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Sample records for actin cytoskeletal rearrangement

  1. Organelle-cytoskeletal interactions: actin mutations inhibit meiosis-dependent mitochondrial rearrangement in the budding yeast Saccharomyces cerevisiae.

    PubMed Central

    Smith, M G; Simon, V R; O'Sullivan, H; Pon, L A

    1995-01-01

    During early stages of meiosis I, yeast mitochondria fuse to form a single continuous thread. Thereafter, portions of the mitochondrial thread are equally distributed to daughter cells. Using time-lapse fluorescence microscopy and a membrane potential sensing dye, mitochondria are resolved as small particles at the cell periphery in pre-meiotic, living yeast. These organelles display low levels of movement. During meiosis I, we observed a threefold increase in mitochondrial motility. Mitochondrial movements were linear, occurred at a maximum velocity of 25 +/- 6.7 nm/s, and resulted in organelle collision and fusion to form elongated tubular structures. Mitochondria do not co-localize with microtubules. Destabilization of microtubules by nocodazole treatment has no significant effect on the rate and extent of thread formation. In contrast, yeast bearing temperature-sensitive mutations in the actin-encoding ACT1 gene (act1-3 and act1-133) exhibit abnormal mitochondrial aggregation, fragmentation, and enlargement as well as loss of mitochondrial motility. In act1-3 cells, mitochondrial defects and actin delocalization occur only at restrictive temperatures. The act1-133 mutation, which perturbs the myosin-binding site of actin without significantly affecting actin cytoskeletal structure in meiotic yeast, results in mitochondrial morphology and motility defects at restrictive and permissive temperatures. These studies support a role for the actin cytoskeleton in the control of mitochondrial position and movements in meiotic yeast. Images PMID:8573793

  2. Calponin 3 regulates actin cytoskeleton rearrangement in trophoblastic cell fusion.

    PubMed

    Shibukawa, Yukinao; Yamazaki, Natsuko; Kumasawa, Keiichi; Daimon, Etsuko; Tajiri, Michiko; Okada, Yuka; Ikawa, Masahito; Wada, Yoshinao

    2010-11-15

    Cell-cell fusion is an intriguing differentiation process, essential for placental development and maturation. A proteomic approach identified a cytoplasmic protein, calponin 3 (CNN3), related to the fusion of BeWo choriocarcinoma cells. CNN3 was expressed in cytotrophoblasts in human placenta. CNN3 gene knockdown promoted actin cytoskeletal rearrangement and syncytium formation in BeWo cells, suggesting CNN3 to be a negative regulator of trophoblast fusion. Indeed, CNN3 depletion promoted BeWo cell fusion. CNN3 at the cytoplasmic face of cytoskeleton was dislocated from F-actin with forskolin treatment and diffused into the cytoplasm in a phosphorylation-dependent manner. Phosphorylation sites were located at Ser293/296 in the C-terminal region, and deletion of this region or site-specific disruption of Ser293/296 suppressed syncytium formation. These CNN3 mutants were colocalized with F-actin and remained there after forskolin treatment, suggesting that dissociation of CNN3 from F-actin is modulated by the phosphorylation status of the C-terminal region unique to CNN3 in the CNN family proteins. The mutant missing these phosphorylation sites displayed a dominant negative effect on cell fusion, while replacement of Ser293/296 with aspartic acid enhanced syncytium formation. These results indicated that CNN3 regulates actin cytoskeleton rearrangement which is required for the plasma membranes of trophoblasts to become fusion competent. PMID:20861310

  3. Structural Rearrangements in CHO Cells After Disruption of Individual Cytoskeletal Elements and Plasma Membrane.

    PubMed

    Jokhadar, Špela Zemljič; Derganc, Jure

    2015-04-01

    Cellular structural integrity is provided primarily by the cytoskeleton, which comprises microtubules, actin filaments, and intermediate filaments. The plasma membrane has been also recognized as a mediator of physical forces, yet its contribution to the structural integrity of the cell as a whole is less clear. In order to investigate the relationship between the plasma membrane and the cytoskeleton, we selectively disrupted the plasma membrane and each of the cytoskeletal elements in Chinese hamster ovary cells and assessed subsequent changes in cellular structural integrity. Confocal microscopy was used to visualize cytoskeletal rearrangements, and optical tweezers were utilized to quantify membrane tether extraction. We found that cholesterol depletion from the plasma membrane resulted in rearrangements of all cytoskeletal elements. Conversely, the state of the plasma membrane, as assessed by tether extraction, was affected by disruption of any of the cytoskeletal elements, including microtubules and intermediate filaments, which are located mainly in the cell interior. The results demonstrate that, besides the cytoskeleton, the plasma membrane is an important contributor to cellular integrity, possibly by acting as an essential framework for cytoskeletal anchoring. In agreement with the tensegrity model of cell mechanics, our results support the notion of the cell as a prestressed structure. PMID:25395197

  4. TREK-1 Regulates Cytokine Secretion from Cultured Human Alveolar Epithelial Cells Independently of Cytoskeletal Rearrangements

    PubMed Central

    Schwingshackl, Andreas; Roan, Esra; Teng, Bin; Waters, Christopher M.

    2015-01-01

    Background TREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements. Methods We determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA. Results Cytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells. Conclusion Although cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the

  5. Simulated Microgravity Induced Cytoskeletal Rearrangements are Modulated by Protooncogenes

    NASA Technical Reports Server (NTRS)

    Melhado, C. D.; Sanford, G. L.; Bosah, F.; Harris-Hooker, S.

    1998-01-01

    Microgravity is the environment living systems encounter during space flight and gravitational unloading is the effect of this environment on living systems. The cell, being a multiphasic chemical system, is a useful starting point to study the potential impact of gravity unloading on physiological function. In the absence of gravity, sedimentation of organelles including chromosomes, mitochondria, nuclei, the Golgi apparatus, vacuoles, and the endoplasmic reticulum may be affected. Most of these organelles, however, are somewhat held in place by cytoskeleton. Hansen and Igber suggest that intermediate filaments act to stabilize the nuleus against rotational movement, and integrate cell and nuclear structure. The tensegrity theory supports the idea that mechanical or physical forces alters the cytoskeletal structures of a cell resulting in the changes in cell: matrix interactions and receptor-signaling coupling. This type of stress to the cytoskeleton may be largely responsible regulating cell shape, growth, movement and metabolism. Mouse MC3T3 El cells under microgravity exhibited significant cytoskeletal changes and alterations in cell growth. The alterations in cytoskeleton architecture may be due to changes in the expression of actin related proteins or integrins. Philopott and coworkers reported on changes in the distribution of microtubule and cytoskeleton elements in the cells of heart tissue from space flight rats and those centrifuged at 1.7g. Other researchers have showed that microgravity reduced EGF-induced c-fos and c-jun expression compared to 1 g controls. Since c-fos and c-jun are known regulators of cell growth, it is likely that altered signal transduction involving protooncogenes may play a crucial role in the reduced growth and alterations in cytoskeletal arrangements found during space flight. It is clear that a microgravity environment induces a number of changes in cell shape, cell surface molecules, gene expression, and cytoskeletal

  6. Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

    SciTech Connect

    Huang, Xionggao; Wei, Yantao; Ma, Haizhi; Zhang, Shaochong

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. Black-Right-Pointing-Pointer Rac1 is activated in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous

  7. Corticosterone treatment results in enhanced release of peptidergic vesicles in astrocytes via cytoskeletal rearrangements.

    PubMed

    Chatterjee, Sreejata; Sikdar, Sujit K

    2013-12-01

    While the effect of stress on neuronal physiology is widely studied, its effect on the functionality of astrocytes is not well understood. We studied the effect of high doses of stress hormone corticosterone, on two physiological properties of astrocytes, i.e., gliotransmission and interastrocytic calcium waves. To study the release of peptidergic vesicles from astrocytes, hippocampal astrocyte cultures were transfected with a plasmid to express pro-atrial natriuretic peptide (ANP) fused with the emerald green fluorescent protein (ANP.emd). The rate of decrease in fluorescence of ANP.emd on application of ionomycin, a calcium ionophore was monitored. Significant increase in the rate of calcium-dependent exocytosis of ANP.emd was observed with the 100 nM and 1 μM corticosterone treatments for 3 h, which depended on the activation of the glucocorticoid receptor. ANP.emd tagged vesicles exhibited increased mobility in astrocyte culture upon corticosterone treatment. Increasing corticosterone concentrations also resulted in concomitant increase in the calcium wave propagation velocity, initiated by focal ATP application. Corticosterone treatment also resulted in increased GFAP expression and F-actin rearrangements. FITC-Phalloidin immunostaining revealed increased formation of cross linked F-actin networks with the 100 nM and 1 μM corticosterone treatment. Alternatively, blockade of actin polymerization and disruption of microtubules prevented the corticosterone-mediated increase in ANP.emd release kinetics. This study reports for the first time the effect of corticosterone on gliotransmission via modulation of cytoskeletal elements. As ANP acts on both neurons and blood vessels, modulation of its release could have functional implications in neurovascular coupling under pathophysiological conditions of stress. PMID:24123181

  8. Purinoreceptor P2X7 Regulation of Ca(2+) Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury.

    PubMed

    Minns, Martin S; Teicher, Gregory; Rich, Celeste B; Trinkaus-Randall, Vickery

    2016-02-01

    The process of wound healing involves a complex network of signaling pathways working to promote rapid cell migration and wound closure. Activation of purinergic receptors by secreted nucleotides plays a major role in calcium mobilization and the subsequent calcium-dependent signaling that is essential for proper healing. The role of the purinergic receptor P2X7 in wound healing is still relatively unknown. We demonstrate that P2X7 expression increases at the leading edge of corneal epithelium after injury in an organ culture model, and that this change occurs despite an overall decrease in P2X7 expression throughout the epithelium. Inhibition of P2X7 prevents this change in localization after injury and impairs wound healing. In cell culture, P2X7 inhibition attenuates the amplitude and duration of injury-induced calcium mobilization in cells at the leading edge. Immunofluorescence analysis of scratch-wounded cells reveals that P2X7 inhibition results in an overall decrease in the number of focal adhesions along with a concentration of focal adhesions at the wound margin. Live cell imaging of green fluorescent protein-labeled actin and talin shows that P2X7 inhibition alters actin cytoskeletal rearrangements and focal adhesion dynamics after injury. Together, these data demonstrate that P2X7 plays a critical role in mediating calcium signaling and coordinating cytoskeletal rearrangement at the leading edge, both of which processes are early signaling events necessary for proper epithelial wound healing. PMID:26683661

  9. HMGB1 induces human lung endothelial cell cytoskeletal rearrangement and barrier disruption.

    PubMed

    Wolfson, Rachel K; Chiang, Eddie T; Garcia, Joe G N

    2011-03-01

    Acute lung injury (ALI) results from loss of alveolar-capillary barrier integrity and the evolution of high-permeability pulmonary edema resulting in alveolar flooding and significant morbidity and mortality. HMGB1 is a late mediator of sepsis which uniquely participates in the evolution of sepsis and sepsis-induced ALI. The molecular events by which HMGB1 contributes to ALI remain poorly characterized. We characterized the role of HMGB1 in endothelial cell (EC) cytoskeletal rearrangement and vascular permeability, events essential to paracellular gap formation and barrier dysfunction characteristic of ALI. Initial experiments demonstrated HMGB1-mediated dose-dependent (5-20 μg/ml) decreases in transendothelial cell electrical resistance (TER) in the human pulmonary artery EC, a reflection of loss of barrier integrity. Furthermore, HMGB1 produced dose-dependent increases in paracellular gap formation in concert with loss of peripheral organized actin fibers, dissociation of cell-cell junctional cadherins, and the development of central stress fibers, a phenotypic change associated with increased contractile activity and increased EC permeability. Using siRNA strategies directed against known HMGB1 receptors (RAGE, TLR2, TLR4), we systematically determined that the receptor for advanced glycation end products (RAGE) is the primary receptor signaling HMGB1-induced TER decreases and paracellular gap formation via p38 MAP kinase activation and phosphorylation of the actin-binding protein, Hsp27. These studies add to the understanding of HMGB1-induced inflammatory events and vascular barrier disruption and offer the potential for clinical intervention in sepsis-induced ALI. PMID:21146549

  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. Quantifying morphological features of actin cytoskeletal filaments in plant cells based on mathematical morphology.

    PubMed

    Kimori, Yoshitaka; Hikino, Kazumi; Nishimura, Mikio; Mano, Shoji

    2016-01-21

    By quantifying the morphological properties of biological structures, we can better evaluate complex shapes and detect subtle morphological changes in organisms. In this paper, we propose a shape analysis method based on morphological image processing, and apply it to image analysis of actin cytoskeletal filaments in root hair cells of Arabidopsis thaliana. In plant cells, the actin cytoskeletal filaments have critical roles in various cellular processes such as vesicle trafficking and organelle motility. The dynamics of vesicles and organelles in plant cells depend on actin cytoskeletal filaments, regulating cell division and cell enlargement. To better understand the actin-dependent organelle motility, we attempted to quantify the organization of actin filaments in the root hair cells of the root hair defective 3 (rhd3) mutant. RHD3 is involved in actin organization, and its defect has been reported to affect the dynamics of various vesicles and organelles. We measured three shape features of the actin filaments in wild-type and mutant plants. One feature (thickness) was depicted on a grayscale; the others (describing the complexity of the filament network patterns in two-dimensional space) were depicted as binary features. The morphological phenotypes of the cytoskeletal filaments clearly differed between wild-type and mutant. Subtle variations of filament morphology among the mutants were detected and statistically quantified. PMID:26551157

  12. Actin-cytoskeleton rearrangement modulates proton-induced uptake

    SciTech Connect

    Ben-Dov, Nadav; Korenstein, Rafi

    2013-04-15

    Recently it has been shown that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesicles accompanied by an enhanced uptake of macromolecules. While the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the mechanisms underlying vesicle formation and its scission are still unknown. In light of the critical role of actin in vesicle formation during endocytosis, the present study addresses the involvement of cytoskeletal actin in proton-induced uptake (PIU). The uptake of dextran-FITC is used as a measure for the factual fraction of inward invaginations that undergo scission from the cell's plasma membrane. Our findings show that the rate of PIU in suspended cells is constant, whereas the rate of PIU in adherent cells is gradually increased in time, saturating at the level possessed by suspended cells. This is consistent with pH induced gradual degradation of stress-fibers in adherent cells. Wortmannin and calyculin-A are able to elevate PIU by 25% in adherent cells but not in suspended cells, while cytochalasin-D, rapamycin and latrunculin-A elevate PIU both in adherent and suspended cells. However, extensive actin depolymerization by high concentrations of latrunculin-A is able to inhibit PIU. We conclude that proton-induced membrane vesiculation is restricted by the actin structural resistance to the plasma membrane bending. Nevertheless, a certain degree of cortical actin restructuring is required for the completion of the scission process. - Highlights: ► Acidification of cells' exterior enhances uptake of macromolecules by the cells. ► Disruption of actin stress fibers leads to enhancement of proton induced uptake. ► Extensive depolymerization of cellular actin attenuates proton-induced uptake.

  13. α-Synuclein and Its A30P Mutant Affect Actin Cytoskeletal Structure and Dynamics

    PubMed Central

    Sousa, Vítor L.; Bellani, Serena; Giannandrea, Maila; Yousuf, Malikmohamed; Valtorta, Flavia; Meldolesi, Jacopo

    2009-01-01

    The function of α-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, α-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson's disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that α-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type α-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant α-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant α-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the α-synuclein gene, electroporation of wild-type α-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P α-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, α-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration. PMID:19553474

  14. Actin Grips: Circular Actin-Rich Cytoskeletal Structures that Mediate the Wrapping of Polymeric Microfibers by Endothelial Cells

    PubMed Central

    Jones, Desiree; Park, DoYoung; Anghelina, Mirela; Pecot, Thierry; Machiraju, Raghu; Xue, Ruipeng; Lannutti, John; Thomas, Jessica; Cole, Sara; Moldovan, Leni; Moldovan, Nicanor I.

    2015-01-01

    Interaction of endothelial-lineage cells with three-dimensional substrates was much less studied than that with flat culture surfaces. We investigated the in vitro attachment of both mature endothelial cells (ECs) and of less differentiated EC colony-forming cells to poly-e-capro-lactone (PCL) fibers with diameters in 5–20 μm range (‘scaffold microfibers’, SMFs). We found that notwithstanding the poor intrinsic adhesiveness to PCL, both cell types completely wrapped the SMFs after long-term cultivation, thus attaining a cylindrical morphology. In this system, both EC types grew vigorously for more than a week and became increasingly more differentiated, as shown by multiplexed gene expression. Three-dimensional reconstructions from multiphoton confocal microscopy images using custom software showed that the filamentous (F) actin bundles took a conspicuous ring-like organization around the SMFs. Unlike the classical F-actin-containing stress fibers, these rings were not associated with either focal adhesions or intermediate filaments. We also demonstrated that plasma membrane boundaries adjacent to these circular cytoskeletal structures were tightly yet dynamically apposed to the SMFs, for which reason we suggest to call them ‘actin grips’. In conclusion, we describe a particular form of F-actin assembly with relevance for cytoskeletal organization in response to biomaterials, for endothelial-specific cell behavior in vitro and in vivo, and for tissue engineering. PMID:25818446

  15. A POROELASTIC MODEL FOR CELL CRAWLING INCLUDING MECHANICAL COUPLING BETWEEN CYTOSKELETAL CONTRACTION AND ACTIN POLYMERIZATION.

    PubMed

    Taber, L A; Shi, Y; Yang, L; Bayly, P V

    2011-01-01

    Much is known about the biophysical mechanisms involved in cell crawling, but how these processes are coordinated to produce directed motion is not well understood. Here, we propose a new hypothesis whereby local cytoskeletal contraction generates fluid flow through the lamellipodium, with the pressure at the front of the cell facilitating actin polymerization which pushes the leading edge forward. The contraction, in turn, is regulated by stress in the cytoskeleton. To test this hypothesis, finite element models for a crawling cell are presented. These models are based on nonlinear poroelasticity theory, modified to include the effects of active contraction and growth, which are regulated by mechanical feedback laws. Results from the models agree reasonably well with published experimental data for cell speed, actin flow, and cytoskeletal deformation in migrating fish epidermal keratocytes. The models also suggest that oscillations can occur for certain ranges of parameter values. PMID:21765817

  16. Cytoskeletal proteins in cortical development and disease: actin associated proteins in periventricular heterotopia

    PubMed Central

    Lian, Gewei; Sheen, Volney L.

    2015-01-01

    The actin cytoskeleton regulates many important cellular processes in the brain, including cell division and proliferation, migration, and cytokinesis and differentiation. These developmental processes can be regulated through actin dependent vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape. Many of these processes are mediated by extensive and intimate interactions of actin with cellular membranes and proteins. Disruption in the actin cytoskeleton in the brain gives rise to periventricular heterotopia (PH), a malformation of cortical development, characterized by abnormal neurons clustered deep in the brain along the lateral ventricles. This disorder can give rise to seizures, dyslexia and psychiatric disturbances. Anatomically, PH is characterized by a smaller brain (impaired proliferation), heterotopia (impaired initial migration) and disruption along the neuroependymal lining (impaired cell-cell adhesion). Genes causal for PH have also been implicated in actin-dependent processes. The current review provides mechanistic insight into actin cytoskeletal regulation of cortical development in the context of this malformation of cortical development. PMID:25883548

  17. Ratiometric Imaging of the T-Cell Actin Cytoskeleton Reveals the Nature of Receptor-Induced Cytoskeletal Enrichment

    PubMed Central

    Smoligovets, Alexander A.; Smith, Adam W.; Groves, Jay T.

    2013-01-01

    The T-cell actin cytoskeleton mediates adaptive immune system responses to peptide antigens by physically directing the motion and clustering of T-cell receptors (TCRs) on the cell surface. When TCR movement is impeded by externally applied physical barriers, the actin network exhibits transient enrichment near the trapped receptors. The coordinated nature of the actin density fluctuations suggests that they are composed of filamentous actin, but it has not been possible to eliminate de novo polymerization at TCR-associated actin polymerizing factors as an alternative cause. Here, we use a dual-probe cytoskeleton labeling strategy to distinguish between stable and polymerizing pools of actin. Our results suggest that TCR-associated actin consists of a relatively high proportion of the stable cytoskeletal fraction and extends away from the cell membrane into the cell. This implies that actin enrichment at mechanically trapped TCRs results from three-dimensional bunching of the existing filamentous actin network. PMID:23931330

  18. A Legionella effector modulates host cytoskeletal structure by inhibiting actin polymerization

    PubMed Central

    Guo, Zhenhua; Stephenson, Robert; Qiu, Jiazhang; Zheng, Shijun; Luo, Zhao-Qing

    2014-01-01

    Successful infection by the opportunistic pathogen Legionella pneumophila requires the collective activity of hundreds of virulence proteins delivered into the host cell by the Dot/Icm type IV secretion system. These virulence proteins, also called effectors modulate distinct host cellular processes to create a membrane-bound niche called the Legionella containing vacuole (LCV) supportive of bacterial growth. We found that Ceg14(Lpg0437), a Dot/Icm substrate is toxic to yeast and such toxicity can be alleviated by overexpression of profilin, a protein involved in cytoskeletal structure in eukaryotes. We further showed that mutations in profilin affect actin binding but not other functions such as interactions with poly-L-proline or phosphatidylinositol, abolish its suppressor activity. Consistent with the fact the profilin suppresses its toxicity, expression of Ceg14 but not its non-toxic mutants in yeast affects actin distribution and budding of daughter cells. Although Ceg14 does not detectably interact with profilin, it co-sediments with filamentous actin and inhibits actin polymerization, causing the accumulation of short actin filaments. These results reveal that multiple L. pneumophila effectors target components of the host cytoskeleton. PMID:24286927

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

    SciTech Connect

    Choong, Grace; Liu, Ying; Xiao, Weiqun; Templeton, Douglas M.

    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 sufficient 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.

  20. 2',3'-Cyclic nucleotide 3'-phosphodiesterase binds to actin-based cytoskeletal elements in an isoprenylation-independent manner.

    PubMed

    De Angelis, D A; Braun, P E

    1996-09-01

    2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is an isoprenylated protein enriched in myelin and oligodendrocytes but also present in several other tissues at low levels. CNP binds avidly to membranes and in addition possesses several characteristics of cytoskeletal proteins. The role of isoprenylation in the association of CNP with the cytoskeleton was analyzed by ectopic expression in L cells of epitope-tagged CNP1 and a non-isoprenylated mutant CNP1. Using nonionic detergent extraction, drug-mediated cytoskeletal disruption, and coimmunoprecipitation with an anti-actin antibody, we show that CNP1 is associated with actin-based cytoskeletal elements independently of its isoprenylation status. A control protein, p21c-H-ras, which is also modified by isoprenylation at its carboxyl-terminus, does not bind to cytoskeletal structures as judged by the same criteria. We present a model that accounts for the association of CNP1 with membranes and the cytoskeleton. PMID:8752099

  1. Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice

    PubMed Central

    Morikawa, Yuka; Zhang, Min; Heallen, Todd; Leach, John; Tao, Ge; Xiao, Yang; Bai, Yan; Li, Wei; Willerson, James T.; Martin, James F.

    2015-01-01

    The mammalian heart regenerates poorly, and damage commonly leads to heart failure. Hippo signaling is an evolutionarily conserved kinase cascade that regulates organ size during development and prevents adult mammalian cardiomyocyte regeneration by inhibiting the transcriptional coactivator Yap, which also responds to mechanical signaling in cultured cells to promote cell proliferation. To identify Yap target genes that are activated during cardiomyocyte renewal and regeneration, we performed Yap chromatin immunoprecipitation sequencing (ChIP-Seq) and mRNA expression profiling in Hippo signaling-deficient mouse hearts. We found that Yap directly regulated genes encoding cell cycle progression proteins, as well as genes encoding proteins that promote F-actin polymerization and that link the actin cytoskeleton to the extracellular matrix. Included in the latter group were components of the dystrophin glycoprotein complex (DGC), a large molecular complex that, when defective, results in muscular dystrophy in humans. Cardiomyocytes near scar tissue of injured Hippo signaling-deficient mouse hearts showed cellular protrusions suggestive of cytoskeletal remodeling. The hearts of mdx mutant mice, which lack functional dystrophin and are a model for muscular dystrophy, showed impaired regeneration and cytoskeleton remodeling, but normal cardiomyocyte proliferation after injury. Our data showed that, in addition to genes encoding cell cycle progression proteins, Yap regulated genes that enhance cytoskeletal remodeling Thus, blocking the Hippo pathway input to Yap may tip the balance so that Yap responds to the mechanical changes associated with heart injury to promote repair. PMID:25943351

  2. Region-Specific Involvement of Actin Rearrangement-Related Synaptic Structure Alterations in Conditioned Taste Aversion Memory

    ERIC Educational Resources Information Center

    Bi, Ai-Ling; Wang, Yue; Li, Bo-Qin; Wang, Qian-Qian; Ma, Ling; Yu, Hui; Zhao, Ling; Chen, Zhe-Yu

    2010-01-01

    Actin rearrangement plays an essential role in learning and memory; however, the spatial and temporal regulation of actin dynamics in different phases of associative memory has not been fully understood. Here, using the conditioned taste aversion (CTA) paradigm, we investigated the region-specific involvement of actin rearrangement-related…

  3. Involvement of Rab6a in organelle rearrangement and cytoskeletal organization during mouse oocyte maturation

    PubMed Central

    Ma, Rujun; Zhang, Jiaqi; Liu, Xiaohui; Li, Ling; Liu, Honglin; Rui, Rong; Gu, Ling; Wang, Qiang

    2016-01-01

    Rab GTPases have been reported to define the identity and transport routes of vesicles. Rab6 is one of the most extensively studied Rab proteins involved in regulating organelle trafficking and integrity maintenance. However, to date, the function of Rab6 in mammalian oocytes has not been addressed. Here we report severe disorganization of endoplasmic reticulum upon specific knockdown of Rab6a in mouse oocytes. In line with this finding, intracellular Ca2+ stores are accordingly reduced in Rab6a-depleted oocytes. Furthermore, in these oocytes, we observe the absence of cortical granule free domain, which is a kind of special organelle in matured oocytes and its exocytosis is calcium dependent. On the other hand, following Rab6a knockdown, the prominent defects of cytoskeletal structures are detected during oocyte meiosis. In particular, the majority of Rab6a-depleted oocytes fail to form the actin cap, and the frequency of spindle defects and chromosome misalignment is significantly elevated. In summary, our data reveal that Rab6a not only participates in modulating the organization of oocyte organelles, but also is a novel regulator of meiotic apparatus in mammalian oocytes. PMID:27030207

  4. Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis

    PubMed Central

    Kasioulis, Ioannis; Syred, Heather M.; Tate, Peri; Finch, Andrew; Shaw, Joseph; Seawright, Anne; Fuszard, Matt; Botting, Catherine H.; Shirran, Sally; Adams, Ian R.; Jackson, Ian J.; van Heyningen, Veronica; Yeyati, Patricia L.

    2014-01-01

    The lysine demethylase Kdm3a (Jhdm2a, Jmjd1a) is required for male fertility, sex determination, and metabolic homeostasis through its nuclear role in chromatin remodeling. Many histone-modifying enzymes have additional nonhistone substrates, as well as nonenzymatic functions, contributing to the full spectrum of events underlying their biological roles. We present two Kdm3a mouse models that exhibit cytoplasmic defects that may account in part for the globozoospermia phenotype reported previously. Electron microscopy revealed abnormal acrosome and manchette and the absence of implantation fossa at the caudal end of the nucleus in mice without Kdm3a demethylase activity, which affected cytoplasmic structures required to elongate the sperm head. We describe an enzymatically active new Kdm3a isoform and show that subcellular distribution, protein levels, and lysine demethylation activity of Kdm3a depended on Hsp90. We show that Kdm3a localizes to cytoplasmic structures of maturing spermatids affected in Kdm3a mutant mice, which in turn display altered fractionation of β-actin and γ-tubulin. Kdm3a is therefore a multifunctional Hsp90 client protein that participates directly in the regulation of cytoskeletal components. PMID:24554764

  5. Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis.

    PubMed

    Kasioulis, Ioannis; Syred, Heather M; Tate, Peri; Finch, Andrew; Shaw, Joseph; Seawright, Anne; Fuszard, Matt; Botting, Catherine H; Shirran, Sally; Adams, Ian R; Jackson, Ian J; van Heyningen, Veronica; Yeyati, Patricia L

    2014-04-01

    The lysine demethylase Kdm3a (Jhdm2a, Jmjd1a) is required for male fertility, sex determination, and metabolic homeostasis through its nuclear role in chromatin remodeling. Many histone-modifying enzymes have additional nonhistone substrates, as well as nonenzymatic functions, contributing to the full spectrum of events underlying their biological roles. We present two Kdm3a mouse models that exhibit cytoplasmic defects that may account in part for the globozoospermia phenotype reported previously. Electron microscopy revealed abnormal acrosome and manchette and the absence of implantation fossa at the caudal end of the nucleus in mice without Kdm3a demethylase activity, which affected cytoplasmic structures required to elongate the sperm head. We describe an enzymatically active new Kdm3a isoform and show that subcellular distribution, protein levels, and lysine demethylation activity of Kdm3a depended on Hsp90. We show that Kdm3a localizes to cytoplasmic structures of maturing spermatids affected in Kdm3a mutant mice, which in turn display altered fractionation of β-actin and γ-tubulin. Kdm3a is therefore a multifunctional Hsp90 client protein that participates directly in the regulation of cytoskeletal components. PMID:24554764

  6. Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

    SciTech Connect

    Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko; Dunham, Ian; Murai, Kasumi; Jones, Philip H.

    2011-07-01

    Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, but F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.

  7. Xenopus cytoskeletal actin and human c-fos gene promoters share a conserved protein-binding site.

    PubMed

    Mohun, T; Garrett, N; Treisman, R

    1987-03-01

    Xenopus laevis cytoskeletal actin gene promoters contain a 20-bp sequence homologous to the serum response element (SRE) required for transient human c-fos gene transcription in response to serum factors. Both sequences bind the same factor in HeLa cell extracts, as shown by binding competition, DNase I and dimethylsulphate (DMS) protection and DMS interference assays. A similar protein is present in Xenopus laevis oocytes. Sequences containing the SRE homology are essential for constitutive activity of the actin promoter in both Xenopus and mouse cells, and a synthetic SRE functions as a promoter element in these cells. In mouse cells, transcription of both transfected Xenopus actin and actin/c-fos fusion genes is activated following serum stimulation. These data suggest that the SRE and its cognate protein form part of a regulatory pathway that has been highly conserved during evolution. PMID:3582369

  8. Xenopus cytoskeletal actin and human c-fos gene promoters share a conserved protein-binding site.

    PubMed Central

    Mohun, T; Garrett, N; Treisman, R

    1987-01-01

    Xenopus laevis cytoskeletal actin gene promoters contain a 20-bp sequence homologous to the serum response element (SRE) required for transient human c-fos gene transcription in response to serum factors. Both sequences bind the same factor in HeLa cell extracts, as shown by binding competition, DNase I and dimethylsulphate (DMS) protection and DMS interference assays. A similar protein is present in Xenopus laevis oocytes. Sequences containing the SRE homology are essential for constitutive activity of the actin promoter in both Xenopus and mouse cells, and a synthetic SRE functions as a promoter element in these cells. In mouse cells, transcription of both transfected Xenopus actin and actin/c-fos fusion genes is activated following serum stimulation. These data suggest that the SRE and its cognate protein form part of a regulatory pathway that has been highly conserved during evolution. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3582369

  9. Ror2 regulates branching, differentiation, and actin-cytoskeletal dynamics within the mammary epithelium

    PubMed Central

    Roarty, Kevin; Shore, Amy N.; Creighton, Chad J.

    2015-01-01

    Wnt signaling encompasses β-catenin–dependent and –independent networks. How receptor context provides Wnt specificity in vivo to assimilate multiple concurrent Wnt inputs throughout development remains unclear. Here, we identified a refined expression pattern of Wnt/receptor combinations associated with the Wnt/β-catenin–independent pathway in mammary epithelial subpopulations. Moreover, we elucidated the function of the alternative Wnt receptor Ror2 in mammary development and provided evidence for coordination of this pathway with Wnt/β-catenin–dependent signaling in the mammary epithelium. Lentiviral short hairpin RNA (shRNA)-mediated depletion of Ror2 in vivo increased branching and altered the differentiation of the mammary epithelium. Microarray analyses identified distinct gene level alterations within the epithelial compartments in the absence of Ror2, with marked changes observed in genes associated with the actin cytoskeleton. Modeling of branching morphogenesis in vitro defined specific defects in cytoskeletal dynamics accompanied by Rho pathway alterations downstream of Ror2 loss. The current study presents a model of Wnt signaling coordination in vivo and assigns an important role for Ror2 in mammary development. PMID:25624393

  10. Hic-5 Regulates Actin Cytoskeletal Reorganization and Expression of Fibrogenic Markers and Myocilin in Trabecular Meshwork Cells

    PubMed Central

    Pattabiraman, Padmanabhan Paranji; Rao, Ponugoti Vasantha

    2015-01-01

    Purpose To explore the role of inducible focal adhesion (FA) protein Hic-5 in actin cytoskeletal reorganization, FA formation, fibrogenic activity, and expression of myocilin in trabecular meshwork (TM) cells. Methods Using primary cultures of human TM (HTM) cells, the effects of various external factors on Hic-5 protein levels, as well as the effects of recombinant Hic-5 and Hic-5 small interfering RNA (siRNA) on actin cytoskeleton, FAs, myocilin, α-smooth muscle actin (αSMA), and collagen-1 were determined by immunofluorescence and immunoblot analyses. Results Hic-5 distributes discretely to the FAs in HTM cells and throughout the TM and Schlemm's canal of the human aqueous humor (AH) outflow pathway. Transforming growth factor-β2 (TGF-β2), endothelin-1, lysophosphatidic acid, hydrogen peroxide, and RhoA significantly increased Hic-5 protein levels in HTM cells in association with reorganization of actin cytoskeleton and FAs. While recombinant Hic-5 induced actin stress fibers, FAs, αv integrin redistribution to the FAs, increased levels of αSMA, collagen-1, and myocilin, Hic-5 siRNA suppressed most of these responses in HTM cells. Hic-5 siRNA also suppressed TGF-β2-induced fibrogenic activity and dexamethasone-induced myocilin expression in HTM cells. Conclusions Taken together, these results reveal that Hic-5, whose levels were increased by various external factors implicated in elevated intraocular pressure, induces actin cytoskeletal reorganization, FAs, expression of fibrogenic markers, and myocilin in HTM cells. These characteristics of Hic-5 in TM cells indicate its importance in regulation of AH outflow through the TM in both normal and glaucomatous eyes. PMID:26313302

  11. 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

  12. Topography Design Concept of a Tissue Engineering Scaffold for Controlling Cell Function and Fate Through Actin Cytoskeletal Modulation

    PubMed Central

    Adachi, Taiji

    2014-01-01

    The physiological role of the actin cytoskeleton is well known: it provides mechanical support and endogenous force generation for formation of a cell shape and for migration. Furthermore, a growing number of studies have demonstrated another significant role of the actin cytoskeleton: it offers dynamic epigenetic memory for guiding cell fate, in particular, proliferation and differentiation. Because instantaneous imbalance in the mechanical homeostasis is adjusted through actin remodeling, a synthetic extracellular matrix (ECM) niche as a source of topographical and mechanical cues is expected to be effective at modulation of the actin cytoskeleton. In this context, the synthetic ECM niche determines cell migration, proliferation, and differentiation, all of which have to be controlled in functional tissue engineering scaffolds to ensure proper regulation of tissue/organ formation, maintenance of tissue integrity and repair, and regeneration. Here, with an emphasis on the epigenetic role of the actin cytoskeletal system, we propose a design concept of micro/nanotopography of a tissue engineering scaffold for control of cell migration, proliferation, and differentiation in a stable and well-defined manner, both in vitro and in vivo. PMID:24720435

  13. Topography design concept of a tissue engineering scaffold for controlling cell function and fate through actin cytoskeletal modulation.

