2015.

    PubMed

    Fischer, Robert S; Fowler, Velia M

    2015-07-10

    The study of cytoskeletal polymers has been an active area of research for more than 70 years. However, despite decades of pioneering work by some of the brightest scientists in biochemistry, cell biology, and physiology, many central questions regarding the polymers themselves are only now starting to be answered. For example, although it has long been appreciated that the actin cytoskeleton provides contractility and couples biochemical responses with mechanical stresses in cells, only recently have we begun to understand how the actin polymer itself responds to mechanical loads. Likewise, although it has long been appreciated that the microtubule cytoskeleton can be post-translationally modified, only recently have the enzymes responsible for these modifications been characterized, so that we can now begin to understand how these modifications alter the polymerization and regulation of microtubule structures. Even the septins in eukaryotes and the cytoskeletal polymers of prokaryotes have yielded new insights due to recent advances in microscopy techniques. In this thematic series of minireviews, these topics are covered by some of the very same scientists who generated these recent insights, thereby providing us with an overview of the State of the Cytoskeleton in 2015. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Rapid Glucose Depletion Immobilizes Active Myosin-V on Stabilized Actin Cables

    PubMed Central

    Xu, Li; Bretscher, Anthony

    2014-01-01

    Summary Polarization of eukaryotic cells requires organelles and protein complexes to be transported to their proper destinations along the cytoskeleton [1]. When nutrients are abundant, budding yeast grows rapidly transporting secretory vesicles for localized growth and actively segregating organelles [2, 3]. This is mediated by myosin-Vs transporting cargos along F-actin bundles known as actin cables [4]. Actin cables are dynamic structures regulated by assembly, stabilization and disassembly [5]. Polarized growth and actin filament dynamics consume energy. For most organisms, glucose is the preferred energy source and generally represses alternative carbon source usage [6]. Thus upon abrupt glucose depletion, yeast shuts down pathways consuming large amounts of energy, including the vacuolar-ATPase [7, 8], translation [9] and phosphoinositide metabolism [10]. Here we show that glucose withdrawal rapidly (<1 min) depletes ATP levels and the yeast myosin V, Myo2, responds by relocalizing to actin cables, making it the fastest response documented. Myo2 immobilized on cables releases its secretory cargo, defining a new rigor-like state of a myosin-V in vivo. Only actively transporting Myo2 can be converted to the rigor-like state. Glucose depletion has differential effects on the actin cytoskeleton resulting in disassembly of actin patches with concomitant inhibition of endocytosis, and strong stabilization of actin cables, thereby revealing a selective and previously unappreciated ATP requirement for actin cable disassembly. A similar response is seen in HeLa cells to ATP depletion. These findings reveal a new fast-acting energy conservation strategy halting growth by immobilizing myosin-V in a newly described state on selectively stabilized actin cables. PMID:25308080

  7. 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

  8. [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.

  9. New functions for alpha-catenins in health and disease: from cancer to heart regeneration.

    PubMed

    Vite, Alexia; Li, Jifen; Radice, Glenn L

    2015-06-01

    Strong cell-cell adhesion mediated by adherens junctions is dependent on anchoring the transmembrane cadherin molecule to the underlying actin cytoskeleton. To do this, the cadherin cytoplasmic domain interacts with catenin proteins, which include α-catenin that binds directly to filamentous actin. Originally thought to be a static structure, the connection between the cadherin/catenin adhesion complex and the actin cytoskeleton is now considered to be dynamic and responsive to both intercellular and intracellular signals. Alpha-catenins are mechanosensing proteins that undergo conformational change in response to cytoskeletal tension thus modifying the linkage between the cadherin and the actin cytoskeleton. There are three α-catenin isoforms expressed in mouse and human: αE-catenin (CTNNA1), αN-catenin (CTNNA2) and αT-catenin (CTNNA3). This review summarizes recent progress in understanding the in vivo function(s) of α-catenins in tissue morphogenesis, homeostasis and disease. The role of α-catenin in the regulation of cellular proliferation will be discussed in the context of cancer and regeneration.

  10. Mechanics of composite actin networks: in vitro and cellular perspectives

    NASA Astrophysics Data System (ADS)

    Upadhyaya, Arpita

    2014-03-01

    Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not well understood. The ABP, palladin, is essential for the integrity of cell morphology and movement during development. Palladin coexists with alpha-actinin in stress fibers and focal adhesions and binds to both actin and alpha-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we have characterized the micro-structure and mechanics of actin networks crosslinked with palladin and alpha-actinin. Our studies on composite networks of alpha-actinin/palladin/actin show that palladin and alpha-actinin synergistically determine network viscoelasticity. We have further examined the role of palladin in cellular force generation and mechanosensing. Traction force microscopy revealed that TAFs are sensitive to substrate stiffness as they generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells, and also inhibited the ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in the actin organization and adhesion dynamics of palladin knock down cells. Perturbation experiments also suggest altered myosin activity in palladin KD cells. Our results suggest that the actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

  11. The CAMSAP3-ACF7 Complex Couples Noncentrosomal Microtubules with Actin Filaments to Coordinate Their Dynamics.

    PubMed

    Ning, Wenxiu; Yu, Yanan; Xu, Honglin; Liu, Xiaofei; Wang, Daiwei; Wang, Jing; Wang, Yingchun; Meng, Wenxiang

    2016-10-10

    For adaptation to complex cellular functions, dynamic cytoskeletal networks are required. There are two major components of the cytoskeleton, microtubules and actin filaments, which form an intricate network maintaining an exquisite cooperation to build the physical basis for their cellular function. However, little is known about the molecular mechanism underlying their synergism. Here, we show that in Caco2 epithelial cells, noncentrosomal microtubules crosstalk with F-actin through their minus ends and contribute to the regulation of focal adhesion size and cell migration. We demonstrate that ACF7, a member of the spectraplakin family of cytoskeletal crosslinking proteins, interacts with Nezha (also called CAMSAP3) at the minus ends of noncentrosomal microtubules and anchors them to actin filaments. Those noncentrosomal microtubules cooperate with actin filaments through retrograde flow to keep their length and orientation perpendicular to the cell edge as well as regulate focal adhesion size and cell migration. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. 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.

  13. 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.

  14. F-actin and G-actin binding are uncoupled by mutation of conserved tyrosine residues in maize actin depolymerizing factor (ZmADF)

    PubMed Central

    Jiang, Chang-Jie; Weeds, Alan G.; Khan, Safina; Hussey, Patrick J.

    1997-01-01

    Actin depolymerizing factors (ADF) are stimulus responsive actin cytoskeleton modulating proteins. They bind both monomeric actin (G-actin) and filamentous actin (F-actin) and, under certain conditions, F-actin binding is followed by filament severing. In this paper, using mutant maize ADF3 proteins, we demonstrate that the maize ADF3 binding of F-actin can be spatially distinguished from that of G-actin. One mutant, zmadf3–1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3. A second mutant, zmadf3–2, in which both Tyr-67 and Tyr-70 are replaced by phenylalanine, shows an affinity for G-actin similar to maize ADF3, but F-actin binding is abolished. The two tyrosines, Tyr-67 and Tyr-70, are in the equivalent position to Tyr-82 and Tyr-85 of destrin, respectively. Using the tertiary structure of destrin, yeast cofilin, and Acanthamoeba actophorin, we discuss the implications of removing the aromatic hydroxyls of Tyr-82 and Tyr-85 (i.e., the effect of substituting phenylalanine for tyrosine) and conclude that Tyr-82 plays a critical role in stabilizing the tertiary structure that is essential for F-actin binding. We propose that this tertiary structure is maintained as a result of a hydrogen bond between the hydroxyl of Tyr-82 and the carbonyl of Tyr-117, which is located in the long α-helix; amino acid components of this helix (Leu-111 to Phe-128) have been implicated in G-actin and F-actin binding. The structures of human destrin and yeast cofilin indicate a hydrogen distance of 2.61 and 2.77 Å, respectively, with corresponding bond angles of 99.5° and 113°, close to the optimum for a strong hydrogen bond. PMID:9275236

  15. 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

  16. 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.

  17. Microtubule-actin crosslinking factor 1 (Macf1) domain function in Balbiani body dissociation and nuclear positioning.

    PubMed

    Escobar-Aguirre, Matias; Zhang, Hong; Jamieson-Lucy, Allison; Mullins, Mary C

    2017-09-01

    Animal-vegetal (AV) polarity of most vertebrate eggs is established during early oogenesis through the formation and disassembly of the Balbiani Body (Bb). The Bb is a structure conserved from insects to humans that appears as a large granule, similar to a mRNP granule composed of mRNA and proteins, that in addition contains mitochondria, ER and Golgi. The components of the Bb, which have amyloid-like properties, include germ cell and axis determinants of the embryo that are anchored to the vegetal cortex upon Bb disassembly. Our lab discovered in zebrafish the only gene known to function in Bb disassembly, microtubule-actin crosslinking factor 1a (macf1a). Macf1 is a conserved, giant multi-domain cytoskeletal linker protein that can interact with microtubules (MTs), actin filaments (AF), and intermediate filaments (IF). In macf1a mutant oocytes the Bb fails to dissociate, the nucleus is acentric, and AV polarity of the oocyte and egg fails to form. The cytoskeleton-dependent mechanism by which Macf1a regulates Bb mRNP granule dissociation was unknown. We found that disruption of AFs phenocopies the macf1a mutant phenotype, while MT disruption does not. We determined that cytokeratins (CK), a type of IF, are enriched in the Bb. We found that Macf1a localizes to the Bb, indicating a direct function in regulating its dissociation. We thus tested if Macf1a functions via its actin binding domain (ABD) and plectin repeat domain (PRD) to integrate cortical actin and Bb CK, respectively, to mediate Bb dissociation at the oocyte cortex. We developed a CRISPR/Cas9 approach to delete the exons encoding these domains from the macf1a endogenous locus, while maintaining the open reading frame. Our analysis shows that Macf1a functions via its ABD to mediate Bb granule dissociation and nuclear positioning, while the PRD is dispensable. We propose that Macf1a does not function via its canonical mechanism of linking two cytoskeletal systems together in dissociating the Bb. Instead

  18. Microtubule-actin crosslinking factor 1 (Macf1) domain function in Balbiani body dissociation and nuclear positioning

    PubMed Central

    Zhang, Hong; Jamieson-Lucy, Allison

    2017-01-01

    Animal-vegetal (AV) polarity of most vertebrate eggs is established during early oogenesis through the formation and disassembly of the Balbiani Body (Bb). The Bb is a structure conserved from insects to humans that appears as a large granule, similar to a mRNP granule composed of mRNA and proteins, that in addition contains mitochondria, ER and Golgi. The components of the Bb, which have amyloid-like properties, include germ cell and axis determinants of the embryo that are anchored to the vegetal cortex upon Bb disassembly. Our lab discovered in zebrafish the only gene known to function in Bb disassembly, microtubule-actin crosslinking factor 1a (macf1a). Macf1 is a conserved, giant multi-domain cytoskeletal linker protein that can interact with microtubules (MTs), actin filaments (AF), and intermediate filaments (IF). In macf1a mutant oocytes the Bb fails to dissociate, the nucleus is acentric, and AV polarity of the oocyte and egg fails to form. The cytoskeleton-dependent mechanism by which Macf1a regulates Bb mRNP granule dissociation was unknown. We found that disruption of AFs phenocopies the macf1a mutant phenotype, while MT disruption does not. We determined that cytokeratins (CK), a type of IF, are enriched in the Bb. We found that Macf1a localizes to the Bb, indicating a direct function in regulating its dissociation. We thus tested if Macf1a functions via its actin binding domain (ABD) and plectin repeat domain (PRD) to integrate cortical actin and Bb CK, respectively, to mediate Bb dissociation at the oocyte cortex. We developed a CRISPR/Cas9 approach to delete the exons encoding these domains from the macf1a endogenous locus, while maintaining the open reading frame. Our analysis shows that Macf1a functions via its ABD to mediate Bb granule dissociation and nuclear positioning, while the PRD is dispensable. We propose that Macf1a does not function via its canonical mechanism of linking two cytoskeletal systems together in dissociating the Bb. Instead

  19. Baculovirus AC102 Is a Nucleocapsid Protein That Is Crucial for Nuclear Actin Polymerization and Nucleocapsid Morphogenesis.

    PubMed

    Hepp, Susan E; Borgo, Gina M; Ticau, Simina; Ohkawa, Taro; Welch, Matthew D

    2018-06-01

    The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was proposed to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex. IMPORTANCE The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also used as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which Ac

  20. 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.

  1. Gamma Interferon-Induced Guanylate Binding Protein 1 Is a Novel Actin Cytoskeleton Remodeling Factor

    PubMed Central

    Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J. K.; Schubert, Dirk W.; Stockinger, Hannes; Herrmann, Christian

    2014-01-01

    Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies. PMID:24190970

  2. Gamma interferon-induced guanylate binding protein 1 is a novel actin cytoskeleton remodeling factor.

    PubMed

    Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J K; Schubert, Dirk W; Stockinger, Hannes; Herrmann, Christian; Stürzl, Michael

    2014-01-01

    Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies.

  3. Actin is an essential component of plant gravitropic signaling pathways

    NASA Astrophysics Data System (ADS)

    Braun, Markus; Hauslage, Jens; Limbach, Christoph

    2003-08-01

    A role of the actin cytoskeleton in the different phases of gravitropism in higher plant organs seems obvious, but experimental evidence is still inconclusive and contradictory. In gravitropically tip-growing rhizoids and protonemata, however, it is well documented that actin is an essential component of the tip-growth machinery and is involved either in the cellular mechanisms that lead to gravity sensing and in the processes of the graviresponses that result in the reorientation of the growth direction. All these processes depend on a complexly organized and highly dynamic organization of actin filaments whose diverse functions are coordinated by numerous associated proteins. Actin filaments and myosins mediate the transport of secretory vehicles to the growing tip and precisely control the delivery of cell wall material. In addition, both cell types use a very efficient actomyosin-based system to control and correct the position of their statoliths and to direct sedimenting statoliths to confined graviperception sites at the plasma membrane. The studies presented in this paper provide evidence for the essential role of actin in plant gravity sensing and the gravitropic responses. A unique actin-organizing center exists in the tip of characean rhizoids and protonemata which is associated with and dynamically regulated by a specific set of actin-dynamizing proteins. It is concluded that this highly dynamic apical actin array is an essential prerequisite for gravity sensing and gravity-oriented tip growth.