    PubMed

    Miyoshi, Hiromi; Adachi, Taiji

    2014-12-01

    The physiological role of the actin cytoskeleton is well known: it provides mechanical support and endogenous force generation for formation of a cell shape and for migration. Furthermore, a growing number of studies have demonstrated another significant role of the actin cytoskeleton: it offers dynamic epigenetic memory for guiding cell fate, in particular, proliferation and differentiation. Because instantaneous imbalance in the mechanical homeostasis is adjusted through actin remodeling, a synthetic extracellular matrix (ECM) niche as a source of topographical and mechanical cues is expected to be effective at modulation of the actin cytoskeleton. In this context, the synthetic ECM niche determines cell migration, proliferation, and differentiation, all of which have to be controlled in functional tissue engineering scaffolds to ensure proper regulation of tissue/organ formation, maintenance of tissue integrity and repair, and regeneration. Here, with an emphasis on the epigenetic role of the actin cytoskeletal system, we propose a design concept of micro/nanotopography of a tissue engineering scaffold for control of cell migration, proliferation, and differentiation in a stable and well-defined manner, both in vitro and in vivo. PMID:24720435

  14. Cell cytoskeletal conformation under reversible thermal control

    NASA Astrophysics Data System (ADS)

    Chang, Ting-Ya; Yang, Chung-Yao; Liao, Kai-Wei; Andrew Yeh, J.; Cheng, Chao-Min

    2013-12-01

    In order to assess the role of cytoskeletal structure in modulating cell surface topography during cell transformation, we investigated cytoskeletal organization of Madin-Darby canine kidney (MDCK) epithelial cells at different thermal gradients. Specifically, we examined actin polymerization as a function of temperature in a controlled thermal environment. After applying an increase in temperature of 5 °C, we observed fewer actin filaments in the network, as these molecular polymers depolymerized. Partial stress fibers of MDCK cells could be rearranged, but some of them were disrupted irreversibly after a second thermal treatment, and MDCK cells underwent apoptosis at higher temperatures as well.

  15. Simultaneous Visualization of Peroxisomes and Cytoskeletal Elements Reveals Actin and Not Microtubule-Based Peroxisome Motility in Plants1[w

    PubMed Central

    Mathur, Jaideep; Mathur, Neeta; Hülskamp, Martin

    2002-01-01

    Peroxisomes were visualized in living plant cells using a yellow fluorescent protein tagged with a peroxisomal targeting signal consisting of the SKL motif. Simultaneous visualization of peroxisomes and microfilaments/microtubules was accomplished in onion (Allium cepa) epidermal cells transiently expressing the yellow fluorescent protein-peroxi construct, a green fluorescent protein-mTalin construct that labels filamentous-actin filaments, and a green fluorescent protein-microtubule-binding domain construct that labels microtubules. The covisualization of peroxisomes and cytoskeletal elements revealed that, contrary to the reports from animal cells, peroxisomes in plants appear to associate with actin filaments and not microtubules. That peroxisome movement is actin based was shown by pharmacological studies. For this analysis we used onion epidermal cells and various cell types of Arabidopsis including trichomes, root hairs, and root cortex cells exhibiting different modes of growth. In transient onion epidermis assay and in transgenic Arabidopsis plants, an interference with the actin cytoskeleton resulted in progressive loss of saltatory movement followed by the aggregation and a complete cessation of peroxisome motility within 30 min of drug application. Microtubule depolymerization or stabilization had no effect. PMID:11891258

  16. The scaffolding protein IQGAP1 co-localizes with actin at the cytoplasmic face of the nuclear envelope: implications for cytoskeletal regulation

    PubMed Central

    Johnson, Michael A.

    2012-01-01

    IQGAP1 is an important cytoskeletal regulator, known to act at the plasma membrane to bundle and cap actin filaments, and to tether the cortical actin meshwork to microtubules via plus-end binding proteins. Here we describe the novel subcellular localization of IQGAP1 at the cytoplasmic face of the nuclear envelope, where it co-located with F-actin. The IQGAP1 and F-actin staining overlapped that of microtubules at the nuclear envelope, revealing a pattern strikingly similar to that observed at the plasma membrane. In detergent-extracted cells IQGAP1 was retained at cytoskeletal structures at the nuclear envelope. This finding has new implications for involvement of IQGAP1 in cell polarization and migration events and potentially in cell cycle-associated nuclear envelope assembly/disassembly. PMID:22964981

  17. The Three-Dimensional Dynamics of Actin Waves, a Model of Cytoskeletal Self-Organization

    PubMed Central

    Bretschneider, Till; Anderson, Kurt; Ecke, Mary; Müller-Taubenberger, Annette; Schroth-Diez, Britta; Ishikawa-Ankerhold, Hellen C.; Gerisch, Günther

    2009-01-01

    Actin polymerization is typically initiated at specific sites in a cell by membrane-bound protein complexes, and the resulting structures are involved in specialized cellular functions, such as migration, particle uptake, or mitotic division. Here we analyze the potential of the actin system to self-organize into waves that propagate on the planar, substrate-attached membrane of a cell. We show that self-assembly involves the ordered recruitment of proteins from the cytoplasmic pool and relate the organization of actin waves to their capacity for applying force. Three proteins are shown to form distinct three-dimensional patterns in the actin waves. Myosin-IB is enriched at the wave front and close to the plasma membrane, the Arp2/3 complex is distributed throughout the waves, and coronin forms a sloping layer on top of them. CARMIL, a protein that links myosin-IB to the Arp2/3 complex, is also recruited to the waves. Wave formation does not depend on signals transmitted by heterotrimeric G-proteins, nor does their propagation require SCAR, a regulator upstream of the Arp2/3 complex. Propagation of the waves is based on an actin treadmilling mechanism, indicating a program that couples actin assembly to disassembly in a three-dimensional pattern. When waves impinge on the cell perimeter, they push the edge forward; when they reverse direction, the cell border is paralyzed. These data show that force-generating, highly organized supramolecular networks are autonomously formed in live cells from molecular motors and proteins controlling actin polymerization and depolymerization. PMID:19348770

  18. Nano-ZnO leads to tubulin macrotube assembly and actin bundling, triggering cytoskeletal catastrophe and cell necrosis

    NASA Astrophysics Data System (ADS)

    García-Hevia, Lorena; Valiente, Rafael; Martín-Rodríguez, Rosa; Renero-Lecuna, Carlos; González, Jesús; Rodríguez-Fernández, Lidia; Aguado, Fernando; Villegas, Juan C.; Fanarraga, Mónica L.

    2016-05-01

    Zinc is a crucial element in biology that plays chief catalytic, structural and protein regulatory roles. Excess cytoplasmic zinc is toxic to cells so there are cell-entry and intracellular buffering mechanisms that control intracellular zinc availability. Tubulin and actin are two zinc-scavenging proteins that are essential components of the cellular cytoskeleton implicated in cell division, migration and cellular architecture maintenance. Here we demonstrate how exposure to different ZnO nanostructures, namely ZnO commercial nanoparticles and custom-made ZnO nanowires, produce acute cytotoxic effects in human keratinocytes (HaCat) and epithelial cells (HeLa) triggering a dose-dependent cell retraction and collapse. We show how engulfed ZnO nanoparticles dissolve intracellularly, triggering actin filament bundling and structural changes in microtubules, transforming these highly dynamic 25 nm diameter polymers into rigid macrotubes of tubulin, severely affecting cell proliferation and survival. Our results demonstrate that nano-ZnO causes acute cytoskeletal collapse that triggers necrosis, followed by a late reactive oxygen species (ROS)-dependent apoptotic process.Zinc is a crucial element in biology that plays chief catalytic, structural and protein regulatory roles. Excess cytoplasmic zinc is toxic to cells so there are cell-entry and intracellular buffering mechanisms that control intracellular zinc availability. Tubulin and actin are two zinc-scavenging proteins that are essential components of the cellular cytoskeleton implicated in cell division, migration and cellular architecture maintenance. Here we demonstrate how exposure to different ZnO nanostructures, namely ZnO commercial nanoparticles and custom-made ZnO nanowires, produce acute cytotoxic effects in human keratinocytes (HaCat) and epithelial cells (HeLa) triggering a dose-dependent cell retraction and collapse. We show how engulfed ZnO nanoparticles dissolve intracellularly, triggering actin

  19. Nano-ZnO leads to tubulin macrotube assembly and actin bundling, triggering cytoskeletal catastrophe and cell necrosis.

    PubMed

    García-Hevia, Lorena; Valiente, Rafael; Martín-Rodríguez, Rosa; Renero-Lecuna, Carlos; González, Jesús; Rodríguez-Fernández, Lidia; Aguado, Fernando; Villegas, Juan C; Fanarraga, Mónica L

    2016-06-01

    Zinc is a crucial element in biology that plays chief catalytic, structural and protein regulatory roles. Excess cytoplasmic zinc is toxic to cells so there are cell-entry and intracellular buffering mechanisms that control intracellular zinc availability. Tubulin and actin are two zinc-scavenging proteins that are essential components of the cellular cytoskeleton implicated in cell division, migration and cellular architecture maintenance. Here we demonstrate how exposure to different ZnO nanostructures, namely ZnO commercial nanoparticles and custom-made ZnO nanowires, produce acute cytotoxic effects in human keratinocytes (HaCat) and epithelial cells (HeLa) triggering a dose-dependent cell retraction and collapse. We show how engulfed ZnO nanoparticles dissolve intracellularly, triggering actin filament bundling and structural changes in microtubules, transforming these highly dynamic 25 nm diameter polymers into rigid macrotubes of tubulin, severely affecting cell proliferation and survival. Our results demonstrate that nano-ZnO causes acute cytoskeletal collapse that triggers necrosis, followed by a late reactive oxygen species (ROS)-dependent apoptotic process. PMID:27228212

  20. Tyrosine-Phosphorylated Caveolin-1 Blocks Bacterial Uptake by Inducing Vav2-RhoA-Mediated Cytoskeletal Rearrangements

    PubMed Central

    Kaushansky, Alexis; Pompaiah, Malvika; Thorn, Hans; Brinkmann, Volker; MacBeath, Gavin; Meyer, Thomas F.

    2010-01-01

    Certain bacterial adhesins appear to promote a pathogen's extracellular lifestyle rather than its entry into host cells. However, little is known about the stimuli elicited upon such pathogen host-cell interactions. Here, we report that type IV pili (Tfp)-producing Neisseria gonorrhoeae (P+GC) induces an immediate recruitment of caveolin-1 (Cav1) in the host cell, which subsequently prevents bacterial internalization by triggering cytoskeletal rearrangements via downstream phosphotyrosine signaling. A broad and unbiased analysis of potential interaction partners for tyrosine-phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2. Both Vav2 and its substrate, the small GTPase RhoA, were found to play a direct role in the Cav1-mediated prevention of bacterial uptake. Our findings, which have been extended to enteropathogenic Escherichia coli, highlight how Tfp-producing bacteria avoid host cell uptake. Further, our data establish a mechanistic link between Cav1 phosphorylation and pathogen-induced cytoskeleton reorganization and advance our understanding of caveolin function. PMID:20808760

  1. Metadherin regulates proliferation and metastasis via actin cytoskeletal remodelling in non-small cell lung cancer

    PubMed Central

    Yao, Y; Gu, X; Liu, H; Wu, G; Yuan, D; Yang, X; Song, Y

    2014-01-01

    Background: Metaderin (MTDH) protein is a novel component part of tight junction complex. The aim of this study was to investigate the correlation between MTDH and prognosis of patients and to explore the role of MTDH on NSCLC development and metastasis. Methods: Relative mRNA expression was evaluated by quantitative real-time PCR, and protein expression was detected using immunohistochemistry staining. The role of MTDH in cancer cell proliferation, migration and invasion was studied by modulation of MTDH expression in NSCLC cell lines. These functions of MTDH were further confirmed in vivo. Results: In NSCLC, low MTDH protein expression was correlated with lymph node metastasis, TNM stage and decreased OS (P=0.001, 0.011 and 0.013, respectively). Overexpression of MTDH reduced anchorage-independent and -dependent growth through arresting cell cycle, inhibited migration and invasion in vitro and further suppressed tumorigenesis, tumour growth and metastasis in vivo. Knockdown of MTDH expression increased cell invasiveness. MTDH overexpression reversed pro-metastatic actin cytoskeleton remodelling and inhibited EMT, supporting that MTDH has a key role on cancer proliferation and metastasis. Conclusions: MTDH has an important role in NSCLC proliferation and metastasis and provides potential in predicting metastasis and prognosis for patients with NSCLC. PMID:24918821

  2. The actin cytoskeleton in presynaptic assembly.

    PubMed

    Nelson, Jessica C; Stavoe, Andrea K H; Colón-Ramos, Daniel A

    2013-01-01

    Dramatic morphogenetic processes underpin nearly every step of nervous system development, from initial neuronal migration and axon guidance to synaptogenesis. Underlying this morphogenesis are dynamic rearrangements of cytoskeletal architecture. Here we discuss the roles of the actin cytoskeleton in the development of presynaptic terminals, from the elaboration of terminal arbors to the recruitment of presynaptic vesicles and active zone components. The studies discussed here underscore the importance of actin regulation at every step in neuronal circuit assembly. PMID:23628914

  3. Reorganization of the actin cytoskeleton via transcriptional regulation of cytoskeletal/focal adhesion genes by myocardin-related transcription factors (MRTFs/MAL/MKLs)

    SciTech Connect

    Morita, Tsuyoshi; Mayanagi, Taira; Sobue, Kenji

    2007-10-01

    RhoA is a crucial regulator of stress fiber and focal adhesion formation through the activation of actin nucleation and polymerization. It also regulates the nuclear translocation of myocardin-related transcription factor-A and -B (MRTF-A/B, MAL or MKL 1/2), which are co-activators of serum response factor (SRF). In dominant-negative MRTF-A (DN-MRTF-A)-expressing NIH 3T3 cell lines, the expressions of several cytoskeletal/focal adhesion genes were down-regulated, and the formation of stress fiber and focal adhesion was severely diminished. MRTF-A/B-knockdown cells also exhibited such cytoskeletal defects. In reporter assays, both RhoA and MRTF-A enhanced promoter activities of these genes in a CArG-box-dependent manner, and DN-MRTF-A inhibited the RhoA-mediated activation of these promoters. In dominant-negative RhoA (RhoA-N19)-expressing NIH 3T3 cell lines, the nuclear translocation of MRTF-A/B was predominantly prevented, resulting in the reduced expression of cytoskeletal/focal adhesion proteins. Further, constitutive-active MRTF-A/B increased the expression of endogenous cytoskeletal/focal adhesion proteins, and thereby rescued the defective phenotype of stress fibers and focal adhesions in RhoA-N19 expressing cells. These results indicate that MRTF-A/B act as pivotal mediators of stress fiber and focal adhesion formation via the transcriptional regulation of a subset of cytoskeletal/focal adhesion genes.

  4. The anti-proliferative agent jasplakinolide rearranges the actin cytoskeleton of plant cells.

    PubMed

    Sawitzky, H; Liebe, S; Willingale-Theune, J; Menzel, D

    1999-06-01

    In the present study, we have characterized the action of the natural cyclodepsipeptide jasplakinolide (JAS) on the cytoplasmic architecture, actin-based cytoplasmic motility, and the organization of the actin cytoskeleton in selected examples of green algae (Acetabularia, Pseudobryopsis and Nitella) and higher plant cells (Allium bulb scale cells and Sinapis root hairs). JAS was capable of influencing the actin cytoskeleton and inhibiting cytoplasmic streaming in a differential, cell type-specific manner. With the exception of Nitella, two consecutive responses were observed upon incubation with 2.5 microM JAS: In the first phase cytoplasmic streaming increased transiently alongside with minor modifications of the actin cytoskeleton in the form of adventitious actin spots and spikes appearing throughout the cell cortex in addition to the normal actin bundle system typical for each cell type. In the second phase, cytoplasmic streaming stopped and the actin cytoskeleton became heavily reorganized into shorter, straight, more and more randomly oriented bundle segments. JAS exerted severe long-term effects on the actin cytoskeleton when treatments exceeded 30min at a concentration of 2.5 microM. An in situ competition assay using equimolar concentrations of JAS and FITC-phalloidin suggested that JAS has a phalloidin-like action. Effects of JAS were significantly different from those of cytochalasin D with respect to the resulting degree of perturbance of cytoplasmic organization, the distribution of actin filaments and the speed of reversibility. PMID:10430024

  5. Rearrangement of Actin Microfilaments in Plant Root Hairs Responding to Rhizobium etli Nodulation Signals1

    PubMed Central

    Cárdenas, Luis; Vidali, Luis; Domínguez, Jimena; Pérez, Héctor; Sánchez, Federico; Hepler, Peter K.; Quinto, Carmen

    1998-01-01

    The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads. PMID:9501120

  6. Retinoids and glucocorticoids have opposite effects on actin cytoskeleton rearrangement in hippocampal HT22 cells.

    PubMed

    Hélène, Roumes; Julie, Brossaud; Aloïs, Lemelletier; Marie-Pierre, Moisan; Véronique, Pallet; Anabelle, Redonnet; Jean-Benoît, Corcuff

    2016-02-01

    A chronic excess of glucocorticoids elicits deleterious effects in the hippocampus. Conversely, retinoic acid plays a major role in aging brain plasticity. As synaptic plasticity depends on mechanisms related to cell morphology, we investigated the involvement of retinoic acid and glucocorticoids in the remodelling of the HT22 neurons actin cytoskeleton. Cells exhibited a significantly more elongated shape with retinoic acid and a rounder shape with dexamethasone; retinoic acid reversed the effects of dexamethasone. Actin expression and abundance were unchanged by retinoic acid or dexamethasone but F-actin organization was dramatically modified. Indeed, retinoic acid and dexamethasone increased (70 ± 7% and 176 ± 5%) cortical actin while retinoic acid suppressed the effect of dexamethasone (90 ± 6%). Retinoic acid decreased (-22 ± 9%) and dexamethasone increased (134 ± 16%) actin stress fibres. Retinoic acid also suppressed the effect of dexamethasone (-21 ± 7%). Spectrin is a key protein in the actin network remodelling. Its abundance was decreased by retinoic acid and increased by dexamethasone (-21 ± 11% and 52 ± 10%). However, retinoic acid did not modify the effect of dexamethasone (48 ± 7%). Calpain activity on spectrin was increased by retinoic acid and decreased by dexamethasone (26 ± 14% and -57 ± 5%); retinoic acid mildly but significantly modified the effect of dexamethasone (-44 ± 7%). The calpain inhibitor calpeptin suppressed the effects of retinoic acid and dexamethasone on cell shape and actin stress fibres remodelling but did not modify the effects on cortical actin. Retinoic acid and dexamethasone have a dramatic but mainly opposite effect on actin cytoskeleton remodelling. These effects originate, at least partly, from calpain activity. PMID:26748244

  7. Cytoskeletal Mechanics

    NASA Astrophysics Data System (ADS)

    Mofrad, Mohammad R. K.; Kamm, Roger D.

    2006-10-01

    1. Introduction and the biological basis for cell mechanics Mohammad R. K. Mofrad and Roger Kamm; 2. Experimental measurements of intracellular mechanics Paul Janmey and Christoph Schmidt; 3. The cytoskeleton as a soft glassy material Jeffrey Fredberg and Ben Fabry; 4. Continuum elastic or viscoelastic models for the cell Mohammad R. K. Mofrad, Helene Karcher and Roger Kamm; 5. Multiphasic models of cell mechanics Farshid Guuilak, Mansoor A. Haider, Lori A. Setton, Tod A. Laursen and Frank P. T. Baaijens; 6. Models of cytoskeletal mechanics based on tensegrity Dimitrije Stamenovic; 7. Cells, gels and mechanics Gerald H. Pollack; 8. Polymer-based models of cytoskeletal networks F. C. MacKintosh; 9. Cell dynamics and the actin cytoskeleton James L. McGrath and C. Forbes Dewey, Jr; 10. Active cellular motion: continuum theories and models Marc Herant and Micah Dembo; 11. Summary Mohammad R. K. Mofrad and Roger Kamm.

  8. Cytoskeletal Mechanics

    NASA Astrophysics Data System (ADS)

    Mofrad, Mohammad R. K.; Kamm, Roger D.

    2011-08-01

    1. Introduction and the biological basis for cell mechanics Mohammad R. K. Mofrad and Roger Kamm; 2. Experimental measurements of intracellular mechanics Paul Janmey and Christoph Schmidt; 3. The cytoskeleton as a soft glassy material Jeffrey Fredberg and Ben Fabry; 4. Continuum elastic or viscoelastic models for the cell Mohammad R. K. Mofrad, Helene Karcher and Roger Kamm; 5. Multiphasic models of cell mechanics Farshid Guuilak, Mansoor A. Haider, Lori A. Setton, Tod A. Laursen and Frank P. T. Baaijens; 6. Models of cytoskeletal mechanics based on tensegrity Dimitrije Stamenovic; 7. Cells, gels and mechanics Gerald H. Pollack; 8. Polymer-based models of cytoskeletal networks F. C. MacKintosh; 9. Cell dynamics and the actin cytoskeleton James L. McGrath and C. Forbes Dewey, Jr; 10. Active cellular motion: continuum theories and models Marc Herant and Micah Dembo; 11. Summary Mohammad R. K. Mofrad and Roger Kamm.

  9. Regulation of AMPA receptor GluR1 subunit surface expression by a 4. 1N-linked actin cytoskeletal association.

    PubMed

    Shen, L; Liang, F; Walensky, L D; Huganir, R L

    2000-11-01

    The synaptic localization, clustering, and immobilization of neurotransmitter receptors and ion channels play important roles in synapse formation and synaptic transmission. Although several proteins have been identified that interact with AMPA receptors and that may regulate their synaptic targeting, little is known about the interaction of AMPA receptors with the cytoskeleton. In studies examining the interaction of the AMPA receptor GluR1 subunit with neuronal proteins, we determined that GluR1 interacts with the 4.1G and 4.1N proteins, homologs of the erythrocyte membrane cytoskeletal protein 4.1. Using the yeast two-hybrid system and a heterologous cell system, we demonstrated that both 4.1G and 4.1N bind to a membrane proximal region of the GluR1 C terminus, and that a region within the C-terminal domain of 4.1G or 4.1N is sufficient to mediate the interaction. We also found that 4.1N can associate with GluR1 in vivo and colocalizes with AMPA receptors at excitatory synapses. Disruption of the interaction of GluR1 with 4.1N or disruption of actin filaments decreased the surface expression of GluR1 in heterologous cells. Moreover, disruption of actin filaments in cultured cortical neurons dramatically reduced the level of surface AMPA receptors. These results suggest that protein 4.1N may link AMPA receptors to the actin cytoskeleton. PMID:11050113

  10. Schip1 Is a Novel Podocyte Foot Process Protein that Mediates Actin Cytoskeleton Rearrangements and Forms a Complex with Nherf2 and Ezrin

    PubMed Central

    Perisic, Ljubica; Rodriguez, Patricia Q.; Hultenby, Kjell; Sun, Ying; Lal, Mark; Betsholtz, Christer; Uhlén, Mathias; Wernerson, Annika; Hedin, Ulf; Pikkarainen, Timo; Tryggvason, Karl; Patrakka, Jaakko

    2015-01-01

    Background Podocyte foot process effacement accompanied by actin cytoskeleton rearrangements is a cardinal feature of many progressive human proteinuric diseases. Results By microarray profiling of mouse glomerulus, SCHIP1 emerged as one of the most highly enriched transcripts. We detected Schip1 protein in the kidney glomerulus, specifically in podocytes foot processes. Functionally, Schip1 inactivation in zebrafish by morpholino knock-down results in foot process disorganization and podocyte loss leading to proteinuria. In cultured podocytes Schip1 localizes to cortical actin-rich regions of lamellipodia, where it forms a complex with Nherf2 and ezrin, proteins known to participate in actin remodeling stimulated by PDGFβ signaling. Mechanistically, overexpression of Schip1 in vitro causes accumulation of cortical F-actin with dissolution of transversal stress fibers and promotes cell migration in response to PDGF-BB stimulation. Upon actin disassembly by latrunculin A treatment, Schip1 remains associated with the residual F-actin-containing structures, suggesting a functional connection with actin cytoskeleton possibly via its interaction partners. A similar assay with cytochalasin D points to stabilization of cortical actin cytoskeleton in Schip1 overexpressing cells by attenuation of actin depolymerisation. Conclusions Schip1 is a novel glomerular protein predominantly expressed in podocytes, necessary for the zebrafish pronephros development and function. Schip1 associates with the cortical actin cytoskeleton network and modulates its dynamics in response to PDGF signaling via interaction with the Nherf2/ezrin complex. Its implication in proteinuric diseases remains to be further investigated. PMID:25807495

  11. The chemokine (C-C motif) ligand protein synthesis inhibitor bindarit prevents cytoskeletal rearrangement and contraction of human mesangial cells.

    PubMed

    Paccosi, Sara; Giachi, Matelda; Di Gennaro, Paola; Guglielmotti, Angelo; Parenti, Astrid

    2016-09-01

    Intraglomerular mesangial cells (MCs) maintain structural and functional integrity of renal glomerular microcirculation and homeostasis of mesangial matrix. Following different types of injury, MCs change their phenotype upregulating the expression of α-smooth muscle actin (α-SMA), changing contractile abilities and increasing the production of matrix proteins, chemokines and cytokines. CCL2 is a chemokine known to be involved in the pathogenesis of renal diseases. Its glomerular upregulation correlates with the extent of renal damage. Bindarit is an indazolic derivative endowed with anti-inflammatory activity when tested in experimental diseases. It selectively inhibits the synthesis of inflammatory C-C chemokines including CCL2, CCL7 and CCL8. This work aims to analyse bindarit effects on ET1-, AngII- and TGFβ-induced mesangial cell dysfunction. Bindarit significantly reduced AngII-, ET1- and TGFβ-induced α-SMA upregulation. In a collagen contraction assay, bindarit reduced AngII-, ET1- and TGFβ-induced HRMC contraction. Within 3-6h stimulation, vinculin organization and phosphorylation was significantly impaired by bindarit in AngII-, ET1- and TGFβ-stimulated cells without any effect on F-actin distribution. Conversely, p38 phosphorylation was not significantly inhibited by bindarit. Our data strengthen the importance of CCL2 on ET-1, AngII- and TGFβ-induced mesangial cell dysfunction, adding new insights into the cellular mechanisms responsible of bindarit protective effects in human MC dysfunction. PMID:27309675

  12. E-Cadherin Suppression Directs Cytoskeletal Rearrangement and Intraepithelial Tumor Cell Migration in 3D Human Skin Equivalents

    PubMed Central

    Alt-Holland, Addy; Shamis, Yulia; Riley, Kathleen N.; DesRochers, Teresa M.; Fusenig, Norbert E.; Herman, Ira M.; Garlick, Jonathan A.

    2010-01-01

    The link between loss of cell–cell adhesion, the activation of cell migration, and the behavior of intraepithelial (IE) tumor cells during the early stages of skin cancer progression is not well understood. The current study characterized the migratory behavior of a squamous cell carcinoma cell line (HaCaT-II-4) upon E-cadherin suppression in both 2D, monolayer cultures and within human skin equivalents that mimic premalignant disease. The migratory behavior of tumor cells was first analyzed in 3D tissue context by developing a model that mimics transepithelial tumor cell migration. We show that loss of cell adhesion enabled migration of single, IE tumor cells between normal keratinocytes as a prerequisite for stromal invasion. To further understand this migratory behavior, E-cadherin-deficient cells were analyzed in 2D, monolayer cultures and displayed altered cytoarchitecture and enhanced membrane protrusive activity that was associated with circumferential actin organization and induction of the nonmuscle, β actin isoform. These features were associated with increased motility and random, individual cell migration in response to scrape-wounding. Thus, loss of E-cadherin-mediated adhesion led to the acquisition of phenotypic properties that augmented cell motility and directed the transition from the precancer to cancer in skin-like tissues. PMID:18528437

  13. Identification of mechanosensitive genes during skeletal development: alteration of genes associated with cytoskeletal rearrangement and cell signalling pathways

    PubMed Central

    2014-01-01

    Background Mechanical stimulation is necessary for regulating correct formation of the skeleton. Here we test the hypothesis that mechanical stimulation of the embryonic skeletal system impacts expression levels of genes implicated in developmentally important signalling pathways in a genome wide approach. We use a mutant mouse model with altered mechanical stimulation due to the absence of limb skeletal muscle (Splotch-delayed) where muscle-less embryos show specific defects in skeletal elements including delayed ossification, changes in the size and shape of cartilage rudiments and joint fusion. We used Microarray and RNA sequencing analysis tools to identify differentially expressed genes between muscle-less and control embryonic (TS23) humerus tissue. Results We found that 680 independent genes were down-regulated and 452 genes up-regulated in humeri from muscle-less Spd embryos compared to littermate controls (at least 2-fold; corrected p-value ≤0.05). We analysed the resulting differentially expressed gene sets using Gene Ontology annotations to identify significant enrichment of genes associated with particular biological processes, showing that removal of mechanical stimuli from muscle contractions affected genes associated with development and differentiation, cytoskeletal architecture and cell signalling. Among cell signalling pathways, the most strongly disturbed was Wnt signalling, with 34 genes including 19 pathway target genes affected. Spatial gene expression analysis showed that both a Wnt ligand encoding gene (Wnt4) and a pathway antagonist (Sfrp2) are up-regulated specifically in the developing joint line, while the expression of a Wnt target gene, Cd44, is no longer detectable in muscle-less embryos. The identification of 84 genes associated with the cytoskeleton that are down-regulated in the absence of muscle indicates a number of candidate genes that are both mechanoresponsive and potentially involved in mechanotransduction, converting a

  14. Deguelin inhibits the migration and invasion of lung cancer A549 and H460 cells via regulating actin cytoskeleton rearrangement.

    PubMed

    Zhao, Honggang; Jiao, Yan; Zhang, Zuncheng

    2015-01-01

    Deguelin, the main components from Mundulea sericea, was reported to suppress the growth of various cancer cells. However, the effect of Deguelin on tumor cell invasion and metastasis and its mechanism still unclear so far. In this study, we investigated the effects of Deguelin on the cell invasion in human lung cancer A549 and H460 cells. Our results demonstrate that Deguelin can significantly inhibited cell proliferation, cell migration and cell invasion. Moreover, Deguelin could also affected reorganization of the actin cytoskeleton and decreased filopodia and lamellipodia formation. Furthermore, deguelin-treated tumors showed decreased the tumor metastasis related genes such as CD44, MMP2 and MMP9 at protein and mRNA levels and the content of CEA, SCC, NSE, CYFAR21-1. In addition, Deguelin down-regulated protein expression of Rac1 and Rock1, which are impotent in actin cytoskeleton rearrangements and cell motility. Together, our results suggest that Deguelin inhibit tumor growth and metastasis of lung cancer cells and might be a candidate compound for curing lung cancer. PMID:26884827

  15. Aegeline from Aegle marmelos stimulates glucose transport via Akt and Rac1 signaling, and contributes to a cytoskeletal rearrangement through PI3K/Rac1.

    PubMed

    Gautam, Sudeep; Ishrat, Nayab; Singh, Rohit; Narender, Tadigoppula; Srivastava, Arvind K

    2015-09-01

    Aegeline is an alkaloidal-amide, isolated from the leaves of Aegle marmelos and have shown antihyperglycemic as well as antidyslipidemic activities in the validated animal models of type 2 diabetes mellitus. Here we delineate, aegeline enhanced GLUT4 translocation mediated 2-deoxy-glucose uptake in both time and concentration-dependent manner. 2-deoxy-glucose uptake was completely stymied by the transport inhibitors (wortmannin and genistein) in C2C12 myotubes. Pharmacological inhibition of Akt (also known as protein kinase B) and Ras-related C3 botulinum toxin substrate 1 (Rac1) suggest that both Akt and Rac1 operate aegeline-stimulated glucose transport via distinct parallel pathways. Moreover, aegeline activates p21 protein-activated kinase 1 (PAK1) and cofilin (an actin polymerization regulator). Rac1 inhibitor (Rac1 inhib II) and PAK1 inhibitor (IPA-3) completely blocked aegeline-induced phosphorylation of cofilin and p21 protein-activated kinase 1 (PAK1). In summary, these findings suggest that aegeline stimulates the glucose transport through Akt and Rac1 dependent distinct parallel pathways and have cytoskeletal roles via stimulation of the PI3-kinase-Rac1-PAK1-cofilin pathway in the skeletal muscle cells. Therefore, multiple targets of aegeline in the improvement of insulin sensitivity of the skeletal muscle cells may be suggested. PMID:26102565

  16. Cytoskeletal F-actin polymerization from cytosolic G-actin occurs in the phagocytosing immunocytes of arthropods (Limulus polyphemus and Gromphadorhina portentosa): does [cAMP]i play any role?

    PubMed

    Gupta, A P; Campenot, E S

    1996-09-01

    Phagocytosis is a major defense reaction in arthropods and is accomplished by two blood cells (hemocytes), the granulocyte (GRs) and plasmatocytes (PLs), collectively called immunocytes. Immunocytes (principally the GRs) from two arthropods, Limulus polyphemus (horseshoe crab) and Gromphadorhina portentosa (Madagascar hissing cockroach) effectively phagocytose fluorescein isothiocyanate (FITC)-conjugated fluoresbrite microspheres (FITC-FM) and chicken (Gallus domesticus) erythrocytes within 1 hr of incubation. Although actin polymerization and changes in intracellular cAMP ([cAMP]i) levels occur during the early stages of phagocytosis in vertebrates, these two phenomena have not been studied in arthropod immunocytes. Using the DNase I inhibition assay, we found a decrease in cytosolic G-actin and an increase in the cytoskeletal F-actin in the phagocytosing immunocytes; the total actin in both resting and phagocytosing immunocytes remained constant. These results showed an 86% increase in F-actin in G. portentosa immunocytes and a 29% increase in those of L. polyphemus after 1 hr of initial incubation with FITC-FM. As in some vertebrates, the role of [cAMP]i in the early stages of phagocytosis in these two animals- and perhaps in arthropods in general-is variable; although we detected some negligible amounts of [cAMP]i (0.10-0.80 pmol/cell at different time intervals) in L. polyphemus immunocytes, it was inconclusive whether those in G. portentosa also contained [cAMP]i. Even in L. polyphemus, the difference in the amounts of [cAMP]i in resting and phagocytosing cells was insignificant (P > 0.05). It was also inconclusive whether [Ca2+]i and/or [Mg2+]i play any roles in the early stages of phagocytosis in the two arthropods in this study. These results suggest that the two phenomena (F-actin polymerization and levels of [cAMP]i in arthropods) are basically similar to those in vertebrate neutrophils and macrophages, which suggests that certain immunological

  17. Coronin 1C-free primary mouse fibroblasts exhibit robust rearrangements in the orientation of actin filaments, microtubules and intermediate filaments.

    PubMed

    Behrens, Juliane; Solga, Roxana; Ziemann, Anja; Rastetter, Raphael H; Berwanger, Carolin; Herrmann, Harald; Noegel, Angelika A; Clemen, Christoph S

    2016-08-01

    Coronin 1C is an established modulator of actin cytoskeleton dynamics. It has been shown to be involved in protrusion formation, cell migration and invasion. Here, we report the generation of primary fibroblasts from coronin 1C knock-out mice in order to investigate the impact of the loss of coronin 1C on cellular structural organisation. We demonstrate that the lack of coronin 1C not only affects the actin system, but also the microtubule and the vimentin intermediate filament networks. In particular, we show that the knock-out cells exhibit a reduced proliferation rate, impaired cell migration and protrusion formation as well as an aberrant subcellular localisation and function of mitochondria. Moreover, we demonstrate that coronin 1C specifically interacts with the non-α-helical amino-terminal domain ("head") of vimentin. Our data suggest that coronin 1C acts as a cytoskeletal integrator of actin filaments, microtubules and intermediate filaments. PMID:27178841

  18. In vivo evidence for mTORC2-mediated actin cytoskeleton rearrangement in neurons.

    PubMed

    Angliker, Nico; Rüegg, Markus A

    2013-01-01

    The mammalian target of rapamycin (mTOR) assembles into two distinct multi-protein complexes called mTORC1 and mTORC2. While mTORC1 controls the signaling pathways important for cell growth, the physiological function of mTORC2 is only partially known. Here we comment on recent work on gene-targeted mice lacking mTORC2 in the cerebellum or the hippocampus that provided strong evidence that mTORC2 plays an important role in neuron morphology and synapse function. We discuss that this phenotype might be based on the perturbed regulation of the actin cytoskeleton and the lack of activation of several PKC isoforms. The fact that PKC isoforms and their targets have been implicated in neurological disease including spinocerebellar ataxia and that they have been shown to affect learning and memory, suggests that aberration of mTORC2 signaling might be involved in diseases of the brain. PMID:24721730

  19. Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

    NASA Technical Reports Server (NTRS)

    Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

    2000-01-01

    DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

  20. Alternative cytoskeletal landscapes: cytoskeletal novelty and evolution in basal excavate protists

    PubMed Central

    Dawson, Scott C.; Paredez, Alexander R.

    2016-01-01

    Microbial eukaryotes encompass the majority of eukaryotic evolutionary and cytoskeletal diversity. The cytoskeletal complexity observed in multicellular organisms appears to be an expansion of components present in genomes of diverse microbial eukaryotes such as the basal lineage of flagellates, the Excavata. Excavate protists have complex and diverse cytoskeletal architectures and life cycles – essentially alternative cytoskeletal “landscapes” – yet still possess conserved microtubule- and actin-associated proteins. Comparative genomic analyses have revealed that a subset of excavates, however, lack many canonical actin-binding proteins central to actin cytoskeleton function in other eukaryotes. Overall, excavates possess numerous uncharacterized and “hypothetical” genes, and may represent an undiscovered reservoir of novel cytoskeletal genes and cytoskeletal mechanisms. The continued development of molecular genetic tools in these complex microbial eukaryotes will undoubtedly contribute to our overall understanding of cytoskeletal diversity and evolution. PMID:23312067

  1. Forcing it on: Cytoskeletal dynamics during lymphocyte activation

    NASA Astrophysics Data System (ADS)

    Upadhyaya, Arpita

    2012-02-01

    Formation of the immune synapse during lymphocyte activation involves cell spreading driven by large scale physical rearrangements of the actin cytoskeleton and the cell membrane. Several recent observations suggest that mechanical forces are important for efficient T cell activation. How forces arise from the dynamics of the cytoskeleton and the membrane during contact formation, and their effect on signaling activation is not well understood. We have imaged membrane topography, actin dynamics and the spatiotemporal localization of signaling clusters during the very early stages of spreading. Formation of signaling clusters was closely correlated with the movement and topography of the membrane in contact with the activating surface. Further, we observed membrane waves driven by actin polymerization originating at these signaling clusters. Actin-driven membrane protrusions likely play an important role in force generation at the immune synapse. In order to study cytoskeletal forces during T-cell activation, we studied cell spreading on elastic gels. We found that gel stiffness influences cell morphology, actin dynamics and receptor activation. Efforts to determine the quantitative relationships between cellular forces and signaling are underway. Our results suggest a role for cytoskeleton driven forces during signaling activation in lymphocytes.

  2. Downregulation of the DNA repair enzyme apurinic/apyrimidinic endonuclease 1 stimulates transforming growth factor-β1 production and promotes actin rearrangement.

    PubMed

    Sakai, Yuri; Yamamori, Tohru; Yasui, Hironobu; Inanami, Osamu

    2015-05-22

    The DNA repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1) plays a central role in base excision repair and functions as a reductive activator of various transcription factors. Multiple other functionalities have been ascribed to APE1 in addition to these major functions. A recent study showed that APE1 knockdown upregulated the expression of a set of genes related to extracellular matrix (ECM) production, indicating an additional novel biological role for this enzyme. Based on this finding, we have investigated the effect of APE1 downregulation on ECM-related gene expression and its biological consequences. Endogenous APE1 expression was downregulated in human cervical carcinoma HeLa cells and human lung carcinoma A549 cells using siRNA. When the expression of six ECM-related genes (TGFB1, LAMC1, FN1, COL1A1, COL3A1, and COL4A1) was evaluated, we found that APE1 knockdown upregulated the expression of TGFB1 in both cell lines. APE1 downregulation promoted actin rearrangement, inducing F-actin accumulation in HeLa cells and the dissipation of stress fibers in A549 cells. We also discovered that APE1 knockdown enhanced cellular motility in A549 cells, which was suppressed by the inhibition of transforming growth factor (TGF)-β1 signaling. These results suggested that APE1 controls the organization of actin cytoskeleton through the regulation of TGF-β1 expression, providing novel insights into the biological significance of APE1. PMID:25858321

  3. Organized F-actin is essential for normal trichome morphogenesis in Arabidopsis.

    PubMed Central

    Szymanski, D B; Marks, M D; Wick, S M

    1999-01-01

    Actin microfilaments form a three-dimensional cytoskeletal network throughout the cell and constitute an essential throughway for organelle and vesicle transport. Development of Arabidopsis trichomes, unicellular structures derived from the epidermis, is being used as a genetic system in which to study actin-dependent growth in plant cells. The present study indicates that filamentous actin (F-actin) plays an important role during Arabidopsis trichome morphogenesis. For example, immunolocalization of actin filaments during trichome morphogenesis identified rearrangements of the cytoskeletal structure during the development of the mature cell. Moreover, pharmacological experiments indicate that there are distinct requirements for actin- and microtubule-dependent function during trichome morphogenesis. The F-actin-disrupting drug cytochalasin D does not affect the establishment of polarity during trichome development; however, maintenance and coordination of the normal pattern of cell growth are very sensitive to this drug. In contrast, oryzalin, an agent that depolymerizes microtubules, severely inhibits cell polarization. Furthermore, cytochalasin D treatment phenocopies a known class of mutations that cause distorted trichome morphology. Results of an analysis of cell shape and microfilament structure in wild-type, mutant, and drug-treated trichomes are consistent with a role for actin in the maintenance and coordination of an established growth pattern. PMID:10590162

  4. Structural interaction of cytoskeletal components.

    PubMed

    Schliwa, M; van Blerkom, J

    1981-07-01

    Three-dimensional cytoskeletal organization of detergent-treated epithelial African green monkey kidney cells (BSC-1) and chick embryo fibroblasts was studied in whole-mount preparations visualized in a high voltage electron microscope. Stereo images are generated at both low and high magnification to reveal both overall cytoskeletal morphology and details of the structural continuity of different filament types. By the use of an improved extraction procedure in combination with heavy meromyosin subfragment 1 decoration of actin filaments, several new features of filament organization are revealed that suggest that the cytoskeleton is a highly interconnected structural unit. In addition to actin filaments, intermediate filaments, and microtubules, a new class of filaments of 2- to 3-nm diameter and 30- to 300-nm length that do not bind heavy merymyosin is demonstrated. They form end-to-side contacts with other cytoskeletal filaments, thereby acting as linkers between various fibers, both like (e.g., actin- actin) and unlike (e.g., actin-intermediate filament, intermediate filament-microtubule). Their nature is unknown. In addition to 2- to 3-nm filaments, actin filaments are demonstrated to form end-to-side contacts with other filaments. Y-shaped actin filament "branches" are observed both in the cell periphery close to ruffles and in more central cell areas also populated by abundant intermediate filaments and microtubules. Arrowhead complexes formed by subfragment 1 decoration of actin filaments point towards the contact site. Actin filaments also form end-to-side contacts with microtubules and intermediate filaments. Careful inspection of numerous actin-microtubule contacts shows that microtubules frequently change their course at sites of contact. A variety of experimentally induced modifications of the frequency of actin-microtubule contacts can be shown to influence the course of microtubules. We conclude that bends in microtubules are imposed by structural

  5. Frutalin, a galactose-binding lectin, induces chemotaxis and rearrangement of actin cytoskeleton in human neutrophils: involvement of tyrosine kinase and phosphoinositide 3-kinase.

    PubMed

    Brando-Lima, Aline C; Saldanha-Gama, Roberta F; Henriques, Maria das Graças M O; Monteiro-Moreira, Ana C O; Moreira, Renato A; Barja-Fidalgo, Christina

    2005-10-15

    Several lectin-like molecules have been shown as potent activators of leukocytes. Galactose-binding lectins are of special interest since they could interact with several endogenous molecules involved in the innate and specific immune responses. The effects of Frutalin (FTL), an alpha-D-galactose (Gal)-binding plant lectin, on the modulation of neutrophil (PMN) functions were investigated. FTL induced a dose-dependent PMN migration in mice pleural cavity. Moreover, FTL was also a potent direct chemotactic for human PMN, in vitro, and triggered oxidative burst in these cells. These effects were accompanied by a rearrangement of the actin cytoskeleton dynamic, activation of tyrosine kinase (TK) pathways, increase in focal adhesion kinase (FAK) phosphorylation, and its subsequent association to phosphoinositide3-kinase (PI3K). All those effects were inhibited in the presence of Gal, suggesting specific carbohydrate recognition for FTL effects. The activations of TK and PI3K pathways are essential events for FTL-induced chemotaxis, since inhibitors of these pathways, genistein and LY294002, inhibited neutrophil migration in vitro. The data indicate that sugar-protein interactions between a soluble lectin and galacto-components on neutrophil surface trigger the TK pathway, inducing FAK and PI3K activation, interfering with cell motility and oxidative response. PMID:16183388

  6. Actin restructuring during Salmonella typhimurium infection investigated by confocal and super-resolution microscopy

    NASA Astrophysics Data System (ADS)

    Han, Jason J.; Kunde, Yuliya A.; Hong-Geller, Elizabeth; Werner, James H.

    2014-01-01

    We have used super-resolution optical microscopy and confocal microscopy to visualize the cytoskeletal restructuring of HeLa cells that accompanies and enables Salmonella typhimurium internalization. Herein, we report the use of confocal microscopy to verify and explore infection conditions that would be compatible with super-resolution optical microscopy, using Alexa-488 labeled phalloidin to stain the actin cytoskeletal network. While it is well known that actin restructuring and cytoskeletal rearrangements often accompany and assist in bacterial infection, most studies have employed conventional diffraction-limited fluorescence microscopy to explore these changes. Here we show that the superior spatial resolution provided by single-molecule localization methods (such as direct stochastic optical reconstruction microscopy) enables more precise visualization of the nanoscale changes in the actin cytoskeleton that accompany bacterial infection. In particular, we found that a thin (100-nm) ring of actin often surrounds an invading bacteria 10 to 20 min postinfection, with this ring being transitory in nature. We estimate that a few hundred monofilaments of actin surround the S. typhimurium in this heretofore unreported bacterial internalization intermediate.

  7. PEGylated-thymoquinone-nanoparticle mediated retardation of breast cancer cell migration by deregulation of cytoskeletal actin polymerization through miR-34a.

    PubMed

    Bhattacharya, Saurav; Ahir, Manisha; Patra, Prasun; Mukherjee, Sudeshna; Ghosh, Swatilekha; Mazumdar, Minakshi; Chattopadhyay, Sreya; Das, Tanya; Chattopadhyay, Dhrubajyoti; Adhikary, Arghya

    2015-05-01

    Thymoquinone (TQ), a major active constituent of black seeds of Nigella sativa, has potential medical applications including spectrum of therapeutic properties against different cancers. However, little is known about their effect on breast cancer cell migration, which is the cause of over 90% of deaths worldwide. Herein, we have synthesized TQ-encapsulated nanoparticles using biodegradable, hydrophilic polymers like polyvinylpyrrolidone (PVP) and polyethyleneglycol (PEG) to overcome TQ's poor aqueous solubility, thermal and light sensitivity as well as consequently, minimal systemic bioavailability which can greatly improve the cancer treatment efficiency. Sizes of synthesized TQ-Nps were found to be below 50 nm and they were mostly spherical in shape with smooth surface texture. Estimation of the zeta potential also revealed that all the three TQ-Nps were negatively charged which also facilitated their cellular uptake. In the present investigation, we provide direct evidence that TQ-Nps showed more efficiency in killing cancer cells as well as proved to be less toxic to normal cells at a significantly lower dose than TQ. Interestingly, evaluation of the anti-migratory effect of the TQ-Nps, revealed that PEG4000-TQ-Nps showed much potent anti-migratory properties than the other types. Further studies indicated that PEG4000-TQ-Nps could significantly increase the expression of miR-34a through p53. Moreover, NPs mediated miR-34a up-regulation directly down-regulated Rac1 expression followed by actin depolymerisation thereby disrupting the actin cytoskeleton which leads to significant reduction in the lamellipodia and filopodia formation on cell surfaces thus retarding cell migration. Considering the biodegradability, non-toxicity and effectivity of PEG4000-TQ-Nps against cancer cell migration, TQ-Nps may provide new insights into specific therapeutic approach for cancer treatment. PMID:25771001

  8. A Novel Human Cytomegalovirus Glycoprotein, gpUS9, Which Promotes Cell-to-Cell Spread in Polarized Epithelial Cells, Colocalizes with the Cytoskeletal Proteins E-Cadherin and F-Actin

    PubMed Central

    Maidji, Ekaterina; Tugizov, Sharof; Abenes, Gerardo; Jones, Thomas; Pereira, Lenore

    1998-01-01

    Processes by which human herpesviruses penetrate and are released from polarized epithelial cells, which have distinct apical and basolateral membrane domains differing in protein and lipid content, are poorly understood. We recently reported that human cytomegalovirus (CMV) mutants with deletions of the gene US9 formed wild-type plaques in cultures of human fibroblasts but were impaired in the capacity for cell-to-cell spread in polarized human retinal pigment epithelial cells. Unlike the glycoproteins that are required for infection, the protein encoded by CMV US9 plays an accessory role by promoting dissemination of virus across cell-cell junctions of polarized epithelial cells. To identify the product and investigate its specialized functions, we selected Madine-Darby canine kidney II (MDCK) epithelial cells that constitutively express CMV US9 or, as a control, US8. The gene products, designated gpUS9 and gpUS8, were glycosylated proteins of comparable molecular masses but differed considerably in intracellular distribution and solubility. Immunofluorescence laser scanning confocal microscopy indicated that, like gpUS8, gpUS9 was present in the endoplasmic reticulum and Golgi compartments of nonpolarized cells. In polarized epithelial cells, gpUS9 also accumulated along lateral membranes, colocalizing with cadherin and actin, and was insoluble in Triton X-100, a property shared with proteins that associate with the cytoskeleton. We hypothesize that gpUS9 may enhance the dissemination of CMV in infected epithelial tissues by associating with the cytoskeletal matrix. PMID:9621030

  9. Dynamic ordering of nuclei in syncytial embryos: a quantitative analysis of the role of cytoskeletal networks.

    PubMed

    Kanesaki, Takuma; Edwards, Carina M; Schwarz, Ulrich S; Grosshans, Jörg

    2011-11-01

    In syncytial embryos nuclei undergo cycles of division and rearrangement within a common cytoplasm. It is presently unclear to what degree and how the nuclear array maintains positional order in the face of rapid cell divisions. Here we establish a quantitative assay, based on image processing, for analysing the dynamics of the nuclear array. By tracking nuclear trajectories in Drosophila melanogaster embryos, we are able to define and evaluate local and time-dependent measures for the level of geometrical order in the array. We find that after division, order is re-established in a biphasic manner, indicating the competition of different ordering processes. Using mutants and drug injections, we show that the order of the nuclear array depends on cytoskeletal networks organised by centrosomes. While both f-actin and microtubules are required for re-establishing order after mitosis, only f-actin is required to maintain the stability of this arrangement. Furthermore, f-actin function relies on myosin-independent non-contractile filaments that suppress individual nuclear mobility, whereas microtubules promote mobility and attract adjacent nuclei. Actin caps are shown to act to prevent nuclear incorporation into adjacent microtubule baskets. Our data demonstrate that two principal ordering mechanisms thus simultaneously contribute: (1) a passive crowding mechanism in which nuclei and actin caps act as spacers and (2) an active self-organisation mechanism based on a microtubule network. PMID:22001900

  10. Dynamics of active actin networks

    NASA Astrophysics Data System (ADS)

    Koehler, Simone

    2014-03-01

    Local mechanical and structural properties of a eukaryotic cell are determined by its cytoskeleton. To adapt to their environment, cells rely on constant self-organized rearrangement processes of their actin cytoskeleton. To shed light on the principles underlying these dynamic self-organization processes we investigate a minimal reconstituted active system consisting of actin filaments, crosslinking molecules and molecular motor filaments. Using quantitative fluorescence microscopy and image analysis, we show, that these minimal model systems exhibit a generic structure formation mechanism. The competition between force generation by molecular motors and the stabilization of the network by crosslinking proteins results in a highly dynamic reorganization process which is characterized by anomalous transport dynamics with a superdiffusive behavior also found in intracellular dynamics. In vitro, these dynamics are governed by chemical and physical parameters that alter the balance of motor and crosslinking proteins, such as pH. These findings can be expected to have broad implications in our understanding of cytoskeletal regulation in vivo.

  11. Non-lytic, actin-based exit of intracellular parasites from C. elegans intestinal cells.

    PubMed

    Estes, Kathleen A; Szumowski, Suzannah C; Troemel, Emily R

    2011-09-01

    The intestine is a common site for invasion by intracellular pathogens, but little is known about how pathogens restructure and exit intestinal cells in vivo. The natural microsporidian parasite N. parisii invades intestinal cells of the nematode C. elegans, progresses through its life cycle, and then exits cells in a transmissible spore form. Here we show that N. parisii causes rearrangements of host actin inside intestinal cells as part of a novel parasite exit strategy. First, we show that N. parisii infection causes ectopic localization of the normally apical-restricted actin to the basolateral side of intestinal cells, where it often forms network-like structures. Soon after this actin relocalization, we find that gaps appear in the terminal web, a conserved cytoskeletal structure that could present a barrier to exit. Reducing actin expression creates terminal web gaps in the absence of infection, suggesting that infection-induced actin relocalization triggers gap formation. We show that terminal web gaps form at a distinct stage of infection, precisely timed to precede spore exit, and that all contagious animals exhibit gaps. Interestingly, we find that while perturbations in actin can create these gaps, actin is not required for infection progression or spore formation, but actin is required for spore exit. Finally, we show that despite large numbers of spores exiting intestinal cells, this exit does not cause cell lysis. These results provide insight into parasite manipulation of the host cytoskeleton and non-lytic escape from intestinal cells in vivo. PMID:21949650

  12. Non-Lytic, Actin-Based Exit of Intracellular Parasites from C. elegans Intestinal Cells

    PubMed Central

    Estes, Kathleen A.; Szumowski, Suzannah C.; Troemel, Emily R.

    2011-01-01

    The intestine is a common site for invasion by intracellular pathogens, but little is known about how pathogens restructure and exit intestinal cells in vivo. The natural microsporidian parasite N. parisii invades intestinal cells of the nematode C. elegans, progresses through its life cycle, and then exits cells in a transmissible spore form. Here we show that N. parisii causes rearrangements of host actin inside intestinal cells as part of a novel parasite exit strategy. First, we show that N. parisii infection causes ectopic localization of the normally apical-restricted actin to the basolateral side of intestinal cells, where it often forms network-like structures. Soon after this actin relocalization, we find that gaps appear in the terminal web, a conserved cytoskeletal structure that could present a barrier to exit. Reducing actin expression creates terminal web gaps in the absence of infection, suggesting that infection-induced actin relocalization triggers gap formation. We show that terminal web gaps form at a distinct stage of infection, precisely timed to precede spore exit, and that all contagious animals exhibit gaps. Interestingly, we find that while perturbations in actin can create these gaps, actin is not required for infection progression or spore formation, but actin is required for spore exit. Finally, we show that despite large numbers of spores exiting intestinal cells, this exit does not cause cell lysis. These results provide insight into parasite manipulation of the host cytoskeleton and non-lytic escape from intestinal cells in vivo. PMID:21949650

  13. A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation.

    PubMed Central

    Rosenshine, I; Ruschkowski, S; Stein, M; Reinscheid, D J; Mills, S D; Finlay, B B

    1996-01-01

    Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce actin accumulation beneath adherent bacteria. We found that EPEC adherence to epithelial cells mediates the formation of fingerlike pseudopods (up to 10 microm) beneath bacteria. These actin-rich structures also contain tyrosine phosphorylated host proteins concentrated at the pseudopod tip beneath adherent EPEC. Intimate bacterial adherence (and pseudopod formation) occurred only after prior bacterial induction of tyrosine phosphorylation of an epithelial membrane protein, Hp90, which then associates directly with an EPEC adhesin, intimin. These interactions lead to cytoskeletal nucleation and pseudopod formation. This is the first example of a bacterial pathogen that triggers signals in epithelial cells which activates receptor binding activity to a specific bacterial ligand and subsequent cytoskeletal rearrangement. Images PMID:8654358

  14. Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia

    PubMed Central

    Bury, Loredana; Falcinelli, Emanuela; Chiasserini, Davide; Springer, Timothy A.; Italiano, Joseph E.; Gresele, Paolo

    2016-01-01

    Several patients have been reported to have variant dominant forms of Glanzmann thrombasthenia, associated with macrothrombocytopenia and caused by gain-of-function mutations of ITGB3 or ITGA2B leading to reduced surface expression and constitutive activation of integrin αIIbβ3. The mechanisms leading to a bleeding phenotype of these patients have never been addressed. The aim of this study was to unravel the mechanism by which ITGB3 mutations causing activation of αIIbβ3 lead to platelet dysfunction and macrothrombocytopenia. Using platelets from two patients carrying the β3 del647-686 mutation and Chinese hamster ovary cells expressing different αIIbβ3-activating mutations, we showed that reduced surface expression of αIIbβ3 is due to receptor internalization. Moreover, we demonstrated that permanent triggering of αIIbβ3-mediated outside-in signaling causes an impairment of cytoskeletal reorganization arresting actin turnover at the stage of polymerization. The induction of actin polymerization by jasplakinolide, a natural toxin that promotes actin nucleation and prevents depolymerization of stress fibers, in control platelets produced an impairment of platelet function similar to that of patients with variant forms of dominant Glanzmann thrombasthenia. del647-686β3-transduced murine megakaryocytes generated proplatelets with a reduced number of large tips and asymmetric barbell-proplatelets, suggesting that impaired cytoskeletal rearrangement is the cause of macrothrombocytopenia. These data show that impaired cytoskeletal remodeling caused by a constitutively activated αIIbβ3 is the main effector of platelet dysfunction and macrothrombocytopenia, and thus of bleeding, in variant forms of dominant Glanzmann thrombasthenia. PMID:26452979

  15. MICAL-Family Proteins: Complex Regulators of the Actin Cytoskeleton

    PubMed Central

    Giridharan, Sai Srinivas Panapakkam

    2014-01-01

    Abstract Significance: The molecules interacting with CasL (MICAL) family members participate in a multitude of activities, including axonal growth cone repulsion, membrane trafficking, apoptosis, and bristle development in flies. An interesting feature of MICAL proteins is the presence of an N-terminal flavo-mono-oxygenase domain. This mono-oxygenase domain generates redox potential with which MICALs can either oxidize proteins or produce reactive oxygen species (ROS). Actin is one such protein that is affected by MICAL function, leading to dramatic cytoskeletal rearrangements. This review describes the MICAL-family members, and discusses their mechanisms of actin-binding and regulation of actin cytoskeleton organization. Recent Advances: Recent studies show that MICALs directly induce oxidation of actin molecules, leading to actin depolymerization. ROS production by MICALs also causes oxidation of collapsin response mediator protein-2, a microtubule assembly promoter, which subsequently undergoes phosphorylation. Critical Issues: MICAL proteins oxidize proteins through two mechanisms: either directly by oxidizing methionine residues or indirectly via the production of ROS. It remains unclear whether MICAL proteins employ both mechanisms or whether the activity of MICAL-family proteins might vary with different substrates. Future Directions: The identification of additional substrates oxidized by MICAL will shed new light on MICAL protein function. Additional directions include expanding studies toward the MICAL-like homologs that lack flavin adenine dinucleotide domains and oxidation activity. Antioxid. Redox Signal. 20, 2059–2073. PMID:23834433

  16. Mechanoaccumulative Elements of the Mammalian Actin Cytoskeleton.

    PubMed

    Schiffhauer, Eric S; Luo, Tianzhi; Mohan, Krithika; Srivastava, Vasudha; Qian, Xuyu; Griffis, Eric R; Iglesias, Pablo A; Robinson, Douglas N

    2016-06-01

    To change shape, divide, form junctions, and migrate, cells reorganize their cytoskeletons in response to changing mechanical environments [1-4]. Actin cytoskeletal elements, including myosin II motors and actin crosslinkers, structurally remodel and activate signaling pathways in response to imposed stresses [5-9]. Recent studies demonstrate the importance of force-dependent structural rearrangement of α-catenin in adherens junctions [10] and vinculin's molecular clutch mechanism in focal adhesions [11]. However, the complete landscape of cytoskeletal mechanoresponsive proteins and the mechanisms by which these elements sense and respond to force remain to be elucidated. To find mechanosensitive elements in mammalian cells, we examined protein relocalization in response to controlled external stresses applied to individual cells. Here, we show that non-muscle myosin II, α-actinin, and filamin accumulate to mechanically stressed regions in cells from diverse lineages. Using reaction-diffusion models for force-sensitive binding, we successfully predicted which mammalian α-actinin and filamin paralogs would be mechanoaccumulative. Furthermore, a "Goldilocks zone" must exist for each protein where the actin-binding affinity must be optimal for accumulation. In addition, we leveraged genetic mutants to gain a molecular understanding of the mechanisms of α-actinin and filamin catch-bonding behavior. Two distinct modes of mechanoaccumulation can be observed: a fast, diffusion-based accumulation and a slower, myosin II-dependent cortical flow phase that acts on proteins with specific binding lifetimes. Finally, we uncovered cell-type- and cell-cycle-stage-specific control of the mechanosensation of myosin IIB, but not myosin IIA or IIC. Overall, these mechanoaccumulative mechanisms drive the cell's response to physical perturbation during proper tissue development and disease. PMID:27185555

  17. Barley MLO Modulates Actin-Dependent and Actin-Independent Antifungal Defense Pathways at the Cell Periphery1[W][OA

    PubMed Central

    Miklis, Marco; Consonni, Chiara; Bhat, Riyaz A.; Lipka, Volker; Schulze-Lefert, Paul; Panstruga, Ralph

    2007-01-01

    Cell polarization is a crucial process during plant development, as well as in plant-microbe interactions, and is frequently associated with extensive cytoskeletal rearrangements. In interactions of plants with inappropriate fungal pathogens (so-called non-host interactions), the actin cytoskeleton is thought to contribute to the establishment of effective barriers at the cell periphery against fungal ingress. Here, we impeded actin cytoskeleton function in various types of disease resistance using pharmacological inhibitors and genetic interference via ectopic expression of an actin-depolymerizing factor-encoding gene, ADF. We demonstrate that barley (Hordeum vulgare) epidermal cells require actin cytoskeleton function for basal defense to the appropriate powdery mildew pathogen Blumeria graminis f. sp. hordei and for mlo-mediated resistance at the cell wall, but not for several tested race-specific immune responses. Analysis of non-host resistance to two tested inappropriate powdery mildews, Erysiphe pisi and B. graminis f. sp. tritici, revealed the existence of actin-dependent and actin-independent resistance pathways acting at the cell periphery. These pathways act synergistically and appear to be under negative control by the plasma membrane-resident MLO protein. PMID:17449647

  18. Identification of actin as a 15-deoxy-Delta12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement.

    PubMed

    Aldini, Giancarlo; Carini, Marina; Vistoli, Giulio; Shibata, Takahiro; Kusano, Yuri; Gamberoni, Luca; Dalle-Donne, Isabella; Milzani, Aldo; Uchida, Koji

    2007-03-13

    A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization. PMID:17297918

  19. Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.

    PubMed Central

    Fasano, A; Fiorentini, C; Donelli, G; Uzzau, S; Kaper, J B; Margaretten, K; Ding, X; Guandalini, S; Comstock, L; Goldblum, S E

    1995-01-01

    The intracellular signaling involved in the mechanism of action of zonula occludens toxin (ZOT) was studied using several in vitro and ex vivo models. ZOT showed a selective effect among various cell lines tested, suggesting that it may interact with a specific receptor, whose surface expression on various cells differs. When tested in IEC6 cell monolayers, ZOT-containing supernatants induced a redistribution of the F-actin cytoskeleton. Similar results were obtained with rabbit ileal mucosa, where the reorganization of F-actin paralleled the increase in tissue permeability. In endothelial cells, the cytoskeletal rearrangement involved a decrease of the soluble G-actin pool (-27%) and a reciprocal increase in the filamentous F-actin pool (+22%). This actin polymerization was time- and dose-dependent, and was reversible. Pretreatment with a specific protein kinase C inhibitor, CGP41251, completely abolished the ZOT effects on both tissue permeability and actin polymerization. In IEC6 cells ZOT induced a peak increment of the PKC-alpha isoform after 3 min incubation. Taken together, these results suggest that ZOT activates a complex intracellular cascade of events that regulate tight junction permeability, probably mimicking the effect of physiologic modulator(s) of epithelial barrier function. Images PMID:7635964

  20. Mechanical and spatial determinants of cytoskeletal geodesic dome formation in cardiac fibroblasts.

    PubMed

    Entcheva, Emilia; Bien, Harold

    2009-02-01

    This study tests the hypothesis that the cell cytoskeletal (CSK) network can rearrange from geodesic dome type structures to stress fibers in response to microenvironmental cues. The CSK geodesic domes are highly organized actin microarchitectures within the cell, consisting of ordered polygonal elements. We studied primary neonatal rat cardiac fibroblasts. The cues used to trigger the interconversion between the two CSK architectures (geodesic domes and stress fibers) included factors affecting spatial order and the degree of CSK tension in the cells. Microfabricated three-dimensional substrates with micrometre sized grooves and peaks were used to alter the spatial order of cell growth in culture. CSK tension was modified by 2,3-butanedione 2-monoxime (BDM), cytochalasin D and the hyphae of Candida albicans. CSK geodesic domes occurred spontaneously in about 20% of the neonatal rat cardiac fibroblasts used in this study. Microfabricated structured surfaces produced anisotropy in the cell CSK and effectively converted geodesic domes into stress fibers in a dose-dependent manner (dependence on the period of the features). Affectors of actin structure, inhibitors of CSK tension and cell motility, e.g. BDM, cytochalasin D and the hyphae of C. albicans, suppressed or eliminated the geodesic domes. Our data suggest that the geodesic domes, similar to actin stress fibers, require maintenance of CSK integrity and tension. However, microenvironments that promote structural anisotropy in tensed cells cause the transformation of the geodesic domes into stress fibers, consistent with topographic cell guidance and some previous CSK model predictions. PMID:20023805

  1. Mechanical and spatial determinants of cytoskeletal geodesic dome formation in cardiac fibroblasts

    PubMed Central

    Bien, Harold

    2015-01-01

    This study tests the hypothesis that the cell cytoskeletal (CSK) network can rearrange from geodesic dome type structures to stress fibers in response to microenvironmental cues. The CSK geodesic domes are highly organized actin microarchitectures within the cell, consisting of ordered polygonal elements. We studied primary neonatal rat cardiac fibroblasts. The cues used to trigger the interconversion between the two CSK architectures (geodesic domes and stress fibers) included factors affecting spatial order and the degree of CSK tension in the cells. Microfabricated three-dimensional substrates with micrometre sized grooves and peaks were used to alter the spatial order of cell growth in culture. CSK tension was modified by 2,3-butanedione 2-monoxime (BDM), cytochalasin D and the hyphae of Candida albicans. CSK geodesic domes occurred spontaneously in about 20% of the neonatal rat cardiac fibroblasts used in this study. Microfabricated structured surfaces produced anisotropy in the cell CSK and effectively converted geodesic domes into stress fibers in a dose-dependent manner (dependence on the period of the features). Affectors of actin structure, inhibitors of CSK tension and cell motility, e.g. BDM, cytochalasin D and the hyphae of C. albicans, suppressed or eliminated the geodesic domes. Our data suggest that the geodesic domes, similar to actin stress fibers, require maintenance of CSK integrity and tension. However, microenvironments that promote structural anisotropy in tensed cells cause the transformation of the geodesic domes into stress fibers, consistent with topographic cell guidance and some previous CSK model predictions. PMID:20023805

  2. Quality control of cytoskeletal proteins and human disease.

    PubMed

    Lundin, Victor F; Leroux, Michel R; Stirling, Peter C

    2010-05-01

    Actins and tubulins are abundant cytoskeletal proteins that support diverse cellular processes. Owing to the unique properties of these filament-forming proteins, an intricate cellular machinery consisting minimally of the chaperonin CCT, prefoldin, phosducin-like proteins, and tubulin cofactors has evolved to facilitate their biogenesis. More recent evidence also suggests that regulated degradation pathways exist for actin (via TRIM32) and tubulin (via parkin or cofactor E-like). Collectively, these pathways maintain the quality control of cytoskeletal proteins ('proteostasis'), ensuring the appropriate function of microfilaments and microtubules. Here, we focus on the molecular mechanisms of the quality control of actin and tubulin, and discuss emerging links between cytoskeletal proteostasis and human diseases. PMID:20116259

  3. Bidirectional actin transport is influenced by microtubule and actin stability.

    PubMed

    Chetta, Joshua; Love, James M; Bober, Brian G; Shah, Sameer B

    2015-11-01

    Local and long-distance transport of cytoskeletal proteins is vital to neuronal maintenance and growth. Though recent progress has provided insight into the movement of microtubules and neurofilaments, mechanisms underlying the movement of actin remain elusive, in large part due to rapid transitions between its filament states and its diverse cellular localization and function. In this work, we integrated live imaging of rat sensory neurons, image processing, multiple regression analysis, and mathematical modeling to perform the first quantitative, high-resolution investigation of GFP-actin identity and movement in individual axons. Our data revealed that filamentous actin densities arise along the length of the axon and move short but significant distances bidirectionally, with a net anterograde bias. We directly tested the role of actin and microtubules in this movement. We also confirmed a role for actin densities in extension of axonal filopodia, and demonstrated intermittent correlation of actin and mitochondrial movement. Our results support a novel mechanism underlying slow component axonal transport, in which the stability of both microtubule and actin cytoskeletal components influence the mobility of filamentous actin. PMID:26043972

  4. Rab1 recruits WHAMM during membrane remodeling but limits actin nucleation

    PubMed Central

    Russo, Ashley J.; Mathiowetz, Alyssa J.; Hong, Steven; Welch, Matthew D.; Campellone, Kenneth G.

    2016-01-01

    Small G-proteins are key regulatory molecules that activate the actin nucleation machinery to drive cytoskeletal rearrangements during plasma membrane remodeling. However, the ability of small G-proteins to interact with nucleation factors on internal membranes to control trafficking processes has not been well characterized. Here we investigated roles for members of the Rho, Arf, and Rab G-protein families in regulating WASP homologue associated with actin, membranes, and microtubules (WHAMM), an activator of Arp2/3 complex–mediated actin nucleation. We found that Rab1 stimulated the formation and elongation of WHAMM-associated membrane tubules in cells. Active Rab1 recruited WHAMM to dynamic tubulovesicular structures in fibroblasts, and an active prenylated version of Rab1 bound directly to an N-terminal domain of WHAMM in vitro. In contrast to other G-protein–nucleation factor interactions, Rab1 binding inhibited WHAMM-mediated actin assembly. This ability of Rab1 to regulate WHAMM and the Arp2/3 complex represents a distinct strategy for membrane remodeling in which a Rab G-protein recruits the actin nucleation machinery but dampens its activity. PMID:26823012

  5. Bacterial actins and their diversity

    PubMed Central

    Ozyamak, Ertan; Kollman, Justin M.; Komeili, Arash

    2015-01-01

    For many years bacteria were considered rather simple organisms, but the dogmatic notion that subcellular organization is a eukaryotic trait has been overthrown for more than a decade. The discovery of homologs of the eukaryotic cytoskeletal proteins actin, tubulin, and intermediate filaments in bacteria has been instrumental in changing this view. Over the recent years we gained an incredible level of insight into the diverse family of bacterial actins and their molecular workings. Here we review the functional, biochemical and structural features of the most well-studied bacterial actins. PMID:24015924

  6. Cytoskeletal coherence requires myosin-IIA contractility

    PubMed Central

    Cai, Yunfei; Rossier, Olivier; Gauthier, Nils C.; Biais, Nicolas; Fardin, Marc-Antoine; Zhang, Xian; Miller, Lawrence W.; Ladoux, Benoit; Cornish, Virginia W.; Sheetz, Michael P.

    2010-01-01

    Maintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in ‘C’ or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading. PMID:20067993

  7. Cytoskeletal regulation of dermal regeneration.

    PubMed

    Strudwick, Xanthe L; Cowin, Allison J

    2012-01-01

    Wound healing results in the repair of injured tissues however fibrosis and scar formation are, more often than not the unfortunate consequence of this process. The ability of lower order vertebrates and invertebrates to regenerate limbs and tissues has been all but lost in mammals; however, there are some instances where glimpses of mammalian regenerative capacity do exist. Here we describe the unlocked potential that exists in mammals that may help us understand the process of regeneration post-injury and highlight the potential role of the actin cytoskeleton in this process. The precise function and regulation of the cytoskeleton is critical to the success of the healing process and its manipulation may therefore facilitate regenerative healing. The gelsolin family of actin remodelling proteins in particular has been shown to have important functions in wound healing and family member Flightless I (Flii) is involved in both regeneration and repair. Understanding the interactions between different cytoskeletal proteins and their dynamic control of processes including cellular adhesion, contraction and motility may assist the development of therapeutics that will stimulate regeneration rather than repair. PMID:24710556

  8. Cytoskeletal Regulation of Dermal Regeneration

    PubMed Central

    Strudwick, Xanthe L.; Cowin, Allison J.