  4. Cytoskeleton in gravisensing and signal transductionof lower plants

    NASA Astrophysics Data System (ADS)

    Braun, M.

    Characean rhizoids and protonemata are favourable cell types for studying tip growth and gravisensing. Both processes are highly dependent on the actin cytoskeleton. The multiple functions and different arrangements of actin in both cell types are regulated by the concerted action of actin-binding proteins. Monomer- binding profilin is distributed evenly throughout the cytoplasm and is likely to be involved in the regulation of the polymerization state of actin. Actin-severing ADF, spectrin- and actinin-like epitopes concentrate in a central prominent spot in the apex of both cell types, where they colocalize with a dense, spherical actin array and a unique aggregation of endoplasmic reticulum (ER), the structural center of the tip - growth organizing Spitzenkörper. The ER aggregate disintegrates and immuno- localization of the actin-binding proteins fails when tip growth is arrested; the epitopes reappear when tip growth resumes. Actin filaments form a meshwork of axially oriented filaments in the subapical zone and focus in this central apical area which seems to represent their apical polymerization site. The rapid turn-over and rearrangement of actin might be under control of ADF and profilin. Spectrin- and actinin-like proteins are candidates for establishing the actin-mediated anchoring and maintaining of the ER aggregate. They could also provide a mechanism for recruiting specific membrane proteins that create the particular physiological environment for gravity-oriented tip growth. The positioning and sedimentation of statoliths in the subapical region (crucial for gravisensing) is highly coordinated by actomyosin. Non-invasive infrared laser micromanipulation techniques, centri- fugation and experiments in microgravity revealed that reorientation of the growth direction was initiated when at least 2-3 statoliths were directed to specific areas of the plasma membrane by actomyosin and gravitational forces. The statolith-sensitive area is confined to the

  5. The carboxyl terminus of the alpha-subunit of the amiloride-sensitive epithelial sodium channel binds to F-actin.

    PubMed

    Mazzochi, Christopher; Bubien, James K; Smith, Peter R; Benos, Dale J

    2006-03-10

    The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.

  6. 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

  7. Regulation of the Pollen-Specific Actin-Depolymerizing Factor LlADF1

    PubMed Central

    Allwood, Ellen G.; Anthony, Richard G.; Smertenko, Andrei P.; Reichelt, Stefanie; Drobak, Bjorn K.; Doonan, John H.; Weeds, Alan G.; Hussey, Patrick J.

    2002-01-01

    Pollen tube growth is dependent on a dynamic actin cytoskeleton, suggesting that actin-regulating proteins are involved. We have examined the regulation of the lily pollen-specific actin-depolymerizing factor (ADF) LlADF1. Its actin binding and depolymerizing activity is pH sensitive, inhibited by certain phosphoinositides, but not controlled by phosphorylation. Compared with its F-actin binding properties, its low activity in depolymerization assays has been used to explain why pollen ADF decorates F-actin in pollen grains. This low activity is incompatible with a role in increasing actin dynamics necessary to promote pollen tube growth. We have identified a plant homolog of actin-interacting protein, AIP1, which enhances the depolymerization of F-actin in the presence of LlADF1 by ∼60%. Both pollen ADF and pollen AIP1 bind F-actin in pollen grains but are mainly cytoplasmic in pollen tubes. Our results suggest that together these proteins remodel actin filaments as pollen grains enter and exit dormancy. PMID:12417710

  8. 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.

  9. 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.

  10. 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.

  11. Dynamic interaction between actin and nesprin2 maintain the cell nucleus in a prestressed state

    NASA Astrophysics Data System (ADS)

    Kumar, Abhishek; Shivashankar, G. V.

    2016-12-01

    Mechanical coupling between the nucleus and the cytoskeleton is indispensable for direct force transduction from the extra cellular matrix (ECM) to the chromatin. Although this physical coupling has been shown to be crucial for nuclear positioning and its function, the quantification of nuclear-cytoskeleton interaction has been lacking. In this paper, using various quantitative fluorescence spectroscopy techniques, we investigate the nature of this connection. High-resolution 3D imaging shows that nesprin2G forms short linear structures along actin stress fibers (ASFs) in the apical region of the nucleus. Fluorescence recovery after photobleaching (FRAP) revealed that the alignment of nesprin2G becomes heterogeneous when cell shape is engineered from elongated rectangular shape to square using micropatterned substrates. Further, fluorescence cross-correlation spectroscopy (FCCS) revealed that actin interacts transiently with outer nuclear membrane protein nesprin2G with a time scale of 12 ms. In addition, fluorescence resonance energy transfer (FRET) experiments show that the apical ASFs and nesprin2G are in close physical proximity. This interaction is spatially heterogeneous with high FRET along the ASFs. Lastly, we show that the disruption of actin to nuclear connection by over-expression of Dominant Negative Klarsicht, ANC-1, Syne Homology (DNKASH) leads to an increase in nuclear height. These results not only reveal the characteristics of actin-nesprin2G interaction and its significance in regulating nuclear morphology, but also validate the utility of quantitative fluorescence techniques in deciphering physical connections that are essential for mechanotransduction.

  12. Arp2/3 complex–dependent actin networks constrain myosin II function in driving retrograde actin flow

    PubMed Central

    Yang, Qing; Zhang, Xiao-Feng; Pollard, Thomas D.

    2012-01-01

    The Arp2/3 complex nucleates actin filaments to generate networks at the leading edge of motile cells. Nonmuscle myosin II produces contractile forces involved in driving actin network translocation. We inhibited the Arp2/3 complex and/or myosin II with small molecules to investigate their respective functions in neuronal growth cone actin dynamics. Inhibition of the Arp2/3 complex with CK666 reduced barbed end actin assembly site density at the leading edge, disrupted actin veils, and resulted in veil retraction. Strikingly, retrograde actin flow rates increased with Arp2/3 complex inhibition; however, when myosin II activity was blocked, Arp2/3 complex inhibition now resulted in slowing of retrograde actin flow and veils no longer retracted. Retrograde flow rate increases induced by Arp2/3 complex inhibition were independent of Rho kinase activity. These results provide evidence that, although the Arp2/3 complex and myosin II are spatially segregated, actin networks assembled by the Arp2/3 complex can restrict myosin II–dependent contractility with consequent effects on growth cone motility. PMID:22711700

  13. Actin-membrane interactions mediated by NETWORKED2 in Arabidopsis pollen tubes through associations with Pollen Receptor-Like Kinase 4 and 5.

    PubMed

    Duckney, Patrick; Deeks, Michael J; Dixon, Martin R; Kroon, Johan; Hawkins, Timothy J; Hussey, Patrick J

    2017-12-01

    During fertilization, Pollen Receptor-Like Kinases (PRKs) control pollen tube growth through the pistil in response to extracellular signals, and regulate the actin cytoskeleton at the tube apex to drive tip growth. We investigated a novel link between membrane-integral PRKs and the actin cytoskeleton, mediated through interactions between PRKs and NET2A; a pollen-specific member of the NETWORKED superfamily of actin-binding proteins. We characterize NET2A as a novel actin-associated protein that localizes to punctae at the plasma membrane of the pollen tube shank, which are stably associated with cortical longitudinal actin cables. NET2A was demonstrated to interact specifically with PRK4 and PRK5 in Nicotiana benthamiana transient expression assays, and associated at discreet foci at the shank membrane of Arabidopsis pollen tubes. Our data indicate that NET2A is recruited to the plasma membrane by PRK4 and PRK5, and that PRK kinase activity is important in facilitating its interaction with NET2A. We conclude that NET2A-PRK interactions mediate discreet sites of stable interactions between the cortical longitudinal actin cables and plasma membrane in the shank region of growing pollen tubes, which we have termed Actin-Membrane Contact Sites (AMCSs). Interactions between PRKs and NET2A implicate a role for NET2A in signal transduction to the actin cytoskeleton during fertilization. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  14. Tip-localized actin polymerization and remodeling, reflected by the localization of ADF, profilin and villin, are fundamental for gravity-sensing and polar growth in characean rhizoids.

    PubMed

    Braun, Markus; Hauslage, Jens; Czogalla, Aleksander; Limbach, Christoph

    2004-07-01

    Polar organization and gravity-oriented, polarized growth of characean rhizoids are dependent on the actin cytoskeleton. In this report, we demonstrate that the prominent center of the Spitzenkörper serves as the apical actin polymerization site in the extending tip. After cytochalasin D-induced disruption of the actin cytoskeleton, the regeneration of actin microfilaments (MFs) starts with the reappearance of a flat, brightly fluorescing actin array in the outermost tip. The actin array rounds up, produces actin MFs that radiate in all directions and is then relocated into its original central position in the center of the Spitzenkörper. The emerging actin MFs rearrange and cross-link to form the delicate, subapical meshwork, which then controls the statolith positioning, re-establishes the tip-high calcium gradient and mediates the reorganization of the Spitzenkörper with its central ER aggregate and the accumulation of secretory vesicles. Tip growth and gravitropic sensing, which includes control of statolith positioning and gravity-induced sedimentation, are not resumed until the original polar actin organization is completely restored. Immunolocalization of the actin-binding proteins, actin-depolymerizing factor (ADF) and profilin, which both accumulate in the center of the Spitzenkörper, indicates high actin turnover and gives additional support for the actin-polymerizing function of this central, apical area. Association of villin immunofluorescence with two populations of thick undulating actin cables with uniform polarity underlying rotational cytoplasmic streaming in the basal region suggests that villin is the major actin-bundling protein in rhizoids. Our results provide evidence that the precise coordination of apical actin polymerization and dynamic remodeling of actin MFs by actin-binding proteins play a fundamental role in cell polarization, gravity sensing and gravity-oriented polarized growth of characean rhizoids.

  15. Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration.

    PubMed

    Croft, Daniel R; Coleman, Mathew L; Li, Shuixing; Robertson, David; Sullivan, Teresa; Stewart, Colin L; Olson, Michael F

    2005-01-17

    Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization.

  16. Actin-myosin–based contraction is responsible for apoptotic nuclear disintegration

    PubMed Central

    Croft, Daniel R.; Coleman, Mathew L.; Li, Shuixing; Robertson, David; Sullivan, Teresa; Stewart, Colin L.; Olson, Michael F.

    2005-01-01

    Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization. PMID:15657395

  17. Kindler surprise: mutations in a novel actin-associated protein cause Kindler syndrome.

    PubMed

    White, Sharon J; McLean, W H Irwin

    2005-06-01

    Kindler syndrome is an autosomal recessive genodermatosis characterized by acral blistering in neonates and diffuse, progressive poikiloderma in later life. Other clinical features include photosensitivity, premature skin ageing and severe periodontal disease. Two groups have recently shown that the molecular basis of Kindler syndrome is loss of a novel epidermal protein, kindlin-1, encoded by the gene KIND1. Two additional kindlin proteins, kindlin-2 and kindlin-3, have also been described. Kindlin-1 is considered to be a component in the linkage of the actin cytoskeleton to the extracellular matrix and as such is proposed to have both structural and cell-signalling functions. Kindler syndrome is therefore the first skin fragility syndrome due to disruption of the actin-extracellular matrix system.

  18. The actin-like MreB proteins in Bacillus subtilis: a new turn.

    PubMed

    Chastanet, Arnaud; Carballido-Lopez, Rut

    2012-06-01

    A decade ago, two breakthrough descriptions were reported: 1) the first helix-like protein localization pattern of MreB and its paralog Mbl in Bacillus subtilis and 2) the crystal structure of Thermotoga maritima MreB1, which was remarkably similar to that of actin. These discoveries strongly stimulated the field of bacterial development, leading to the identification of many new cytoskeletal proteins (1) and the publication of many studies describing the helical patterns of protein, DNA and even lipid domains. However, today, new breakthroughs are shaking up what had become a dogma. Instead of helical structures, MreBs appear to form discrete patches that move circumferentially around the cell, questioning the idea of MreB cables forming an actin-like cytoskeleton. Furthermore, increasing evidence of biochemical properties that are unlike the properties of actin suggest that the molecular behavior of MreB proteins may be different. The aim of this review is to summarize the current knowledge of the so-called "actin-like" MreB cytoskeleton through a discussion of the model Gram-positive bacterium B. subtilis and the most recent findings in this rapidly evolving research field.

  19. The subcortical maternal complex controls symmetric division of mouse zygotes by regulating F-actin dynamics.

    PubMed

    Yu, Xing-Jiang; Yi, Zhaohong; Gao, Zheng; Qin, Dandan; Zhai, Yanhua; Chen, Xue; Ou-Yang, Yingchun; Wang, Zhen-Bo; Zheng, Ping; Zhu, Min-Sheng; Wang, Haibin; Sun, Qing-Yuan; Dean, Jurrien; Li, Lei

    2014-09-11

    Maternal effect genes play critical roles in early embryogenesis of model organisms where they have been intensively investigated. However, their molecular function in mammals remains largely unknown. Recently, we identified a subcortical maternal complex (SCMC) that contains four proteins encoded by maternal effect genes (Mater, Filia, Floped and Tle6). Here we report that TLE6, similar to FLOPED and MATER, stabilizes the SCMC and is necessary for cleavage beyond the two-cell stage of development. We document that the SCMC is required for formation of the cytoplasmic F-actin meshwork that controls the central position of the spindle and ensures symmetric division of mouse zygotes. We further demonstrate that the SCMC controls formation of the actin cytoskeleton specifically via Cofilin, a key regulator of F-actin assembly. Our results provide molecular insight into the physiological function of TLE6, its interaction with the SCMC and their roles in the symmetric division of the zygote in early mouse development.

  20. The F-BAR protein Hof1 tunes formin activity to sculpt actin cables during polarized growth

    PubMed Central

    Graziano, Brian R.; Yu, Hoi-Ying E.; Alioto, Salvatore L.; Eskin, Julian A.; Ydenberg, Casey A.; Waterman, David P.; Garabedian, Mikael; Goode, Bruce L.