    2012-01-01

    Wound healing results in the repair of injured tissues however fibrosis and scar formation are, more often than not the unfortunate consequence of this process. The ability of lower order vertebrates and invertebrates to regenerate limbs and tissues has been all but lost in mammals; however, there are some instances where glimpses of mammalian regenerative capacity do exist. Here we describe the unlocked potential that exists in mammals that may help us understand the process of regeneration post-injury and highlight the potential role of the actin cytoskeleton in this process. The precise function and regulation of the cytoskeleton is critical to the success of the healing process and its manipulation may therefore facilitate regenerative healing. The gelsolin family of actin remodelling proteins in particular has been shown to have important functions in wound healing and family member Flightless I (Flii) is involved in both regeneration and repair. Understanding the interactions between different cytoskeletal proteins and their dynamic control of processes including cellular adhesion, contraction and motility may assist the development of therapeutics that will stimulate regeneration rather than repair. PMID:24710556

  9. Biotechnological aspects of cytoskeletal regulation in plants.

    PubMed

    Komis, George; Luptovciak, Ivan; Doskocilova, Anna; Samaj, Jozef

    2015-11-01

    The cytoskeleton is a protein-based intracellular superstructure that evolved early after the appearance of bacterial prokaryotes. Eventually cytoskeletal proteins and their macromolecular assemblies were established in eukaryotes and assumed critical roles in cell movements, intracellular organization, cell division and cell differentiation. In biomedicine the small-molecules targeting cytoskeletal elements are in the frontline of anticancer research with plant-derived cytoskeletal drugs such as Vinca alkaloids and toxoids, being routinely used in the clinical practice. Moreover, plants are also major material, food and energy resources for human activities ranging from agriculture, textile industry, carpentry, energy production and new material development to name some few. Most of these inheritable traits are associated with cell wall synthesis and chemical modification during primary and secondary plant growth and inevitably are associated with the dynamics, organization and interactions of the plant cytoskeleton. Taking into account the vast intracellular spread of microtubules and actin microfilaments the cytoskeleton collectively assumed central roles in plant growth and development, in determining the physical stance of plants against the forces of nature and becoming a battleground between pathogenic invaders and the defense mechanisms of plant cells. This review aims to address the role of the plant cytoskeleton in manageable features of plants including cellulose biosynthesis with implications in wood and fiber properties, in biofuel production and the contribution of plant cytoskeletal elements in plant defense responses against pathogens or detrimental environmental conditions. Ultimately the present work surveys the potential of cytoskeletal proteins as platforms of plant genetic engineering, nominating certain cytoskeletal proteins as vectors of favorable traits in crops and other economically important plants. PMID:25784147

  10. Remodeling of the fibroblast cytoskeletal architecture during the replication cycle of Ectromelia virus: A morphological in vitro study in a murine cell line.

    PubMed

    Szulc-Dabrowska, Lidia; Gregorczyk, Karolina P; Struzik, Justyna; Boratynska-Jasinska, Anna; Szczepanowska, Joanna; Wyzewski, Zbigniew; Toka, Felix N; Gierynska, Malgorzata; Ostrowska, Agnieszka; Niemialtowski, Marek G

    2016-08-01

    Ectromelia virus (ECTV, the causative agent of mousepox), which represents the same genus as variola virus (VARV, the agent responsible for smallpox in humans), has served for years as a model virus for studying mechanisms of poxvirus-induced disease. Despite increasing knowledge on the interaction between ECTV and its natural host-the mouse-surprisingly, still little is known about the cell biology of ECTV infection. Because pathogen interaction with the cytoskeleton is still a growing area of research in the virus-host cell interplay, the aim of the present study was to evaluate the consequences of ECTV infection on the cytoskeleton in a murine fibroblast cell line. The viral effect on the cytoskeleton was reflected by changes in migration of the cells and rearrangement of the architecture of tubulin, vimentin, and actin filaments. The virus-induced cytoskeletal rearrangements observed in these studies contributed to the efficient cell-to-cell spread of infection, which is an important feature of ECTV virulence. Additionally, during later stages of infection L929 cells produced two main types of actin-based cellular protrusions: short (actin tails and "dendrites") and long (cytoplasmic corridors). Due to diversity of filopodial extensions induced by the virus, we suggest that ECTV represents a valuable new model for studying processes and pathways that regulate the formation of cytoskeleton-based cellular structures. © 2016 Wiley Periodicals, Inc. PMID:27169394

  11. Actinic Keratosis

    MedlinePlus

    ... rashes clinical tools newsletter | contact Share | Actinic Keratosis (Solar Keratosis) Information for adults A A A Actinic ... the touch. Overview Actinic keratoses, also known as solar keratoses, are small rough or scaly areas of ...

  12. Cucurbitacin covalent bonding to cysteine thiols: the filamentous-actin severing protein Cofilin1 as an exemplary target

    PubMed Central

    2013-01-01

    Background Cucurbitacins are a class of triterpenoid natural compounds with potent bioactivities that led to their use as traditional remedies, and which continue to attract considerable attention as chemical biology tools and potential therapeutics. One obvious target is the actin-cytoskeleton; treatment with cucurbitacins results in cytoskeletal rearrangements that impact upon motility and cell morphology. Findings Cucurbitacin reacted with protein cysteine thiols as well as dithiothreitol, and we propose that the cucurbitacin mechanism of action is through broad protein thiol modifications that could result in inhibition of numerous protein targets. An example of such a target protein is Cofilin1, whose filamentous actin severing activity is inhibited by cucurbitacin conjugation. Conclusions The implications of these results are that cucurbitacins are unlikely to be improved for selectivity by medicinal chemistry and that their use as chemical biology probes to analyse the role of specific signalling pathways should be undertaken with caution. PMID:23945128

  13. The cytoskeletal arrangements necessary to neurogenesis

    PubMed Central

    Compagnucci, Claudia; Piemonte, Fiorella; Sferra, Antonella; Piermarini, Emanuela; Bertini, Enrico

    2016-01-01

    During the process of neurogenesis, the stem cell committed to the neuronal cell fate starts a series of molecular and morphological changes. The understanding of the physio-pathology of mechanisms controlling the molecular and morphological changes occurring during neuronal differentiation is fundamental to the development of effective therapies for many neurologic diseases. Unfortunately, our knowledge of the biological events occurring in the cell during neuronal differentiation is still poor. In this study, we focus preliminarily on the relevance of the cytoskeletal rearrangements, which earlier drive the morphology of the neuronal precursors, and later the migrating/mature neurons. In fact, neuritogenesis, neurite branching, outgrowth and retraction are seminal to the development of a fully functional nervous system. With this in mind, we highlight the importance of iPSC technology to study the processes of cytoskeletal-driven morphological changes during neuronal differentiation. PMID:26760504

  14. Cytoskeletal reorganization dependence of signaling by the gonadotropin-releasing hormone receptor.

    PubMed

    Davidson, Lindsay; Pawson, Adam J; Millar, Robert P; Maudsley, Stuart

    2004-01-16

    Activation of classical G protein-coupled receptors (GPCRs) like the mammalian gonadotropin-releasing hormone receptor (GnRHR) typically stimulates heterotrimeric G protein molecules that subsequently activate downstream effectors. Receptor activation of heterotrimeric G protein pathways primarily controls intermediary cell metabolism by elevation or diminution of soluble cytoplasmic second messenger molecules. We have demonstrated here that stimulation of the GnRHR also results in a dramatic change in both cell adhesion and superstructural morphology. Gonadotropin-releasing hormone (GnRH) receptor activation rapidly increases the capacity of HEK293 cells expressing the GnRHR to remain matrix-adherent in the face of fluid insults. Coinciding with this profound elevation in matrix adherence, we demonstrated a GnRH-induced alteration in both cell morphology and the de novo generation of polymerized actin structures. GnRH induction of cytoskeletal remodeling was correlated with significant increases in the tyrosine phosphorylation status of a series of cytoskeletal associated proteins, e.g. focal adhesion kinase (FAK), c-Src, and microtubule-associated protein kinase (MAPK or ERK1/2). The activation of the distal downstream effector ERK1/2 was demonstrated to be sensitive to the disrupters of cytoskeletal rearrangement, cytochalasin D and latrunculin B. In addition to the sensitivity of ERKs to cytoskeletal integrity, GnRH-induced FAK and c-Src kinase activation were sensitive to these agents and the fibronectin-integrin antagonistic RGDS peptide. Activation of ERK was dependent on its protein-protein assembly with FAK and c-Src at focal adhesion complexes. Induction of the cell remodeling event leading to this signaling complex assembly occurred primarily via GnRHR activation of the monomeric G protein Rac but not RhoA. These findings demonstrated a clear divergence of GnRHR signaling via the Rac monomeric G protein focal adhesion signaling complex assembly and

  15. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression

    PubMed Central

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the “status” of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

  16. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression.

    PubMed

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the "status" of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

  17. Cell Forces and Cytoskeletal Order Parameters

    NASA Astrophysics Data System (ADS)

    Discher, Dennis

    2012-02-01

    Nematic, Smectic and Isotropic Order parameters have found wide-spread use in characterizing all manner of soft matter systems, but have not yet been applied to characterize and understand the structures within living cells, particularly cytoskeletal structures. Several examples will be used to illustrate the utility of such analyses, ranging from experiments on stem cells attached to or in various elastic matrices to embryonic heart tissue and simulations of membrane cytoskeletons under all manner of stressing. Recently developed theory will be shown to apply in general with account of cell contractility, matrix elasticity and dimensionality as well as cell shape and a newly defined ``cytoskeletal polarizability.'' The latter property of cells is likely different between different cell types due to different amounts of key cytoskeletal components with some types of stem cells being more polarizable than others. Evidence of coupling to the nucleus as a viscoelastic inclusion will also be presented. [4pt] References: (1) P. Dalhaimer, D.E. Discher, T. Lubensky. Crosslinked actin networks exhibit liquid crystal elastomer behavior, including soft-mode elasticity. Nature Physics 3: 354-360 (2007). (2) A. Zemel, F.Rehfeldt, A.E.X. Brown, D.E. Discher, and S.A. Safran. Optimal matrix rigidity in the self-polarization of stem cells. Nature Physics 6: 468 - 473 (2010).

  18. Luteinizing Hormone Receptor-Stimulated Progesterone Production by Preovulatory Granulosa Cells Requires Protein Kinase A-Dependent Activation/Dephosphorylation of the Actin Dynamizing Protein Cofilin

    PubMed Central

    Karlsson, Amelia B.; Maizels, Evelyn T.; Flynn, Maxfield P.; Jones, Jonathan C.; Shelden, Eric A.; Bamburg, James R.; Hunzicker-Dunn, Mary

    2010-01-01

    Activation of the LH receptor (LHR) on preovulatory granulosa cells stimulates the cAMP/protein kinase A (PKA) pathway to regulate expression of genes required for ovulation and luteinization. LHR signaling also initiates rearrangement of the actin cytoskeleton. Because disruption of the actin cytoskeleton has been causally linked to steroidogenesis in various cell models, we sought to identify the cellular mechanisms that may modulate reorganization of the actin cytoskeleton and to determine whether cytoskeletal reorganization is required for steroidogenesis. Herein we report that LHR signaling in preovulatory granulosa cells promotes rapid dephosphorylation of the actin-depolymerizing factor cofilin at Ser3 that is dependent on PKA. The LHR-stimulated dephosphorylation of cofilin(Ser3) switches on cofilin activity to bind actin filaments and enhance their dynamics. Basal phosphorylation of cofilin(Ser3) is mediated by active/GTP-bound Rho and downstream protein kinases; LHR signaling promotes a decrease in active/GTP-bound Rho by a PKA-dependent mechanism. LHR-dependent Rho inactivation and subsequent activation of cofilin does not involve ERK, epidermal growth factor receptor, or phosphatidylinositol 3-kinase pathways downstream of PKA. To understand the biological significance of cofilin activation, preovulatory granulosa cells were transduced with a mutant cofilin adenoviral vector in which Ser3 was mutated to Glu (S-E cofilin). Inactive S-E cofilin abolished LHR-mediated reorganization of the actin cytoskeleton and caused a 70% decrease in LHR-stimulated progesterone that is obligatory for ovulation. Taken together, these results show that LHR signaling via PKA activates a cofilin-regulated rearrangement of the actin cytoskeleton and that active cofilin is required to initiate progesterone secretion by preovulatory granulosa cells. PMID:20610540

  19. Binding of WIP to Actin Is Essential for T Cell Actin Cytoskeleton Integrity and Tissue Homing

    PubMed Central

    Massaad, Michel J.; Oyoshi, Michiko K.; Kane, Jennifer; Koduru, Suresh; Alcaide, Pilar; Nakamura, Fumihiko; Ramesh, Narayanaswamy; Luscinskas, Francis W.; Hartwig, John

    2014-01-01

    The Wiskott-Aldrich syndrome protein (WASp) is important for actin polymerization in T cells and for their migration. WASp-interacting protein (WIP) binds to and stabilizes WASp and also interacts with actin. Cytoskeletal and functional defects are more severe in WIP−/− T cells, which lack WASp, than in WASp−/− T cells, suggesting that WIP interaction with actin may be important for T cell cytoskeletal integrity and function. We constructed mice that lack the actin-binding domain of WIP (WIPΔABD mice). WIPΔABD associated normally with WASp but not F-actin. T cells from WIPΔABD mice had normal WASp levels but decreased cellular F-actin content, a disorganized actin cytoskeleton, impaired chemotaxis, and defective homing to lymph nodes. WIPΔABD mice exhibited a T cell intrinsic defect in contact hypersensitivity and impaired responses to cutaneous challenge with protein antigen. Adoptively transferred antigen-specific CD4+ T cells from WIPΔABD mice had decreased homing to antigen-challenged skin of wild-type recipients. These findings show that WIP binding to actin, independently of its binding to WASp, is critical for the integrity of the actin cytoskeleton in T cells and for their migration into tissues. Disruption of WIP binding to actin could be of therapeutic value in T cell-driven inflammatory diseases. PMID:25246631

  20. Measurements and models of cytoskeletal rheology

    NASA Astrophysics Data System (ADS)

    Kamm, Roger

    2006-11-01

    Much attention has recently focused on understanding the rheology of living cells and reconstituted actin gels using a variety of experimental methods (e.g., single- and multi-particle tracking, magnetic twisting cytometry, AFM indentation) and several different models or descriptors (e.g., biopolymer models, tensegrity, cellular solids, power-law rheology), but the debate continues regarding the fundamental basis for the experimental observations. Our recent studies examine the time-dependent behavior of neutrophils as they deform to enter a narrow channel with capillary-scale dimensions. A sudden drop in the shear modulus is observed, followed by recovery to pre-deformation values in < 1 minute. These rheological changes coincide with a reduction in f-actin content and a transient increase in calcium ion concentration [Ca^++], and the change in storage modulus can be prevented by calcium chelation, suggesting that these observations are causally linked. Cells lacking the ability to increase [Ca^++] also become activated more rapidly following deformation, and the time to activation is independent of intracellular strain rates, contrary to experiments lacking the chelating agent. To better understand these processes and the nature of cytoskeletal rheology in general, we have developed a Brownian dynamics model for cytoskeletal self-assembly and subsequent rheological measurement by single particle tracking. Cross-linking proteins are included possessing a range of properties that lead to a variety of cytoskeletal structures from a fine, homogeneous mesh to a structure containing large stress fibers of varying thickness. These results are described in a multi-dimensional phase space that takes into account the geometry, dimensions and stiffness of the cross-linkers.

  1. Cytoskeletal Mechanics Regulating Amoeboid Cell Locomotion

    PubMed Central

    Álvarez-González, Begoña; Meili, Ruedi; Firtel, Richard; Bastounis, Effie; del Álamo, Juan C.; Lasheras, Juan C.

    2014-01-01

    Migrating cells exert traction forces when moving. Amoeboid cell migration is a common type of cell migration that appears in many physiological and pathological processes and is performed by a wide variety of cell types. Understanding the coupling of the biochemistry and mechanics underlying the process of migration has the potential to guide the development of pharmacological treatment or genetic manipulations to treat a wide range of diseases. The measurement of the spatiotemporal evolution of the traction forces that produce the movement is an important aspect for the characterization of the locomotion mechanics. There are several methods to calculate the traction forces exerted by the cells. Currently the most commonly used ones are traction force microscopy methods based on the measurement of the deformation induced by the cells on elastic substrate on which they are moving. Amoeboid cells migrate by implementing a motility cycle based on the sequential repetition of four phases. In this paper we review the role that specific cytoskeletal components play in the regulation of the cell migration mechanics. We investigate the role of specific cytoskeletal components regarding the ability of the cells to perform the motility cycle effectively and the generation of traction forces. The actin nucleation in the leading edge of the cell, carried by the ARP2/3 complex activated through the SCAR/WAVE complex, has shown to be fundamental to the execution of the cyclic movement and to the generation of the traction forces. The protein PIR121, a member of the SCAR/WAVE complex, is essential to the proper regulation of the periodic movement and the protein SCAR, also included in the SCAR/WAVE complex, is necessary for the generation of the traction forces during migration. The protein Myosin II, an important F-actin cross-linker and motor protein, is essential to cytoskeletal contractility and to the generation and proper organization of the traction forces during

  2. Actinic keratosis

    MedlinePlus

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar) ... Some actinic keratoses become squamous cell skin cancer . Have your health care provider look at all skin growths as soon as you find them. Your provider will ...

  3. Bacterial actin and tubulin homologs in cell growth and division.

    PubMed

    Busiek, Kimberly K; Margolin, William

    2015-03-16

    In contrast to the elaborate cytoskeletal machines harbored by eukaryotic cells, such as mitotic spindles, cytoskeletal structures detectable by typical negative stain electron microscopy are generally absent from bacterial cells. As a result, for decades it was thought that bacteria lacked cytoskeletal machines. Revolutions in genomics and fluorescence microscopy have confirmed the existence not only of smaller-scale cytoskeletal structures in bacteria, but also of widespread functional homologs of eukaryotic cytoskeletal proteins. The presence of actin, tubulin, and intermediate filament homologs in these relatively simple cells suggests that primitive cytoskeletons first arose in bacteria. In bacteria such as Escherichia coli, homologs of tubulin and actin directly interact with each other and are crucial for coordinating cell growth and division. The function and direct interactions between these proteins will be the focus of this review. PMID:25784047

  4. Mechanosensitive kinetic preference of actin-binding protein to actin filament

    NASA Astrophysics Data System (ADS)

    Inoue, Yasuhiro; Adachi, Taiji

    2016-04-01

    The kinetic preference of actin-binding proteins to actin filaments is altered by external forces on the filament. Such an altered kinetic preference is largely responsible for remodeling the actin cytoskeletal structure in response to intracellular forces. During remodeling, actin-binding proteins and actin filaments interact under isothermal conditions, because the cells are homeostatic. In such a temperature homeostatic state, we can rigorously and thermodynamically link the chemical potential of actin-binding proteins to stresses on the actin filaments. From this relationship, we can construct a physical model that explains the force-dependent kinetic preference of actin-binding proteins to actin filaments. To confirm the model, we have analyzed the mechanosensitive alternation of the kinetic preference of Arp2/3 and cofilin to actin filaments. We show that this model captures the qualitative responses of these actin-binding proteins to the forces, as observed experimentally. Moreover, our theoretical results demonstrate that, depending on the structural parameters of the binding region, actin-binding proteins can show different kinetic responses even to the same mechanical signal tension, in which the double-helix nature of the actin filament also plays a critical role in a stretch-twist coupling of the filament.

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

    PubMed

    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

  6. 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

  7. Mechanisms of cytoskeletal regulation. Modulation of aortic endothelial cell spectrin by the extracellular matrix.

    PubMed Central

    Pratt, B. M.; Harris, A. S.; Morrow, J. S.; Madri, J. A.

    1984-01-01

    Endothelial cells have a complex cytoskeleton that is responsive to a variety of stimuli such as shear and desquamative injury. The extracellular matrix is known to influence several aspects of cellular behavior, including attachment, spreading, and migration and may, in part, initiate and control vascular responses in growth, differentiation, wound repair, and neoplasia. It is likely that linkage between surface receptors responsible for sensing the matrix and the cytoskeleton may be relevant to understanding the mechanisms of these responses. Spectrin is a high-molecular-weight heterodimer recently identified in many cells that appears to link surface receptors to cortical actin filaments. We have confirmed the existence of spectrin in cultured aortic endothelial cells by metabolic labeling and immunoprecipitation and demonstrated that its organization and intracellular distribution is sensitive to the extracellular matrix. When bovine calf aortic endothelial cells (BAEC) are cultured to confluency on a fibronectin (Fn) substrate, they assume a flattened, spread morphology and exhibit a punctate spectrin distribution with no discernible peripheral localization. In contrast, BAECs cultured on a Type I/III collagen (I/III) substrate exhibit a fibrillar spectrin pattern with significant peripheral localization. When migrating cells were examined, the distribution of spectrin was strikingly different. The cells on the Fn substrate showed no changes in spectrin localization, whereas the cells on I/III exhibited a significant rearrangement, with spectrin being in a coarse fibrillar form, with the fibrils aligned parallel to the direction of migration. The differences in arrangement of this cytoskeletal component on the two substrata reflect the ability of the substrate to perturb the cytoskeletal organization and modulate some aspects of cell behavior such as spreading, proliferation, and migration. These data are consistent with the concept that the nonerythroid

  8. [Cytoskeletal control of cell length regulation].

    PubMed

    Kharitonova, M A; Levina, C M; Rovenskii, I A

    2002-01-01

    It was shown that mouse embryo fibroblasts and human foreskin diploid fibroblasts of AGO 1523 line cultivated on specially prepared substrates with narrow (15 +/- 3 microns) linear adhesive strips were elongated and oriented along the strips, but the mean lengths of the fibroblasts of each type on the strips differed from those on the standard culture substrates. In contrast to the normal fibroblasts, the length of mouse embryonic fibroblasts with inactivated gene-suppresser Rb responsible for negative control of cell proliferation (MEF Rb-/-), ras-transformed mouse embryonic fibroblasts (MEF Rb-/-ras), or normal rat epitheliocytes of IAR2 line significantly exceeded those of the same cells on the standard culture substrates. The results of experiments with the drugs specifically affecting the cytoskeleton (colcemid and cytochalasin D) suggest that the constant mean length of normal fibroblasts is controlled by a dynamic equilibrium between two forces: centripetal tension of contractile actin-myosin microfilaments and centrifugal force generated by growing microtubules. This cytoskeletal mechanism is disturbed in MEF Rb-/- or MEF Rb-/-ras, probably, because of an impaired actin cytoskeleton and also in IAR2 epitheliocytes due to the different organization of the actin-myosin system in these cells, as compared to that in the fibroblasts. PMID:11862697

  9. Mertk deficiency affects macrophage directional migration via disruption of cytoskeletal organization.

    PubMed

    Tang, Yong; Wu, Shen; Liu, Qian; Xie, Jiayi; Zhang, Jingxue; Han, Dong; Lu, Qingxian; Lu, Qingjun

    2015-01-01

    Mertk belongs to the Tyro3, Axl and Mertk (TAM) family of receptor tyrosine kinases, and plays a pivotal role in regulation of cytoskeletal rearrangement during phagocytosis. Phagocytosis by either professional or non-professional phagocytes is impaired in the Mertk deficient individual. In the present study, we further investigated the effects of Mertk mutation on peritoneal macrophage morphology, attachment, spreading and movement. Mertk-mutated macrophages exhibited decreased attachment, weak spreading, loss of spindle-like body shape and lack of clear leading and trailing edges within the first few hours of culture, as observed by environmental scanning electron microscopy. Time-lapse video photography recording showed that macrophage without Mertk conducted mainly random movement with oscillating swing around the cell body, and lost the directional migration action seen on the WT cells. Western blotting showed a decreased phosphorylation of focal adhesion kinase (FAK). Immunocytochemistry revealed that actin filaments and dynamic protein myosin II failed to concentrate in the leading edge of migrating cells. Microtubules were localized mainly in one side of mutant cell body, with no clear MTOC and associated radially-distributed microtubule bundles, which were clearly evident in the WT cells. Our results suggest that Mertk deficiency affects not only phagocytosis but also cell shape and migration, likely through a common regulatory mechanism on cytoskeletons. PMID:25617898

  10. Effect of collagen I and fibronectin on the adhesion, elasticity and cytoskeletal organization of prostate cancer cells

    SciTech Connect

    Docheva, Denitsa; Padula, Daniela; Schieker, Matthias; Clausen-Schaumann, Hauke

    2010-11-12

    Research highlights: {yields} Depending on the metastatic origin, prostate cancer cells differ in their affinity to COL1. {yields} COL1 affects specifically the F-actin and cell elasticity of bone-derived prostate cancer cells. {yields} Cell elasticity can be used as a biomarker for cancer cells from different metastases. -- Abstract: Despite of intensive research efforts, the precise mechanism of prostate cancer metastasis in bone is still not fully understood. Several studies have suggested that specific matrix production by the bone cells, such as collagen I, supports cancer cell invasion. The aim of this study was to investigate the effect of collagen I (COL1) and fibronectin (FN) on cell adhesion, cell elasticity and cytoskeletal organization of prostate cancer cells. Two cell lines, bone marrow- (PC3) and lymph node-derived (LNCaP) were cultivated on COL1 and FN (control protein). By using a quantitative adhesion assay and time-lapse analysis, it was found that PC3, but not LNCaP, adhered strongly and were more spread on COL1. Next, PC3 and LNCaP were evaluated by atomic force microscopy (AFM) and flatness shape factor and cellular Young's modulus were calculated. The shape analysis revealed that PC3 were significantly flatter when grown on COL1 in comparison to LNCaP. In general, PC3 were also significantly stiffer than LNCaP and furthermore, their stiffness increased upon interaction with COL1. Since cell stiffness is strongly dependent on actin organization, phalloidin-based actin staining was performed and revealed that, of the two cell types as well as the two different matrix proteins, only PC3 grown on COL1 formed robust actin cytoskeleton. In conclusion, our study showed that PC3 cells have a strong affinity towards COL1. On this matrix protein, the cells adhered strongly and underwent a specific cell flattening. Moreover, with the establishment of PC3 contact to COL1 a significant increase of PC3 stiffness was observed due to a profound cytoskeletal

  11. Biophysical models of length control of cytoskeletal structures

    NASA Astrophysics Data System (ADS)

    Mohapatra, Lishibanya

    Cells contain elaborate and interconnected networks of protein polymers which make up the cytoskeleton. The cytoskeleton governs the internal positioning and movement of vesicles and organelles, and controls dynamic changes in cell polarity, shape and movement. Many of these processes require tight control of the size and shape of these cytoskeletal structures. A key question in cell biology is how these structures maintain a particular size and shape despite the rapid turnover of their components. In this thesis I show that the emerging mechanisms by which cells control and regulate the size of filamentous cytoskeletal structures can be classified using key parameters related to their assembly and disassembly kinetics. First, I examine quantitative models based on these specific molecular mechanisms of length control and make experimentally testable predictions that can be used to distinguish different mechanisms of length-control. Second, I study the length control of actin cables in budding yeast cells. Inspired by recent experimental observations in cells, I propose a novel antenna mechanism for cable length control which involves three key proteins: formins, which polymerize actin, Smy1 proteins, which bind formins and inhibit actin polymerization, and myosin motors, which deliver Smy1 to formins, leading to a length-dependent actin polymerization rate. My results provide testable predictions of the antenna mechanism of actin-cable length control. Next I consider the question of how different sized structures can co-exist in the same cytoplasm while making use of the same building blocks. Using theory, I discover limitations imposed by physics on the finite monomer pool as a mechanism of size control and conclude that additional length control mechanisms are required if a cell is to maintain multiple structures. While the primary focus of this thesis is on cytoskeletal structures, the broader principles and mechanisms discussed herein will apply to a range of

  12. Computational model of polarized actin cables and cytokinetic actin ring formation in budding yeast

    PubMed Central

    Tang, Haosu; Bidone, Tamara C.

    2015-01-01

    The budding yeast actin cables and contractile ring are important for polarized growth and division, revealing basic aspects of cytoskeletal function. To study these formin-nucleated structures, we built a 3D computational model with actin filaments represented as beads connected by springs. Polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling, are included. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosin stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which we suggest as a model for the assembly of actin filaments to the contractile ring. PMID:26538307

  13. Cytoskeletal mechanisms regulating vascular endothelial barrier function in response to acute lung injury.

    PubMed

    Kása, Anita; Csortos, Csilla; Verin, Alexander D

    2015-01-01

    Endothelial cells (EC) form a semi-permeable barrier between the interior space of blood vessels and the underlying tissues. In acute lung injury (ALI) the EC barrier is weakened leading to increased vascular permeability. It is widely accepted that EC barrier integrity is critically dependent upon intact cytoskeletal structure and cell junctions. Edemagenic agonists, like thrombin or endotoxin lipopolysaccharide (LPS), induced cytoskeletal rearrangement, and EC contractile responses leading to disruption of intercellular contacts and EC permeability increase. The highly clinically-relevant cytoskeletal mechanisms of EC barrier dysfunction are currently under intense investigation and will be described and discussed in the current review. PMID:25838980

  14. Cytoskeletal mechanisms regulating vascular endothelial barrier function in response to acute lung injury

    PubMed Central

    Kása, Anita; Csortos, Csilla; Verin, Alexander D

    2014-01-01

    Endothelial cells (EC) form a semi-permeable barrier between the interior space of blood vessels and the underlying tissues. In acute lung injury (ALI) the EC barrier is weakened leading to increased vascular permeability. It is widely accepted that EC barrier integrity is critically dependent upon intact cytoskeletal structure and cell junctions. Edemagenic agonists, like thrombin or endotoxin lipopolysaccharide (LPS), induced cytoskeletal rearrangement, and EC contractile responses leading to disruption of intercellular contacts and EC permeability increase. The highly clinically-relevant cytoskeletal mechanisms of EC barrier dysfunction are currently under intense investigation and will be described and discussed in the current review. PMID:25838980

  15. Cytoskeletal Proteins of Actinobacteria

    PubMed Central

    Letek, Michal; Fiuza, María; Villadangos, Almudena F.; Mateos, Luís M.; Gil, José A.

    2012-01-01

    Although bacteria are considered the simplest life forms, we are now slowly unraveling their cellular complexity. Surprisingly, not only do bacterial cells have a cytoskeleton but also the building blocks are not very different from the cytoskeleton that our own cells use to grow and divide. Nonetheless, despite important advances in our understanding of the basic physiology of certain bacterial models, little is known about Actinobacteria, an ancient group of Eubacteria. Here we review current knowledge on the cytoskeletal elements required for bacterial cell growth and cell division, focusing on actinobacterial genera such as Mycobacterium, Corynebacterium, and Streptomyces. These include some of the deadliest pathogens on earth but also some of the most prolific producers of antibiotics and antitumorals. PMID:22481946

  16. Segmentation and Tracking of Cytoskeletal Filaments Using Open Active Contours

    PubMed Central

    Smith, Matthew B.; Li, Hongsheng; Shen, Tian; Huang, Xiaolei; Yusuf, Eddy; Vavylonis, Dimitrios

    2010-01-01

    We use open active contours to quantify cytoskeletal structures imaged by fluorescence microscopy in two and three dimensions. We developed an interactive software tool for segmentation, tracking, and visualization of individual fibers. Open active contours are parametric curves that deform to minimize the sum of an external energy derived from the image and an internal bending and stretching energy. The external energy generates (i) forces that attract the contour toward the central bright line of a filament in the image, and (ii) forces that stretch the active contour toward the ends of bright ridges. Images of simulated semiflexible polymers with known bending and torsional rigidity are analyzed to validate the method. We apply our methods to quantify the conformations and dynamics of actin in two examples: actin filaments imaged by TIRF microscopy in vitro, and actin cables in fission yeast imaged by spinning disk confocal microscopy. PMID:20814909

  17. Post-polymerization crosstalk between the actin cytoskeleton and microtubule network.

    PubMed

    Joo, E Emily; Yamada, Kenneth M

    2016-05-01

    Cellular cytoskeletal systems play many pivotal roles in living organisms by controlling cell shape, division, and migration, which ultimately govern morphology, physiology, and functions of animals. Although the cytoskeletal systems are distinct and play different roles, there is growing evidence that these diverse cytoskeletal systems coordinate their functions with each other. This coordination between cytoskeletal systems, often termed cytoskeletal crosstalk, has been identified when the dynamic state of one individual system affects the other system. In this review, we briefly describe some well-established examples of crosstalk between cytoskeletal systems and then introduce a newly discovered form of crosstalk between the actin cytoskeleton and microtubule network that does not appear to directly alter polymerization or depolymerization of either system. The biological impact and possible significance of this post-polymerization crosstalk between actin and microtubules will be discussed in detail. PMID:27058810

  18. Actinic keratosis

    MedlinePlus

    ... example, if you work outdoors) Had many severe sunburns early in life Are older Symptoms Actinic keratosis ... and tanning salons. Other things to know about sun exposure: Sun exposure is stronger in or near surfaces ...

  19. Actinic Cheilitis

    MedlinePlus

    ... is a precancerous condition related to cumulative lifetime sun exposure. The lower lip is most often affected. Individuals ... Wearing barrier clothing (eg, wide-brimmed hats) and sunscreen-containing lip balms can aid in preventing actinic ...

  20. Regulation of cellular actin architecture by S100A10.

    PubMed

    Jung, M Juliane; Murzik, Ulrike; Wehder, Liane; Hemmerich, Peter; Melle, Christian

    2010-04-15

    Actin structures are involved in several biological processes and the disruption of actin polymerisation induces impaired motility of eukaryotic cells. Different factors are involved in regulation and maintenance of the cytoskeletal actin architecture. Here we show that S100A10 participates in the particular organisation of actin filaments. Down-regulation of S100A10 by specific siRNA triggered a disorganisation of filamentous actin structures without a reduction of the total cellular actin concentration. In contrast, the formation of cytoskeleton structures containing tubulin was unhindered in S100A10 depleted cells. Interestingly, the cellular distribution of annexin A2, an interaction partner of S100A10, was unaffected in S100A10 depleted cells. Cells lacking S100A10 showed an impaired migration activity and were unable to close a scratched wound. Our data provide first insights of S100A10 function as a regulator of the filamentous actin network. PMID:20100475

  1. Formin3 is required for assembly of the F-actin structure that mediates tracheal fusion in Drosophila.

    PubMed

    Tanaka, Hiromasa; Takasu, Etsuko; Aigaki, Toshiro; Kato, Kagayaki; Hayashi, Shigeo; Nose, Akinao

    2004-10-15

    During tracheal development in Drosophila, some branches join to form a continuous luminal network. Specialized cells at the branch tip, called fusion cells, extend filopodia to make contact and become doughnut shaped to allow passage of the lumen. These morphogenetic processes accompany the highly regulated cytoskeletal reorganization of fusion cells. We identified the Drosophila formin3 (form3) gene that encodes a novel formin and plays a role in tracheal fusion. Formins are a family of proteins characterized by highly conserved formin homology (FH) domains. The formin family functions in various actin-based processes, including cytokinesis and cell polarity. During embryogenesis, form3 mRNA is expressed mainly in the tracheal system. In form3 mutant embryos, the tracheal fusion does not occur at some points. This phenotype is rescued by the forced expression of form3 in the trachea. We used live imaging of GFP-moesin during tracheal fusion to show that an F-actin structure that spans the adjoining fusion cells and mediates the luminal connection does not form at abnormal anastomosis sites in form3 mutants. These results suggested that Form3 plays a role in the F-actin assembly, which is essential for cellular rearrangement during tracheal fusion. PMID:15385168

  2. Continuum descriptions of cytoskeletal dynamics

    PubMed Central

    2013-01-01

    This tutorial presents an introduction into continuum descriptions of cytoskeletal dynamics. In contrast to discrete models in which each molecule keeps its identity, such descriptions are given in terms of averaged quantities per unit volume like the number density of a certain molecule. Starting with a discrete description for the assembly dynamics of cytoskeletal filaments, we derive the continuity equation, which serves as the basis of many continuum theories. We illustrate the use of this approach with an investigation of spontaneous cytoskeletal polymerization waves. Such waves have by now been observed in various cell types and might help to orchestrate cytoskeletal dynamics during cell spreading and locomotion. Our analysis shows how processes at the scale of single molecules, namely, the nucleation of new filaments and filament treadmilling, can lead to the spontaneous appearance of coherent traveling waves on scales spanning many filament lengths. For readers less familiar with calculus, we include an informal introduction to the Taylor expansion. PMID:24565412

  3. The Role of Formin Tails in Actin Nucleation, Processive Elongation, and Filament Bundling*

    PubMed Central

    Vizcarra, Christina L.; Bor, Batbileg; Quinlan, Margot E.