    2014-01-01

    Asymmetric cell growth and division rely on polarized actin cytoskeleton remodeling events, the regulation of which is poorly understood. In budding yeast, formins stimulate the assembly of an organized network of actin cables that direct polarized secretion. Here we show that the Fer/Cip4 homology–Bin amphiphysin Rvs protein Hof1, which has known roles in cytokinesis, also functions during polarized growth by directly controlling the activities of the formin Bnr1. A mutant lacking the C-terminal half of Hof1 displays misoriented and architecturally altered cables, along with impaired secretory vesicle traffic. In vitro, Hof1 inhibits the actin nucleation and elongation activities of Bnr1 without displacing the formin from filament ends. These effects depend on the Src homology 3 domain of Hof1, the formin homology 1 (FH1) domain of Bnr1, and Hof1 dimerization, suggesting a mechanism by which Hof1 “restrains” the otherwise flexible FH1-FH2 apparatus. In vivo, loss of inhibition does not alter actin levels in cables but, instead, cable shape and functionality. Thus Hof1 tunes formins to sculpt the actin cable network. PMID:24719456

  1. Multiple forms of Spire-actin complexes and their functional consequences.

    PubMed

    Chen, Christine K; Sawaya, Michael R; Phillips, Martin L; Reisler, Emil; Quinlan, Margot E

    2012-03-23

    Spire is a WH2 domain-containing actin nucleator essential for establishing an actin mesh during oogenesis. In vitro, in addition to nucleating filaments, Spire can sever them and sequester actin monomers. Understanding how Spire is capable of these disparate functions and which are physiologically relevant is an important goal. To study severing, we examined the effect of Drosophila Spire on preformed filaments in bulk and single filament assays. We observed rapid depolymerization of actin filaments by Spire, which we conclude is largely due to its sequestration activity and enhanced by its weak severing activity. We also studied the solution and crystal structures of Spire-actin complexes. We find structural and functional differences between constructs containing four WH2 domains (Spir-ABCD) and two WH2 domains (Spir-CD) that may provide insight into the mechanisms of nucleation and sequestration. Intriguingly, we observed lateral interactions between actin monomers associated with Spir-ABCD, suggesting that the structures built by these four tandem WH2 domains are more complex than originally imagined. Finally, we propose that Spire-actin mixtures contain both nuclei and sequestration structures.

  2. Distinct cytoskeleton populations and extensive crosstalk control Ciona notochord tubulogenesis.

    PubMed

    Dong, Bo; Deng, Wei; Jiang, Di

    2011-04-01

    Cell elongation is a fundamental process that allows cells and tissues to adopt new shapes and functions. During notochord tubulogenesis in the ascidian Ciona intestinalis, a dramatic elongation of individual cells takes place that lengthens the notochord and, consequently, the entire embryo. We find a novel dynamic actin- and non-muscle myosin II-containing constriction midway along the anteroposterior aspect of each notochord cell during this process. Both actin polymerization and myosin II activity are required for the constriction and cell elongation. Discontinuous localization of myosin II in the constriction indicates that the actomyosin network produces local contractions along the circumference. This reveals basal constriction by the actomyosin network as a novel mechanism for cell elongation. Following elongation, the notochord cells undergo a mesenchymal-epithelial transition and form two apical domains at opposite ends. Extracellular lumens then form at the apical surfaces. We show that cortical actin and Ciona ezrin/radixin/moesin (ERM) are essential for lumen formation and that a polarized network of microtubules, which contributes to lumen development, forms in an actin-dependent manner at the apical cortex. Later in notochord tubulogenesis, when notochord cells initiate a bi-directional crawling movement on the notochordal sheath, the microtubule network rotates 90° and becomes organized as parallel bundles extending towards the leading edges of tractive lamellipodia. This process is required for the correct organization of actin-based protrusions and subsequent lumen coalescence. In summary, we establish the contribution of the actomyosin and microtubule networks to notochord tubulogenesis and reveal extensive crosstalk and regulation between these two cytoskeleton components.

  3. Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions

    NASA Technical Reports Server (NTRS)

    Pavalko, F. M.; Chen, N. X.; Turner, C. H.; Burr, D. B.; Atkinson, S.; Hsieh, Y. F.; Qiu, J.; Duncan, R. L.

    1998-01-01

    Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.

  4. 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.

  5. Direct membrane binding by bacterial actin MreB.

    PubMed

    Salje, Jeanne; van den Ent, Fusinita; de Boer, Piet; Löwe, Jan

    2011-08-05

    Bacterial actin MreB is one of the key components of the bacterial cytoskeleton. It assembles into short filaments that lie just underneath the membrane and organize the cell wall synthesis machinery. Here we show that MreB from both T. maritima and E. coli binds directly to cell membranes. This function is essential for cell shape determination in E. coli and is proposed to be a general property of many, if not all, MreBs. We demonstrate that membrane binding is mediated by a membrane insertion loop in TmMreB and by an N-terminal amphipathic helix in EcMreB and show that purified TmMreB assembles into double filaments on a membrane surface that can induce curvature. This, the first example of a membrane-binding actin filament, prompts a fundamental rethink of the structure and dynamics of MreB filaments within cells. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. 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.

  7. Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane - a minimally invasive investigation by STED-FCS

    NASA Astrophysics Data System (ADS)

    Andrade, Débora M.; Clausen, Mathias P.; Keller, Jan; Mueller, Veronika; Wu, Congying; Bear, James E.; Hell, Stefan W.; Lagerholm, B. Christoffer; Eggeling, Christian

    2015-06-01

    Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.

  8. Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane--a minimally invasive investigation by STED-FCS.

    PubMed

    Andrade, Débora M; Clausen, Mathias P; Keller, Jan; Mueller, Veronika; Wu, Congying; Bear, James E; Hell, Stefan W; Lagerholm, B Christoffer; Eggeling, Christian

    2015-06-29

    Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.

  9. 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.

  10. 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.

  11. Vesicular transport protein Arf6 modulates cytoskeleton dynamics for polar body extrusion in mouse oocyte meiosis.

    PubMed

    Duan, Xing; Zhang, Hao-Lin; Pan, Meng-Hao; Zhang, Yu; Sun, Shao-Chen

    2018-02-01

    Arf6 (ADP-ribosylation factor 6) is known to play important roles in membrane dynamics through the regulation of actin filament reorganization for multiple cellular processes such as cytokinesis, phagocytosis, cell migration and tumor cell invasion. However, the functions of Arf6 in mammalian oocyte meiosis have not been clarified. In present study we showed that Arf6 expressed in mouse oocytes and was mainly distributed around the spindle during meiosis. Depletion of Arf6 by morpholino microinjection caused oocytes failing to extrude first polar body. Further analysis indicated that Arf6 knock down caused the aberrant actin distribution, which further induced the failure of meiotic spindle movement. And the loss of oocyte polarity also confirmed this. The regulation of Arf6 on actin filaments in mouse oocytes might be due to its effects on the phosphorylation level of cofilin and the expression of Arp2/3 complex. Moreover, we found that the decrease of Arf6 caused the disruption of spindle formation, indicating the multiple roles of Arf6 on cytoskeleton dynamics in meiosis. In summary, our results indicated that Arf6 was involved in mouse oocyte meiosis through its functional roles in actin-mediated spindle movement and spindle organization. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. The actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

    We report evidence that the actin machinery of chemotactic Dictyostelium cells operates close to an oscillatory instability. The averaged F-actin response of many cells to a short-time pulse of cAMP is 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. We systematically exposed a large number of cells to periodic pulse trains. The results indicate a resonance peak at periodic inputs of around 20 s. We propose a delayed feedback model that explains our experimental findings based on a time-delay in the actin regulatory network. To quantitatively test the model, we performed stimulation experiments with cells that express GFP-tagged fusion proteins of Coronin and Aip1. These served as markers of the F-actin disassembly process and thus allow us to estimate the delay time. Based on this independent estimate, our model predicts an intrinsic period of 20 s, which agrees with the resonance observed experimentally. Financial support by the Max-Planck Society and the DFG (SFB 937).

  13. Electrostatics Control Actin Filament Nucleation and Elongation Kinetics*

    PubMed Central

    Crevenna, Alvaro H.; Naredi-Rainer, Nikolaus; Schönichen, André; Dzubiella, Joachim; Barber, Diane L.; Lamb, Don C.; Wedlich-Söldner, Roland

    2013-01-01

    The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment. PMID:23486468

  14. AtFH1 formin mutation affects actin filament and microtubule dynamics in Arabidopsis thaliana.

    PubMed

    Rosero, Amparo; Žársky, Viktor; Cvrčková, Fatima

    2013-01-01

    Plant cell growth and morphogenesis depend on remodelling of both actin and microtubule cytoskeletons. AtFH1 (At5g25500), the main housekeeping Arabidopsis formin, is targeted to membranes and known to nucleate and bundle actin. The effect of mutations in AtFH1 on root development and cytoskeletal dynamics was examined. Consistent with primarily actin-related formin function, fh1 mutants showed increased sensitivity to the actin polymerization inhibitor latrunculin B (LatB). LatB-treated mutants had thicker, shorter roots than wild-type plants. Reduced cell elongation and morphological abnormalities were observed in both trichoblasts and atrichoblasts. Fluorescently tagged cytoskeletal markers were used to follow cytoskeletal dynamics in wild-type and mutant plants using confocal microscopy and VAEM (variable-angle epifluorescence microscopy). Mutants exhibited more abundant but less dynamic F-actin bundles and more dynamic microtubules than wild-type seedlings. Treatment of wild-type seedlings with a formin inhibitor, SMIFH2, mimicked the root growth and cell expansion phenotypes and cytoskeletal structure alterations observed in fh1 mutants. The results suggest that besides direct effects on actin organization, the in vivo role of AtFH1 also includes modulation of microtubule dynamics, possibly mediated by actin-microtubule cross-talk.

  15. A WAVE2-Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens.

    PubMed

    Verma, Suzie; Han, Siew Ping; Michael, Magdalene; Gomez, Guillermo A; Yang, Zhe; Teasdale, Rohan D; Ratheesh, Aparna; Kovacs, Eva M; Ali, Radiya G; Yap, Alpha S

    2012-12-01

    The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2-Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2-Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2-Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin.

  16. MAP18 regulates the direction of pollen tube growth in Arabidopsis by modulating F-actin organization.

    PubMed

    Zhu, Lei; Zhang, Yan; Kang, Erfang; Xu, Qiangyi; Wang, Miaoying; Rui, Yue; Liu, Baoquan; Yuan, Ming; Fu, Ying

    2013-03-01

    For fertilization to occur in plants, the pollen tube must be guided to enter the ovule via the micropyle. Previous reports have implicated actin filaments, actin binding proteins, and the tip-focused calcium gradient as key contributors to polar growth of pollen tubes; however, the regulation of directional pollen tube growth is largely unknown. We reported previously that Arabidopsis thaliana MICROTUBULE-ASSOCIATED PROTEIN18 (MAP18) contributes to directional cell growth and cortical microtubule organization. The preferential expression of MAP18 in pollen and in pollen tubes suggests that MAP18 also may function in pollen tube growth. In this study, we demonstrate that MAP18 functions in pollen tubes by influencing actin organization, rather than microtubule assembly. In vitro biochemical results indicate that MAP18 exhibits Ca(2+)-dependent filamentous (F)-actin-severing activity. Abnormal expression of MAP18 in map18 and MAP18 OX plants was associated with disorganization of the actin cytoskeleton in the tube apex, resulting in aberrant pollen tube growth patterns and morphologies, inaccurate micropyle targeting, and fewer fertilization events. Experiments with MAP18 mutants created by site-directed mutagenesis suggest that F-actin-severing activity is essential to the effects of MAP18 on pollen tube growth direction. Our study demonstrates that in Arabidopsis, MAP18 guides the direction of pollen tube growth by modulating actin filaments.

  17. Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics.

    PubMed

    Ono, Shoichiro; Ono, Kanako

    2002-03-18

    Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B-induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.

  18. 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

  19. 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.

  20. Highly Dynamic Host Actin Reorganization around Developing Plasmodium Inside Hepatocytes

    PubMed Central

    Gomes-Santos, Carina S. S.; Itoe, Maurice A.; Afonso, Cristina; Henriques, Ricardo; Gardner, Rui; Sepúlveda, Nuno; Simões, Pedro D.; Raquel, Helena; Almeida, António Paulo; Moita, Luis F.; Frischknecht, Friedrich; Mota, Maria M.

    2012-01-01

    Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver. PMID:22238609

  1. Force Exertion and Transmission in Cross-Linked Actin Networks

    NASA Astrophysics Data System (ADS)

    Stam, Samantha

    Cells are responsive to external cues in their environment telling them to proliferate or migrate within their surrounding tissue. Sensing of cues that are mechanical in nature, such stiffness of a tissue or forces transmitted from other cells, is believed to involve the cytoskeleton of a cell. The cytoskeleton is a complex network of proteins consisting of polymers that provide structural support, motor proteins that remodel these structures, and many others. We do not yet have a complete understanding of how cytoskeletal components respond to either internal or external mechanical force and stiffness. Such an understanding should involve mechanisms by which constituent molecules, such as motor proteins, are responsive to mechanics. Additionally, physical models of how forces are transmitted through biopolymer networks are necessary. My research has focused on networks formed by the cytoskeletal filament actin and the molecular motor protein myosin II. Actin filaments form networks and bundles that form a structural framework of the cell, and myosin II slides actin filaments. In this thesis, we show that stiffness of an elastic load that opposes myosin-generated actin sliding has a very sharp effect on the myosin force output in simulations. Secondly, we show that the stiffness and connectivity of cytoskeletal filaments regulates the contractility and anisotropy of network deformations that transmit force on material length scales. Together, these results have implications for predicting and interpreting the deformations and forces in biopolymeric active materials.

  2. The amino acid motif L/IIxxFE defines a novel actin-binding sequence in PDZ-RhoGEF

    PubMed Central

    Banerjee, Jayashree; Fischer, Christopher C.; Wedegaertner, Philip B.

    2009-01-01

    PDZ-RhoGEF is a member of the regulator of G protein signaling (RGS) domain-containing RhoGEFs (RGS-RhoGEFs) that link activated heterotrimeric G protein α subunits of the G12 family to activation of the small GTPase RhoA. Unique among the RGS-RhoGEFs, PDZ-RhoGEF contains a short sequence that localizes the protein to the actin cytoskeleton. In this report, we demonstrate that the actin-binding domain, located between amino acids 561–585, directly binds to F-actin in vitro. Extensive mutagenesis identifies isoleucine 568, isoleucine 569, phenylalanine 572, and glutamic acid 573 as necessary for binding to actin and for co-localization with the actin cytoskeleton in cells. These results define a novel actin-binding sequence in PDZ-RhoGEF with a critical amino acid motif of IIxxFE. Moreover, sequence analysis identifies a similar actin-binding motif in the N-terminus of the RhoGEF frabin, and, as with PDZ-RhoGEF, mutagenesis and actin interaction experiments demonstrate a motif of LIxxFE, consisting of the key amino acids leucine 23, isoleucine 24, phenylalanine 27, and glutamic acid 28. Taken together, results with PDZ-RhoGEF and frabin identify a novel actin binding sequence. Lastly, inducible dimerization of the actin-binding region of PDZ-RhoGEF revealed a dimerization-dependent actin bundling activity in vitro. PDZ-RhoGEF exists in cells as a dimer, raising the possibility that PDZ-RhoGEF could influence actin structure independent of its ability to activate RhoA. PMID:19618964

  3. Single-point ACT2 gene mutation in the Arabidopsis root hair mutant der1-3 affects overall actin organization, root growth and plant development.

    PubMed

    Vaškebová, L; Šamaj, J; Ovecka, M

    2017-12-27

    The actin cytoskeleton forms a dynamic network in plant cells. A single-point mutation in the DER1 (deformed root hairs1) locus located in the sequence of ACTIN2, a gene for major actin in vegetative tissues of Arabidopsis thaliana, leads to impaired root hair development (Ringli C, Baumberger N, Diet A, Frey B, Keller B. 2002. ACTIN2 is essential for bulge site selection and tip growth during root hair development of Arabidopsis. Plant Physiology129: 1464-1472). Only root hair phenotypes have been described so far in der1 mutants, but here we demonstrate obvious aberrations in the organization of the actin cytoskeleton and overall plant development. Organization of the actin cytoskeleton in epidermal cells of cotyledons, hypocotyls and roots was studied qualitatively and quantitatively by live-cell imaging of transgenic lines carrying the GFP-FABD2 fusion protein and in fixed cells after phalloidin labelling. Patterns of root growth were characterized by FM4-64 vital staining, light-sheet microscopy imaging and microtubule immunolabelling. Plant phenotyping included analyses of germination, root growth and plant biomass. Speed of germination, plant fresh weight and total leaf area were significantly reduced in the der1-3 mutant in comparison with the C24 wild-type. Actin filaments in root, hypocotyl and cotyledon epidermal cells of the der1-3 mutant were shorter, thinner and arranged in more random orientations, while actin bundles were shorter and had altered orientations. The wavy pattern of root growth in der1-3 mutant was connected with higher frequencies of shifted cell division planes (CDPs) in root cells, which was consistent with the shifted positioning of microtubule-based preprophase bands and phragmoplasts. The organization of cortical microtubules in the root cells of the der1-3 mutant, however, was not altered. Root growth rate of the der1-3 mutant is not reduced, but changes in the actin cytoskeleton organization can induce a wavy root growth pattern

  4. 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.

  5. Structure-function relationships in the stem cell's mechanical world B: emergent anisotropy of the cytoskeleton correlates to volume and shape changing stress exposure.

    PubMed

    Chang, Hana; Knothe Tate, Melissa L

    2011-12-01

    In the preceding study (Part A), we showed that prescribed seeding conditions as well as seeding density can be used to subject multipotent stem cells (MSCs) to volume changing stresses and that changes in volume of the cell are associated with changes in shape, but not volume, of the cell nucleus. In the current study, we aim to control the mechanical milieu of live cells using these prescribed seeding conditions concomitant to delivery of shape changing stresses via fluid flow, while observing adaptation of the cytoskeleton, a major cellular transducer that modulates cell shape, stiffness and remodeling. We hypothesize that the spatiotemporal organization of tubulin and actin elements of the cytoskeleton changes in response to volume and shape changing stresses emulating those during development, prior to the first beating of the heart or twitching of muscle. Our approach was to quantify the change over baseline in spatiotemporal distribution of actin and tubulin in live C3H/10T1/2 model stem cells subjected to volume changing stresses induced by seeding at density as well as low magnitude, short duration, shape changing (shear) stresses induced by fluid flow (0.5 or 1.0 dyne/cm2 for 30/60/90 minutes). Upon exposure to fluid flow, both tubulin thickness (height) and concentration (fluorescence intensity) change significantly over baseline, as a function of proximity to neighboring cells (density) and the substrate (apical-basal height). Given our recently published studies showing amplification of stress gradients (flow velocity) with increasing distance to nearest neighbors and the substrate, i.e. with decreasing density and toward the apical side of the cell, tubulin adaptation appears to depend significantly on the magnitude of the stress to which the cell is exposed locally. In contrast, adaptation of actin to the changing mechanical milieu is more global, exhibiting less significant differences attributable to nearest neighbors or boundaries than differences

  6. 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.

  7. 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.

  8. 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

  9. A Feedback Loop between Dynamin and Actin Recruitment during Clathrin-Mediated Endocytosis

    PubMed Central

    Taylor, Marcus J.; Lampe, Marko; Merrifield, Christien J.

    2012-01-01

    Clathrin-mediated endocytosis proceeds by a sequential series of reactions catalyzed by discrete sets of protein machinery. The final reaction in clathrin-mediated endocytosis is membrane scission, which is mediated by the large guanosine triophosphate hydrolase (GTPase) dynamin and which may involve the actin-dependent recruitment of N-terminal containing BIN/Amphiphysin/RVS domain containing (N-BAR) proteins. Optical microscopy has revealed a detailed picture of when and where particular protein types are recruited in the ∼20–30 s preceding scission. Nevertheless, the regulatory mechanisms and functions that underpin protein recruitment are not well understood. Here we used an optical assay to investigate the coordination and interdependencies between the recruitment of dynamin, the actin cytoskeleton, and N-BAR proteins to individual clathrin-mediated endocytic scission events. These measurements revealed that a feedback loop exists between dynamin and actin at sites of membrane scission. The kinetics of dynamin, actin, and N-BAR protein recruitment were modulated by dynamin GTPase activity. Conversely, acute ablation of actin dynamics using latrunculin-B led to a ∼50% decrease in the incidence of scission, an ∼50% decrease in the amplitude of dynamin recruitment, and abolished actin and N-BAR recruitment to scission events. Collectively these data suggest that dynamin, actin, and N-BAR proteins work cooperatively to efficiently catalyze membrane scission. Dynamin controls its own recruitment to scission events by modulating the kinetics of actin and N-BAR recruitment to sites of scission. Conversely actin serves as a dynamic scaffold that concentrates dynamin and N-BAR proteins at sites of scission. PMID:22505844

  10. A WAVE2–Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens

    PubMed Central

    Verma, Suzie; Han, Siew Ping; Michael, Magdalene; Gomez, Guillermo A.; Yang, Zhe; Teasdale, Rohan D.; Ratheesh, Aparna; Kovacs, Eva M.; Ali, Radiya G.; Yap, Alpha S.

    2012-01-01

    The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2–Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2–Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2–Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin. PMID:23051739

  11. Mechanosensitive channels are activated by stress in the actin stress fibres, and could be involved in gravity sensing in plants.

    PubMed

    Tatsumi, H; Furuichi, T; Nakano, M; Toyota, M; Hayakawa, K; Sokabe, M; Iida, H

    2014-01-01

    Mechanosensitive (MS) channels are expressed in a variety of cells. The molecular and biophysical mechanism involved in the regulation of MS channel activities is a central interest in basic biology. MS channels are thought to play crucial roles in gravity sensing in plant cells. To date, two mechanisms have been proposed for MS channel activation. One is that tension development in the lipid bilayer directly activates MS channels. The second mechanism proposes that the cytoskeleton is involved in the channel activation, because MS channel activities are modulated by pharmacological treatments that affect the cytoskeleton. We tested whether tension in the cytoskeleton activates MS channels. Mammalian endothelial cells were microinjected with phalloidin-conjugated beads, which bound to stress fibres, and a traction force to the actin cytoskeleton was applied by dragging the beads with optical tweezers. MS channels were activated when the force was applied, demonstrating that a sub-pN force to the actin filaments activates a single MS channel. Plants may use a similar molecular mechanism in gravity sensing, since the cytoplasmic Ca(2+) concentration increase induced by changes in the gravity vector was attenuated by potential MS channel inhibitors, and by actin-disrupting drugs. These results support the idea that the tension increase in actin filaments by gravity-dependent sedimentation of amyloplasts activates MS Ca(2+) -permeable channels, which can be the molecular mechanism of a Ca(2+) concentration increase through gravistimulation. We review recent progress in the study of tension sensing by actin filaments and MS channels using advanced biophysical methods, and discuss their possible roles in gravisensing. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

  12. A role for γS-crystallin in the organization of actin and fiber cell maturation in the mouse lens

    PubMed Central

    Fan, Jianguo; Dong, Lijin; Mishra, Sanghamitra; Chen, Yingwei; FitzGerald, Paul; Wistow, Graeme

    2012-01-01

    γS-crystallin (γS) is a highly conserved component of the eye lens. To gain insights into the functional role(s) of this protein, the mouse gene (Crygs) was deleted. Although mutations in γS can cause severe cataracts, loss of function of γS in knockout (KO) mice produced no obvious lens opacity, but was associated with focusing defects. Electron microscopy showed no major differences in lens cell organization, suggesting that the optical defects are primarily cytoplasmic in origin. KO lenses were also grossly normal by light microscopy but showed evidence of incomplete clearance of cellular organelles in maturing fiber cells. Phalloidin labeling showed an unusual distribution of F-actin in a band of mature fiber cells in KO lenses, suggesting a defect in the organization or processing of the actin cytoskeleton. Indeed, in wild-type lenses, γS and F-actin colocalize along the fiber cell plasma membrane. Relative levels of F-actin and G-actin in wild-type and KO lenses were estimated from fluorescent staining profiles and from isolation of actin fractions from whole lenses. Both methods showed a twofold reduction in the F-actin/G-actin ratio in KO lenses, whereas no difference in tubulin organization was detected. In vitro experiments showed that recombinant mouse γS can directly stabilize F-actin. This suggests that γS may have a functional role related to actin, perhaps in ‘shepherding’ filaments to maintain the optical properties of the lens cytoplasm and normal fiber cell maturation. PMID:22715935

  13. Perfluorooctanesulfonate (PFOS) Perturbs Male Rat Sertoli Cell Blood-Testis Barrier Function by Affecting F-Actin Organization via p-FAK-Tyr407: An in Vitro Study

    PubMed Central

    Wan, Hin-Ting; Mruk, Dolores D.; Wong, Chris K. C.

    2014-01-01

    Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality, in humans. However, the underlying mechanism(s) remains unknown. Herein PFOS at 10–20 μM (∼5–10 μg/mL) was found to be more potent than bisphenol A (100 μM) in perturbing the blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory and adhesion proteins at the cell-cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43, a GJ integral membrane protein. Third, phosphorylated focal adhesion kinase (FAK)-Tyr407 was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific microRNA, miR-135b, to knock down the expression of phosphorylated FAK-Tyr407 was found to worsen PFOS-mediated Sertoli cell tight junction disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated FAK-Tyr407 and connexin-43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mislocalization of actin-regulatory and adhesion proteins at the BTB. PMID:24169556

  14. Two Functionally Distinct Sources of Actin Monomers Supply the Leading Edge of Lamellipodia

    PubMed Central

    Vitriol, Eric A.; McMillen, Laura M.; Kapustina, Maryna; Gomez, Shawn M.; Vavylonis, Dimitrios; Zheng, James Q.

    2015-01-01

    Summary Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin) regulated by the ordered assembly from and disassembly into actin monomers (G-actin). Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer binding protein thymosin β4 (Tβ4) for optimal leading edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it doesn’t interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions. PMID:25865895

  15. GRP75 upregulates clathrin-independent endocytosis through actin cytoskeleton reorganization mediated by the concurrent activation of Cdc42 and RhoA.

    PubMed

    Chen, Hang; Gao, Zhihui; He, Changzheng; Xiang, Rong; van Kuppevelt, Toin H; Belting, Mattias; Zhang, Sihe

    2016-05-01

    Therapeutic macromolecules are internalized into the cell by either clathrin-mediated endocytosis (CME) or clathrin-independent endocytosis (CIE). Although some chaperone proteins play an essential role in CME (e.g. Hsc70 in clathrin uncoating), relatively few of these proteins are functionally involved in CIE. We previously revealed a role for the mitochondrial chaperone protein GRP75 in heparan sulfate proteoglycan (HSPG)-mediated, membrane raft-associated macromolecule endocytosis. However, the mechanism underlying this process remains unclear. In this study, using a mitochondrial signal peptide-directed protein trafficking expression strategy, we demonstrate that wild-type GRP75 expression enhanced the uptakes of HSPG and CIE marker cholera toxin B subunit but impaired the uptake of CME marker transferrin. The endocytosis regulation function of GRP75 is largely mediated by its subcellular location in mitochondria and is essentially determined by its ATPase domain. Interestingly, the mitochondrial expression of GRP75 or its ATPase domain significantly stimulates increases in both RhoA and Cdc42 activation, remarkably induces stress fibers and enhances filopodia formation, which collectively results in the promotion of CIE, but the inhibition of CME. Furthermore, silencing of Cdc42 or RhoA impaired the ability of GRP75 overexpression to increase CIE. Therefore, these results suggest that endocytosis vesicle enrichment of GRP75 by mitochondria trafficking upregulates CIE through an actin cytoskeleton reorganization mechanism mediated by the concurrent activation of Cdc42 and RhoA. This finding provides novel insight into organelle-derived chaperone signaling and the regulation of different endocytosis pathways in cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Assembly kinetics determine the architecture of α-actinin crosslinked F-actin networks.

    PubMed

    Falzone, Tobias T; Lenz, Martin; Kovar, David R; Gardel, Margaret L

    2012-05-29

    The actin cytoskeleton is organized into diverse meshworks and bundles that support many aspects of cell physiology. Understanding the self-assembly of these actin-based structures is essential for developing predictive models of cytoskeletal organization. Here we show that the competing kinetics of bundle formation with the onset of dynamic arrest arising from filament entanglements and crosslinking determine the architecture of reconstituted actin networks formed with α-actinin crosslinks. Crosslink-mediated bundle formation only occurs in dilute solutions of highly mobile actin filaments. As actin polymerization proceeds, filament mobility and bundle formation are arrested concomitantly. By controlling the onset of dynamic arrest, perturbations to actin assembly kinetics dramatically alter the architecture of biochemically identical samples. Thus, the morphology of reconstituted F-actin networks is a kinetically determined structure similar to those formed by physical gels and glasses. These results establish mechanisms controlling the structure and mechanics in diverse semiflexible biopolymer networks.