    2014-01-01

    Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements. PMID:25246531

  4. Actin engine in immunological synapse.

    PubMed

    Piragyte, Indre; Jun, Chang-Duk

    2012-06-01

    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

  5. Accumulation of Glucosylceramide in the Absence of the Beta-Glucosidase GBA2 Alters Cytoskeletal Dynamics

    PubMed Central

    Raju, Diana; Schonauer, Sophie; Hamzeh, Hussein; Flynn, Kevin C.; Bradke, Frank; vom Dorp, Katharina; Dörmann, Peter; Yildiz, Yildiz; Trötschel, Christian; Poetsch, Ansgar; Breiden, Bernadette; Sandhoff, Konrad; Körschen, Heinz G.; Wachten, Dagmar

    2015-01-01

    Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types. PMID:25803043

  6. Accumulation of glucosylceramide in the absence of the beta-glucosidase GBA2 alters cytoskeletal dynamics.

    PubMed

    Raju, Diana; Schonauer, Sophie; Hamzeh, Hussein; Flynn, Kevin C; Bradke, Frank; Vom Dorp, Katharina; Dörmann, Peter; Yildiz, Yildiz; Trötschel, Christian; Poetsch, Ansgar; Breiden, Bernadette; Sandhoff, Konrad; Körschen, Heinz G; Wachten, Dagmar

    2015-03-01

    Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types. PMID:25803043

  7. The Role of Nox-Mediated Oxidation in the Regulation of Cytoskeletal Dynamics

    PubMed Central

    Valdivia, Alejandra; Duran, Charity; Martin, Alejandra San

    2015-01-01

    Nox generated ROS, particularly those derived from Nox1, Nox2 and Nox4, have emerged as important regulators of the actin cytoskeleton and cytoskeleton-supported cell functions, such as migration and adhesion. The effects of Nox-derived ROS on cytoskeletal remodeling may be largely attributed to the ability of ROS to directly modify proteins that constitute or are associated with the cytoskeleton. Additionally, Nox-derived ROS may participate in signaling pathways governing cytoskeletal remodeling. In addition to these more extensively studied signaling pathways involving Nox-derived ROS, there also exist redox sensitive pathways for which the source of ROS is unclear. ROS from as of yet undetermined sources play a role in modifying, and thus regulating, the activity of several proteins critical for remodeling of the actin cytoskeleton. In this review we discuss ROS sensitive targets that are likely to affect cytoskeletal dynamics, as well as the potential involvement of Nox proteins. PMID:26510432

  8. Cell elasticity with altered cytoskeletal architectures across multiple cell types.

    PubMed

    Grady, Martha E; Composto, Russell J; Eckmann, David M

    2016-08-01

    The cytoskeleton is primarily responsible for providing structural support, localization and transport of organelles, and intracellular trafficking. The structural support is supplied by actin filaments, microtubules, and intermediate filaments, which contribute to overall cell elasticity to varying degrees. We evaluate cell elasticity in five different cell types with drug-induced cytoskeletal derangements to probe how actin filaments and microtubules contribute to cell elasticity and whether it is conserved across cell type. Specifically, we measure elastic stiffness in primary chondrocytes, fibroblasts, endothelial cells (HUVEC), hepatocellular carcinoma cells (HUH-7), and fibrosarcoma cells (HT 1080) subjected to two cytoskeletal destabilizers: cytochalasin D and nocodazole, which disrupt actin and microtubule polymerization, respectively. Elastic stiffness is measured by atomic force microscopy (AFM) and the disruption of the cytoskeleton is confirmed using fluorescence microscopy. The two cancer cell lines showed significantly reduced elastic moduli values (~0.5kPa) when compared to the three healthy cell lines (~2kPa). Non-cancer cells whose actin filaments were disrupted using cytochalasin D showed a decrease of 60-80% in moduli values compared to untreated cells of the same origin, whereas the nocodazole-treated cells showed no change in elasticity. Overall, we demonstrate actin filaments contribute more to elastic stiffness than microtubules but this result is cell type dependent. Cancer cells behaved differently, exhibiting increased stiffness as well as stiffness variability when subjected to nocodazole. We show that disruption of microtubule dynamics affects cancer cell elasticity, suggesting therapeutic drugs targeting microtubules be monitored for significant elastic changes. PMID:26874250

  9. Processive cytoskeletal motors studied with single-molecule fluorescence techniques

    PubMed Central

    Belyy, Vladislav; Yildiz, Ahmet

    2014-01-01

    Processive cytoskeletal motors from the myosin, kinesin, and dynein families walk on actin filaments and microtubules to drive cellular transport and organization in eukaryotic cells. These remarkable molecular machines are able to take hundreds of successive steps at speeds of up to several microns per second, allowing them to effectively move vesicles and organelles throughout the cytoplasm. Here, we focus on single-molecule fluorescence techniques and discuss their wide-ranging applications to the field of cytoskeletal motor research. We cover both traditional fluorescence and sub-diffraction imaging of motors, providing examples of how fluorescence data can be used to measure biophysical parameters of motors such as coordination, stepping mechanism, gating, and processivity. We also outline some remaining challenges in the field and suggest future directions. PMID:24882363

  10. F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

    PubMed

    Masters, Thomas A; Sheetz, Michael P; Gauthier, Nils C

    2016-04-01

    Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks. PMID:26915738

  11. The interplay of nonlinearity and architecture in equilibrium cytoskeletal mechanics.

    PubMed

    Wang, Shenshen; Shen, Tongye; Wolynes, Peter G

    2011-01-01

    The interplay between cytoskeletal architecture and the nonlinearity of the interactions due to bucklable filaments plays a key role in modulating the cell's mechanical stability and affecting its structural rearrangements. We study a model of cytoskeletal structure treating it as an amorphous network of hard centers rigidly cross-linked by nonlinear elastic strings, neglecting the effects of motorization. Using simulations along with a self-consistent phonon method, we show that this minimal model exhibits diverse thermodynamically stable mechanical phases that depend on excluded volume, cross-link concentration, filament length, and stiffness. Within the framework set by the free energy functional formulation and making use of the random first order transition theory of structural glasses, we further estimate the characteristic densities for a kinetic glass transition to occur in this model system. Network connectivity strongly modulates the transition boundaries between various equilibrium phases, as well as the kinetic glass transition density. PMID:21219010

  12. Viscoelastic Retraction of Single Living Stress Fibers and Its Impact on Cell Shape, Cytoskeletal Organization, and Extracellular Matrix Mechanics

    PubMed Central

    Kumar, Sanjay; Maxwell, Iva Z.; Heisterkamp, Alexander; Polte, Thomas R.; Lele, Tanmay P.; Salanga, Matthew; Mazur, Eric; Ingber, Donald E.

    2006-01-01

    Cells change their form and function by assembling actin stress fibers at their base and exerting traction forces on their extracellular matrix (ECM) adhesions. Individual stress fibers are thought to be actively tensed by the action of actomyosin motors and to function as elastic cables that structurally reinforce the basal portion of the cytoskeleton; however, these principles have not been directly tested in living cells, and their significance for overall cell shape control is poorly understood. Here we combine a laser nanoscissor, traction force microscopy, and fluorescence photobleaching methods to confirm that stress fibers in living cells behave as viscoelastic cables that are tensed through the action of actomyosin motors, to quantify their retraction kinetics in situ, and to explore their contribution to overall mechanical stability of the cell and interconnected ECM. These studies reveal that viscoelastic recoil of individual stress fibers after laser severing is partially slowed by inhibition of Rho-associated kinase and virtually abolished by direct inhibition of myosin light chain kinase. Importantly, cells cultured on stiff ECM substrates can tolerate disruption of multiple stress fibers with negligible overall change in cell shape, whereas disruption of a single stress fiber in cells anchored to compliant ECM substrates compromises the entire cellular force balance, induces cytoskeletal rearrangements, and produces ECM retraction many microns away from the site of incision; this results in large-scale changes of cell shape (> 5% elongation). In addition to revealing fundamental insight into the mechanical properties and cell shape contributions of individual stress fibers and confirming that the ECM is effectively a physical extension of the cell and cytoskeleton, the technologies described here offer a novel approach to spatially map the cytoskeletal mechanics of living cells on the nanoscale. PMID:16500961

  13. A dynamic formin-dependent deep F-actin network in axons

    PubMed Central

    Ganguly, Archan; Tang, Yong; Wang, Lina; Ladt, Kelsey; Loi, Jonathan; Dargent, Bénédicte; Leterrier, Christophe

    2015-01-01

    Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles. PMID:26216902

  14. Actinic reticuloid

    SciTech Connect

    Marx, J.L.; Vale, M.; Dermer, P.; Ragaz, A.; Michaelides, P.; Gladstein, A.H.

    1982-09-01

    A 58-year-old man has his condition diagnosed as actinic reticuloid on the basis of clinical and histologic findings and phototesting data. He had clinical features resembling mycosis fungoides in light-exposed areas. Histologic findings disclosed a bandlike infiltrate with atypical mononuclear cells in the dermis and scattered atypical cells in the epidermis. Electron microscopy disclosed mononuclear cells with bizarre, convoluted nuclei, resembling cerebriform cells of Lutzner. Phototesting disclosed a diminished minimal erythemal threshold to UV-B and UV-A. Microscopic changes resembling actinic reticuloid were reproduced in this patient 24 and 72 hours after exposure to 15 minimal erythemal doses of UV-B.

  15. Regulation of cytoskeletal dynamics by redox signaling and oxidative stress: implications for neuronal development and trafficking

    PubMed Central

    Wilson, Carlos; González-Billault, Christian

    2015-01-01

    A proper balance between chemical reduction and oxidation (known as redox balance) is essential for normal cellular physiology. Deregulation in the production of oxidative species leads to DNA damage, lipid peroxidation and aberrant post-translational modification of proteins, which in most cases induces injury, cell death and disease. However, physiological concentrations of oxidative species are necessary to support important cell functions, such as chemotaxis, hormone synthesis, immune response, cytoskeletal remodeling, Ca2+ homeostasis and others. Recent evidence suggests that redox balance regulates actin and microtubule dynamics in both physiological and pathological contexts. Microtubules and actin microfilaments contain certain amino acid residues that are susceptible to oxidation, which reduces the ability of microtubules to polymerize and causes severing of actin microfilaments in neuronal and non-neuronal cells. In contrast, inhibited production of reactive oxygen species (ROS; e.g., due to NOXs) leads to aberrant actin polymerization, decreases neurite outgrowth and affects the normal development and polarization of neurons. In this review, we summarize emerging evidence suggesting that both general and specific enzymatic sources of redox species exert diverse effects on cytoskeletal dynamics. Considering the intimate relationship between cytoskeletal dynamics and trafficking, we also discuss the potential effects of redox balance on intracellular transport via regulation of the components of the microtubule and actin cytoskeleton as well as cytoskeleton-associated proteins, which may directly impact localization of proteins and vesicles across the soma, dendrites and axon of neurons. PMID:26483635

  16. High molecular weight tropomyosins regulate osteoclast cytoskeletal morphology.

    PubMed

    Kotadiya, Preeyal; McMichael, Brooke K; Lee, Beth S

    2008-11-01

    Tropomyosins are coiled-coil dimers that bind to the major groove of F-actin and regulate its accessibility to actin-modifying proteins. Although approximately 40 tropomyosin isoforms have been identified in mammals, they can broadly be classified into two groups based on protein size, that is, high molecular weight and low molecular weight isoforms. Osteoclasts, which undergo rounds of polarization and depolarization as they progress through the resorptive cycle, possess an unusual and highly dynamic actin cytoskeleton. To further define some of the actin regulatory proteins involved in osteoclast activity, we previously performed a survey of tropomyosin isoforms in resting and resorbing osteoclasts. Osteoclasts were found to express two closely related tropomyosins of the high molecular weight type, which are not expressed in monocytic and macrophage precursors. These isoforms, Tm-2 and Tm-3, are not strongly associated with actin-rich adhesion structures, but are instead distributed diffusely throughout the cell. In this study, we found that Tm-2/3 expression occurs late in osteoclastogenesis and continues to increase as cells mature. Knockdown of these isoforms via RNA interference results in flattening and increased spreading of osteoclasts, accompanied by diminished motility and altered resorptive capacity. In contrast, overexpression of Tm-2, but not Tm-3, caused morphological changes that include decreased spreading of the cells and induction of actin patches or stress fiber-like actin filaments, also with effects on motility and resorption. Suppression of Tm-2/3 or overexpression of Tm-2 resulted in altered distribution of gelsolin and microfilament barbed ends. These data suggest that high molecular weight tropomyosins are expressed in fusing osteoclasts to regulate the cytoskeletal scaffolding of these large cells, due at least in part by moderating accessibility of gelsolin to these microfilaments. PMID:18674650

  17. Hydroxyapatite-Coated Sillicone Rubber Enhanced Cell Adhesion and It May Be through the Interaction of EF1β and γ-Actin

    PubMed Central

    Zhang, Yi-ming; Wang, Yi-cheng; Yang, Zhi; Zhou, Xin; Lei, Ze-yuan; Fan, Dong-li

    2014-01-01

    Silicone rubber (SR) is a common soft tissue filler material used in plastic surgery. However, it presents a poor surface for cellular adhesion and suffers from poor biocompatibility. In contrast, hydroxyapatite (HA), a prominent component of animal bone and teeth, can promote improved cell compatibility, but HA is an unsuitable filler material because of the brittleness in mechanism. In this study, using a simple and economical method, two sizes of HA was applied to coat on SR to counteract the poor biocompatibility of SR. Surface and mechanical properties of SR and HA/SRs confirmed that coating with HA changes the surface topology and material properties. Analysis of cell proliferation and adhesion as well as measurement of the expression levels of adhesion related molecules indicated that HA-coated SR significantly increased cell compatibility. Furthermore, mass spectrometry proved that the biocompatibility improvement may be related to elongation factor 1-beta (EF1β)/γ-actin adjusted cytoskeletal rearrangement. PMID:25386892

  18. Probing mechanics and activity of cytoskeletal networks using carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Fakhri, Nikta

    2013-03-01

    We use single-walled carbon nanotubes (SWNTs) as multi-scale micro-probes to monitor transport and fluctuations in cytoskeletal networks. SWNTs are nanometer-diameter hollow carbon filaments with micrometer lengths and a tunable bending stiffness. Their persistence length varies between 20-100 microns. We study the motion of individual SWNTs in reconstituted actin networks by near-infrared fluorescence microscopy. At long times, SWNTs reptate through the networks. At short times, SWNTs sample the spectrum of thermal fluctuations in the networks. We can calculate complex shear moduli from recorded fluctuations and observe power-law scaling in equilibrium actin networks. In the non-equilibrium cytoskeleton of cells we have targeted SWNTs to kinesin motors and thereby to their microtubule tracks. We observe both transport along the tracks as well as active fluctuations of the tracks themselves. Human Frontier Science Program Cross-Disciplinary Fellow

  19. Emp is a component of the nuclear matrix of mammalian cells and undergoes dynamic rearrangements during cell division

    SciTech Connect

    Bala, Shashi; Kumar, Ajay; Soni, Shivani; Sinha, Sudha; Hanspal, Manjit . E-mail: manjit.hanspal@tufts.edu

    2006-04-21

    Emp, originally detected in erythroblastic islands, is expressed in numerous cell types and tissues suggesting a functionality not limited to hematopoiesis. To study the function of Emp in non-hematopoietic cells, an epitope-tagged recombinant human Emp was expressed in HEK cells. Preliminary studies revealed that Emp partitioned into both the nuclear and Triton X-100-insoluble cytoskeletal fractions in approximately a 4:1 ratio. In this study, we report investigations of Emp in the nucleus. Sequential extractions of interphase nuclei showed that recombinant Emp was present predominantly in the nuclear matrix. Immunofluorescence microscopy showed that Emp was present in typical nuclear speckles enriched with the spliceosome assembly factor SC35 and partially co-localized with actin staining. Coimmunoprecipitation and GST-pull-down assays confirmed the apparent close association of Emp with nuclear actin. During mitosis, Emp was detected at the mitotic spindle/spindle poles, as well as in the contractile ring during cytokinesis. These results suggest that Emp undergoes dynamic rearrangements within the nuclear architecture that are correlated with cell division.

  20. Analysis of Cytoskeletal and Motility Proteins in the Sea Urchin Genome Assembly

    PubMed Central

    RL, Morris; MP, Hoffman; RA, Obar; SS, McCafferty; IR, Gibbons; AD, Leone; J, Cool; EL, Allgood; AM, Musante; KM, Judkins; BJ, Rossetti; AP, Rawson; DR, Burgess

    2007-01-01

    The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development. PMID:17027957

  1. Conformational phases of membrane bound cytoskeletal filaments

    NASA Astrophysics Data System (ADS)

    Quint, David A.; Grason, Gregory; Gopinathan, Ajay

    2013-03-01

    Membrane bound cytoskeletal filaments found in living cells are employed to carry out many types of activities including cellular division, rigidity and transport. When these biopolymers are bound to a membrane surface they may take on highly non-trivial conformations as compared to when they are not bound. This leads to the natural question; What are the important interactions which drive these polymers to particular conformations when they are bound to a surface? Assuming that there are binding domains along the polymer which follow a periodic helical structure set by the natural monomeric handedness, these bound conformations must arise from the interplay of the intrinsic monomeric helicity and membrane binding. To probe this question, we study a continuous model of an elastic filament with intrinsic helicity and map out the conformational phases of this filament for various mechanical and structural parameters in our model, such as elastic stiffness and intrinsic twist of the filament. Our model allows us to gain insight into the possible mechanisms which drive real biopolymers such as actin and tubulin in eukaryotes and their prokaryotic cousins MreB and FtsZ to take on their functional conformations within living cells.

  2. Pathogenic microbes manipulate cofilin activity to subvert actin cytoskeleton.

    PubMed

    Zheng, Kai; Kitazato, Kaio; Wang, Yifei; He, Zhendan

    2016-09-01

    Actin-depolymerizing factor (ADF)/cofilin proteins are key players in controlling the temporal and spatial extent of actin dynamics, which is crucial for mediating host-pathogen interactions. Pathogenic microbes have evolved molecular mechanisms to manipulate cofilin activity to subvert the actin cytoskeletal system in host cells, promoting their internalization into the target cells, modifying the replication niche and facilitating their intracellular and intercellular dissemination. The study of how these pathogens exploit cofilin pathways is crucial for understanding infectious disease and providing potential targets for drug therapies. PMID:25853495

  3. Chemotaxis and Actin Oscillations

    NASA Astrophysics Data System (ADS)

    Bodenschatz, Eberhard; Hsu, Hsin-Fang; Negrete, Jose; Beta, Carsten; Pumir, Alain; Gholami, Azam; Tarantola, Marco; Westendorf, Christian; Zykov, Vladimir

    Recently, self-oscillations of the cytoskeletal actin have been observed in Dictyostelium, a model system for studying chemotaxis. Here we report experimental results on the self-oscillation mechanism and the role of regulatory proteins and myosin II. We stimulate cells rapidly and periodically by using photo un-caging of the chemoattractant in a micro-fluidic device and measured the cellular responses. We found that the response amplitude grows with stimulation strength only in a very narrow region of stimulation, after which the response amplitude reaches a plateau. Moreover, the frequency-response is not constant but rather varies with the strength of external stimuli. To understand the underlying mechanism, we analyzed the polymerization and de-polymerization time in the single cell level. Despite of the large cell-to-cell variability, we found that the polymerization time is independent of external stimuli and the de-polymerization time is prolonged as the stimulation strength increases. Our conclusions will be summarized and the role of noise in the signaling network will be discussed. German Science Foundation CRC 937.

  4. Shape remodeling and blebbing of active cytoskeletal vesicles

    PubMed Central

    Loiseau, Etienne; Schneider, Jochen A. M.; Keber, Felix C.; Pelzl, Carina; Massiera, Gladys; Salbreux, Guillaume; Bausch, Andreas R.

    2016-01-01

    Morphological transformations of living cells, such as shape adaptation to external stimuli, blebbing, invagination, or tethering, result from an intricate interplay between the plasma membrane and its underlying cytoskeleton, where molecular motors generate forces. Cellular complexity defies a clear identification of the competing processes that lead to such a rich phenomenology. In a synthetic biology approach, designing a cell-like model assembled from a minimal set of purified building blocks would allow the control of all relevant parameters. We reconstruct actomyosin vesicles in which the coupling of the cytoskeleton to the membrane, the topology of the cytoskeletal network, and the contractile activity can all be precisely controlled and tuned. We demonstrate that tension generation of an encapsulated active actomyosin network suffices for global shape transformation of cell-sized lipid vesicles, which are reminiscent of morphological adaptations in living cells. The observed polymorphism of our cell-like model, such as blebbing, tether extrusion, or faceted shapes, can be qualitatively explained by the protein concentration dependencies and a force balance, taking into account the membrane tension, the density of anchoring points between the membrane and the actin network, and the forces exerted by molecular motors in the actin network. The identification of the physical mechanisms for shape transformations of active cytoskeletal vesicles sets a conceptual and quantitative benchmark for the further exploration of the adaptation mechanisms of cells. PMID:27152328

  5. Shape remodeling and blebbing of active cytoskeletal vesicles.

    PubMed

    Loiseau, Etienne; Schneider, Jochen A M; Keber, Felix C; Pelzl, Carina; Massiera, Gladys; Salbreux, Guillaume; Bausch, Andreas R

    2016-04-01

    Morphological transformations of living cells, such as shape adaptation to external stimuli, blebbing, invagination, or tethering, result from an intricate interplay between the plasma membrane and its underlying cytoskeleton, where molecular motors generate forces. Cellular complexity defies a clear identification of the competing processes that lead to such a rich phenomenology. In a synthetic biology approach, designing a cell-like model assembled from a minimal set of purified building blocks would allow the control of all relevant parameters. We reconstruct actomyosin vesicles in which the coupling of the cytoskeleton to the membrane, the topology of the cytoskeletal network, and the contractile activity can all be precisely controlled and tuned. We demonstrate that tension generation of an encapsulated active actomyosin network suffices for global shape transformation of cell-sized lipid vesicles, which are reminiscent of morphological adaptations in living cells. The observed polymorphism of our cell-like model, such as blebbing, tether extrusion, or faceted shapes, can be qualitatively explained by the protein concentration dependencies and a force balance, taking into account the membrane tension, the density of anchoring points between the membrane and the actin network, and the forces exerted by molecular motors in the actin network. The identification of the physical mechanisms for shape transformations of active cytoskeletal vesicles sets a conceptual and quantitative benchmark for the further exploration of the adaptation mechanisms of cells. PMID:27152328

  6. Spiral actin-polymerization waves can generate amoeboidal cell crawling

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

  7. Formation of actin networks in microfluidic concentration gradients

    NASA Astrophysics Data System (ADS)

    Strelnikova, Natalja; Herren, Florian; Schoenenberger, Cora-Ann; Pfohl, Thomas

    2016-05-01

    The physical properties of cytoskeletal networks are contributors in a number of mechanical responses of cells including cellular deformation and locomotion, and are crucial for the proper action of living cells. Local chemical gradients modulate cytoskeletal functionality including the interactions of the cytoskeleton with other cellular components. Actin is a major constituent of the cytoskeleton. Introducing a microfluidic-based platform, we explored the impact of concentration gradients on the formation and structural properties of actin networks. Microfluidics-controlled flow-free steady state experimental conditions allow for the generation of chemical gradients of different profiles, such as linear or step-like. We discovered specific features of actin networks emerging in defined gradients. In particular, we analyzed the effects of spatial conditions on network properties, bending rigidities of network links, and the network elasticity.

  8. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    PubMed

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition. PMID:26240174

  9. Isolation of a strawberry gene fragment encoding an actin depolymerizing factor-like protein from genotypes resistant to Colletotrichum acutatum.

    PubMed

    Ontivero, Marta; Zamora, Gustavo Martínez; Salazar, Sergio; Ricci, Juan Carlos Díaz; Castagnaro, Atilio Pedro

    2011-12-01

    Actin depolymerizing factors (ADFs) have been recently implicated in plant defense against pathogenic fungi, associated with the cytoskeletal rearrangements that contribute to establish an effective barrier against fungal ingress. In this work, we identified a DNA fragment corresponding to a part of a gene predicted to encode an ADF-like protein in genotypes of Fragaria ananassa resistant to the fungus Colletotrichum acutatum. Bulked segregant analysis combined with AFLP was used to identify polymorphisms linked to resistance in hybrids derived from the cross between the resistant cultivar 'Sweet Charlie' and the susceptible cultivar 'Pájaro'. The sequence of one out of three polymorphic bands detected showed significant BLASTX hits to ADF proteins from other plants. Two possible exons were identified and bioinformatic analysis revealed the presence of the ADF homology domain with two actin-binding sites, an N-terminal phosphorylation site, and a nuclear localization signal. In addition to its possible application in strawberry breeding programs, these finding may contribute to investigate the role of ADFs in plant resistance against fungi. PMID:22107362

  10. Actin-associated Proteins in the Pathogenesis of Podocyte Injury

    PubMed Central

    He, Fang-Fang; Chen, Shan; Su, Hua; Meng, Xian-Fang; Zhang, Chun

    2013-01-01

    Podocytes have a complex cellular architecture with interdigitating processes maintained by a precise organization of actin filaments. The actin-based foot processes of podocytes and the interposed slit diaphragm form the final barrier to proteinuria. The function of podocytes is largely based on the maintenance of the normal foot process structure with actin cytoskeleton. Cytoskeletal dynamics play important roles during normal podocyte development, in maintenance of the healthy glomerular filtration barrier, and in the pathogenesis of glomerular diseases. In this review, we focused on recent findings on the mechanisms of organization and reorganization of these actin-related molecules in the pathogenesis of podocyte injury and potential therapeutics targeting the regulation of actin cytoskeleton in podocytopathies. PMID:24396279

  11. Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

    PubMed Central

    Müller, Mirco; Diensthuber, Ralph P.; Chizhov, Igor; Claus, Peter; Heissler, Sarah M.; Preller, Matthias; Taft, Manuel H.; Manstein, Dietmar J.

    2013-01-01

    Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments. PMID:23923011

  12. Sensing actin dynamics: Structural basis for G-actin-sensitive nuclear import of MAL

    SciTech Connect

    Hirano, Hidemi; Matsuura, Yoshiyuki

    2011-10-22

    Highlights: {yields} MAL has a bipartite NLS that binds to Imp{alpha} in an extended conformation. {yields} Mutational analyses verified the functional significance of MAL-Imp{alpha} interactions. {yields} Induced folding and NLS-masking by G-actins inhibit nuclear import of MAL. -- Abstract: The coordination of cytoskeletal actin dynamics with gene expression reprogramming is emerging as a crucial mechanism to control diverse cellular processes, including cell migration, differentiation and neuronal circuit assembly. The actin-binding transcriptional coactivator MAL (also known as MRTF-A/MKL1/BSAC) senses G-actin concentration and transduces Rho GTPase signals to serum response factor (SRF). MAL rapidly shuttles between the cytoplasm and the nucleus in unstimulated cells but Rho-induced depletion of G-actin leads to MAL nuclear accumulation and activation of transcription of SRF:MAL-target genes. Although the molecular and structural basis of actin-regulated nucleocytoplasmic shuttling of MAL is not understood fully, it is proposed that nuclear import of MAL is mediated by importin {alpha}/{beta} heterodimer, and that G-actin competes with importin {alpha}/{beta} for the binding to MAL. Here we present structural, biochemical and cell biological evidence that MAL has a classical bipartite nuclear localization signal (NLS) in the N-terminal 'RPEL' domain containing Arg-Pro-X-X-X-Glu-Leu (RPEL) motifs. The NLS residues of MAL adopt an extended conformation and bind along the surface groove of importin-{alpha}, interacting with the major- and minor-NLS binding sites. We also present a crystal structure of wild-type MAL RPEL domain in complex with five G-actins. Comparison of the importin-{alpha}- and actin-complexes revealed that the binding of G-actins to MAL is associated with folding of NLS residues into a helical conformation that is inappropriate for importin-{alpha} recognition.

  13. Cytoskeletal reorganization during process of apoptosis induced by cytostatic drugs in K-562 and HL-60 leukemia cell lines.

    PubMed

    Grzanka, A; Grzanka, D; Orlikowska, M

    2003-10-15

    The aim of the present study was to investigate the reorganization of F-actin, vimentin and tubulin in K-562 and HL-60 cell lines during apoptosis induced by etoposide, doxorubicin and taxol. The distribution of cytoskeletal proteins was analyzed by fluorescence microscopy. Actin was also studied by confocal microscopy and at the ultrastructural level. Changes in the distribution of cytoskeletal proteins were found to be dose-dependent and appeared to be more intense in HL-60 cells. Etoposide- and doxorubicin-treated cells showed similar changes in the distribution of F-actin, vimentin and tubulin. The reorganization of cytoskeletal proteins seemed to be consistent with features of apoptosis. An increase in bright staining of F-actin, vimentin and tubulin at the site of apoptotic bodies formation was observed. Immunogold labeling of actin in HL-60 cells was associated with features typical for apoptosis, i.e. compaction and margination of nuclear chromatin. K-562 cells showed cytoplasmic actin-positivity in the cytoplasm. Significant changes in morphology of HL-60 cells were found in the following concentrations: etoposide 20, 200 microM; doxorubicin 5, 10 microM and taxol 2-10 microM. The investigated proteins seemed to be involved in the above-reported apoptotic changes. Bright staining of F-actin, vimentin and tubulin, concentrated at the site of apoptotic bodies formation might suggested importance of these proteins for this process. Moreover, the increase in actin labeling in areas of chromatin compaction and margination of nuclear chromatin especially in HL-60 cells, which are more susceptible to apoptosis might implicate that actin might be involved in the chromatin remodeling during apoptosis. PMID:14555241

  14. An Excitable Signal Integrator Couples to an Idling Cytoskeletal Oscillator to Drive Cell Migration

    PubMed Central

    Huang, Chuan-Hsiang; Tang, Ming; Shi, Changji; Iglesias, Pablo A.; Devreotes, Peter N.

    2013-01-01

    It is generally believed that cytoskeletal activities drive random cell migration while signal transduction events initiated by receptors regulate the cytoskeleton to guide cells. However, we find that the cytoskeletal network, involving Scar/Wave, Arp 2/3, and actin binding proteins, is only capable of generating rapid oscillations and undulations of the cell boundary. The signal transduction network, comprising multiple pathways that include Ras GTPases, PI3K, and Rac GTPases, is required to generate the sustained protrusions of migrating cells. The signal transduction network is excitable, displaying wave propagation, refractoriness, and maximal response to suprathreshold stimuli, even in the absence of the cytoskeleton. We suggest that cell motility results from coupling of “pacemaker” signal transduction and “idling motor” cytoskeletal networks, and various guidance cues that modulate the threshold for triggering signal transduction events are integrated to control the mode and direction of migration. PMID:24142103

  15. Cytoskeletal involvement during hypo-osmotic swelling and volume regulation in cultured chick cardiac myocytes.

    PubMed

    Larsen, T H; Dalen, H; Boyle, R; Souza, M M; Lieberman, M

    2000-06-01

    The membrane skeleton in spherical cardiac myocytes subjected to hypo-osmotic challenge was examined by laser scanning confocal microscopy. A distinct cortical layer intimately localized under the plasmalemma was revealed for spectrin and actin (including filamentous actin and alpha-sarcomeric actin). Desmin filaments were abundant and in close contact with the plasmalemma. During swelling and subsequent regulatory volume decrease (RVD) the structural integrity of these cytoskeletal elements remained intact, and the close association between actin and plasmalemma persisted as confirmed by double immunolabeling. Subplasmalemmal beta-tubulin labeling was sparse. Hypo-osmotic conditions disrupted the microtubules and depolymerized tubulin. Neither pretreatment with taxol nor with colchicine, resulted in any effect on cell volume regulation. The present results show that actin, desmin, and spectrin contribute to a subplasmalemmal cytoskeletal network in spherical cardiac myocytes, and that this membrane skeleton remains structurally intact during swelling and RVD. It is suggested that the integrity of this membrane skeleton is important for stabilization of the plasmalemma and the membrane-integrated proteins during hypo-osmotic challenge, and that it may participate in the regulation of the cell volume. PMID:10933224

  16. Which way to go? Cytoskeletal organization and polarized transport in neurons.

    PubMed

    Kapitein, Lukas C; Hoogenraad, Casper C

    2011-01-01

    To establish and maintain their polarized morphology, neurons employ active transport driven by cytoskeletal motor proteins to sort cargo between axons and dendrites. These motors can move in a specific direction over either microtubules (kinesins, dynein) or actin filaments (myosins). The basic traffic rules governing polarized transport on the neuronal cytoskeleton have long remained unclear, but recent work has revealed several fundamental sorting principles based on differences in the cytoskeletal organization in axons versus dendrites. We will highlight the basic characteristics of the neuronal cytoskeleton and review existing evidence for microtubule and actin based traffic rules in polarized neuronal transport. We will propose a model in which polarized sorting of cargo is established by recruiting or activating the proper subset of motor proteins, which are subsequently guided to specific directions by the polarized organization of the neuronal cytoskeleton. PMID:20817096

  17. Actinic Prurigo.

    PubMed

    Rodríguez-Carreón, Alma Angélica; Rodríguez-Lobato, Erika; Rodríguez-Gutiérrez, Georgina; Cuevas-González, Juan Carlos; Mancheno-Valencia, Alexandra; Solís-Arias, Martha Patricia; Vega-Memije, María Elisa; Hojyo-Tomoka, María Teresa; Domínguez-Soto, Luciano

    2015-01-01

    Actinic prurigo is an idiopathic photodermatosis that affects the skin, as well as the labial and conjunctival mucosa in indigenous and mestizo populations of Latin America. It starts predominantly in childhood, has a chronic course, and is exacerbated with solar exposure. Little is known of its pathophysiology, including the known mechanisms of the participation of HLA-DR4 and an abnormal immunologic response with increase of T CD4+ lymphocytes. The presence of IgE, eosinophils, and mast cells suggests that it is a hypersensitivity reaction (likely type IVa or b). The diagnosis is clinical, and the presence of lymphoid follicles in the mucosal histopathologic study of mucosa is pathognomonic. The best available treatment to date is thalidomide, despite its secondary effects. PMID:26861426

  18. [Actinic Keratosis].

    PubMed

    Dejaco, D; Hauser, U; Zelger, B; Riechelmann, H

    2015-07-01

    Actinic keratosis is a cutaneous lesion characterized by proliferation of atypical epidermal keratinocytes due to prolonged exposure to exogenous factors such as ultraviolet radiation. AKs are in-situ-squamous cell carcinomas (PEC) of the skin. AK typically presents as erythematous, scaly patch or papule (classic AK), occasionally as thick, adherent scale on an erythematous base. Mostly fair-skinned adults are affected. AKs typically occur in areas of frequent sun exposure (balding scalp, face, "H-region", lateral neck, décolleté, dorsum of the hand and lower extremities). Actinic Cheilitis is the term used for AKs appearing on the lips. The diagnosis of AK is based on clinical examination including inspection and palpation. The typical palpable rough surface of AK often precedes a visible lesion. Dermoscopy may provide additional information. If diagnosis is uncertain and invasion suspected, biopsy and histopathologic evaluation should be performed. The potential for progression to invasive PECs mandates therapeutic intervention. Treatment options include topical and systemic therapies. Topical therapies are classified into physical, medical and combined physical-chemical approaches and a sequential combination of treatment modalities is possible. Topical-physical cryotherapy is the treatment of choice for isolated, non-hypertrophic AK. Topical-medical treatment, e. g. 5-fluoruracil (5FU) cream or Imiquomod or Ingenolmebutat application is used for multiple, non-hypertrophic AKs. For hypertrophic AKs, a dehorning pretreatment with salicinated vaseline is recommended. Isolated hypertrophic AKs often need cryotherapy with prolonged freezing time or several consecutive applications. Sequentially combined approaches are recommended for multiple, hypertrophic AKs. Photodynamic therapy (PDT) as example for a combined physical-chemical approach is an established treatment for multiple, non-hypertrophic and hypertrophic AKs. Prevention includes avoidance of sun and

  19. Molecular rearrangements of superelectrophiles

    PubMed Central

    2011-01-01

    Summary Superelectrophiles are multiply charged cationic species (dications, trications, etc.) which are characterized by their reactions with weak nucleophiles. These reactive intermediates may also undergo a wide variety of rearrangement-type reactions. Superelectrophilic rearrangements are often driven by charge–charge repulsive effects, as these densely charged ions react so as to maximize the distances between charge centers. These rearrangements involve reaction steps similar to monocationic rearrangements, such as alkyl group shifts, Wagner–Meerwein shifts, hydride shifts, ring opening reactions, and other skeletal rearrangements. This review will describe these types of superelectrophilic reactions. PMID:21512598

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

    PubMed

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

    1998-02-01

    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. PMID:11536873

  1. 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.