  17. Novel actin crosslinker superfamily member identified by a two step degenerate PCR procedure.

    PubMed

    Byers, T J; Beggs, A H; McNally, E M; Kunkel, L M

    1995-07-24

    Actin-crosslinking proteins link F-actin into the bundles and networks that constitute the cytoskeleton. Dystrophin, beta-spectrin, alpha-actinin, ABP-120, ABP-280, and fimbrin share homologous actin-binding domains and comprise an actin crosslinker superfamily. We have identified a novel member of this superfamily (ACF7) using a degenerate primer-mediated PCR strategy that was optimized to resolve less-abundant superfamily sequences. The ACF7 gene is on human chromosome 1 and hybridizes to high molecular weight bands on northern blots. Sequence comparisons argue that ACF7 does not fit into one of the existing families, but represents a new class within the superfamily.

  18. Actin cytoskeleton as a putative target of the neem limonoid Azadirachtin A.

    PubMed

    Anuradha, Aritakula; Annadurai, Ramaswamy S; Shashidhara, L S

    2007-06-01

    Limonoids isolated from the Indian neem tree (Azadirachta indica) have been gaining global acceptance in agricultural applications and in contemporary medicine for their myriad but discrete properties. However, their mode of action is still not very well understood. We have studied the mode of action of Azadirachtin A, the major limonoid of neem seed extracts, using Drosophila melanogaster as the model system. Azadirachtin A induces moderate-to-severe phenotypes in different tissues in a dose-dependent manner. At the cellular level, Azadirachtin A induces depolymerization of Actin leading to arrest of cells and subsequently apoptosis in a caspase-independent manner. Azadirachtin A-induced phenotypes were rescued by the over-expression of Cyclin E in a tissue-dependent manner. Cyclin E, which caused global rescue of Azadirachtin A-induced phenotypes, also effected rearrangement of the actin filaments. These results suggest that probably actin is a target of Azadirachtin A activity.

  19. 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.

  20. 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.

  1. Filament formation of the Escherichia coli actin-related protein, MreB, in fission yeast.

    PubMed

    Srinivasan, Ramanujam; Mishra, Mithilesh; Murata-Hori, Maki; Balasubramanian, Mohan K

    2007-02-06

    Proteins structurally related to eukaryotic actins have recently been identified in several prokaryotic organisms. These actin-like proteins (MreB and ParM) and the deviant Walker A ATPase (SopA) play a key role in DNA segregation and assemble into polymers in vitro and in vivo. MreB also plays a role in cellular morphogenesis. Whereas the dynamic properties of eukaryotic actins have been extensively characterized, those of bacterial actins are only beginning to emerge. We have established the fission yeast Schizosaccharomyces pombe as a cellular model for the functional analysis of the Escherichia coli actin-related protein MreB. We show that MreB organizes into linear bundles that grow in a symmetrically bidirectional manner at 0.46 +/- 0.03 microm/min, with new monomers and/or oligomers being added along the entire length of the bundle. Organization of linear arrays was dependent on the ATPase activity of MreB, and their alignment along the cellular long axis was achieved by sliding along the cortex of the cylindrical part of the cell. The cell ends appeared to provide a physical barrier for bundle elongation. These experiments provide new insights into the mechanism of assembly and organization of the bacterial actin cytoskeleton.

  2. AtFH1 formin mutation affects actin filament and microtubule dynamics in Arabidopsis thaliana

    PubMed Central

    Cvrčková, Fatima

    2013-01-01

    Plant cell growth and morphogenesis depend on remodelling of both actin and microtubule cytoskeletons. AtFH1 (At5g25500), the main housekeeping Arabidopsis formin, is targeted to membranes and known to nucleate and bundle actin. The effect of mutations in AtFH1 on root development and cytoskeletal dynamics was examined. Consistent with primarily actin-related formin function, fh1 mutants showed increased sensitivity to the actin polymerization inhibitor latrunculin B (LatB). LatB-treated mutants had thicker, shorter roots than wild-type plants. Reduced cell elongation and morphological abnormalities were observed in both trichoblasts and atrichoblasts. Fluorescently tagged cytoskeletal markers were used to follow cytoskeletal dynamics in wild-type and mutant plants using confocal microscopy and VAEM (variable-angle epifluorescence microscopy). Mutants exhibited more abundant but less dynamic F-actin bundles and more dynamic microtubules than wild-type seedlings. Treatment of wild-type seedlings with a formin inhibitor, SMIFH2, mimicked the root growth and cell expansion phenotypes and cytoskeletal structure alterations observed in fh1 mutants. The results suggest that besides direct effects on actin organization, the in vivo role of AtFH1 also includes modulation of microtubule dynamics, possibly mediated by actin–microtubule cross-talk. PMID:23202131

  3. Stochastic Severing of Actin Filaments by Actin Depolymerizing Factor/Cofilin Controls the Emergence of a Steady Dynamical Regime

    PubMed Central

    Roland, Jeremy; Berro, Julien; Michelot, Alphée; Blanchoin, Laurent; Martiel, Jean-Louis

    2008-01-01

    Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, 〈L〉, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: −2/3). We also showed that the average subunit residence time inside the filament, 〈T〉, depends on the actin monomer (power law: −1/6) and ADF/cofilin (power law: −2/3) concentrations. In addition, filament length fluctuations are ∼20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-Pi versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo. PMID:18065447

  4. Root cytoskeleton: its role in perception of and response to gravity

    NASA Technical Reports Server (NTRS)

    Baluska, F.; Hasenstein, K. H.

    1997-01-01

    We have critically evaluated the possible functions of the plant cytoskeleton in root gravisensing and graviresponse and discussed the evidence that microtubules (MTs) and actin microfilaments (MFs) do not control differential cell growth during bending of roots. On the other hand, MF and MT networks are envisaged to participate in gravisensing because of the mechanical properties of the cytoskeletal structures that interconnect plant cell organelles with the plasma membrane. In restrained gravisensing, forces are suggested to be transmitted to membranes because large-scale gravity-dependent repositioning of organelles is effectively prevented due to the cytoskeleton-mediated anchorage of their envelopes at the plasma membrane. From the cytoskeletal point of view, we can also envisage an unrestrained gravity sensing when cytoskeletal tethers are not strong enough to preserve the tight control over distribution of organelles and the latter, if heavy enough, are allowed to sediment towards the physical bottom of cells. This situation obviously occurs in root cap statocytes because these uniquely organized cells are depleted of prominent actin MF bundles, endoplasmic MT arrays, and ER elements in their internal cytoplasm. Nevertheless, indirect evidence clearly indicates that sedimented root cap statoliths are enmeshed within fine but dynamic MF networks and that their behaviour is obviously under, at least partial, cytoskeletal control. The actomyosin-enriched domain among and around amyloplasts is proposed to increase the perception of gravity due to the grouping effect of sedimenting statoliths. Cytoskeletal links between myosin-rich statoliths, and cell peripheries well equipped with dense cortical MTs, membrane-associated cytoskeleton, as well as with ER elements, would allow efficient restrained gravisensing only at the statocyte cell cortex. As a consequence of cytoskeletal depletion in the internal statocyte cytoplasm and bulk sedimentation of large

  5. 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.

  6. Acting on Actin: Rac and Rho Played by Yersinia.

    PubMed

    Aepfelbacher, Martin; Wolters, Manuel

    2017-01-01

    Pathogenic bacteria of the genus Yersinia include Y. pestis-the agent of plaque-and two enteropathogens, Y. enterocolitica, and Y. pseudotuberculosis. These pathogens have developed an array of virulence factors aimed at manipulating Rho GTP-binding proteins and the actin cytoskeleton in host cells to cross the intestinal barrier and suppress the immune system. Yersinia virulence factors include outer membrane proteins triggering cell invasion by binding to integrins, effector proteins injected into host cells to manipulate Rho protein functions and a Rho protein-activating exotoxin. Here, we present an overview of how Yersinia and host factors are integrated in a regulatory network that orchestrates the subversion of host defense.

  7. Actin Interacting Protein1 and Actin Depolymerizing Factor Drive Rapid Actin Dynamics in Physcomitrella patens[W

    PubMed Central

    Augustine, Robert C.; Pattavina, Kelli A.; Tüzel, Erkan; Vidali, Luis; Bezanilla, Magdalena

    2011-01-01

    The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics. PMID:22003077

  8. 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

  9. Assembly Kinetics Determine the Architecture of α-actinin Crosslinked F-actin Networks

    PubMed Central

    Falzone, Tobias T.; Lenz, Martin; Kovar, David R.; Gardel, Margaret L.

    2013-01-01

    The actin cytoskeleton is organized into diverse meshworks and bundles that support many aspects of cell physiology. Understanding the self-assembly of these actin-based structures is essential for developing predictive models of cytoskeletal organization. Here we show that the competing kinetics of bundle formation with the onset of dynamic arrest arising from filament entanglements and cross-linking determine the architecture of reconstituted actin networks formed with α-actinin cross-links. Cross-link mediated bundle formation only occurs in dilute solutions of highly mobile actin filaments. As actin polymerization proceeds, filament mobility and bundle formation are arrested concomitantly. By controlling the onset of dynamic arrest, perturbations to actin assembly kinetics dramatically alter the architecture of biochemically identical samples. Thus, the morphology of reconstituted F-actin networks is a kinetically determined structure similar to those formed by physical gels and glasses. These results establish mechanisms controlling the structure and mechanics in diverse semi-flexible biopolymer networks. PMID:22643888

  10. Increased Training Intensity Induces Proper Membrane Localization of Actin Remodeling Proteins in the Hippocampus Preventing Cognitive Deficits: Implications for Fragile X Syndrome.

    PubMed

    Martinez, L A; Tejada-Simon, Maria Victoria

    2018-06-01

    Behavioral intervention therapy has proven beneficial in the treatment of autism and intellectual disabilities (ID), raising the possibility of certain changes in molecular mechanisms activated by these interventions that may promote learning. Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by autistic features and intellectual disability and can serve as a model to examine mechanisms that promote learning. FXS results from mutations in the fragile X mental retardation 1 gene (Fmr1) that prevents expression of the Fmr1 protein (FMRP), a messenger RNA (mRNA) translation regulator at synapses. Among many other functions, FMRP organizes a complex with the actin cytoskeleton-regulating small Rho GTPase Rac1. As in humans, Fmr1 KO mice lacking FMRP display autistic-like behaviors and deformities of actin-rich synaptic structures in addition to impaired hippocampal learning and synaptic plasticity. These features have been previously linked to proper function of actin remodeling proteins that includes Rac1. An important step in Rac1 activation and function is its translocation to the membrane, where it can influence synaptic actin cytoskeleton remodeling during hippocampus-dependent learning. Herein, we report that Fmr1 KO mouse hippocampus exhibits increased levels of membrane-bound Rac1, which may prevent proper learning-induced synaptic changes. We also determine that increasing training intensity during fear conditioning (FC) training restores contextual memory in Fmr1 KO mice and reduces membrane-bound Rac1 in Fmr1 KO hippocampus. Increased training intensity also results in normalized long-term potentiation in hippocampal slices taken from Fmr1 KO mice. These results point to interventional treatments providing new therapeutic options for FXS-related cognitive dysfunction.

  11. Mechanism for CARMIL Protein Inhibition of Heterodimeric Actin-capping Protein*

    PubMed Central

    Kim, Taekyung; Ravilious, Geoffrey E.; Sept, David; Cooper, John A.

    2012-01-01

    Capping protein (CP) controls the polymerization of actin filaments by capping their barbed ends. In lamellipodia, CP dissociates from the actin cytoskeleton rapidly, suggesting the possible existence of an uncapping factor, for which the protein CARMIL (capping protein, Arp2/3 and myosin-I linker) is a candidate. CARMIL binds to CP via two motifs. One, the CP interaction (CPI) motif, is found in a number of unrelated proteins; the other motif is unique to CARMILs, the CARMIL-specific interaction motif. A 115-aa CARMIL fragment of CARMIL with both motifs, termed the CP-binding region (CBR), binds to CP with high affinity, inhibits capping, and causes uncapping. We wanted to understand the structural basis for this function. We used a collection of mutants affecting the actin-binding surface of CP to test the possibility of a steric-blocking model, which remained open because a region of CBR was not resolved in the CBR/CP co-crystal structure. The CP actin-binding mutants bound CBR normally. In addition, a CBR mutant with all residues of the unresolved region changed showed nearly normal binding to CP. Having ruled out a steric blocking model, we tested an allosteric model with molecular dynamics. We found that CBR binding induces changes in the conformation of the actin-binding surface of CP. In addition, ∼30-aa truncations on the actin-binding surface of CP decreased the affinity of CBR for CP. Thus, CARMIL promotes uncapping by binding to a freely accessible site on CP bound to a filament barbed end and inducing a change in the conformation of the actin-binding surface of CP. PMID:22411988

  12. 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.

  13. Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane – a minimally invasive investigation by STED-FCS

    PubMed Central

    Andrade, Débora M.; Clausen, Mathias P.; Keller, Jan; Mueller, Veronika; Wu, Congying; Bear, James E.; Hell, Stefan W.; Lagerholm, B. Christoffer; Eggeling, Christian

    2015-01-01

    Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes. PMID:26118385

  14. Mercury exposure induces cytoskeleton disruption and loss of renal function through epigenetic modulation of MMP9 expression.

    PubMed

    Khan, Hafizurrahman; Singh, Radha Dutt; Tiwari, Ratnakar; Gangopadhyay, Siddhartha; Roy, Somendu Kumar; Singh, Dhirendra; Srivastava, Vikas

    2017-07-01

    Mercury is one of the major heavy metal pollutants occurring in elemental, inorganic and organic forms. Due to ban on most inorganic mercury containing products, human exposure to mercury generally occurs as methylmercury (MeHg) by consumption of contaminated fish and other sea food. Animal and epidemiological studies indicate that MeHg affects neural and renal function. Our study is focused on nephrotoxic potential of MeHg. In this study, we have shown for the first time how MeHg could epigenetically modulate matrix metalloproteinase 9(MMP9) to promote nephrotoxicity using an animal model of sub chronic MeHg exposure. MeHg caused renal toxicity as was seen by increased levels of serum creatinine and expression of early nephrotoxicity markers (KIM-1, Clusterin, IP-10, and TIMP). MeHg exposure also correlated strongly with induction of MMP9 mRNA and protein in a dose dependent manner. Further, while induction of MMP9 promoted cytoskeleton disruption and loss of cell-cell adhesion (loss of F-actin, Vimentin and Fibronectin), inhibition of MMP9 was found to reduce these disruptions. Mechanistic studies by ChIP analysis showed that MeHg modulated MMP9 by promoting demethylation of its regulatory region to increase its expression. Bisulfite sequencing identified critical CpGs in the first exon of MMP9 which were demethylated following MeHg exposure. ChIP studies also showed loss of methyl binding protein, MeCP2 and transcription factor PEA3 at the demethylated site confirming decreased CpG methylation. Our studies thus show how MeHg could epigenetically modulate MMP9 to promote cytoskeleton disruption leading to loss of renal function. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Lateral Membrane Diffusion Modulated by a Minimal Actin Cortex

    PubMed Central

    Heinemann, Fabian; Vogel, Sven K.; Schwille, Petra

    2013-01-01

    Diffusion of lipids and proteins within the cell membrane is essential for numerous membrane-dependent processes including signaling and molecular interactions. It is assumed that the membrane-associated cytoskeleton modulates lateral diffusion. Here, we use a minimal actin cortex to directly study proposed effects of an actin meshwork on the diffusion in a well-defined system. The lateral diffusion of a lipid and a protein probe at varying densities of membrane-bound actin was characterized by fluorescence correlation spectroscopy (FCS). A clear correlation of actin density and reduction in mobility was observed for both the lipid and the protein probe. At high actin densities, the effect on the protein probe was ∼3.5-fold stronger compared to the lipid. Moreover, addition of myosin filaments, which contract the actin mesh, allowed switching between fast and slow diffusion in the minimal system. Spot variation FCS was in accordance with a model of fast microscopic diffusion and slower macroscopic diffusion. Complementing Monte Carlo simulations support the analysis of the experimental FCS data. Our results suggest a stronger interaction of the actin mesh with the larger protein probe compared to the lipid. This might point toward a mechanism where cortical actin controls membrane diffusion in a strong size-dependent manner. PMID:23561523

  16. Oxidation of F-actin controls the terminal steps of cytokinesis

    PubMed Central

    Frémont, Stéphane; Hammich, Hussein; Bai, Jian; Wioland, Hugo; Klinkert, Kerstin; Rocancourt, Murielle; Kikuti, Carlos; Stroebel, David; Romet-Lemonne, Guillaume; Pylypenko, Olena; Houdusse, Anne; Echard, Arnaud

    2017-01-01

    Cytokinetic abscission, the terminal step of cell division, crucially depends on the local constriction of ESCRT-III helices after cytoskeleton disassembly. While the microtubules of the intercellular bridge are cut by the ESCRT-associated enzyme Spastin, the mechanism that clears F-actin at the abscission site is unknown. Here we show that oxidation-mediated depolymerization of actin by the redox enzyme MICAL1 is key for ESCRT-III recruitment and successful abscission. MICAL1 is recruited to the abscission site by the Rab35 GTPase through a direct interaction with a flat three-helix domain found in MICAL1 C terminus. Mechanistically, in vitro assays on single actin filaments demonstrate that MICAL1 is activated by Rab35. Moreover, in our experimental conditions, MICAL1 does not act as a severing enzyme, as initially thought, but instead induces F-actin depolymerization from both ends. Our work reveals an unexpected role for oxidoreduction in triggering local actin depolymerization to control a fundamental step of cell division. PMID:28230050

  17. Cadmium-induced glutathionylation of actin occurs through a ROS-independent mechanism: Implications for cytoskeletal integrity

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

    Choong, Grace; Liu, Ying; Xiao, Weiqun

    2013-10-15

    Cadmium disrupts the actin cytoskeleton in rat mesangial cells, and we have previously shown that this involves a complex interplay involving activation of kinase signaling, protein translocation, and disruption of focal adhesions. Here we investigate the role that glutathionylation of actin plays in Cd{sup 2+}-associated cytoskeletal reorganization. Low concentrations of Cd{sup 2+} (0.5–2 μM) caused an increase in actin glutathionylation by 6 h, whereas at higher concentrations glutathionylation remained at basal levels. Although oxidation with diamide increased glutathionylation, reactive oxygen species (ROS) were not involved in the Cd{sup 2+}-dependent effect, as only Cd{sup 2+} concentrations above 2 μM were sufficientmore » to increase ROS. However, low [Cd{sup 2+}] increased total glutathione levels without affecting the ratio of reduced/oxidized glutathione, and inhibition of glutathione synthesis suppressed actin glutathionylation. Cadmium increased the activity of the enzyme glutaredoxin, which influences the equilibrium between glutathionylated and deglutathionylated proteins and thus may influence levels of glutathionylated actin. Together these observations show that cadmium-dependent effects on actin glutathionylation are affected by glutathione metabolism and not by direct effects of ROS on thiol chemistry. In vitro polymerization assays with glutathionylated actin show a decreased rate of polymerization. In contrast, immunofluorescence of cytoskeletal structure in intact cells suggests that increases in actin glutathionylation accompanying increased glutathione levels occurring under low Cd{sup 2+} exposure are protective in vivo, with cytoskeletal disruption ensuing only when higher Cd{sup 2+} concentrations increase ROS levels and prevent an increase in actin–glutathione conjugates. - Highlights: • Cadmium disrupts the actin cytoskeleton in mesangial cells. • Cadmium induces glutathionylation of actin at low concentrations.

  18. Decidable and undecidable arithmetic functions in actin filament networks

    NASA Astrophysics Data System (ADS)

    Schumann, Andrew

    2018-01-01

    The plasmodium of Physarum polycephalum is very sensitive to its environment, and reacts to stimuli with appropriate motions. Both the sensory and motor stages of these reactions are explained by hydrodynamic processes, based on fluid dynamics, with the participation of actin filament networks. This paper is devoted to actin filament networks as a computational medium. The point is that actin filaments, with contributions from many other proteins like myosin, are sensitive to extracellular stimuli (attractants as well as repellents), and appear and disappear at different places in the cell to change aspects of the cell structure—e.g. its shape. By assembling and disassembling actin filaments, some unicellular organisms, like Amoeba proteus, can move in response to various stimuli. As a result, these organisms can be considered a simple reversible logic gate—extracellular signals being its inputs and motions its outputs. In this way, we can implement various logic gates on amoeboid behaviours. These networks can embody arithmetic functions within p-adic valued logic. Furthermore, within these networks we can define the so-called diagonalization for deducing undecidable arithmetic functions.

  19. Effects of Microtubule and Actin Inhibitors on Cryptococcus neoformans Examined by Scanning and Transmission Electron Microscopy.

    PubMed

    Kopecká, Marie

    2014-01-01

    Cryptococcus neoformans is one of the most important human fungal pathogens. Its cells contain rich microtubules required for nuclear division and rich F-actin cytoskeletons for cell division. Disruption of microtubules by a microtubule inhibitor should block nuclear division, and disruption of F-actin by an actin inhibitor should block cell division. We investigated the effects of microtubule and actin inhibitors to find out whether the cytoskeletons of C. neoformans can become a new anti-fungal target for the inhibition of cell division, when examined at the ultrastructural level. Cells treated with the microtubule inhibitors vincristine (VIN) and methyl benzimidazole-2-ylcarbamate (BCM) and the actin inhibitor latrunculin A (LA), in yeast extract peptone dextrose medium, were examined by scanning (SEM) and transmission electron microscopy (TEM), and the cell number was counted using a Bürker chamber. After 2 days of inhibition with VIN, BCM or LA, the cells did not divide, but later, resistant, proliferating cells appeared in all samples. With combined microtubule and actin inhibitors (VIN + LA or BCM + LA), cells did not divide during 6 or even 14 days, and no resistant cells originated. TEM showed that the inhibited cells were without cytoplasm and were dead; only empty cell walls persisted with reduced capsules, shown on SEM. Combined microtubule and actin inhibitors (VIN + LA or BCM + LA), have lethal effects on C. neoformans cells and no resistant cells originate. © 2015 S. Karger AG, Basel

  20. 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

  1. Mutations in actin used for structural studies partially disrupt β-thymosin/WH2 domains interaction.

    PubMed

    Deville, Célia; Girard-Blanc, Christine; Assrir, Nadine; Nhiri, Naïma; Jacquet, Eric; Bontems, François; Renault, Louis; Petres, Stéphane; van Heijenoort, Carine

    2016-10-01

    Understanding the structural basis of actin cytoskeleton remodeling requires stabilization of actin monomers, oligomers, and filaments in complex with partner proteins, using various biochemical strategies. Here, we report a dramatic destabilization of the dynamic interaction with a model β-thymosin/WH2 domain induced by mutations in actin. This result underlines that mutant actins should be used with prudence to characterize interactions with intrinsically disordered partners as destabilization of dynamic interactions, although identifiable by NMR, may be invisible to other structural techniques. It also highlights how both β-thymosin/WH2 domains and actin tune local structure and dynamics in regulatory processes involving intrinsically disordered domains. © 2016 Federation of European Biochemical Societies.

  2. New Aspects of Progesterone Interactions with the Actin Cytoskeleton and Neurosteroidogenesis in the Cerebellum and the Neuronal Growth Cone

    PubMed Central

    Wessel, Lisa; Olbrich, Laura; Brand-Saberi, Beate

    2014-01-01

    The impact of progesterone on neuronal tissues in the central (CNS) and peripheral (PNS) nervous system is of significant scientific and therapeutic interest. Glial and neuronal cells of vertebrates express steroidogenic enzymes, and are able to synthesize progesterone de novo from cholesterol. Progesterone is described to have neuroprotective, neuroreparative, anti-degenerative, and anti-apoptotic effects in the CNS and the PNS. Thus, the first clinical studies promise new therapeutic options using progesterone in the treatment of patients with traumatic brain injury. Additionally, experimental data from different animal models suggest further positive effects of progesterone on neurological diseases such as cerebral ischemia, peripheral nerve injury and amyothropic lateral sclerosis. In regard to this future clinical use of progesterone, we discuss in this review the underlying physiological principles of progesterone effects in neuronal tissues. Mechanisms leading to morphological reorganizations of neurons in the CNS and PNS affected by progesterone are addressed, with special focus on the actin cytoskeleton. Furthermore, new aspects of a progesterone-dependent regulation of neurosteroidogenesis mediated by the recently described progesterone binding protein PGRMC1 in the nervous system are discussed. PMID:25141866

  3. Actin, actin-binding proteins, and actin-related proteins in the nucleus.

    PubMed

    Kristó, Ildikó; Bajusz, Izabella; Bajusz, Csaba; Borkúti, Péter; Vilmos, Péter

    2016-04-01

    Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.

  4. 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.

  5. Symbiont-Induced Changes in Host Actin during the Onset of a Beneficial Animal-Bacterial Association

    PubMed Central

    Kimbell, Jennifer R.; McFall-Ngai, Margaret J.

    2004-01-01

    The influence of bacteria on the cytoskeleton of animal cells has been studied extensively only in pathogenic associations. We characterized changes in host cytoskeletal actin induced by the bacterial partner during the onset of a cooperative animal-bacteria association using the squid-vibrio model. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that Vibrio fischeri induced a dramatic increase in actin protein abundance in the bacteria-associated host tissues during the onset of the symbiosis. Immunocytochemistry revealed that this change in actin abundance correlated with a two- to threefold increase in actin in the apical cell surface of the epithelium-lined ducts, the route of entry of symbionts into host tissues. Real-time reverse transcriptase PCR and in situ hybridization did not detect corresponding changes in actin mRNA. Temporally correlated with the bacteria-induced changes in actin levels was a two- to threefold decrease in duct circumference, a 20% loss in the average number of cells interfacing with the duct lumina, and dramatic changes in duct cell shape. When considered with previous studies of the biomechanical and biochemical characteristics of the duct, these findings suggest that the bacterial symbionts, upon colonizing the host organ, induce modifications that physically and chemically limit the opportunity for subsequent colonizers to pass through the ducts. Continued study of the squid-vibrio system will allow further comparisons of the mechanisms by which pathogenic and cooperative bacteria influence cytoskeleton dynamics in host cells. PMID:15006763

  6. 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

  7. Molecular Characterization of abLIM, a Novel Actin-binding and Double Zinc Finger Protein

    PubMed Central

    Roof, Dorothy J.; Hayes, Annmarie; Adamian, Michael; Chishti, Athar H.; Li, Tiansen

    1997-01-01

    Molecules that couple the actin-based cytoskeleton to intracellular signaling pathways are central to the processes of cellular morphogenesis and differentiation. We have characterized a novel protein, the actin-binding LIM (abLIM) protein, which could mediate such interactions between actin filaments and cytoplasmic targets. abLIM protein consists of a COOH-terminal cytoskeletal domain that is fused to an NH2-terminal domain consisting of four double zinc finger motifs. The cytoskeletal domain is ∼50% identical to erythrocyte dematin, an actin-bundling protein of the red cell membrane skeleton, while the zinc finger domains conform to the LIM motif consensus sequence. In vitro expression studies demonstrate that abLIM protein can bind to F-actin through the dematin-like domain. Transcripts corresponding to three distinct isoforms have a widespread tissue distribution. However, a polypeptide corresponding to the full-length isoform is found exclusively in the retina and is enriched in biochemical extracts of retinal rod inner segments. abLIM protein also undergoes extensive phosphorylation in light-adapted retinas in vivo, and its developmental expression in the retina coincides with the elaboration of photoreceptor inner and outer segments. Based on the composite primary structure of abLIM protein, actin-binding capacity, potential regulation via phosphorylation, and isoform expression pattern, we speculate that abLIM may play a general role in bridging the actin-based cytoskeleton with an array of potential LIM protein-binding partners. The developmental time course of abLIM expression in the retina suggests that the retina-specific isoform may have a specialized role in the development or elaboration of photoreceptor inner and outer segments. PMID:9245787

  8. A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division.

    PubMed

    Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

    2015-08-25

    Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.

  9. Kindlin-2 directly binds actin and regulates integrin outside-in signaling

    PubMed Central

    Bledzka, Kamila; Bialkowska, Katarzyna; Sossey-Alaoui, Khalid; Vaynberg, Julia; Pluskota, Elzbieta

    2016-01-01

    Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2+/− mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK47/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK47/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK47/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses. PMID:27044892

  10. Questioning the role of actinfree Gc-Globulin as actin scavenger in neurodegenerative central nervous system disease: relationship to S-100B levels and blood-brain barrier function.