  2. Hierarchical self-organization of cytoskeletal active networks

    NASA Astrophysics Data System (ADS)

    Gordon, Daniel; Bernheim-Groswasser, Anne; Keasar, Chen; Farago, Oded

    2012-04-01

    The structural reorganization of the actin cytoskeleton is facilitated through the action of motor proteins that crosslink the actin filaments and transport them relative to each other. Here, we present a combined experimental-computational study that probes the dynamic evolution of mixtures of actin filaments and clusters of myosin motors. While on small spatial and temporal scales the system behaves in a very noisy manner, on larger scales it evolves into several well distinct patterns such as bundles, asters and networks. These patterns are characterized by junctions with high connectivity, whose formation is possible due to the organization of the motors in ‘oligoclusters’ (intermediate-size aggregates). The simulations reveal that the self-organization process proceeds through a series of hierarchical steps, starting from local microscopic moves and ranging up to the macroscopic large scales where the steady-state structures are formed. Our results shed light on the mechanisms involved in processes such as cytokinesis and cellular contractility, where myosin motors organized in clusters operate cooperatively to induce the structural organization of cytoskeletal networks.

  3. On the significance of microtubule flexural behavior in cytoskeletal mechanics.

    PubMed

    Mehrbod, Mehrdad; Mofrad, Mohammad R K

    2011-01-01

    Quantitative description of cell mechanics has challenged biological scientists for the past two decades. Various structural models have been attempted to analyze the structure of the cytoskeleton. One important aspect that has been largely ignored in all these modeling approaches is related to the flexural and buckling behavior of microtubular filaments. The objective of this paper is to explore the influence of this flexural and buckling behavior in cytoskeletal mechanics.In vitro the microtubules are observed to buckle in the first mode, reminiscent of a free, simply-supported beam. In vivo images of microtubules, however, indicate that the buckling mostly occurs in higher modes. This buckling mode switch takes place mostly because of the lateral support of microtubules via their connections to actin and intermediate filaments. These lateral loads are exerted throughout the microtubule length and yield a considerable bending behavior that, unless properly accounted for, would produce erroneous results in the modeling and analysis of the cytoskeletal mechanics.One of the promising attempts towards mechanical modeling of the cytoskeleton is the tensegrity model, which simplifies the complex network of cytoskeletal filaments into a combination merely of tension-bearing actin filaments and compression-bearing microtubules. Interestingly, this discrete model can qualitatively explain many experimental observations in cell mechanics. However, evidence suggests that the simplicity of this model may undermine the accuracy of its predictions, given the model's underlying assumption that "every single member bears solely either tensile or compressive behavior," i.e. neglecting the flexural behavior of the microtubule filaments. We invoke an anisotropic continuum model for microtubules and compare the bending energy stored in a single microtubule with its axial strain energy at the verge of buckling. Our results suggest that the bending energy can exceed the axial energy

  4. On the Significance of Microtubule Flexural Behavior in Cytoskeletal Mechanics

    PubMed Central

    Mehrbod, Mehrdad; Mofrad, Mohammad R. K.

    2011-01-01

    Quantitative description of cell mechanics has challenged biological scientists for the past two decades. Various structural models have been attempted to analyze the structure of the cytoskeleton. One important aspect that has been largely ignored in all these modeling approaches is related to the flexural and buckling behavior of microtubular filaments. The objective of this paper is to explore the influence of this flexural and buckling behavior in cytoskeletal mechanics. In vitro the microtubules are observed to buckle in the first mode, reminiscent of a free, simply-supported beam. In vivo images of microtubules, however, indicate that the buckling mostly occurs in higher modes. This buckling mode switch takes place mostly because of the lateral support of microtubules via their connections to actin and intermediate filaments. These lateral loads are exerted throughout the microtubule length and yield a considerable bending behavior that, unless properly accounted for, would produce erroneous results in the modeling and analysis of the cytoskeletal mechanics. One of the promising attempts towards mechanical modeling of the cytoskeleton is the tensegrity model, which simplifies the complex network of cytoskeletal filaments into a combination merely of tension-bearing actin filaments and compression-bearing microtubules. Interestingly, this discrete model can qualitatively explain many experimental observations in cell mechanics. However, evidence suggests that the simplicity of this model may undermine the accuracy of its predictions, given the model's underlying assumption that “every single member bears solely either tensile or compressive behavior,” i.e. neglecting the flexural behavior of the microtubule filaments. We invoke an anisotropic continuum model for microtubules and compare the bending energy stored in a single microtubule with its axial strain energy at the verge of buckling. Our results suggest that the bending energy can exceed the axial

  5. Cdc42 and p190RhoGAP activation by CCN2 regulates cell spreading and polarity and induces actin disassembly in migrating keratinocytes.

    PubMed

    Kiwanuka, Elizabeth; Lee, Cameron Cy; Hackl, Florian; Caterson, Edward J; Junker, Johan Pe; Gerdin, Bengt; Eriksson, Elof

    2016-06-01

    Cell migration requires spatiotemporal integration of signals that regulate cytoskeletal dynamics. In response to a migration-promoting agent, cells begin to polarise and extend protrusions in the direction of migration. These cytoskeletal rearrangements are orchestrated by a variety of proteins, including focal adhesion kinase (FAK) and the Rho family of GTPases. CCN2, also known as connective tissue growth factor, has emerged as a regulator of cell migration but the mechanism by which CCN2 regulates keratinocyte function is not well understood. In this article, we sought to elucidate the basic mechanism of CCN2-induced cell migration in human keratinocytes. Immunohistochemical staining was used to demonstrate that treatment with CCN2 induces a migratory phenotype through actin disassembly, spreading of lamellipodia and re-orientation of the Golgi. In vitro assays were used to show that CCN2-induced cell migration is dependent on FAK, RhoA and Cdc42, but independent of Rac1. CCN2-treated keratinocytes displayed increased Cdc42 activity and decreased RhoA activity up to 12 hours post-treatment, with upregulation of p190RhoGAP. An improved understanding of how CCN2 regulates cell migration may establish the foundation for future therapeutics in fibrotic and neoplastic diseases. PMID:25185742

  6. Closure of supporting cell scar formations requires dynamic actin mechanisms.

    PubMed

    Hordichok, Andrew J; Steyger, Peter S

    2007-10-01

    In many vertebrate inner ear sensory epithelia, dying sensory hair cells are extruded, and the apices of surrounding supporting cells converge to re-seal the epithelial barrier between the electrochemically-distinct endolymph and perilymph. These cellular mechanisms remain poorly understood. Dynamic microtubular mechanisms have been proposed for hair cell extrusion; while contractile actomyosin-based mechanisms are required for cellular extrusion and closure in epithelial monolayers. The hypothesis that cytoskeletal mechanisms are required for hair cell extrusion and supporting cell scar formation was tested using bullfrog saccules incubated with gentamicin (6h), and allowed to recover (18h). Explants were then fixed, labeled for actin and cytokeratins, and viewed with confocal microscopy. To block dynamic cytoskeletal processes, disruption agents for microtubules (colchicine, paclitaxel) myosin (Y-27632, ML-9) or actin (cytochalasin D, latrunculin A) were added during treatment and recovery. Microtubule disruption agents had no effect on hair cell extrusion or supporting cell scar formation. Myosin disruption agents appeared to slow down scar formation but not hair cell extrusion. Actin disruption agents blocked scar formation, and largely prevented hair cell extrusion. These data suggest that actin-based cytoskeletal processes are required for hair cell extrusion and supporting cell scar formation in bullfrog saccules. PMID:17716843

  7. CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor.

    PubMed

    Zhao, Shuangshuang; Jiang, Yuxiang; Zhao, Yang; Huang, Shanjin; Yuan, Ming; Zhao, Yanxiu; Guo, Yan

    2016-06-01

    The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, but the underlying mechanism for this cytoskeletal reorganization remains largely unclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2 does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actin filaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actin depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive to ABA Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filament reorganization and stomatal closure mainly through phosphorylation of ADF. PMID:27268429

  8. The multi-faceted role of the actin cap in cellular mechanosensation and mechanotransduction.

    PubMed

    Kim, Dong-Hwee; Chambliss, Allison B; Wirtz, Denis

    2013-06-21

    The perinuclear actin cap (or actin cap) is a recently characterized cytoskeletal organelle composed of thick, parallel, and highly contractile acto-myosin filaments that are specifically anchored to the apical surface of the interphase nucleus. The actin cap is present in a wide range of adherent eukaryotic cells, but is disrupted in several human diseases, including laminopathies and cancer. Through its large terminating focal adhesions and anchorage to the nuclear lamina and nuclear envelope through LINC complexes, the perinuclear actin cap plays a critical role both in mechanosensation and mechanotransduction, the ability of cells to sense changes in matrix compliance and to respond to mechanical forces, respectively. PMID:23930135

  9. The Rho-GEF Rom2p Localizes to Sites of Polarized Cell Growth and Participates in Cytoskeletal Functions in Saccharomyces cerevisiae

    PubMed Central

    Manning, Brendan D.; Padmanabha, Ramesh; Snyder, Michael

    1997-01-01

    Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 were identified in a screen for high-copy number suppressors of cik1Δ, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11°C and 37°C. rom2Δ cells exhibit morphological defects. At permissive temperatures, rom2Δ cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1Δ and kar3Δ strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. Furthermore, rom2Δ strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton. PMID:9348527

  10. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    SciTech Connect

    Purdy, Kirstin R.; Wong, Gerard C. L.; Bartles, James R.

    2007-02-02

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system's phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  11. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    NASA Astrophysics Data System (ADS)

    Purdy, Kirstin R.; Bartles, James R.; Wong, Gerard C. L.

    2007-02-01

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system’s phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  12. Actin-mediated motion of meiotic chromosomes

    PubMed Central

    Koszul, R.; Kim, K. P.; Prentiss, M.; Kleckner, N.; Kameoka, S.

    2008-01-01

    Summary Chromosome movement is prominent during meiosis. Here, using a combination of in vitro and in vivo approaches, we elucidate the basis for dynamic mid-prophase chromosome movement in budding yeast. Diverse finding reveal a process in which, at the pachytene stage, individual telomere/nuclear envelope (NE) ensembles attach passively to, and then move in concert with, nucleus-hugging actin cables that are continuous with the global cytoskeletal actin network. Other chromosomes move in concert with lead chromosome(s). The same process, in modulated form, explains the zygotene "bouquet" configuration in which, immediately preceding pachytene, chromosome ends colocalize dynamically in a restricted region of the NE. Mechanical properties of the system and biological roles of mid-prophase movement for meiosis, including recombination, are discussed. PMID:18585353

  13. Cortactin Branches Out: Roles in Regulating Protrusive Actin Dynamics

    PubMed Central

    Ammer, Amanda Gatesman; Weed, Scott A.

    2008-01-01

    Since its discovery in the early 1990’s, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cytoskeletal organization, impacting the regulation and formation of actin-rich motility structures. PMID:18615630

  14. Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins

    PubMed Central

    Jitoboam, Kunlakanya; Phaonakrop, Narumon; Libsittikul, Sirikwan; Thepparit, Chutima; Roytrakul, Sittiruk; Smith, Duncan R.

    2016-01-01

    Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component. PMID:27010925

  15. Buckling of Branched Cytoskeletal Filaments

    NASA Astrophysics Data System (ADS)

    Quint, D. A.; Schwarz, J. M.

    2011-03-01

    In vitro experiments of growing dendritic actin networks demonstrate reversible stress-softening at high loads, above some critical load. The transition to the stress-softening regime has been attributed to the elastic buckling of individual actin filaments. To estimate the critical load above which softening should occur, we extend the elastic theory of buckling of individual filaments embedded in a network to include the buckling of branched filaments, a signature trait of growing dendritic actin networks. Under certain assumptions, there will be approximately a seven-fold increase in the classical critical bucking load, when compared to the unbranched filament, which is entirely due to the presence of a branch. Moreover, we go beyond the classical buckling regime to investigate the effect of entropic fluctuations. The result of compressing the filament in this case leads to an increase in these fluctuations and eventually the harmonic approximation breaks down signifying the onset of the buckling transition. We compute corrections to the classical critical buckling load near this breakdown.

  16. Continuum modeling of forces in growing viscoelastic cytoskeletal networks.

    PubMed

    Kim, Jin Seob; Sun, Sean X

    2009-02-21

    Mechanical properties of the living cell are important in cell movement, cell division, cancer development and cell signaling. There is considerable interest in measuring local mechanical properties of living materials and the living cytoskeleton using micromechanical techniques. However, living materials are constantly undergoing internal dynamics such as growth and remodeling. A modeling framework that combines mechanical deformations with cytoskeletal growth dynamics is necessary to describe cellular shape changes. The present paper develops a general finite deformation modeling approach that can treat the viscoelastic cytoskeleton. Given the growth dynamics in the cytoskeletal network and the relationship between deformation and stress, the shape of the network is computed in an incremental fashion. The growth dynamics of the cytoskeleton can be modeled as stress dependent. The result is a consistent treatment of overall cell deformation. The framework is applied to a growing 1-d bundle of actin filaments against an elastic cantilever, and a 2-d cell undergoing wave-like protrusion dynamics. In the latter example, mechanical forces on the cell adhesion are examined as a function of the protrusion dynamics. PMID:19041329

  17. Cytoskeletal protein kinases: titin and its relations in mechanosensing.

    PubMed

    Gautel, Mathias

    2011-07-01

    Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca(2+)-calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other "MLCKs", is not Ca(2+)-calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation. PMID:21416260

  18. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity

    PubMed Central

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; Camacho-Arroyo, Ignacio

    2015-01-01

    In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone. PMID:26635640

  19. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity.

    PubMed

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; Camacho-Arroyo, Ignacio

    2015-01-01

    In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone. PMID:26635640

  20. Continuum Modeling of Forces in Growing Viscoelastic Cytoskeletal Networks

    PubMed Central

    Kim, Jin Seob; Sun, Sean X.

    2013-01-01

    Mechanical properties of the living cell are important in cell movement, cell division, cancer development and cell signaling. There is considerable interest in measuring local mechanical properties of living materials and the living cytoskeleton using micromechanical techniques. However, living materials are constantly undergoing internal dynamics such as growth and remodeling. A modeling framework that combines mechanical deformations with cytoskeletal growth dynamics is necessary to describe cellular shape changes. The present paper develops a general finite deformation modeling approach that can treat the viscoelastic cytoskeleton. Given the growth dynamics in the cytoskeletal network and the relationship between deformation and stress, the shape of the network is computed in an incremental fashion. The growth dynamics of the cytoskeleton can be modeled as stress dependent. The result is a consistent treatment of overall cell deformation. The framework is applied to a growing 1-d bundle of actin filaments against an elastic cantilever, and a 2-d cell undergoing wave-like protrusion dynamics. In the latter example, mechanical forces on the cell adhesion are examined as a function the protrusion dynamics. PMID:19041329

  1. Implementing cell contractility in filament-based cytoskeletal models.

    PubMed

    Fallqvist, B

    2016-02-01

    Cells are known to respond over time to mechanical stimuli, even actively generating force at longer times. In this paper, a microstructural filament-based cytoskeletal network model is extended to incorporate this active response, and a computational study to assess the influence on relaxation behaviour was performed. The incorporation of an active response was achieved by including a strain energy function of contractile activity from the cross-linked actin filaments. A four-state chemical model and strain energy function was adopted, and generalisation to three dimensions and the macroscopic deformation field was performed by integration over the unit sphere. Computational results in MATLAB and ABAQUS/Explicit indicated an active cellular response over various time-scales, dependent on contractile parameters. Important features such as force generation and increasing cell stiffness due to prestress are qualitatively predicted. The work in this paper can easily be extended to encompass other filament-based cytoskeletal models as well. © 2016 Wiley Periodicals, Inc. PMID:26899417

  2. Intracellular calcium rise is not a necessary step for the stimulated actin polymerization

    SciTech Connect

    Yassin, R.

    1986-03-01

    Stimulation of rabbit peritoneal neutrophils by many chemotactic (formyl Methionyl-Leucyl-Phenylalanine (fMLP), Leukotriene B/sub 4/ (LTB/sub 4/)) and non-chemotactic (phorbol 12-myristate, 13-acetate (PMA), platelet activating factor (PAF), and the calcium ionophore A23187) factors produces rapid and dose dependent increases in the amount of actin associated with the cytoskeleton. The stimulated increase in cytoskeletal actin does not appear to require a rise in the intracellular concentration of free calcium. The increase in cytoskeletal actin produced by A23187 is transient and does not depend on the presence of calcium in the suspending medium. In the presence of extracellular calcium, the effect of the ionophore is biphasic with respect to concentration. The increases in actin association with cytoskeletal produced by fMLP, LTB/sub 4/, and A23187 but not by PMA, are inhibited by hyperosmolarity and pertussis toxin pretreatment. On the other hand, the addition of hyperosmolarity or pertussis toxin has small effect on the rise in the intracellular calcium produced by A23187. The results presented here suggest that an increase in the intracellular concentration of free calcium is not necessary for the stimulated increases in cytoskeletal actin.

  3. 3D Actin Network Centerline Extraction with Multiple Active Contours

    PubMed Central

    Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

    2013-01-01

    Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and actin cables. While these cytoskeletal structures are often dilute enough to allow imaging of individual filaments or bundles of them, quantitative analysis of these images is challenging. To facilitate quantitative, reproducible and objective analysis of the image data, we propose a semi-automated method to extract actin networks and retrieve their topology in 3D. Our method uses multiple Stretching Open Active Contours (SOACs) that are automatically initialized at image intensity ridges and then evolve along the centerlines of filaments in the network. SOACs can merge, stop at junctions, and reconfigure with others to allow smooth crossing at junctions of filaments. The proposed approach is generally applicable to images of curvilinear networks with low SNR. We demonstrate its potential by extracting the centerlines of synthetic meshwork images, actin networks in 2D Total Internal Reflection Fluorescence Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy. Quantitative evaluation of the method using synthetic images shows that for images with SNR above 5.0, the average vertex error measured by the distance between our result and ground truth is 1 voxel, and the average Hausdorff distance is below 10 voxels. PMID:24316442

  4. Polycystin-1 regulates actin cytoskeleton organization and directional cell migration through a novel PC1-Pacsin 2-N-Wasp complex.

    PubMed

    Yao, Gang; Su, Xuefeng; Nguyen, Vy; Roberts, Kristina; Li, Xiaogang; Takakura, Ayumi; Plomann, Markus; Zhou, Jing

    2014-05-15

    How epithelial cells form a tubule with defined length and lumen diameter remains a fundamental question in cell and developmental biology. Loss of control of tubule lumen size in multiple organs including the kidney, liver and pancreas features polycystic kidney disease (PKD). To gain insights into autosomal dominant polycystic kidney disease, we performed yeast two-hybrid screens using the C-terminus of polycystin-1 (PC1) as bait. Here, we report that PC1 interacts with Pacsin 2, a cytoplasmic phosphoprotein that has been implicated in cytoskeletal organization, vesicle trafficking and more recently in cell intercalation during gastrulation. PC1 binds to a 107-residue fragment containing the α3 helix of the F-BAR domain of Pacsin 2 via a coiled-coil domain in its C-tail. PC1 and Pacsin 2 co-localize on the lamellipodia of migrating kidney epithelial cells. PC1 and Pacsin 2-deficient kidney epithelial cells migrate at a slower speed with reduced directional persistency. We further demonstrate that PC1, Pacsin 2 and N-Wasp are in the same protein complex, and both PC1 and Pacsin 2 are required for N-Wasp/Arp2/3-dependent actin remodeling. We propose that PC1 modulates actin cytoskeleton rearrangements and directional cell migration through the Pacsin 2/N-Wasp/Arp2/3 complex, which consequently contributes to the establishment and maintenance of the sophisticated tubular architecture. Disruption of this complex contributes to cyst formation in PKD. PMID:24385601

  5. Cortactin involvement in the keratinocyte growth factor and fibroblast growth factor 10 promotion of migration and cortical actin assembly in human keratinocytes

    SciTech Connect

    Ceccarelli, Simona; Cardinali, Giorgia; Aspite, Nicaela; Picardo, Mauro; Marchese, Cinzia; Torrisi, Maria Rosaria; Mancini, Patrizia . E-mail: patrizia.mancini@uniroma1.it

    2007-05-15

    Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10.

  6. The WAVE Regulatory Complex Links Diverse Receptors to the Actin Cytoskeleton

    PubMed Central

    Chen, Baoyu; Chen, Zhucheng; Brinkmann, Klaus; Pak, Chi W.; Liao, Yuxing; Shi, Shuoyong; Henry, Lisa; Grishin, Nick V.; Bogdan, Sven; Rosen, Michael K.

    2014-01-01

    SUMMARY The WAVE regulatory complex (WRC) controls actin cytoskeletal dynamics throughout the cell by stimulating the actin nucleating activity of the Arp2/3 complex at distinct membrane sites. However, the factors that recruit the WRC to specific locations remain poorly understood. Here we have identified a large family of potential WRC ligands, consisting of ~120 diverse membrane proteins including protocadherins, ROBOs, netrin receptors, Neuroligins, GPCRs and channels. Structural, biochemical and cellular studies reveal that a novel sequence motif that defines these ligands binds to a highly conserved interaction surface of the WRC formed by the Sra and Abi subunits. Mutating this binding surface in flies resulted in defects in actin cytoskeletal organization and egg morphology during oogenesis, leading to female sterility. Our findings directly link diverse membrane proteins to the WRC and actin cytoskeleton, and have broad physiological and pathological ramifications in metazoans. PMID:24439376

  7. Cytoskeletal Regulation of Inflammation and Its Impact on Skin Blistering Disease Epidermolysis Bullosa Acquisita.

    PubMed

    Kopecki, Zlatko; Ludwig, Ralf J; Cowin, Allison J

    2016-01-01

    Actin remodelling proteins regulate cytoskeletal cell responses and are important in both innate and adaptive immunity. These responses play a major role in providing a fine balance in a cascade of biological events that results in either protective acute inflammation or chronic inflammation that leads to a host of diseases including autoimmune inflammation mediated epidermolysis bullosa acquisita (EBA). This review describes the role of the actin cytoskeleton and in particular the actin remodelling protein called Flightless I (Flii) in regulating cellular inflammatory responses and its subsequent effect on the autoimmune skin blistering disease EBA. It also outlines the potential of an antibody based therapy for decreasing Flii expression in vivo to ameliorate the symptoms associated with EBA. PMID:27420054

  8. Cytoskeletal Regulation of Inflammation and Its Impact on Skin Blistering Disease Epidermolysis Bullosa Acquisita

    PubMed Central

    Kopecki, Zlatko; Ludwig, Ralf J.; Cowin, Allison J.

    2016-01-01

    Actin remodelling proteins regulate cytoskeletal cell responses and are important in both innate and adaptive immunity. These responses play a major role in providing a fine balance in a cascade of biological events that results in either protective acute inflammation or chronic inflammation that leads to a host of diseases including autoimmune inflammation mediated epidermolysis bullosa acquisita (EBA). This review describes the role of the actin cytoskeleton and in particular the actin remodelling protein called Flightless I (Flii) in regulating cellular inflammatory responses and its subsequent effect on the autoimmune skin blistering disease EBA. It also outlines the potential of an antibody based therapy for decreasing Flii expression in vivo to ameliorate the symptoms associated with EBA. PMID:27420054

  9. Degradations and Rearrangement Reactions

    NASA Astrophysics Data System (ADS)

    Zhang, Jianbo

    This section deals with recent reports concerning degradation and rearrangement reactions of free sugars as well as some glycosides. The transformations are classified in chemical and enzymatic ways. In addition, the Maillard reaction will be discussed as an example of degradation and rearrangement transformation and its application in current research in the fields of chemistry and biology.

  10. Symmetry breaking in reconstituted actin cortices.

    PubMed

    Abu Shah, Enas; Keren, Kinneret

    2014-01-01

    The actin cortex plays a pivotal role in cell division, in generating and maintaining cell polarity and in motility. In all these contexts, the cortical network has to break symmetry to generate polar cytoskeletal dynamics. Despite extensive research, the mechanisms responsible for regulating cortical dynamics in vivo and inducing symmetry breaking are still unclear. Here we introduce a reconstituted system that self-organizes into dynamic actin cortices at the inner interface of water-in-oil emulsions. This artificial system undergoes spontaneous symmetry breaking, driven by myosin-induced cortical actin flows, which appears remarkably similar to the initial polarization of the embryo in many species. Our in vitro model system recapitulates the rich dynamics of actin cortices in vivo, revealing the basic biophysical and biochemical requirements for cortex formation and symmetry breaking. Moreover, this synthetic system paves the way for further exploration of artificial cells towards the realization of minimal model systems that can move and divide.DOI: http://dx.doi.org/10.7554/eLife.01433.001. PMID:24843007

  11. Symmetry breaking in reconstituted actin cortices

    PubMed Central

    Abu Shah, Enas; Keren, Kinneret

    2014-01-01

    The actin cortex plays a pivotal role in cell division, in generating and maintaining cell polarity and in motility. In all these contexts, the cortical network has to break symmetry to generate polar cytoskeletal dynamics. Despite extensive research, the mechanisms responsible for regulating cortical dynamics in vivo and inducing symmetry breaking are still unclear. Here we introduce a reconstituted system that self-organizes into dynamic actin cortices at the inner interface of water-in-oil emulsions. This artificial system undergoes spontaneous symmetry breaking, driven by myosin-induced cortical actin flows, which appears remarkably similar to the initial polarization of the embryo in many species. Our in vitro model system recapitulates the rich dynamics of actin cortices in vivo, revealing the basic biophysical and biochemical requirements for cortex formation and symmetry breaking. Moreover, this synthetic system paves the way for further exploration of artificial cells towards the realization of minimal model systems that can move and divide. DOI: http://dx.doi.org/10.7554/eLife.01433.001 PMID:24843007

  12. Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae

    PubMed Central

    Casolari, Jason M.; Thompson, Michael A.; Salzman, Julia; Champion, Lowry M.; Moerner, W. E.; Brown, Patrick O.

    2012-01-01

    Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe her e a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches. PMID:22359641

  13. Formin-mediated actin polymerization at endothelial junctions is required for vessel lumen formation and stabilization.

    PubMed

    Phng, Li-Kun; Gebala, Véronique; Bentley, Katie; Philippides, Andrew; Wacker, Andrin; Mathivet, Thomas; Sauteur, Loïc; Stanchi, Fabio; Belting, Heinz-Georg; Affolter, Markus; Gerhardt, Holger

    2015-01-12

    During blood vessel formation, endothelial cells (ECs) establish cell-cell junctions and rearrange to form multicellular tubes. Here, we show that during lumen formation, the actin nucleator and elongation factor, formin-like 3 (fmnl3), localizes to EC junctions, where filamentous actin (F-actin) cables assemble. Fluorescent actin reporters and fluorescence recovery after photobleaching experiments in zebrafish embryos identified a pool of dynamic F-actin with high turnover at EC junctions in vessels. Knockdown of fmnl3 expression, chemical inhibition of formin function, and expression of dominant-negative fmnl3 revealed that formin activity maintains a stable F-actin content at EC junctions by continual polymerization of F-actin cables. Reduced actin polymerization leads to destabilized endothelial junctions and consequently to failure in blood vessel lumenization and lumen instability. Our findings highlight the importance of formin activity in blood vessel morphogenesis. PMID:25584798

  14. Cofilin-2 controls actin filament length in muscle sarcomeres

    PubMed Central

    Kremneva, Elena; Makkonen, Maarit H.; Skwarek-Maruszewska, Aneta; Gateva, Gergana; Michelot, Alphee; Dominguez, Roberto; Lappalainen, Pekka

    2014-01-01

    SUMMARY ADF/cofilins drive cytoskeletal dynamics by promoting the disassembly of ‘aged’ ADP-actin filaments. Mammals express several ADF/cofilin isoforms, but their specific biochemical activities and cellular functions have not been studied in detail. Here we demonstrate that the muscle-specific isoform cofilin-2 promotes actin filament disassembly in sarcomeres to control the precise length of thin filaments in the contractile apparatus. In contrast to other isoforms, cofilin-2 efficiently binds and disassembles both ADP- and ATP/ADP-Pi-actin filaments. We mapped surface-exposed cofilin-2-specific residues required for ATP-actin binding and propose that these residues function as an ‘actin nucleotide-state sensor’ among ADF/cofilins. The results suggest that cofilin-2 evolved specific biochemical and cellular properties allowing it to control actin dynamics in sarcomeres, where filament pointed ends may contain a mixture of ADP- and ATP/ADP-Pi-actin subunits. Our findings also offer a rationale for why cofilin-2 mutations in humans lead to myopathies. PMID:25373779

  15. Regulation of T cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm

    PubMed Central

    Beemiller, Peter; Krummel, Matthew F.

    2013-01-01

    Summary The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering, signalosome assembl, y and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has considered actin largely for its ‘scaffolding’ function. We examine the roles of the actin cytoskeleton in TCR signaling and immune synapse formation with an emphasis on how poroelasticity, an ensemble feature of actin dynamics with the cytosol, relates to how T cells respond to stimulation. PMID:24117819

  16. Regulation of T-cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm.

    PubMed

    Beemiller, Peter; Krummel, Matthew F

    2013-11-01

    The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering signalosome assembly and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has considered actin largely for its 'scaffolding' function. We examine the roles of the actin cytoskeleton in TCR signaling and immune synapse formation with an emphasis on how poroelasticity, an ensemble feature of actin dynamics with the cytosol, relates to how T cells respond to stimulation. PMID:24117819

  17. Differential Actin-regulatory Activities of Tropomodulin1 and Tropomodulin3 with Diverse Tropomyosin and Actin Isoforms*

    PubMed Central

    Yamashiro, Sawako; Gokhin, David S.; Sui, Zhenhua; Bergeron, Sarah E.; Rubenstein, Peter A.; Fowler, Velia M.

    2014-01-01

    Tropomodulins (Tmods) are F-actin pointed end capping proteins that interact with tropomyosins (TMs) and cap TM-coated filaments with higher affinity than TM-free filaments. Here, we tested whether differences in recognition of TM or actin isoforms by Tmod1 and Tmod3 contribute to the distinct cellular functions of these Tmods. We found that Tmod3 bound ∼5-fold more weakly than Tmod1 to α/βTM, TM5b, and TM5NM1. However, surprisingly, Tmod3 was as effective as Tmod1 at capping pointed ends of skeletal muscle α-actin (αsk-actin) filaments coated with α/βTM, TM5b, or TM5NM1. Tmod3 only capped TM-coated αsk-actin filaments more weakly than Tmod1 in the presence of recombinant αTM2, which is unacetylated at its NH2 terminus, binds F-actin weakly, and has a disabled Tmod-binding site. Moreover, both Tmod1 and Tmod3 were similarly effective at capping pointed ends of platelet β/cytoplasmic γ (γcyto)-actin filaments coated with TM5NM1. In the absence of TMs, both Tmod1 and Tmod3 had similarly weak abilities to nucleate β/γcyto-actin filament assembly, but only Tmod3 could sequester cytoplasmic β- and γcyto-actin (but not αsk-actin) monomers and prevent polymerization under physiological conditions. Thus, differences in TM binding by Tmod1 and Tmod3 do not appear to regulate the abilities of these Tmods to cap TM-αsk-actin or TM-β/γcyto-actin pointed ends and, thus, are unlikely to determine selective co-assembly of Tmod, TM, and actin isoforms in different cell types and cytoskeletal structures. The ability of Tmod3 to sequester β- and γcyto-actin (but not αsk-actin) monomers in the absence of TMs suggests a novel function for Tmod3 in regulating actin remodeling or turnover in cells. PMID:24644292

  18. Arabidopsis Actin Depolymerizing Factor4 Modulates the Stochastic Dynamic Behavior of Actin Filaments in the Cortical Array of Epidermal Cells[C][W

    PubMed Central

    Henty, Jessica L.; Bledsoe, Samuel W.; Khurana, Parul; Meagher, Richard B.; Day, Brad; Blanchoin, Laurent; Staiger, Christopher J.

    2011-01-01

    Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells. PMID:22010035

  19. Quantitative Evaluation of Stomatal Cytoskeletal Patterns during the Activation of Immune Signaling in Arabidopsis thaliana

    PubMed Central

    Shimono, Masaki; Higaki, Takumi; Kaku, Hanae; Shibuya, Naoto; Hasezawa, Seiichiro

    2016-01-01

    Historically viewed as primarily functioning in the regulation of gas and water vapor exchange, it is now evident that stomata serve an important role in plant immunity. Indeed, in addition to classically defined functions related to cell architecture and movement, the actin cytoskeleton has emerged as a central component of the plant immune system, underpinning not only processes related to cell shape and movement, but also receptor activation and signaling. Using high resolution quantitative imaging techniques, the temporal and spatial changes in the actin microfilament array during diurnal cycling of stomatal guard cells has revealed a highly orchestrated transition from random arrays to ordered bundled filaments. While recent studies have demonstrated that plant stomata close in response to pathogen infection, an evaluation of stimulus-induced changes in actin cytoskeletal dynamics during immune activation in the guard cell, as well as the relationship of these changes to the function of the actin cytoskeleton and stomatal aperture, remains undefined. In the current study, we employed quantitative cell imaging and hierarchical clustering analyses to define the response of the guard cell actin cytoskeleton to pathogen infection and the elicitation of immune signaling. Using this approach, we demonstrate that stomatal-localized actin filaments respond rapidly, and specifically, to both bacterial phytopathogens and purified pathogen elicitors. Notably, we demonstrate that higher order temporal and spatial changes in the filament array show distinct patterns of organization during immune activation, and that changes in the naïve diurnal oscillations of guard cell actin filaments are perturbed by pathogens, and that these changes parallel pathogen-induced stomatal gating. The data presented herein demonstrate the application of a highly tractable and quantifiable method to assign transitions in actin filament organization to the activation of immune signaling in

  20. Dynamic morphology and cytoskeletal protein changes during spontaneous inside-out vesiculation of red blood cell membranes.

    PubMed

    Tiffert, Teresa; Lew, Virgilio L

    2014-12-01

    Vesicle preparations from cell plasma membranes, red blood cells in particular, are extensively used in transport and enzymic studies and in the fields of drug delivery and drug-transport interactions. Here we investigated the role of spectrin-actin, the main components of the red cell cortical cytoskeleton, in a particular mechanism of vesicle generation found to be relevant to the egress process of Plasmodium falciparum merozoites from infected red blood cells. Plasma membranes from red blood cells lysed in ice-cold media of low ionic strength and free of divalent cations spontaneously and rapidly vesiculate upon incubation at 37 °C rendering high yields of inside-out vesicles. We tested the working hypothesis that the dynamic shape transformations resulted from changes in spectrin-actin configuration within a disintegrating cytoskeletal mesh. We showed that cytoskeletal-free membranes behave like a two-dimensional fluid lacking shape control, that spectrin-actin remain attached to vesiculating membranes for as long as spontaneous movement persists, that most of the spectrin-actin detachment occurs terminally at the time of vesicle sealing and that naked membrane patches increasingly appear during vesiculation. These results support the proposed role of spectrin-actin in spontaneous vesiculation. The implications of these results to membrane dynamics and to the mechanism of merozoite egress are discussed. PMID:24615169

  1. The cortical cytoskeletal network and cell-wall dynamics in the unicellular charophycean green alga Penium margaritaceum

    PubMed Central

    Ochs, Julie; LaRue, Therese; Tinaz, Berke; Yongue, Camille; Domozych, David S.