    PubMed

    Gressner, Olav A; Schifflers, Marie-Claire; Kim, Philipp; Heuts, Leo; Lahme, Birgit; Gressner, Axel M

    2009-02-01

    Preliminary studies report on significantly higher levels of the major cytoskeleton protein actin in CSF of patients with neurodegenerative conditions and that the dynamics of these levels obviously correlates with disease progression and clinical disability. One of the primary functions of actinfree Gc-Globulin is to bind and neutralize extracellular monomeric actin, released into the circulation by necrotic or ruptured cells, and thus ameliorating the clinical outcome in situations of severe organ damage. This is the first study to investigate actinfree Gc-Globulin and S100-B levels (as reliable marker of neurodegeneration) in paired CSF and serum samples of patients with multietiological CNS diseases. 42% of all patients with CNS disease displayed serum concentrations of actinfree Gc-Globulin above the established reference range. CSF concentrations of actinfree Gc-Globulin and S100-B were positively correlated with the severity of blood-brain barrier (BBB) dysfunction. Furthermore, patients with severe BBB dysfunction presented a higher percentage of intrathecal synthesis of actinfree Gc-Globulin compared to patients with mild to moderate dysfunction and to patients with normal BBB function. Representative longitudinal data from selected patients demonstrated an inverse behaviour of actinfree Gc-Globulin and S100-B CSF concentrations, suggesting a consumption of the actin scavenger capacity of Gc-Globulin in times of increased neuronal damage. This presumption was supported by the fact that those conditions associated with a severe neuronal damage, in particular CNS trauma, and highest S100-B concentrations simultaneously displayed lowest actinfree Gc-Globulin levels, and thus residual actin binding capacity of Gc-Globulin. In summary, our data propose a function of actinfree Gc-Globulin also in the clearance of actin filaments from CSF of patients with neuronal damage. However, active recruitment of hepatic derived actinfree Gc-Globulin to the site of CNS

  11. 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.

  12. Marginal Iodine Deficiency Affects Dendritic Spine Development by Disturbing the Function of Rac1 Signaling Pathway on Cytoskeleton.

    PubMed

    Min, Hui; Dong, Jing; Wang, Yi; Wang, Yuan; Yu, Ye; Shan, Zhongyan; Xi, Qi; Teng, Weiping; Chen, Jie

    2017-01-01

    Iodine deficiency (ID)-induced thyroid hormone (TH) insufficient during development leads to impairments of brain function, such as learning and memory. Marginal ID has been defined as subtle insufficiency of TH, characterized as low thyroxine (T 4 ) levels, whether marginal ID potentially had adverse effects on the development of hippocampus and the underlying mechanisms remain unclear. Thus, in the present study, we established Wistar rat models with ID diet during pregnancy and lactation. The effects of marginal ID on long-term potentiation (LTP) were investigated in the hippocampal CA1 region. To study the development of dendritic spines in pyramidal cells, Golgi-Cox staining was conducted on postnatal day (PN) 7, PN14, PN21, and PN28. The activation of Rac1 signaling pathway, which is essential for dendritic spine development by regulating actin cytoskeleton, was also investigated. Our results showed that marginal ID slightly reduced the field-excitatory postsynaptic potential (f-EPSP) slope and the population spike (PS) amplitude. Besides, the density of dendritic spines during the critical period of rat postnatal development was mildly decreased, and we found no significant change of spine morphology in marginal ID group. We also observed decreased activation of the Rac1 signaling pathway in pups subjected to maternal marginal ID. Our study may support the hypothesis that decreased T 4 induced by marginal ID results in slight impairments of LTP and leads to mild damage of dendritic spine development, which may be due to abnormal regulation of Rac1 signaling pathway on cytoskeleton.

  13. Lights, camera, actin.

    PubMed

    Rubenstein, Peter A; Wen, Kuo-Kuang

    2005-10-01

    Actin participates in many important biological processes. Currently, intensive investigation is being carried out in a number of laboratories concerning the function of actin in these processes and the molecular basis of its functions. We present a glimpse into four of these areas: actin-like proteins in bacterial cells, actin in the eukaryotic nucleus, the conformational plasticity of the actin filament, and finally, Arp2/3-dependent regulation of actin filament branching and creation of new filament barbed ends. IUBMB Life, 57: 683-687, 2005.

  14. Protective effects of lysophosphatidic acid (LPA) on chronic ethanol-induced injuries to the cytoskeleton and on glucose uptake in rat astrocytes.

    PubMed

    Tomás, Mónica; Lázaro-Diéguez, Francisco; Durán, Juan M; Marín, Pilar; Renau-Piqueras, Jaime; Egea, Gustavo

    2003-10-01

    Ethanol induces severe alterations in membrane trafficking in hepatocytes and astrocytes, the molecular basis of which is unclear. One of the main candidates is the cytoskeleton and the molecular components that regulate its organization and dynamics. Here, we examine the effect of chronic exposure to ethanol on the organization and dynamics of actin and microtubule cytoskeletons and glucose uptake in rat astrocytes. Ethanol-treated cells cultured in either the presence or absence of fetal calf serum showed a significant increase in 2-deoxyglucose uptake. Ethanol also caused alterations in actin organization, consisting of the dissolution of stress fibres and the appearance of circular filaments beneath the plasma membrane. When lysophosphatidic acid (LPA), which is a normal constituent of serum and a potent intercellular lipid mediator with growth factor and actin rearrangement activities, was added to ethanol-treated astrocytes cultured without fetal calf serum, it induced the re-appearance of actin stress fibres and the normalization of 2-deoxyglucose uptake. Furthermore, ethanol also perturbed the microtubule dynamics, which delayed the recovery of the normal microtubule organization following removal of the microtubule-disrupting agent nocodazole. Again, pre-treatment with LPA prevented this alteration. Ethanol-treated rodent fibroblast NIH3T3 cells that constitutively express an activated Rho mutant protein (GTP-bound form) were insensitive to ethanol, as they showed no alteration either in actin stress-fibre organization or in 2-deoxyglucose uptake. We discuss the putative signalling targets by which ethanol could alter the cytoskeleton and hexose uptake and the cytoprotective effect of LPA against ethanol-induced damages. The latter opens the possibility that LPA or a similar non-hydrolysable lipid derivative could be used as a cytoprotective agent against the noxious effects of ethanol.

  15. Localization and function of KLF4 in cytoplasm of vascular smooth muscle cell

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

    Liu, Yan; The Third Hospital of Hebei Medical University, Shijazhuang; Zheng, Bin

    2013-06-28

    Highlights: •PDGF-BB prompts the translocation of KLF4 to the cytoplasm. •PDGF-BB promotes interaction between KLF4 and actin in the cytoplasm. •Phosphorylation and SUMOylation of KLF4 participates in regulation of cytoskeletal organization. •KLF4 regulates cytoskeleton by promoting the expression of contraction-associated genes. -- Abstract: The Krüppel-like factor 4 is a DNA-binding transcriptional regulator that regulates a diverse array of cellular processes, including development, differentiation, proliferation, and apoptosis. The previous studies about KLF4 functions mainly focused on its role as a transcription factor, its functions in the cytoplasm are still unknown. In this study, we found that PDGF-BB could prompt the translocationmore » of KLF4 to the cytoplasm through CRM1-mediated nuclear export pathway in vascular smooth muscle cells (VSMCs) and increased the interaction of KLF4 with actin in the cytoplasm. Further study showed that both KLF4 phosphorylation and SUMOylation induced by PDGF-BB participates in regulation of cytoskeletal organization by stabilizing the actin cytoskeleton in VSMCs. In conclusion, these results identify that KLF4 participates in the cytoskeletal organization by stabilizing cytoskeleton in the cytoplasm of VSMCs.« less

  16. 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

  17. Reconstitution of actin-based motility of Listeria and Shigella using pure proteins

    NASA Astrophysics Data System (ADS)

    Loisel, Thomas P.; Boujemaa, Rajaa; Pantaloni, Dominique; Carlier, Marie-France

    1999-10-01

    Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, α-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

  18. Actin-binding proteins sensitively mediate F-actin bundle stiffness

    NASA Astrophysics Data System (ADS)

    Claessens, Mireille M. A. E.; Bathe, Mark; Frey, Erwin; Bausch, Andreas R.

    2006-09-01

    Bundles of filamentous actin (F-actin) form primary structural components of a broad range of cytoskeletal processes including filopodia, sensory hair cell bristles and microvilli. Actin-binding proteins (ABPs) allow the cell to tailor the dimensions and mechanical properties of the bundles to suit specific biological functions. Therefore, it is important to obtain quantitative knowledge on the effect of ABPs on the mechanical properties of F-actin bundles. Here we measure the bending stiffness of F-actin bundles crosslinked by three ABPs that are ubiquitous in eukaryotes. We observe distinct regimes of bundle bending stiffness that differ by orders of magnitude depending on ABP type, concentration and bundle size. The behaviour observed experimentally is reproduced quantitatively by a molecular-based mechanical model in which ABP shearing competes with F-actin extension/compression. Our results shed new light on the biomechanical function of ABPs and demonstrate how single-molecule properties determine mesoscopic behaviour. The bending mechanics of F-actin fibre bundles are general and have implications for cytoskeletal mechanics and for the rational design of functional materials.

  19. Actin dynamics mediates the changes of calcium level during the pulvinus movement of Mimosa pudica

    PubMed Central

    Yao, Heng; Xu, Qiangyi

    2008-01-01

    The bending movement of the pulvinus of Mimosa pudica is caused by a rapid change in volume of the abaxial motor cells, in response to various environmental stimuli. We investigated the relationship between the actin cytoskeleton and changes in the level of calcium during rapid contractile movement of the motor cells that was induced by electrical stimulation. The bending of the pulvinus was retarded by treatments with actin-affecting reagents and calcium channel inhibitors. The actin filaments in the motor cells were fragmented in response to electrical stimulation. Further investigations were performed using protoplasts from the motor cells of M. pudica pulvini. Calcium-channel inhibitors and EGTA had an inhibitory effect on contractile movement of the protoplasts. The level of calcium increased and became concentrated in the tannin vacuole after electrical stimulation. Ruthenium Red inhibited the increase in the level of calcium in the tannin vacuole and the contractile movement of the protoplasts. However, treatment with latrunculin A abolished the inhibitory effect of Ruthenium Red. Phalloidin inhibited the contractile movement and the increase in the level of calcium in the protoplasts. Our study demonstrates that depolymerization of the actin cytoskeleton in pulvinus motor cells in response to electrical signals results in increased levels of calcium. PMID:19513198

  20. 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...

  1. Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus.

    PubMed

    Kim, So Yeon; Gitai, Zemer; Kinkhabwala, Anika; Shapiro, Lucy; Moerner, W E

    2006-07-18

    The actin cytoskeleton represents a key regulator of multiple essential cellular functions in both eukaryotes and prokaryotes. In eukaryotes, these functions depend on the orchestrated dynamics of actin filament assembly and disassembly. However, the dynamics of the bacterial actin homolog MreB have yet to be examined in vivo. In this study, we observed the motion of single fluorescent MreB-yellow fluorescent protein fusions in living Caulobacter cells in a background of unlabeled MreB. With time-lapse imaging, polymerized MreB [filamentous MreB (fMreB)] and unpolymerized MreB [globular MreB (gMreB)] monomers could be distinguished: gMreB showed fast motion that was characteristic of Brownian diffusion, whereas the labeled molecules in fMreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer provides an indication that, like actin, MreB monomers treadmill through MreB filaments by preferential polymerization at one filament end and depolymerization at the other filament end. From these data, we extract several characteristics of single MreB filaments, including that they are, on average, much shorter than the cell length and that the direction of their polarized assembly seems to be independent of the overall cellular polarity. Thus, MreB, like actin, exhibits treadmilling behavior in vivo, and the long MreB structures that have been visualized in multiple bacterial species seem to represent bundles of short filaments that lack a uniform global polarity.

  2. Effects of basic calponin on the flexural mechanics and stability of F-actin.

    PubMed

    Jensen, Mikkel Herholdt; Watt, James; Hodgkinson, Julie L; Gallant, Cynthia; Appel, Sarah; El-Mezgueldi, Mohammed; Angelini, Thomas E; Morgan, Kathleen G; Lehman, William; Moore, Jeffrey R

    2012-01-01

    The cellular actin cytoskeleton plays a central role in the ability of cells to properly sense, propagate, and respond to external stresses and other mechanical stimuli. Calponin, an actin-binding protein found both in muscle and non-muscle cells, has been implicated in actin cytoskeletal organization and regulation. In this work, we studied the mechanical and structural interaction of actin with basic calponin, a differentiation marker in smooth muscle cells, on a single filament level. We imaged fluorescently labeled thermally fluctuating actin filaments and found that at moderate calponin binding densities, actin filaments were more flexible, evident as a reduction in persistence length from 8.0 to 5.8 μm. When calponin-decorated actin filaments were subjected to shear, we observed a marked reduction of filament lengths after decoration with calponin, which we argue was due to shear-induced filament rupture rather than depolymerization. This increased shear susceptibility was exacerbated with calponin concentration. Cryo-electron microscopy results confirmed previously published negative stain electron microscopy results and suggested alterations in actin involving actin subdomain 2. A weakening of F-actin intermolecular association is discussed as the underlying cause of the observed mechanical perturbations. Copyright © 2011 Wiley Periodicals, Inc.

  3. Long non-coding RNA CRYBG3 blocks cytokinesis by directly binding G-actin.

    PubMed

    Pei, Hailong; Hu, Wentao; Guo, Ziyang; Chen, Huaiyuan; Ma, Ji; Mao, Weidong; Li, Bingyan; Wang, Aiqing; Wan, Jianmei; Zhang, Jian; Nie, Jing; Zhou, Guangming; Hei, Tom K

    2018-06-22

    The dynamic interchange between monomeric globular actin (G-actin) and polymeric filamentous actin filaments (F-actin) is fundamental and essential to many cellular processes including cytokinesis and maintenance of genomic stability. Here we report that the long non-coding RNA LNC CRYBG3 directly binds G-actin to inhibit its polymerization and formation of contractile rings, resulting in M-Phase cell arrest. Knockdown of LNC CRYBG3 in tumor cells enhanced their malignant phenotypes. Nucleotide sequence 228-237 of the full-length LNC CRYBG3 and the ser14 domain of beta-actin are essential for their interaction, and mutation of either of these sites abrogated binding of LNC CRYBG3 to G-actin. Binding of LNC CRYBG3 to G-actin blocked nuclear localization of MAL, which consequently kept serum response factor (SRF) away from the promoter region of several immediate early genes, including JUNB and Arp3, which are necessary for cellular proliferation, tumor growth, adhesion, movement, and metastasis. These findings reveal a novel lncRNA-actin-MAL-SRF pathway and highlight LNC CRYBG3 as a means to block cytokinesis and treat cancer by targeting the actin cytoskeleton. Copyright ©2018, American Association for Cancer Research.

  4. Super-resolution links vinculin localization to function in focal adhesions.

    PubMed

    Giannone, Grégory

    2015-07-01

    Integrin-based focal adhesions integrate biochemical and biomechanical signals from the extracellular matrix and the actin cytoskeleton. The combination of three-dimensional super-resolution imaging and loss- or gain-of-function protein mutants now links the nanoscale dynamic localization of proteins to their activation and function within focal adhesions.

  5. 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.

  6. 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

  7. Quantitative Analysis of Statics and Dynamics of Actin Cables in Fission Yeast

    NASA Astrophysics Data System (ADS)

    Yusuf, Eddy; Wu, Jian-Qiu; Vavylonis, Dimitrios

    2010-03-01

    The assembly of actin and tubulin proteins into long filaments and bundles, i.e. closely-packed filaments, underlies important cellular processes such as cell motility, intracellular transport, and cell division. Recent theoretical and experimental work has addressed the nonequilibrium dynamics of single microtubules within live cells [1]. Actin filaments usually form dense networks that prevents microscopic imaging of individual filaments or bundles. Here, we studied actin dynamics using fission yeast that has low-density actin cytoskeleton consisting of actin cables (actin bundles aligned along the long axis of the cell) and ``actin patches.'' Yeast cells expressing GFP-CHD were imaged by 3D confocal microscopy. Stretching open active contours [2] were used to segment and track individual actin cables. We analyzed their curvature distribution, the tangent correlation, and the temporal bending amplitude fluctuations. We contrast our findings to equilibrium fluctuating semiflexible polymers and to microtubules in cells. We calculate the important time and length scales for the actin cables. We also discuss our findings within the broad context of understanding actin assembly in cells. [1] C. P. Brangwynne et. al., Phys. Rev. Lett. 100, 118104 (2008) [2] H. Li et. al., Proc. of the IEEE Int'l Symposium on Biomedical Imaging: From Nano to Macro, ISBI'09

  8. [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

  9. Nonsteroidal anti-inflammatory drugs attenuate amyloid-β protein-induced actin cytoskeletal reorganization through Rho signaling modulation.

    PubMed

    Ferrera, Patricia; Zepeda, Angélica; Arias, Clorinda

    2017-10-01

    Amyloid-β protein (Aβ) neurotoxicity occurs along with the reorganization of the actin-cytoskeleton through the activation of the Rho GTPase pathway. In addition to the classical mode of action of the non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin, and ibuprofen have Rho-inhibiting effects. In order to evaluate the role of the Rho GTPase pathway on Aβ-induced neuronal death and on neuronal morphological modifications in the actin cytoskeleton, we explored the role of NSAIDS in human-differentiated neuroblastoma cells exposed to Aβ. We found that Aβ induced neurite retraction and promoted the formation of different actin-dependent structures such as stress fibers, filopodia, lamellipodia, and ruffles. In the presence of Aβ, both NSAIDs prevented neurite collapse and formation of stress fibers without affecting the formation of filopodia and lamellipodia. Similar results were obtained when the downstream effector, Rho kinase inhibitor Y27632, was applied in the presence of Aβ. These results demonstrate the potential benefits of the Rho-inhibiting NSAIDs in reducing Aβ-induced effects on neuronal structural alterations.

  10. 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.

  11. A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division

    PubMed Central

    Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

    2015-01-01

    Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division. DOI: http://dx.doi.org/10.7554/eLife.08828.001 PMID:26305500

  12. Actin in Mung Bean Mitochondria and Implications for Its Function[W][OA

    PubMed Central

    Lo, Yih-Shan; Cheng, Ning; Hsiao, Lin-June; Annamalai, Arunachalam; Jauh, Guang-Yuh; Wen, Tuan-Nan; Dai, Hwa; Chiang, Kwen-Sheng

    2011-01-01

    Here, a large fraction of plant mitochondrial actin was found to be resistant to protease and high-salt treatments, suggesting it was protected by mitochondrial membranes. A portion of this actin became sensitive to protease or high-salt treatment after removal of the mitochondrial outer membrane, indicating that some actin is located inside the mitochondrial outer membrane. The import of an actin–green fluorescent protein (GFP) fusion protein into the mitochondria in a transgenic plant, actin:GFP, was visualized in living cells and demonstrated by flow cytometry and immunoblot analyses. Polymerized actin was found in mitochondria of actin:GFP plants and in mung bean (Vigna radiata). Notably, actin associated with mitochondria purified from early-developing cotyledons during seed germination was sensitive to high-salt and protease treatments. With cotyledon ageing, mitochondrial actin became more resistant to both treatments. The progressive import of actin into cotyledon mitochondria appeared to occur in concert with the conversion of quiescent mitochondria into active forms during seed germination. The binding of actin to mitochondrial DNA (mtDNA) was demonstrated by liquid chromatography–tandem mass spectrometry analysis. Porin and ADP/ATP carrier proteins were also found in mtDNA-protein complexes. Treatment with an actin depolymerization reagent reduced the mitochondrial membrane potential and triggered the release of cytochrome C. The potential function of mitochondrial actin and a possible actin import pathway are discussed. PMID:21984697

  13. Actin polymerization mediated by Babesia gibsoni aldolase is required for parasite invasion.

    PubMed

    Goo, Youn-Kyoung; Ueno, Akio; Terkawi, Mohamad Alaa; Aboge, G Oluga; Junya, Yamagishi; Igarashi, Makoto; Kim, Jung-Yeon; Hong, Yeon-Chul; Chung, Dong-Il; Nishikawa, Yoshifumi; Xuan, Xuenan

    2013-09-01

    Host cell invasion by apicomplexan parasites driven by gliding motility and empowered by actin-based movement is essential for parasite survival and pathogenicity. The parasites share a conserved invasion process: actin-based motility led by the coordination of adhesin-cytoskeleton via aldolase. A number of studies of host cell invasion in the Plasmodium species and Toxoplasma gondii have been performed. However, the mechanisms of host cell invasion by Babesia species have not yet been studied. Here, we show that Babesia gibsoni aldolase (BgALD) forms a complex with B. gibsoni thrombospondin-related anonymous protein (BgTRAP) and B. gibsoni actin (BgACT), depending on tryptophan-734 (W-734) in BgTRAP. In addition, actin polymerization is mediated by BgALD. Moreover, cytochalasin D, which disrupts actin polymerization, suppressed B. gibsoni parasite growth and inhibited the host cell invasion by parasites, indicating that actin dynamics are essential for erythrocyte invasion by B. gibsoni. This study is the first molecular approach to determine the invasion mechanisms of Babesia species. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Modulation of NF-kappaB activation in Theileria annulata-infected cloned cell lines is associated with detection of parasite-dependent IKK signalosomes and disruption of the actin cytoskeleton.

    PubMed

    Schmuckli-Maurer, Jacqueline; Kinnaird, Jane; Pillai, Sreerekha; Hermann, Pascal; McKellar, Sue; Weir, William; Dobbelaere, Dirk; Shiels, Brian

    2010-02-01

    Apicomplexan parasites within the genus Theileria have the ability to induce continuous proliferation and prevent apoptosis of the infected bovine leukocyte. Protection against apoptosis involves constitutive activation of the bovine transcription factor NF-kappaB in a parasite-dependent manner. Activation of NF-kappaB is thought to involve recruitment of IKK signalosomes at the surface of the macroschizont stage of the parasite, and it has been postulated that additional host proteins with adaptor or scaffolding function may be involved in signalosome formation. In this study two clonal cell lines were identified that show marked differences in the level of activated NF-kappaB. Further characterization of these lines demonstrated that elevated levels of activated NF-kappaB correlated with increased resistance to cell death and detection of parasite-associated IKK signalosomes, supporting results of our previous studies. Evidence was also provided for the existence of host- and parasite-dependent NF-kappaB activation pathways that are influenced by the architecture of the actin cytoskeleton. Despite this influence, it appears that the primary event required for formation of the parasite-dependent IKK signalosome is likely to be an interaction between a signalosome component and a parasite-encoded surface ligand.

  15. RNAi knockdown of the focal adhesion protein TES reveals its role in actin stress fibre organisation.

    PubMed

    Griffith, Elen; Coutts, Amanda S; Black, Donald M

    2005-03-01

    TES was originally identified as a candidate tumour suppressor gene and has subsequently been found to encode a novel focal adhesion protein. As well as localising to cell-matrix adhesions, TES localises to cell-cell contacts and to actin stress fibres. TES interacts with a variety of cytoskeletal proteins including zyxin, mena, VASP, talin and actin. There is evidence that TES may function in actin-dependent processes as overexpression of TES results in increased cell spreading and decreased cell motility. Together with TES's interacting partners, these data suggest that TES might be involved in regulation of the actin cytoskeleton. Here, for the first time, we have used RNAi to successfully knockdown TES in HeLa cells and we demonstrate that loss of TES from focal adhesions results in loss of actin stress fibres. Similarly, and as previously reported, RNAi-mediated knockdown of zyxin results in loss of actin stress fibres. TES siRNA treated cells show reduced RhoA activity, suggesting that the Rho GTPase pathway may be involved in the TES RNAi-induced loss of stress fibres. We have also used RNAi to examine the requirement of TES and zyxin for each other's localisation at focal adhesions, and we propose a hierarchy of recruitment, with zyxin being first, followed by VASP and then TES. Cell Motil. Copyright 2005 Wiley-Liss, Inc.

  16. Altered osteoblast structure and function in parabolic flight

    NASA Astrophysics Data System (ADS)

    Zhong-Quan, Dai; Ying-Hui, Li; Fen, Yang; Bai, Ding; Ying-Jun, Tan

    Introduction Bone loss has a significant impact on astronauts during spaceflight being one of the main obstacles preventing interplanetary missions However the exact mechanism is not well understood In the present study we investigated the effects of acute gravitational changes generated by parabolic flight on the structure and function of osteoblasts ROS17 2 8 carried by airbus A300 Methods The alteration of microfilament cytoskeleton was observed by the Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I immunofluorescence stain ALP activity and expression COL1A1 expression osteocalcin secrete which presenting the osteoblast function were detected by modified calcium and cobalt method RT-PCR and radioimmunity methods respectively Results The changed gravity induced the reorganization of microfilament cytoskeleton of osteoblast After 3 hours parabolic flight F-actin of osteoblast cytoskeleton became more thickness and directivity whereas G-actin reduced and relatively concentrated at the edge of nucleus observed by confocal fluorescence microscopy This phenomenon is identical with structure alternation observed in hypergravity but the osteoblast function decrease The excretion of osteocalcin the activity and mRNA expression of ALP decrease but the COL1A1 expression has no changes These results were similar to the changes in simulated or real microgravity Conclusion Above results suggest that short time gravity alternative change induce osteoblast structure and function

  17. Pure F-actin networks are distorted and branched by steps in the critical-point drying method.

    PubMed

    Resch, Guenter P; Goldie, Kenneth N; Hoenger, Andreas; Small, J Victor

    2002-03-01

    Elucidation of the ultrastructural organization of actin networks is crucial for understanding the molecular mechanisms underlying actin-based motility. Results obtained from cytoskeletons and actin comets prepared by the critical-point procedure, followed by rotary shadowing, support recent models incorporating actin filament branching as a main feature of lamellipodia and pathogen propulsion. Since actin branches were not evident in earlier images obtained by negative staining, we explored how these differences arise. Accordingly, we have followed the structural fate of dense networks of pure actin filaments subjected to steps of the critical-point drying protocol. The filament networks have been visualized in parallel by both cryo-electron microscopy and negative staining. Our results demonstrate the selective creation of branches and other artificial structures in pure F-actin networks by the critical-point procedure and challenge the reliability of this method for preserving the detailed organization of actin assemblies that drive motility. (c) 2002 Elsevier Science (USA).

  18. ADF Proteins Are Involved in the Control of Flowering and Regulate F-Actin Organization, Cell Expansion, and Organ Growth in Arabidopsis

    PubMed Central

    Dong, Chun-Hai; Xia, Gui-Xian; Hong, Yan; Ramachandran, Srinivasan; Kost, Benedikt; Chua, Nam-Hai

    2001-01-01

    Based mostly on the results of in vitro experiments, ADF (actin-depolymerizing factor) proteins are thought to be key modulators of the dynamic organization of the actin cytoskeleton. The few studies concerned with the in vivo function of ADF proteins that have been reported to date were performed almost exclusively using single-cell systems and have failed to produce consistent results. To investigate ADF functions in vivo and during the development of multicellular organs, we generated transgenic Arabidopsis plants that express a cDNA encoding an ADF protein (AtADF1) in the sense or the antisense orientation under the control of a strong constitutively active promoter. Selected lines with significantly altered levels of AtADF protein expression were characterized phenotypically. Overexpression of AtADF1 resulted in the disappearance of thick actin cables in different cell types, caused irregular cellular and tissue morphogenesis, and reduced the growth of cells and organs. In contrast, reduced AtADF expression promoted the formation of actin cables, resulted in a delay in flowering, and stimulated cell expansion as well as organ growth. These results are consistent with the molecular functions of ADF as predicted by in vitro studies, support the global roles of ADF proteins during the development of a multicellular organism, and demonstrate that these proteins are key regulators of F-actin organization, flowering, and cell and organ expansion in Arabidopsis. PMID:11402164

  19. Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork

    PubMed Central

    Lenne, Pierre-François; Wawrezinieck, Laure; Conchonaud, Fabien; Wurtz, Olivier; Boned, Annie; Guo, Xiao-Jun; Rigneault, Hervé; He, Hai-Tao; Marguet, Didier

    2006-01-01

    It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (∅<120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin-based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane. PMID:16858413

  20. Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork.

    PubMed

    Lenne, Pierre-François; Wawrezinieck, Laure; Conchonaud, Fabien; Wurtz, Olivier; Boned, Annie; Guo, Xiao-Jun; Rigneault, Hervé; He, Hai-Tao; Marguet, Didier

    2006-07-26

    It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (Ø <120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin-based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.