    2014-01-01

    Background and Aims Penium margaritaceum is a unicellular charophycean green alga with a unique bi-directional polar expansion mechanism that occurs at the central isthmus zone prior to cell division. This entails the focused deposition of cell-wall polymers coordinated by the activities of components of the endomembrane system and cytoskeletal networks. The goal of this study was to elucidate the structural organization of the cortical cytoskeletal network during the cell cycle and identify its specific functional roles during key cell-wall developmental events: pre-division expansion and cell division. Methods Microtubules and actin filaments were labelled during various cell cycle phases with an anti-tubulin antibody and rhodamine phalloidin, respectively. Chemically induced disruption of the cytoskeleton was used to elucidate specific functional roles of microtubules and actin during cell expansion and division. Correlation of cytoskeletal dynamics with cell-wall development included live cell labelling with wall polymer-specific antibodies and electron microscopy. Key Results The cortical cytoplasm of Penium is highlighted by a band of microtubules found at the cell isthmus, i.e. the site of pre-division wall expansion. This band, along with an associated, transient band of actin filaments, probably acts to direct the deposition of new wall material and to mark the plane of the future cell division. Two additional bands of microtubules, which we identify as satellite bands, arise from the isthmus microtubular band at the onset of expansion and displace toward the poles during expansion, ultimately marking the isthmus of future daughter cells. Treatment with microtubule and actin perturbation agents reversibly stops cell division. Conclusions The cortical cytoplasm of Penium contains distinct bands of microtubules and actin filaments that persist through the cell cycle. One of these bands, termed the isthmus microtubule band, or IMB, marks the site of both pre

  2. Molecular Pathways: New Signaling Considerations When Targeting Cytoskeletal Balance to Reduce Tumor Growth.

    PubMed

    Chakrabarti, Kristi R; Hessler, Lindsay; Bhandary, Lekhana; Martin, Stuart S

    2015-12-01

    The dynamic balance between microtubule extension and actin contraction regulates mammalian cell shape, division, and motility, which has made the cytoskeleton an attractive and very successful target for cancer drugs. Numerous compounds in clinical use to reduce tumor growth cause microtubule breakdown (vinca alkaloids, colchicine-site, and halichondrins) or hyperstabilization of microtubules (taxanes and epothilones). However, both of these strategies indiscriminately alter the assembly and dynamics of all microtubules, which causes significant dose-limiting toxicities on normal tissues. Emerging data are revealing that posttranslational modifications of tubulin (detyrosination, acetylation) or microtubule-associated proteins (Tau, Aurora kinase) may allow for more specific targeting of microtubule subsets, thereby avoiding the broad disruption of all microtubule polymerization. Developing approaches to reduce tumor cell migration and invasion focus on disrupting actin regulation by the kinases SRC and ROCK. Because the dynamic balance between microtubule extension and actin contraction also regulates cell fate decisions and stem cell characteristics, disrupting this cytoskeletal balance could yield unexpected effects beyond tumor growth. This review will examine recent data demonstrating that cytoskeletal cancer drugs affect wound-healing responses, microtentacle-dependent reattachment efficiency, and stem cell characteristics in ways that could affect the metastatic potential of tumor cells, both beneficially and detrimentally. PMID:26463706

  3. Aluminum Induces Rigor within the Actin Network of Soybean Cells.

    PubMed Central

    Grabski, S.; Schindler, M.

    1995-01-01

    Aluminum is toxic to both plants and animals. Root growth and pollen-tube extension are inhibited after aluminum stress in acidic environments. Incubation of cultured neurons with aluminum results in the formation of neurofibrillar tangles reminiscent of the neural pathology observed in Alzheimer's disease. The present communication demonstrates that aluminum induces a rapid and dramatic increase in the rigidity of the actin network in soybean (Glycine max) root cells. This rigidity can be prevented by either co-incubation with sodium fluoride or magnesium, or pretreatment with cytochalasin D. It is proposed that the growth-inhibitory activity and cytotoxicity of aluminum in plants may be a consequence of a global rigor that is induced within the actin network. This rigor may result from the formation of nonhydrolyzable [Al3+-ADP] or [Al3+-ATP] complexes whose binding to actin/myosin can modify contraction. Additionally, Al3+-mediated interference with the normal kinetics of F-actin filament assembly/disassembly could precipitate subsequent disorganization of associated cytoskeletal structures and promote altered expression of cytoskeletal proteins. PMID:12228515

  4. Heterotypic and homotypic associations between ezrin and moesin, two putative membrane-cytoskeletal linking proteins.

    PubMed Central

    Gary, R; Bretscher, A

    1993-01-01

    Ezrin and moesin are components of actin-rich cell surface structures that are thought to function as membrane-cytoskeletal linking proteins. Here we show that a stable complex of ezrin and moesin can be isolated from cultured cells by immunoprecipitation with specific antibodies. The capacity of these two proteins to interact directly was confirmed with a blot-overlay procedure in which biotin-tagged proteins in solution were incubated with immobilized binding partners. In addition to the heterotypic association of ezrin and moesin, homotypic binding of ezrin to ezrin and of moesin to moesin was also demonstrated in vitro. These results suggest mechanisms by which ezrin and moesin might participate in dynamic aspects of cortical cytoskeletal structure. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8248180

  5. Prostate Specific Membrane Antigen-Targeted Photodynamic Therapy Induces Rapid Cytoskeletal Disruption

    PubMed Central

    Liu, Tiancheng; Wu, Lisa Y.; Berkman, Clifford E.

    2010-01-01

    Prostate-specific membrane antigen (PSMA), an established enzyme-biomarker for prostate cancer, has attracted considerable attention as a target for imaging and therapeutic applications. We aimed to determine the effects of PSMA-targeted photodynamic therapy (PDT) on cytoskeletal networks in prostate cancer cells. PSMA-targeted PDT resulted in rapid disruption of microtubules (α-/β-tubulin), microfilaments (actin), and intermediate filaments (cytokeratin 8/18) in the cytoplasm of LNCaP cells. The collapse of cytoplasmic microtubules and the later nuclear translocation of α-/β-tubulin were the most dramatic alternation. It is likely that these early changes of cytoskeletal networks are partly involved in the initiation of cell death. PMID:20452720

  6. Rearrangements of Cycloalkenyl Aryl Ethers.

    PubMed

    Törincsi, Mercedesz; Nagy, Melinda; Bihari, Tamás; Stirling, András; Kolonits, Pál; Novak, Lajos

    2016-01-01

    Rearrangement reactions of cycloalkenyl phenol and naphthyl ethers and the acid-catalyzed cyclization of the resulting product were investigated. Claisen rearrangement afforded 2-substituted phenol and naphthol derivatives. Combined Claisen and Cope rearrangement resulted in the formation of 4-substituted phenol and naphthol derivatives. In the case of cycloocthylphenyl ether the consecutive Claisen and Cope rearrangements were followed by an alkyl migration. The mechanism of this novel rearrangement reaction is also discussed. PMID:27104504

  7. Cdc42 and PI(4,5)P2-induced actin assembly in Xenopus egg extracts.

    PubMed

    Lebensohn, Andres M; Ma, Le; Ho, Hsin-Yi Henry; Kirschner, Marc W

    2006-01-01

    Xenopus egg cytoplasmic extracts have been used to study a variety of complex cellular processes. Given their amenability to biochemical manipulation and physiological balance of regulatory proteins, these extracts are an ideal system to dissect signal transduction pathways leading to actin assembly. We have developed methods to study Cdc42 and PI(4,5)P2-induced actin assembly in Xenopus egg extracts. In this chapter, we describe detailed procedures to prepare Xenopus egg extracts, Cdc42, and PI(4,5)P2 for use in actin assembly experiments. We also describe a fluorometric pyrene actin assay for quantitative kinetic analysis of actin polymerization and a microscopic rhodamine actin assay for quick measurement of actin rearrangements in extracts. Finally we provide a protocol for immunodepletion of proteins and discuss the use of immunodepletion and rescue experiments for functional analysis of components in the extracts. PMID:16472657

  8. Cytoskeletal remodeling of rat aortic smooth muscle cells in vitro: relationships to culture conditions and analogies to in vivo situations.

    PubMed

    Skalli, O; Bloom, W S; Ropraz, P; Azzarone, B; Gabbiani, G

    1986-07-01

    Cytoskeletal features of arterial smooth muscle cells (SMC) vary characteristically during development and during atheromatous plaque formation (Gabbiani et al., 1984; Kocher et al., 1985). We have analyzed the cytoskeletal features of rat aortic SMC placed in culture in the presence of 10% foetal calf serum (thus containing growth factors probably playing a role in SMC development and atheroma formation), as compared to SMC freshly isolated from the rat aortic media. Under these conditions, SMC show a typical cytoskeletal remodeling characterized by: 1) increased content of vimentin per cell, increased number of cells containing only vimentin, and decreased number of vimentin plus desmin containing cells; 2) decreased contents of actin, tropomyosin and myosin; 3) a switch in the pattern of actin isoforms with the appearance of a beta-type predominance. Some of these changes (e.g. increase of vimentin and decrease of alpha-type actin) are seen already in cells entering for the first time in S-phase after plating. Pulse-chase experiments with 3H-thymidine (3H-TdR) indicate that vimentin containing SMC possess a higher replicative activity than vimentin plus desmin containing SMC, thus explaining the selection of vimentin containing cells during culture. Our results indicate that during culture SMC develop features similar to those observed in normal foetal SMC or in SMC present in atheromatous plaques; this model may be useful for the understanding of mechanisms leading to SMC differentiation and to atheroma formation. PMID:3528513

  9. Actomyosin contractility spatiotemporally regulates actin network dynamics in migrating cells.

    PubMed

    Okeyo, Kennedy Omondi; Adachi, Taiji; Sunaga, Junko; Hojo, Masaki

    2009-11-13

    Coupling interactions among mechanical and biochemical factors are important for the realization of various cellular processes that determine cell migration. Although F-actin network dynamics has been the focus of many studies, it is not yet clear how mechanical forces generated by actomyosin contractility spatiotemporally regulate this fundamental aspect of cell migration. In this study, using a combination of fluorescent speckle microscopy and particle imaging velocimetry techniques, we perturbed the actomyosin system and examined quantitatively the consequence of actomyosin contractility on F-actin network flow and deformation in the lamellipodia of actively migrating fish keratocytes. F-actin flow fields were characterized by retrograde flow at the front and anterograde flow at the back of the lamellipodia, and the two flows merged to form a convergence zone of reduced flow intensity. Interestingly, activating or inhibiting actomyosin contractility altered network flow intensity and convergence, suggesting that network dynamics is directly regulated by actomyosin contractility. Moreover, quantitative analysis of F-actin network deformation revealed that the deformation was significantly negative and predominant in the direction of cell migration. Furthermore, perturbation experiments revealed that the deformation was a function of actomyosin contractility. Based on these results, we suggest that the actin cytoskeletal structure is a mechanically self-regulating system, and we propose an elaborate pathway for the spatiotemporal self-regulation of the actin cytoskeletal structure during cell migration. In the proposed pathway, mechanical forces generated by actomyosin interactions are considered central to the realization of the various mechanochemical processes that determine cell motility. PMID:19665125

  10. Regulators of Actin Dynamics in Gastrointestinal Tract Tumors

    PubMed Central

    Steinestel, Konrad; Wardelmann, Eva; Hartmann, Wolfgang; Grünewald, Inga

    2015-01-01

    Reorganization of the actin cytoskeleton underlies cell migration in a wide variety of physiological and pathological processes, such as embryonic development, wound healing, and tumor cell invasion. It has been shown that actin assembly and disassembly are precisely regulated by intracellular signaling cascades that respond to changes in the cell microenvironment, ligand binding to surface receptors, or oncogenic transformation of the cell. Actin-nucleating and actin-depolymerizing (ANFs/ADFs) and nucleation-promoting factors (NPFs) regulate cytoskeletal dynamics at the leading edge of migrating cells, thereby modulating cell shape; these proteins facilitate cellular movement and mediate degradation of the surrounding extracellular matrix by secretion of lytic proteases, thus eliminating barriers for tumor cell invasion. Accordingly, expression and activity of these actin-binding proteins have been linked to enhanced metastasis and poor prognosis in a variety of malignancies. In this review, we will summarize what is known about expression patterns and the functional role of actin regulators in gastrointestinal tumors and evaluate first pharmacological approaches to prevent invasion and metastatic dissemination of malignant cells. PMID:26345720

  11. Regulators of Actin Dynamics in Gastrointestinal Tract Tumors.

    PubMed

    Steinestel, Konrad; Wardelmann, Eva; Hartmann, Wolfgang; Grünewald, Inga

    2015-01-01

    Reorganization of the actin cytoskeleton underlies cell migration in a wide variety of physiological and pathological processes, such as embryonic development, wound healing, and tumor cell invasion. It has been shown that actin assembly and disassembly are precisely regulated by intracellular signaling cascades that respond to changes in the cell microenvironment, ligand binding to surface receptors, or oncogenic transformation of the cell. Actin-nucleating and actin-depolymerizing (ANFs/ADFs) and nucleation-promoting factors (NPFs) regulate cytoskeletal dynamics at the leading edge of migrating cells, thereby modulating cell shape; these proteins facilitate cellular movement and mediate degradation of the surrounding extracellular matrix by secretion of lytic proteases, thus eliminating barriers for tumor cell invasion. Accordingly, expression and activity of these actin-binding proteins have been linked to enhanced metastasis and poor prognosis in a variety of malignancies. In this review, we will summarize what is known about expression patterns and the functional role of actin regulators in gastrointestinal tumors and evaluate first pharmacological approaches to prevent invasion and metastatic dissemination of malignant cells. PMID:26345720

  12. Endosomal trafficking of the receptor tyrosine kinase MuSK proceeds via clathrin-dependent pathways, Arf6 and actin

    PubMed Central

    Luiskandl, Susan; Woller, Barbara; Schlauf, Marlies; Schmid, Johannes A; Herbst, Ruth

    2013-01-01

    Muscle-specific kinase (MuSK), a receptor tyrosine kinase, is the key player during the formation of the neuromuscular junction. Signal transduction events downstream of MuSK activation induce both pre-and postsynaptic differentiation, which, most prominently, includes the clustering of acetylcholine receptors at synaptic sites. More recently, regulated MuSK endocytosis and degradation have been implicated as crucial events for MuSK signalling activity, implicating a cross-talk between signalling and endocytosis. In the present study, we use a live imaging approach to study MuSK endocytosis. We find that MuSK is internalized via a clathrin-, dynamin-dependent pathway. MuSK is transported to Rab7-positive endosomes for degradation and recycled via Rab4-and Rab11-positive vesicles. MuSK activation by Dok7 mildly affects the localization of MuSK on the cell surface but has no effect on the rate of MuSK internalization. Interestingly, MuSK colocalizes with actin and Arf6 at the cell surface and during endosomal trafficking. Disruption of the actin cytoskeleton or the proper function of Arf6 concentrates MuSK in cell protrusions. Moreover, inhibition of Arf6 or cytoskeletal rearrangements impairs acetylcholine receptor clustering and phosphorylation. These results suggest that MuSK uses both classical and nonclassical endosomal pathways that involve a variety of different components of the endosomal machinery. Structured digital abstract MuSK and Arf6 colocalize by fluorescence microscopy (View Interaction: 1, 2) MuSK and Rab4 colocalize by fluorescence microscopy (View interaction) MuSK and Rab11 colocalize by fluorescence microscopy (View interaction) MuSK and Rab7 colocalize by fluorescence microscopy (View interaction) PMID:23621612

  13. 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

  14. Actin in Herpesvirus Infection

    PubMed Central

    Roberts, Kari L.; Baines, Joel D.

    2011-01-01

    Actin is important for a variety of cellular processes, including uptake of extracellular material and intracellular transport. Several emerging lines of evidence indicate that herpesviruses exploit actin and actin-associated myosin motors for viral entry, intranuclear transport of capsids, and virion egress. The goal of this review is to explore these processes and to highlight potential future directions for this area of research. PMID:21994736

  15. Labial Salivary Glands in Infants: Histochemical Analysis of Cytoskeletal and Antimicrobial Proteins.

    PubMed

    Stoeckelhuber, Mechthild; Loeffelbein, Denys J; Olzowy, Bernhard; Schmitz, Christoph; Koerdt, Steffen; Kesting, Marco R

    2016-08-01

    Human labial glands secrete mucous and serous substances for maintaining oral health. The normal microbial flora of the oral cavity is regulated by the acquired and innate immune systems. The localization and distribution of proteins of the innate immune system were investigated in serous acinar cells and the ductal system by the method of immunohistochemistry. Numerous antimicrobial proteins could be detected in the labial glands: β-defensin-1, -2, -3; lysozyme; lactoferrin; and cathelicidin. Cytoskeletal components such as actin, myosin II, cytokeratins 7 and 19, α- and β-tubulin were predominantly observed in apical cell regions and may be involved in secretory activities. PMID:27439958

  16. Actin from Saccharomyces cerevisiae.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

    Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth. Images PMID:6217414

  17. Actin Rings of Power.

    PubMed

    Schwayer, Cornelia; Sikora, Mateusz; Slováková, Jana; Kardos, Roland; Heisenberg, Carl-Philipp

    2016-06-20

    Circular or ring-like actin structures play important roles in various developmental and physiological processes. Commonly, these rings are composed of actin filaments and myosin motors (actomyosin) that, upon activation, trigger ring constriction. Actomyosin ring constriction, in turn, has been implicated in key cellular processes ranging from cytokinesis to wound closure. Non-constricting actin ring-like structures also form at cell-cell contacts, where they exert a stabilizing function. Here, we review recent studies on the formation and function of actin ring-like structures in various morphogenetic processes, shedding light on how those different rings have been adapted to fulfill their specific roles. PMID:27326928

  18. 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. PMID:11543284

  19. 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.

  20. Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis

    NASA Technical Reports Server (NTRS)

    Ingber, D. E.; Prusty, D.; Sun, Z.; Betensky, H.; Wang, N.

    1995-01-01

    Capillary endothelial cells can be switched between growth and differentiation by altering cell-extracellular matrix interactions and thereby, modulating cell shape. Studies were carried out to determine when cell shape exerts its growth-regulatory influence during cell cycle progression and to explore the role of cytoskeletal structure and mechanics in this control mechanism. When G0-synchronized cells were cultured in basic fibroblast growth factor (FGF)-containing defined medium on dishes coated with increasing densities of fibronectin or a synthetic integrin ligand (RGD-containing peptide), cell spreading, nuclear extension, and DNA synthesis all increased in parallel. To determine the minimum time cells must be adherent and spread on extracellular matrix (ECM) to gain entry into S phase, cells were removed with trypsin or induced to retract using cytochalasin D at different times after plating. Both approaches revealed that cells must remain extended for approximately 12-15 h and hence, most of G1, in order to enter S phase. After this restriction point was passed, normally 'anchorage-dependent' endothelial cells turned on DNA synthesis even when round and in suspension. The importance of actin-containing microfilaments in shape-dependent growth control was confirmed by culturing cells in the presence of cytochalasin D (25-1000 ng ml-1): dose-dependent inhibition of cell spreading, nuclear extension, and DNA synthesis resulted. In contrast, induction of microtubule disassembly using nocodazole had little effect on cell or nuclear spreading and only partially inhibited DNA synthesis. Interestingly, combination of nocodazole with a suboptimal dose of cytochalasin D (100 ng ml-1) resulted in potent inhibition of both spreading and growth, suggesting that microtubules are redundant structural elements which can provide critical load-bearing functions when microfilaments are partially compromised. Similar synergism between nocodazole and cytochalasin D was observed

  1. The cubyl cation rearrangements.

    PubMed

    Jalife, Said; Mondal, Sukanta; Cabellos, Jose Luis; Martinez-Guajardo, Gerardo; Fernandez-Herrera, Maria A; Merino, Gabriel

    2016-02-25

    Born-Oppenheimer molecular dynamics simulations and high-level ab initio computations predict that the cage-opening rearrangement of the cubyl cation to the 7H(+)-pentalenyl cation is feasible in the gas phase. The rate-determining step is the formation of the cuneyl cation with an activation barrier of 25.3 kcal mol(-1) at the CCSD(T)/def2-TZVP//MP2/def2-TZVP level. Thus, the cubyl cation is kinetically stable enough to be formed and trapped at moderate temperatures, but it may be rearranged at higher temperatures. PMID:26880646

  2. Cooperation of the BTB-Zinc finger protein, Abrupt, with cytoskeletal regulators in Drosophila epithelial tumorigenesis

    PubMed Central

    Turkel, Nezaket; Portela, Marta; Poon, Carole; Li, Jason; Brumby, Anthony M.; Richardson, Helena E.

    2015-01-01

    ABSTRACT The deregulation of cell polarity or cytoskeletal regulators is a common occurrence in human epithelial cancers. Moreover, there is accumulating evidence in human epithelial cancer that BTB-ZF genes, such as Bcl6 and ZBTB7A, are oncogenic. From our previous studies in the vinegar fly, Drosophila melanogaster, we have identified a cooperative interaction between a mutation in the apico-basal cell polarity regulator Scribble (Scrib) and overexpression of the BTB-ZF protein Abrupt (Ab). Herein, we show that co-expression of ab with actin cytoskeletal regulators, RhoGEF2 or Src64B, in the developing eye-antennal epithelial tissue results in the formation of overgrown amorphous tumours, whereas ab and DRac1 co-expression leads to non-cell autonomous overgrowth. Together with ab, these genes affect the expression of differentiation genes, resulting in tumours locked in a progenitor cell fate. Finally, we show that the expression of two mammalian genes related to ab, Bcl6 and ZBTB7A, which are oncogenes in mammalian epithelial cancers, significantly correlate with the upregulation of cytoskeletal genes or downregulation of apico-basal cell polarity neoplastic tumour suppressor genes in colorectal, lung and other human epithelial cancers. Altogether, this analysis has revealed that upregulation of cytoskeletal regulators cooperate with Abrupt in Drosophila epithelial tumorigenesis, and that high expression of human BTB-ZF genes, Bcl6 and ZBTB7A, shows significant correlations with cytoskeletal and cell polarity gene expression in specific epithelial tumour types. This highlights the need for further investigation of the cooperation between these genes in mammalian systems. PMID:26187947

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

    PubMed

    Tang, Dale D

    2015-01-01

    Asthma is characterized by airway hyperresponsiveness and airway remodeling, which are largely attributed to increased airway smooth muscle contractility and cell proliferation. It is known that both chemical and mechanical stimulation regulates smooth muscle contraction. Recent studies suggest that contractile activation and mechanical stretch induce actin cytoskeletal remodeling in smooth muscle. However, the mechanisms that control actin cytoskeletal reorganization are not completely elucidated. This review summarizes our current understanding regarding how actin-associated proteins may regulate remodeling of the actin cytoskeleton in airway smooth muscle. In particular, there is accumulating evidence to suggest that Abelson tyrosine kinase (Abl) plays a critical role in regulating airway smooth muscle contraction and cell proliferation in vitro, and airway hyperresponsiveness and remodeling in vivo. These studies indicate that Abl may be a novel target for the development of new therapy to treat asthma. PMID:26517982

  4. A Steric Antagonism of Actin Polymerization by a Salmonella Virulence Protein

    SciTech Connect

    Margarit,S.; Davidson, W.; Frego, L.; Stebbins, F.

    2006-01-01

    Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.

  5. The degree of resistance of erythrocyte membrane cytoskeletal proteins to supra-physiologic concentrations of calcium: an in vitro study.

    PubMed

    Mostafavi, Ebrahim; Nargesi, Arash Aghajani; Ghazizadeh, Zaniar; Larry, Mehrdad; Farahani, Roya Horabad; Morteza, Afsaneh; Esteghamati, Alireza; Vigneron, Claude; Nakhjavani, Manouchehr

    2014-08-01

    Calcium is a key regulator of cell dynamics. Dysregulation of its cytosolic concentration is implicated in the pathophysiology of several diseases. This study aimed to assess the effects of calcium on the network of membrane cytoskeletal proteins. Erythrocyte membranes were obtained from eight healthy donors and incubated with 250 µM and 1.25 mM calcium solutions. Membrane cytoskeletal proteins were quantified using SDS-PAGE at baseline and after 3 and 5 days of incubation. Supra-physiologic concentrations of calcium (1.25 mM) induced a significant proteolysis in membrane cytoskeletal proteins, compared with magnesium (p < 0.001). Actin exhibited the highest sensitivity to calcium-induced proteolysis (6.8 ± 0.3 vs. 5.3 ± 0.6, p < 0.001), while spectrin (39.9 ± 1.0 vs. 40.3 ± 2.0, p = 0.393) and band-6 (6.3 ± 0.3 vs. 6.8 ± 0.8, p = 0.191) were more resistant to proteolysis after incubation with calcium in the range of endoplasmic reticulum concentrations (250 µM). Aggregation of membrane cytoskeletal proteins was determined after centrifugation and was significantly higher after incubation with calcium ions compared with control, EDTA and magnesium solutions (p < 0.001). In a supra-physiologic range of 1.25-10 mM of calcium ions, there was a nearly perfect linear relationship between calcium concentration and aggregation of erythrocyte membrane cytoskeletal proteins (R(2) = 0.971, p < 0.001). Our observation suggests a strong interaction between calcium ions and membrane cytoskeletal network. Cumulative effects of disrupted calcium homeostasis on cytoskeletal proteins need to be further investigated at extended periods of time in disease states. PMID:24930024

  6. The antioxidant function of Bcl-2 preserves cytoskeletal stability of cells with defective respiratory complex I.

    PubMed

    Porcelli, A M; Ghelli, A; Iommarini, L; Mariani, E; Hoque, M; Zanna, C; Gasparre, G; Rugolo, M

    2008-09-01

    Human thyroid carcinoma XTC.UC1 cells harbor a homoplasmic frameshift mutation in the MT-ND1 subunit of respiratory complex I. When forced to use exclusively oxidative phosphorylation for energy production by inhibiting glycolysis, these cells triggered a caspase-independent cell death pathway, which was associated to a significant imbalance in glutathione homeostasis and a cleavage of the actin cytoskeleton. Overexpression of the anti-apoptotic Bcl-2 protein significantly increased the level of endogenous reduced glutathione, thus preventing its oxidation after the metabolic stress. Furthermore, Bcl-2 completely inhibited actin cleavage and increased cell adhesion, but was unable to improve cellular viability. Similar effects were obtained when XTC.UC1 cells were incubated with exogenous glutathione. We hence propose that Bcl-2 can safeguard cytoskeletal stability through an antioxidant function. PMID:18695940

  7. Cytoarchitecture of Kirsten sarcoma virus-transformed rat kidney fibroblasts: butyrate-induced reorganization within the actin microfilament network.

    PubMed

    Ryan, M P; Higgins, P J

    1988-10-01

    Murine sarcoma virus-transformed rat fibroblasts (KNRK cells) undergo marked cytoarchitectural reorganization during in vitro exposure to sodium-n-butyrate (NaB) resulting in restoration of (1) a more typical fibroblastoid morphology, (2) proper cell-to-cell orientation, and (3) substratum adherence. Augmented cell spreading, involving greater than 90% of the population, was a function of culture density and time of exposure to NaB (2 mM final concentration). Induced cell spreading reflected a 2.5- to 3.0-fold increase in both total cellular actin content and deposition of actin into the detergent-resistant cytoskeleton. Cytoskeletal actin deposition in response to NaB was accompanied by the formation of occasionally dense, parallel alignments of F-actin-containing microfilaments and by a dramatic increase in the size and incidence of actin-enriched membrane ruffles. Long-term NaB-treated cells exhibited parallel orientations of microfilaments similar to those found in untransformed fibroblasts. Increased cytoskeletal actin occurred within 24 hr of NaB exposure, correlating with the initial reorganization of actin-containing microfilaments detected microscopically, and reflected concomitant 3-fold increases in cellular alpha-actinin and fibronectin content. In contrast, the amount of vimentin, tropomyosin, and tubulin in NaB-treated cells was significantly decreased. NaB-induced morphologic restructuring of sarcoma virus-transformed fibroblasts, thus, impacts on all three basic cytoskeletal systems. Selective increases, however, were evident in particular cytoskeletal proteins (actin, alpha-actinin, fibronectin) implicated in microfilament networking and cell spreading. PMID:2844835

  8. Direct dynamin–actin interactions regulate the actin cytoskeleton

    PubMed Central

    Gu, Changkyu; Yaddanapudi, Suma; Weins, Astrid; Osborn, Teresia; Reiser, Jochen; Pollak, Martin; Hartwig, John; Sever, Sanja

    2010-01-01

    The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs. PMID:20935625

  9. Cytoskeletal and functional changes in bioreactor assembled thyroid tissue organoids exposed to gamma radiation

    NASA Technical Reports Server (NTRS)

    Green, Lora M.; Patel, Zarana; Murray, Deborah K.; Rightnar, Steven; Burell, Cheryl G.; Gridley, Daila S.; Nelson, Gregory A.

    2002-01-01

    Fischer rat thyroid cells were grown under low-shear stress in a bioreactor to a stage of organization composed of integrated follicles resembling small thyroid glands prior to exposure to 3 Gray-gamma radiation. Bioreactor tissues and controls (both irradiated and non-irradiated) were harvested at 24, 48, 96 and 144 hours post-exposure. Tissue samples were fixed and fluorescently labeled for actin and microtubules. Tissues were assessed for changes in cytoskeletal components induced by radiation and quantified by laser scanning cytometry. ELISA's were used to quantify transforming growth factor-beta and thyroxin released from cells to the culture supernatant. Tissue architecture was disrupted by exposure to radiation with the structural organization of actin and loss of follicular content the most obviously affected. With time post-irradiation the actin appeared disordered and the levels of fluorescence associated with filamentous-actin and microtubules cycled in the tissue analogs, but not in the flask-grown cultures. Active transforming growth factor-beta was higher in supernatants from the irradiated bioreactor tissue. Thyroxin release paralleled cell survival in the bioreactors and control cultures. Thus, the engineered tissue responses to radiation differed from those of conventional tissue culture making it a potentially better mimic of the in vivo situation.

  10. Rapid non-equilibrium turnover fluidizes entangled F-actin solutions

    NASA Astrophysics Data System (ADS)

    McCall, Patrick M.; Kovar, David R.; Gardel, Margaret L.

    The actin cytoskeleton of living cells is a semiflexible polymer network which regulates cell division, motility, and morphogenesis by controlling cell shape. These complex shape-changing processes require both mechanical deformation and remodeling of the actin cytoskeleton. Molecular motors generate internal forces to drive deformation, while cytoskeletal remodeling is regulated by non-equilibrium polymer turnover. Although the mechanical properties of equilibrium actin filament (F-actin) networks are well-described by theories of semiflexible polymers, these theories do not incorporate the effects of non-equilibrium turnover. To address this experimentally, we developed a model system in which both the turnover rate and the length distribution of purified F-actin can be tuned independently at steady-state through the combined action of actin regulatory proteins. Specifically we tune the concentrations of cofilin, profilin, and formin to regulate F-actin severing, recycling, and nucleation, respectively. We find that the actin turnover rate can be tuned by cofilin up to 25-fold (31 +/- 2 subunits/sec/filament). Surprisingly, changes in turnover rate have no effect on the steady-state F-actin length distribution, which is instead set by formin concentration. Passive microrheology measurements show that increased turnover leads to striking fluidization in both entangled and crosslinked networks. Non-equilibrium turnover thus enables modulation of network mechanics, which impacts force transmission and material deformation.

  11. Three-Dimensional Structure of Cofilin Bound to Monomeric Actin Derived by Structural Mass Spectrometry Data

    SciTech Connect

    Amisha Kamal,J.; Benchaar, S.; Takamoto, K.; Reisler, E.; Chance, M.

    2007-01-01

    The cytoskeletal protein, actin, has its structure and function regulated by cofilin. In the absence of an atomic resolution structure for the actin/cofilin complex, the mechanism of cofilin regulation is poorly understood. Theoretical studies based on the similarities of cofilin and gelsolin segment 1 proposed the cleft between subdomains 1 and 3 in actin as the cofilin binding site. We used radiolytic protein footprinting with mass spectrometry and molecular modeling to provide an atomic model of how cofilin binds to monomeric actin. Footprinting data suggest that cofilin binds to the cleft between subdomains 1 and 2 in actin and that cofilin induces further closure of the actin nucleotide cleft. Site-specific fluorescence data confirm these results. The model identifies key ionic and hydrophobic interactions at the binding interface, including hydrogen-bonding between His-87 of actin to Ser-89 of cofilin that may control the charge dependence of cofilin binding. This model and its implications fill an especially important niche in the actin field, owing to the fact that ongoing crystallization efforts of the actin/cofilin complex have so far failed. This 3D binary complex structure is derived from a combination of solution footprinting data and computational approaches and outlines a general method for determining the structure of such complexes.

  12. Molecular Architecture of Synaptic Actin Cytoskeleton in Hippocampal Neurons Reveals a Mechanism of Dendritic Spine Morphogenesis

    PubMed Central

    Korobova, Farida

    2010-01-01

    Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility. PMID:19889835

  13. Cortactin promotes exosome secretion by controlling branched actin dynamics.

    PubMed

    Sinha, Seema; Hoshino, Daisuke; Hong, Nan Hyung; Kirkbride, Kellye C; Grega-Larson, Nathan E; Seiki, Motoharu; Tyska, Matthew J; Weaver, Alissa M

    2016-07-18

    Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites. PMID:27402952

  14. Direct interaction of microtubule- and actin-based transport motors

    NASA Technical Reports Server (NTRS)

    Huang, J. D.; Brady, S. T.; Richards, B. W.; Stenolen, D.; Resau, J. H.; Copeland, N. G.; Jenkins, N. A.

    1999-01-01

    The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.

  15. Phosphonate–phosphinate rearrangement.

    PubMed

    Qian, Renzhe; Roller, Alexander; Hammerschmidt, Friedrich

    2015-01-16

    LiTMP metalated dimethyl N-Boc-phosphoramidates derived from 1-phenylethylamine and 1,2,3,4-tetrahydronaphthalen-1-ylamine highly selectively at the CH3O group to generate short-lived oxymethyllithiums. These isomerized to diastereomeric hydroxymethylphosphonamidates (phosphate–phosphonate rearrangement). However, s-BuLi converted the dimethyl N-Boc-phosphoramidate derived from 1-phenylethylamine to the N-Boc α-aminophosphonate preferentially. Only s-BuLi deprotonated dimethyl hydroxymethylphosphonamidates at the benzylic position and dimethyl N-Boc α-aminophosphonates at the CH3O group to induce phosphonate–phosphinate rearrangements. In the former case, the migration of the phosphorus substituent from the nitrogen to the carbon atom followed a retentive course with some racemization because of the involvement of a benzyllithium as an intermediate. PMID:25525945

  16. Zonal variations in cytoskeletal element organization, mRNA and protein expression in the intervertebral disc

    PubMed Central

    Li, Siyuan; Duance, Victor C; Blain, Emma J

    2008-01-01

    The intervertebral disc is important in maintaining flexibility and dissipating loads applied to the spine. The disc comprises a heterogeneous population of cells, including those of the nucleus pulposus and annulus fibrosus, which are diverse in phenotype, partly due to the different mechanical loads they experience. Several studies have implicated the cytoskeleton in mechanotransduction, but little characterization of the three major cytoskeletal elements – actin, tubulin and vimentin – in the intervertebral disc has been undertaken. In this study we show that there are differences in both the organization and the amounts of these cytoskeletal proteins across the regions of immature bovine intervertebral disc (nucleus pulposus and outer annulus fibrosus), which differs with skeletal maturity. These differences are likely to reflect the diverse mechanical characteristics of the disc regions, and the loads that they experience, i.e. tension in the annulus fibrosus and compression in the nucleus pulposus. Alterations to the organization and amount of cytoskeletal element proteins may change the ability of the cells to respond to mechanical signals, with a loss of tissue homeostasis, suggesting that the cytoskeleton has a potential role in intervertebral disc degeneration. PMID:19094188

  17. Cytoskeletal to Nuclear Strain Transfer Regulates YAP Signaling in Mesenchymal Stem Cells

    PubMed Central

    Driscoll, Tristan P.; Cosgrove, Brian D.; Heo, Su-Jin; Shurden, Zach E.; Mauck, Robert L.

    2015-01-01

    Mechanical forces transduced to cells through the extracellular matrix are critical regulators of tissue development, growth, and homeostasis, and can play important roles in directing stem cell differentiation. In addition to force-sensing mechanisms that reside at the cell surface, there is growing evidence that forces transmitted through the cytoskeleton and to the nuclear envelope are important for mechanosensing, including activation of the Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) pathway. Moreover, nuclear shape, mechanics, and deformability change with differentiation state and have been likewise implicated in force sensing and differentiation. However, the significance of force transfer to the nucleus through the mechanosensing cytoskeletal machinery in the regulation of mesenchymal stem cell mechanobiologic response remains unclear. Here we report that actomyosin-generated cytoskeletal tension regulates nuclear shape and force transmission through the cytoskeleton and demonstrate the differential short- and long-term response of mesenchymal stem cells to dynamic tensile loading based on the contractility state, the patency of the actin cytoskeleton, and the connections it makes with the nucleus. Specifically, we show that while some mechanoactive signaling pathways (e.g., ERK signaling) can be activated in the absence of nuclear strain transfer, cytoskeletal strain transfer to the nucleus is essential for activation of the YAP/TAZ pathway with stretch. PMID:26083918

  18. Loss of cytoskeletal proteins and lens cell opacification in the selenite cataract model.

    PubMed

    Matsushima, H; David, L L; Hiraoka, T; Clark, J I

    1997-03-01

    This study of lens protein composition found that some cytoskeletal proteins were degraded during the earliest stages of cataract formation. Cataract was induced in 13-14 day old rats by a single subcutaneous injection of sodium selenite (19 mumol kg-1). By 24 hr after the injection of selenite, the ratio of insoluble to soluble protein increased as lens opacification began. The increase in insoluble protein aggregates was correlated with an accelerated loss of proteins having molecular weights of 42, 55/57 and 235 kDa which reacted with antibodies to the cytoskeletal proteins actin, tubulin/vimentin and spectrin, respectively. We observed the loss of 49, 60 and 90 kDa proteins which were not identified. In the lenses of animals protected from protein aggregation and opacification by administration of 1.5 mmol kg-1 pantethine, the pattern of proteins in SDS-PAGE gels resembled the pattern for proteins from transparent lenses of normal untreated animals and loss of cytoskeletal proteins was prevented. PMID:9196390

  19. Disruption of cytoskeletal structures mediates shear stress-induced endothelin-1 gene expression in cultured porcine aortic endothelial cells.

    PubMed Central

    Morita, T; Kurihara, H; Maemura, K; Yoshizumi, M; Yazaki, Y

    1993-01-01

    Hemodynamic shear stress alters the architecture and functions of vascular endothelial cells. We have previously shown that the synthesis of endothelin-1 (ET-1) in endothelial cells is increased by exposure to shear stress. Here we examined whether shear stress-induced alterations in cytoskeletal structures are responsible for increases in ET-1 synthesis in cultured porcine aortic endothelial cells. Exposure of endothelial cells to 5 dyn/cm2 of low shear stress rapidly increased monomeric G-actin contents within 5 min without changing total actin contents. The ratio of G- to total actin, 54 +/- 0.8% in quiescent endothelial cells, increased to 87 +/- 4.2% at 6 h and then decreased. Following the disruption of filamentous (F)-actin into G-actin, ET-1 mRNA levels in endothelial cells also increased within 30 min and reached a peak at 6 h. The F-actin stabilizer, phalloidin, abolished shear stress-induced increases in ET-1 mRNA; however, it failed to inhibit increases in ET-1 mRNA secondary to other stimulants. This indicates that shear stress-induced increases in ET-1 mRNA levels may be mediated by the disruption of actin fibers. Furthermore, increases in ET-1 gene expression can be induced by actin-disrupting agents, cytochalasin B and D. Another cytoskeleton-disrupting agent, colchicine, which inhibits dimerization of tubulin, did not affect the basal level of ET-1 mRNA. However, colchicine completely inhibited shear stress- and cytochalasin B-induced increases in ET-1 mRNA levels. These results suggest that shear stress-induced ET-1 gene expression in endothelial cells is mediated by the disruption of actin cytoskeleton and this induction is dependent on the integrity of microtubules. Images PMID:8408624

  20. Epigenetic repression of ribosomal RNA transcription by ROCK-dependent aberrant cytoskeletal organization

    PubMed Central

    Wu, Tse-Hsiang; Kuo, Yuan-Yeh; Lee, Hsiao-Hui; Kuo, Jean-Cheng; Ou, Meng-Hsin; Chang, Zee-Fen

    2016-01-01

    It is known that ribosomal RNA (rRNA) synthesis is regulated by cellular energy and proliferation status. In this study, we investigated rRNA gene transcription in response to cytoskeletal stress. Our data revealed that the cell shape constrained by isotropic but not elongated micropatterns in HeLa cells led to a significant reduction in rRNA transcription dependent on ROCK. Expression of a dominant-active form of ROCK also repressed rRNA transcription. Isotropic constraint and ROCK over-activation led to different types of aberrant F-actin organization, but their suppression effects on rRNA transcription were similarly reversed by inhibition of histone deacetylase (HDAC) or overexpression of a dominant negative form of Nesprin, which shields the signal transmitted from actin filament to the nuclear interior. We further showed that the binding of HDAC1 to the active fraction of rDNA genes is increased by ROCK over-activation, thus reducing H3K9/14 acetylation and suppressing transcription. Our results demonstrate an epigenetic control of active rDNA genes that represses rRNA transcription in response to the cytoskeletal stress. PMID:27350000

  1. Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure

    NASA Technical Reports Server (NTRS)

    Maniotis, A. J.; Chen, C. S.; Ingber, D. E.

    1997-01-01

    We report here that living cells and nuclei are hard-wired such that a mechanical tug on cell surface receptors can immediately change the organization of molecular assemblies in the cytoplasm and nucleus. When integrins were pulled by micromanipulating bound microbeads or micropipettes, cytoskeletal filaments reoriented, nuclei distorted, and nucleoli redistributed along the axis of the applied tension field. These effects were specific for integrins, independent of cortical membrane distortion, and were mediated by direct linkages between the cytoskeleton and nucleus. Actin microfilaments mediated force transfer to the nucleus at low strain; however, tearing of the actin gel resulted with greater distortion. In contrast, intermediate filaments effectively mediated force transfer to the nucleus under both conditions. These filament systems also acted as molecular guy wires to mechanically stiffen the nucleus and anchor it in place, whereas microtubules acted to hold open the intermediate filament lattice and to stabilize the nucleus against lateral compression. Molecular connections between integrins, cytoskeletal filaments, and nuclear scaffolds may therefore provide a discrete path for mechanical signal transfer through cells as well as a mechanism for producing integrated changes in cell and nuclear structure in response to changes in extracellular matrix adhesivity or mechanics.

  2. The phosphorylation status and cytoskeletal remodeling of striatal astrocytes treated with quinolinic acid

    SciTech Connect

    Pierozan, Paula; Ferreira, Fernanda; Ortiz de Lima, Bárbara; Gonçalves Fernandes, Carolina; Totarelli Monteforte, Priscila; Castro Medaglia, Natalia de; Bincoletto, Claudia; Soubhi Smaili, Soraya; Pessoa-Pureur, Regina

    2014-04-01

    Quinolinic acid (QUIN) is a glutamate agonist which markedly enhances the vulnerability of neural cells to excitotoxicity. QUIN is produced from the amino acid tryptophan through the kynurenine pathway (KP). Dysregulation of this pathway is associated with neurodegenerative conditions. In this study we treated striatal astrocytes in culture with QUIN and assayed the endogenous phosphorylating system associated with glial fibrillary acidic protein (GFAP) and vimentin as well as cytoskeletal remodeling. After 24 h incubation with 100 µM QUIN, cells were exposed to {sup 32}P-orthophosphate and/or protein kinase A (PKA), protein kinase dependent of Ca{sup 2+}/calmodulin II (PKCaMII) or protein kinase C (PKC) inhibitors, H89 (20 μM), KN93 (10 μM) and staurosporin (10 nM), respectively. Results showed that hyperphosphorylation was abrogated by PKA and PKC inhibitors but not by the PKCaMII inhibitor. The specific antagonists to ionotropic NMDA and non-NMDA (50 µM DL-AP5 and CNQX, respectively) glutamate receptors as well as to metabotropic glutamate receptor (mGLUR; 50 µM MCPG), mGLUR1 (100 µM MPEP) and mGLUR5 (10 µM 4C3HPG) prevented the hyperphosphorylation provoked by QUIN. Also, intra and extracellular Ca{sup 2+} quelators (1 mM EGTA; 10 µM BAPTA-AM, respectively) prevented QUIN-mediated effect, while Ca{sup 2+} influx through voltage-dependent Ca{sup 2+} channel type L (L-VDCC) (blocker: 10 µM verapamil) is not implicated in this effect. Morphological analysis showed dramatically altered actin cytoskeleton with concomitant change of morphology to fusiform and/or flattened cells with retracted cytoplasm and disruption of the GFAP meshwork, supporting misregulation of actin cytoskeleton. Both hyperphosphorylation and cytoskeletal remodeling were reversed 24 h after QUIN removal. Astrocytes are highly plastic cells and the vulnerability of astrocyte cytoskeleton may have important implications for understanding the neurotoxicity of QUIN in neurodegenerative

  3. Actin Mechanics and Fragmentation*

    PubMed Central

    De La Cruz, Enrique M.; Gardel, Margaret L.

    2015-01-01

    Cell physiological processes require the regulation and coordination of both mechanical and dynamical properties of the actin cytoskeleton. Here we review recent advances in understanding the mechanical properties and stability of actin filaments and how these properties are manifested at larger (network) length scales. We discuss how forces can influence local biochemical interactions, resulting in the formation of mechanically sensitive dynamic steady states. Understanding the regulation of such force-activated chemistries and dynamic steady states reflects an important challenge for future work that will provide valuable insights as to how the actin cytoskeleton engenders mechanoresponsiveness of living cells. PMID:25957404

  4. Actin cytoskeleton organization regulated by the PAK family of protein kinases.

    PubMed

    Eby, J J; Holly, S P; van Drogen, F; Grishin, A V; Peter, M; Drubin, D G; Blumer, K J

    1998-08-27

    Cdc42, Rac1 and other Rho-type GTPases regulate gene expression, cell proliferation and cytoskeletal architecture [1,2]. A challenge is to identify the effectors of Cdc42 and Rac1 that mediate these biological responses. Protein kinases of the p21-activated kinase (PAK) family bind activated Rac1 and Cdc42, and switch on mitogen-activated protein (MAP) kinase pathways; however, their roles in regulating actin cytoskeleton organization have not been clearly established [3-5]. Here, we show that mutants of the budding yeast Saccharomyces cerevisiae lacking the PAK homologs Ste20 and Cla4 exhibit actin cytoskeletal defects, in vivo and in vitro, that resemble those of cdc42-1 mutants. Moreover, STE20 overexpression suppresses cdc42-1 growth defects and cytoskeletal defects in vivo, and Ste20 kinase corrects the actin-assembly defects of permeabilized cdc42-1 cells in vitro. Thus, PAKs are effectors of Cdc42 in pathways that regulate the organization of the cortical actin cytoskeleton. PMID:9742399

  5. Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin

    PubMed Central

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G.; Orlova, Albina; Egelman, Edward H.; Beck, Moriah R.

    2016-01-01

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the coordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. Here we show that the actin binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro crosslinking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of G-actin, akin to metal ions, either through charge neutralization or conformational changes. PMID:26607837

  6. 2E4 (Kaptin): A novel actin-associated protein from human blood platelets found in lamellipodia and the tips of the stereocilia of the inner ear

    PubMed Central

    Bearer, Elaine L.; Abraham, Manoj T.

    2010-01-01

    Actin – 2E4/kaptin – platelet activation – stereocilia – sensory epithelium Platelet activation, crucial for hemostasis, requires actin polymerization, yet the molecular mechanisms by which localized actin polymerization is mediated are not clear. Here we report the characterization of a novel actin-binding protein. 2E4, originally isolated from human blood platelets and likely to be involved in the actin rearrangements occurring during activation. 2E4 binds to filamentous (F)-actin by F-actin affinity chromatography and is eluted from F-actin affinity columns and extracted from cells with ATP. Its presence at the leading edge of platelets spread on glass and in the lamellipodia of motile fibroblasts suggests a role in actin dynamics. Using localization to obtain clues about function, we stained the sensory epithelium of the embryonic inner car to determine whether 2E4 is at the barbed end of actin filaments during their elongation. Indeed, 2E4 was present at the tips of the elongating stereocilium. 2E4 is novel by DNA sequence and has no identifiable structural motifs. Its unusual amino acid sequence, its ATP-sensitive actin association and its location at sites of actin polymerization in cells suggest 2E4 plays a unique role in the actin rearrangements that accompany platelet activation and stereocilia formation. PMID:10099934

  7. Multiscale Modelling for investigating single molecule effects on the mechanics of actin filaments

    NASA Astrophysics Data System (ADS)

    A, Deriu Marco; C, Bidone Tamara; Laura, Carbone; Cristina, Bignardi; M, Montevecchi Franco; Umberto, Morbiducci

    2011-12-01

    This work presents a preliminary multiscale computational investigation of the effects of nucleotides and cations on the mechanics of actin filaments (F-actin). At the molecular level, Molecular Dynamics (MD) simulations are employed to characterize the rearrangements of the actin monomers (G-actin) in terms of secondary structures evolution in physiological conditions. At the mesoscale level, a coarse grain (CG) procedure is adopted where each monomer is represented by means of Elastic Network Modeling (ENM) technique. At the macroscale level, actin filaments up to hundreds of nanometers are assumed as isotropic and elastic beams and characterized via Rotation Translation Block (RTB) analysis. F-actin bound to adenosine triphosphate (ATP) shows a persistence length around 5 μm, while actin filaments bound to adenosine diphosphate (ADP) have a persistence length of about 3 μm. With magnesium bound to the high affinity binding site of G-actin, the persistence length of F-actin decreases to about 2 μm only in the ADP-bound form of the filament, while the same ion has no effects, in terms of stiffness variation, on the ATP-bound form of F-actin. The molecular mechanisms behind these changes in flexibility are herein elucidated. Thus, this study allows to analyze how the local binding of cations and nucleotides on G-actin induce molecular rearrangements that transmit to the overall F-actin, characterizing shifts of mechanical properties, that can be related with physiological and pathological cellular phenomena, as cell migration and spreading. Further, this study provides the basis for upcoming investigating of network and cellular remodelling at higher length scales.

  8. The role of actin networks in cellular mechanosensing

    NASA Astrophysics Data System (ADS)

    Azatov, Mikheil

    behavior as in cancer metastasis. In addition to stiffness, the local geometry or topography of the surface has been shown to modulate the movement, morphology, and cytoskeletal organization of cells. However, the effect of topography on fluctuations of intracellular structures, which arise from motor driven activity on a viscoelastic actin network are not known. I have used nanofabricated substrates with parallel ridges to show that the cell shape, the actin cytoskeleton and focal adhesions all align along the direction of the ridges, exhibiting a biphasic dependence on the spacing between ridges. I further demonstrated that palladin bands along actin stress fibers undergo a complex diffusive motion with velocities aligned along the direction of ridges. These results provide insight into the mechanisms of cellular mechanosensing of the environment, suggesting a complex interplay between the actin cytoskeleton and cellular adhesions in coordinating cellular response to surface topography. Overall, this work has advanced our understanding of mechanisms that govern cellular responses to their physical environment.

  9. Effect of abscisic acid and cold acclimation on the cytoskeletal and phosphorylated proteins in different cultivars of Triticum aestivum L.

    PubMed

    Olinevich, O V; Khokhlova, L P; Raudaskoski, M

    2000-01-01

    In winter wheat, the tubulin and 60 kDa-phosphorylated proteins/actin ratio is considerably higher in the roots than in the leaves. Differences in the content of the main cytoskeletal proteins were also found in the leaves of the different cultivars. It is suggested that the lower amount of the tubulin and 60 kDa-phosphorylated proteins and higher content of actin determine the greater tubulin cytoskeletal stability in the leaves and their higher frost resistance, as compared with the roots. Also, it is possible that the higher content of the tubulin and 60 kDa-phosphorylated proteins defines the lower microtubule (MT) stability in the leaves of the low frost resistant cultivar than in the leaves of the more frost resistant ones. In the roots and leaves of the low frost resistant cultivar, the low stability of the numerous tubulin structures is apparently one reason for the abscisic acid (ABA)-induced reduction of the cytoskeletal and 60 kDa-phosphorylated proteins in the cells. The cold acclimation compensated the ABA effect in the roots of the very frost resistant cultivar in the most extent. This suggests the existence of the different pathways in the increased plant cell frost resistance through the action of ABA and low temperature. PMID:10860572

  10. Cytoskeletal disruption activates the DLK/JNK pathway, which promotes axonal regeneration and mimics a preconditioning injury

    PubMed Central

    Valakh, Vera; Frey, Erin; Babetto, Elisabetta; Walker, Lauren J; DiAntonio, Aaron

    2015-01-01

    Nerve injury can lead to axonal regeneration, axonal degeneration, and/or neuronal cell death. Remarkably, the MAP3K dual leucine zipper kinase, DLK, promotes each of these responses, suggesting that DLK is a sensor of axon injury. In Drosophila, mutations in proteins that stabilize the actin and microtubule cytoskeletons activate the DLK pathway, suggesting that DLK may be activated by cytoskeletal disruption. Here we test this model in mammalian sensory neurons. We find that pharmacological agents designed to disrupt either the actin or microtubule cytoskeleton activate the DLK pathway, and that activation is independent of calcium influx or induction of the axon degeneration program. Moreover, activation of the DLK pathway by targeting the cytoskeleton induces a pro-regenerative state, enhancing axon regeneration in response to a subsequent injury in a process akin to preconditioning. This highlights the potential utility of activating the DLK pathway as a method to improve axon regeneration. Moreover, DLK is required for these responses to cytoskeletal perturbations, suggesting that DLK functions as a key neuronal sensor of cytoskeletal damage. PMID:25726747

  11. Actin Automata with Memory

    NASA Astrophysics Data System (ADS)

    Alonso-Sanz, Ramón; Adamatzky, Andy

    Actin is a globular protein which forms long polar filaments in eukaryotic. The actin filaments play the roles of cytoskeleton, motility units, information processing and learning. We model actin filament as a double chain of finite state machines, nodes, which take states “0” and “1”. The states are abstractions of absence and presence of a subthreshold charge on actin units corresponding to the nodes. All nodes update their state in parallel to discrete time. A node updates its current state depending on states of two closest neighbors in the node chain and two closest neighbors in the complementary chain. Previous models of actin automata consider momentary state transitions of nodes. We enrich the actin automata model by assuming that states of nodes depend not only on the current states of neighboring node but also on their past states. Thus, we assess the effect of memory of past states on the dynamics of acting automata. We demonstrate in computational experiments that memory slows down propagation of perturbations, decrease entropy of space-time patterns generated, transforms traveling localizations to stationary oscillators, and stationary oscillations to still patterns.

  12. The Molecular Evolution of Actin

    PubMed Central

    Hightower, Robin C.; Meagher, Richard B.

    1986-01-01

    We have investigated the molecular evolution of plant and nonplant actin genes comparing nucleotide and amino acid sequences of 20 actin genes. Nucleotide changes resulting in amino acid substitutions (replacement substitutions) ranged from 3–7% for all pairwise comparisons of animal actin genes with the following exceptions. Comparisons between higher animal muscle actin gene sequences and comparisons between higher animal cytoplasmic actin gene sequences indicated <3% divergence. Comparisons between plant and nonplant actin genes revealed, with two exceptions, 11–15% replacement substitution. In the analysis of plant actins, replacement substitution between soybean actin genes SAc1, SAc3, SAc4 and maize actin gene MAc1 ranged from 8–10%, whereas these members within the soybean actin gene family ranged from 6–9% replacement substitution. The rate of sequence divergence of plant actin sequences appears to be similar to that observed for animal actins. Furthermore, these and other data suggest that the plant actin gene family is ancient and that the families of soybean and maize actin genes have diverged from a single common ancestral plant actin gene that originated long before the divergence of monocots and dicots. The soybean actin multigene family encodes at least three classes of actin. These classes each contain a pair of actin genes that have been designated kappa (SAc1, SAc6), lambda (SAc2, SAc4) and mu (SAc3, SAc7). The three classes of soybean actin are more divergent in nucleotide sequence from one another than higher animal cytoplasmic actin is divergent from muscle actin. The location and distribution of amino acid changes were compared between actin proteins from all sources. A comparison of the hydropathy of all actin sequences, except from Oxytricha, indicated a strong similarity in hydropathic character between all plant and nonplant actins despite the greater number of replacement substitutions in plant actins. These protein sequence

  13. Thromboxane-induced actin polymerization in hypoxic neonatal pulmonary arterial myocytes involves Cdc42 signaling.

    PubMed

    Fediuk, Jena; Sikarwar, Anurag S; Nolette, Nora; Dakshinamurti, Shyamala

    2014-12-01

    In hypoxic pulmonary arterial (PA) myocytes, challenge with thromboxane mimetic U46619 induces marked actin polymerization and contraction, phenotypic features of persistent pulmonary hypertension of the newborn (PPHN). Rho GTPases regulate the actin cytoskeleton. We previously reported that U46619-induced actin polymerization in hypoxic PA myocytes occurs independently of the RhoA pathway and hypothesized involvement of the Cdc42 pathway. PA myocytes grown in normoxia or hypoxia for 72 h were stimulated with U46619, then analyzed for Rac/Cdc42 activation by affinity precipitation, phosphatidylinositide-3-kinase (PI3K) activity by phospho-Akt, phospho-p21-activated kinase (PAK) by immunoblot, and association of Cdc42 with neuronal Wiskott Aldrich Syndrome protein (N-WASp) by immunoprecipitation. The effect of Rac or PAK inhibition on filamentous actin was quantified by laser-scanning cytometry and by cytoskeletal fractionation; effects of actin-modifying agents were measured by isometric myography. Basal Cdc42 activity increased in hypoxia, whereas Rac activity decreased. U46619 challenge increased Cdc42 and Rac activity in hypoxic cells, independently of PI3K. Hypoxia increased phospho-PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia but increased after U46619 exposure. Hypoxia doubled filamentous-to-globular ratios of α- and γ-actin isoforms. Jasplakinolide stabilized γ-filaments, increasing force; cytochalasin D depolymerized all actin isoforms, decreasing force. Rac and PAK inhibition decreased filamentous actin in tissues although without decrease in force. Rho inhibition decreased myosin phosphorylation and force. Hypoxia induces actin polymerization in PA myocytes, particularly increasing filamentous α- and γ-actin, contributing to U46619-induced contraction. Hypoxic PA myocytes challenged with a thromboxane mimetic polymerize actin via the Cdc42 pathway, reflecting increased Cdc42 association with N-WASp. Mechanisms

  14. Hydrodynamics of pairs of interacting cytoskeletal filaments

    NASA Astrophysics Data System (ADS)

    Shinar, Tamar; Shelley, Michael

    2011-11-01

    Pairwise filament interactions underlie the dynamics of complex cytoskeletal networks in cells. These networks in turn play a crucial role in many cellular processes such as formation of the mitotic spindle and cell cleavage in cytokinesis. We model interactions of pairs of filaments immersed in a viscous, fluidic environment. The filaments are modeled using a slender body approximation, capturing their indirect interactions mediated by the immersing fluid. Direct filament interactions via molecular motors complexes induce alignment and parallel or anti-parallel sliding. The motor proteins are modeled as simple spring-like structures that walk directionally toward one end of the filament. We examine the resulting stresses in the fluid to better understand how the microscopic interactions lead to bulk behavior of cytoskeletal networks.

  15. Cytoskeletal elements in the bacterium Mycoplasma pneumoniae

    NASA Astrophysics Data System (ADS)

    Hegermann, Jan; Herrmann, Richard; Mayer, Frank

    2002-09-01

    Mycoplasma pneumoniae is a pathogenic eubacterium lacking a cell wall. Three decades ago, a "rod", an intracellular cytoskeletal structure, was discovered that was assumed to define and stabilize the elongated cell shape. Later, by treatment with detergent, a "Triton shell" (i.e. a fraction of detergent-insoluble cell material) could be obtained, believed to contain additional cytoskeletal elements. Now, by application of a modified Triton X-100 treatment, we are able to demonstrate that M. pneumoniae possesses a cytoskeleton consisting of a blade-like rod and a peripheral lining located close to the inner face of the cytoplasmic membrane, exhibiting features of a highly regular network. Attached "stalks" may support the cytoplasmic membrane. The rod was connected to the cell periphery by "spokes" and showed a defined ultrastructure. Its proximal end was found to be attached to a wheel-like complex. Fibrils extended from the proximal end of the rod into the cytoplasm.

  16. Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

    NASA Astrophysics Data System (ADS)

    Appert-Rolland, C.; Ebbinghaus, M.; Santen, L.

    2015-09-01

    Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized. The cytoskeleton, which is composed of three types of filaments (microtubules, actin and intermediate filaments), determines the shape of the cell, and plays a role in cell motion. It also serves as a road network for a special kind of vehicles, namely the cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach. It is a central issue to understand how intracellular transport driven by molecular motors is regulated. The interest for this type of question was enhanced when it was discovered that intracellular transport breakdown is one of the signatures of some neuronal diseases like the Alzheimer. We give a survey of the current knowledge on microtubule based intracellular transport. Our review includes on the one hand an overview of biological facts, obtained from experiments, and on the other hand a presentation of some modeling attempts based on cellular automata. We present some background knowledge on the original and variants of the TASEP (Totally Asymmetric Simple Exclusion Process), before turning to more application oriented models. After addressing microtubule based transport in general, with a focus on in vitro experiments, and on cooperative effects in the

  17. A microstructural approach to cytoskeletal mechanics based on tensegrity

    NASA Technical Reports Server (NTRS)

    Stamenovic, D.; Fredberg, J. J.; Wang, N.; Butler, J. P.; Ingber, D. E.

    1996-01-01

    Mechanical properties of living cells are commonly described in terms of the laws of continuum mechanics. The purpose of this report is to consider the implications of an alternative approach that emphasizes the discrete nature of stress bearing elements in the cell and is based on the known structural properties of the cytoskeleton. We have noted previously that tensegrity architecture seems to capture essential qualitative features of cytoskeletal shape distortion in adherent cells (Ingber, 1993a; Wang et al., 1993). Here we extend those qualitative notions into a formal microstructural analysis. On the basis of that analysis we attempt to identify unifying principles that might underlie the shape stability of the cytoskeleton. For simplicity, we focus on a tensegrity structure containing six rigid struts interconnected by 24 linearly elastic cables. Cables carry initial tension ("prestress") counterbalanced by compression of struts. Two cases of interconnectedness between cables and struts are considered: one where they are connected by pin-joints, and the other where the cables run through frictionless loops at the junctions. At the molecular level, the pinned structure may represent the case in which different cytoskeletal filaments are cross-linked whereas the looped structure represents the case where they are free to slip past one another. The system is then subjected to uniaxial stretching. Using the principal of virtual work, stretching force vs. extension and structural stiffness vs. stretching force relationships are calculated for different prestresses. The stiffness is found to increase with increasing prestress and, at a given prestress, to increase approximately linearly with increasing stretching force. This behavior is consistent with observations in living endothelial cells exposed to shear stresses (Wang & Ingber, 1994). At a given prestress, the pinned structure is found to be stiffer than the looped one, a result consistent with data on

  18. Multiscale modeling of cell shape from the actin cytoskeleton.

    PubMed

    Rangamani, Padmini; Xiong, Granville Yuguang; Iyengar, Ravi

    2014-01-01

    The actin cytoskeleton is a dynamic structure that constantly undergoes complex reorganization events during many cellular processes. Mathematical models and simulations are powerful tools that can provide insight into the physical mechanisms underlying these processes and make predictions that can be experimentally tested. Representation of the interactions of the actin filaments with the plasma membrane and the movement of the plasma membrane for computation remains a challenge. Here, we provide an overview of the different modeling approaches used to study cytoskeletal dynamics and highlight the differential geometry approach that we have used to implement the interactions between the plasma membrane and the cytoskeleton. Using cell spreading as an example, we demonstrate how this approach is able to successfully capture in simulations, experimentally observed behavior. We provide a perspective on how the differential geometry approach can be used for other biological processes. PMID:24560144

  19. 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. PMID:20038822

  20. Proteome rearrangements after auditory learning: high-resolution profiling of synapse-enriched protein fractions from mouse brain.

    PubMed

    Kähne, Thilo; Richter, Sandra; Kolodziej, Angela; Smalla, Karl-Heinz; Pielot, Rainer; Engler, Alexander; Ohl, Frank W; Dieterich, Daniela C; Seidenbecher, Constanze; Tischmeyer, Wolfgang; Naumann, Michael; Gundelfinger, Eckart D

    2016-07-01

    Learning and memory processes are accompanied by rearrangements of synaptic protein networks. While various studies have demonstrated the regulation of individual synaptic proteins during these processes, much less is known about the complex regulation of synaptic proteomes. Recently, we reported that auditory discrimination learning in mice is associated with a relative down-regulation of proteins involved in the structural organization of synapses in various brain regions. Aiming at the identification of biological processes and signaling pathways involved in auditory memory formation, here, a label-free quantification approach was utilized to identify regulated synaptic junctional proteins and phosphoproteins in the auditory cortex, frontal cortex, hippocampus, and striatum of mice 24 h after the learning experiment. Twenty proteins, including postsynaptic scaffolds, actin-remodeling proteins, and RNA-binding proteins, were regulated in at least three brain regions pointing to common, cross-regional mechanisms. Most of the detected synaptic proteome changes were, however, restricted to individual brain regions. For example, several members of the Septin family of cytoskeletal proteins were up-regulated only in the hippocampus, while Septin-9 was down-regulated in the hippocampus, the frontal cortex, and the striatum. Meta analyses utilizing several databases were employed to identify underlying cellular functions and biological pathways. Data are available via ProteomeExchange with identifier PXD003089. How does the protein composition of synapses change in different brain areas upon auditory learning? We unravel discrete proteome changes in mouse auditory cortex, frontal cortex, hippocampus, and striatum functionally implicated in the learning process. We identify not only common but also area-specific biological pathways and cellular processes modulated 24 h after training, indicating individual contributions of the regions to memory processing. PMID

  1. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics.

    PubMed

    Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay

    2015-10-20

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable timescales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design. PMID:26488648

  2. 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

  3. β-Actin protein expression differs in the submandibular glands of male and female mice.

    PubMed

    Chen, Gang; Zou, Ye; Zhang, Xuan; Xu, Lingfei; Hu, Qiaoyun; Li, Ting; Yao, Chenjuan; Yu, Shali; Wang, Xiaoke; Wang, Chun

    2016-07-01

    β-actin, a cytoskeletal protein, is the most widely used housekeeping gene. Although housekeeping genes are expressed in all tissues, the β-actin gene is expressed in certain cell types because of differential binding of transcriptional factors to the regulatory elements of the gene. The expression and localization of β-actin protein in the submandibular glands (SMG) of mice were investigated in this study, using Western blot analysis and immunohistochemistry. In ICR and C57BL/6J mice, the levels of β-actin protein in the SMG of females are significantly higher than those in the SMG of males. β-actin protein is majorly distributed in acinar cells of SMG. There is no significant difference in the expression level of β-actin protein between females and castrated males. After castrated male ICR mice are treated with 10 mg/kg/day testosterone propionate (TP) for 3 weeks, the levels of β-actin protein in SMG decrease. The numbers of duct per unit area increase, whereas the numbers of acinus per unit area decrease after TP administration. These data suggest that β-actin protein is mainly distributed in acinar cells of SMG and results in a marked sexual dimorphism in mice. PMID:27079296

  4. Orchestration of microtubules and the actin cytoskeleton in trichome cell shape determination by a plant-unique kinesin

    PubMed Central

    Tian, Juan; Han, Libo; Feng, Zhidi; Wang, Guangda; Liu, Weiwei; Ma, Yinping; Yu, Yanjun; Kong, Zhaosheng

    2015-01-01

    Microtubules (MTs) and actin filaments (F-actin) function cooperatively to regulate plant cell morphogenesis. However, the mechanisms underlying the crosstalk between these two cytoskeletal systems, particularly in cell shape control, remain largely unknown. In this study, we show that introduction of the MyTH4-FERM tandem into KCBP (kinesin-like calmodulin-binding protein) during evolution conferred novel functions. The MyTH4 domain and the FERM domain in the N-terminal tail of KCBP physically bind to MTs and F-actin, respectively. During trichome morphogenesis, KCBP distributes in a specific cortical gradient and concentrates at the branching sites and the apexes of elongating branches, which lack MTs but have cortical F-actin. Further, live-cell imaging and genetic analyses revealed that KCBP acts as a hub integrating MTs and actin filaments to assemble the required cytoskeletal configuration for the unique, polarized diffuse growth pattern during trichome cell morphogenesis. Our findings provide significant insights into the mechanisms underlying cytoskeletal regulation of cell shape determination. DOI: http://dx.doi.org/10.7554/eLife.09351.001 PMID:26287478

  5. Uncoupling of gate and fence functions of MDCK cells by the actin-depolymerizing reagent mycalolide B.

    PubMed

    Takakuwa, R; Kokai, Y; Kojima, T; Akatsuka, T; Tobioka, H; Sawada, N; Mori, M

    2000-06-15

    The tight junction serves as a paracellular gate to seal the paracellular space of apposing cells and as a molecular fence to prevent diffusion of membrane proteins and lipids in epithelial cells. Although involvement of the actin cytoskeleton has been considered to be important in these two functions, it remains to be elucidated whether both functions are regulated in a coupled manner or differentially by actin. Treatment of highly polarized MDCK cells with mycalolide B (MB), a recently developed actin-depolymerizing reagent, induced a decrease of transepithelial resistance in a dose- and time-dependent manner with reversibility when the reagent was washed out. Changes in cytoskeletal actin, such as a reduction of cortical actin, irregularity of stress fibers, and punctated actin aggregates, were observed after MB treatment. However, the fence function, as studied by diffusion of apically labeled sphingomyelin/BSA complex, remained intact in the MB-treated MDCK cells. Localization of junctional molecules and apical marker proteins such as E-cadherin, ZO-1, and 114-kDa protein was shown to be unaffected. Furthermore, freeze-fracture study showed apparent tight junction strands. Collectively, MB treatment abolished the paracellular gate but not the fence function of MDCK cells, suggesting that cytoskeletal actin may play differential roles in the gate and fence functions of the tight junction. PMID:10837137

  6. Mechanics of composite cytoskeletal and extracellular networks

    NASA Astrophysics Data System (ADS)

    Das, Moumita

    2014-03-01

    Living cells sense and respond to mechanical forces in their surroundings. This mechanical response is mainly due to the cell cytoskeleton, and its interaction with the extracellular matrix (ECM). The cell cytoskeleton is a composite polymeric scaffold made of many different types of protein filaments and crosslinking proteins. Two major filament systems in the cytoskeleton are actin filaments (F-actin) and microtubules (MTs). Actin filaments are semiflexible, while the much stiffer MTs behave as rigid rods. I shall discuss theories that help understand how the direct coupling to the surrounding F-actin matrix allows intracellular MTs to bear large compressive forces and controls the range of force transmission along the MTs, and how the MTs not only enhance the stiffness of the cell cytoskeleton, but can also dramatically endow an initially nearly incompressible F-actin matrix with enhanced compressibility relative to its shear compliance. A second source of compositeness in the cytoskeleton is the presences of different types of crosslinkers that can interact cooperatively leading to enhanced mechanical rigidity and tunable response. Like the cytoskeleton, the ECM is also a polymeric composite. It is primarily composed of a mesh of fibrous proteins, mainly stiff collagen filaments, and a comparatively flexible gel of proteoglycans and hyaluronan. I shall discuss a model that shows how the interplay between the collagen network and the background elastic gel leads to a mechanically robust ECM.

  7. Emerging roles of actin cytoskeleton regulating enzymes in drug addiction: Actin or reactin’?

    PubMed Central

    Rothenfluh, Adrian; Cowan, Christopher W.

    2013-01-01

    Neurons rely on their cytoskeleton to give them shape and stability, and on cytoskeletal dynamics for growth and synaptic plasticity. Because drug addiction is increasingly seen as the inappropriate learning of strongly reinforcing stimuli, the role of the cytoskeleton in shaping drug memories has been of increasing interest in recent years. Does the cytoskeleton have an active role in shaping these memories, and to what extent do alterations in the cytoskeleton reflect the acute actions of drug exposure, or homeostatic reactions to the chronic exposure to drugs of abuse? Here we will review recent advances in understanding the role of the cytoskeleton in the development of drug addiction, with a focus on actin filaments, as they have been studied in greater detail. PMID:23428655

  8. Adhesion controls bacterial actin polymerization-based movement.

    PubMed

    Soo, Frederick S; Theriot, Julie A

    2005-11-01

    As part of its infectious life cycle, the bacterial pathogen Listeria monocytogenes propels itself through the host-cell cytoplasm by triggering the polymerization of host-cell actin near the bacterial surface, harnessing the activity of several cytoskeletal proteins used during actin-based cell crawling. To distinguish among several classes of biophysical models of actin-based bacterial movement, we used a high-throughput tracking technique to record the movement of many individual bacteria during temperature shifts. The speed of each bacterium varied strongly with temperature, closely following the Arrhenius rate law. Among bacteria, the prefactor A of the Arrhenius dependence unexpectedly varied exponentially with apparent activation energy, E(a), over a wide range (8-21 kcal/mol), reminiscent of the "rate compensation effect" of classical catalytic reactions. Average E(a) were increased for mutant bacteria deficient in binding Ena/VASP proteins and bacteria moving in diluted extract. These two effects were additive. The observed temperature and rate compensation effects are consistent with a class of simple kinetic models in which the bacterium advances through the thermally driven, cooperative breakage of groups of adhesive bonds on its surface. The estimated number of coupled adhesive bonds N on the bacterial surface varies between 10 and 40 bonds. In contrast to other models, this model correctly predicts an experimentally observed negative correlation between bacterial speed and actin gel density. The idea that speed depends on adhesion, rather than polymerization, suggests several alternative mechanisms by which known cytoskeletal regulatory proteins could control cellular movement. PMID:16251274

  9. Pharmacologic regulation of AMPK in breast cancer affects cytoskeletal properties involved with microtentacle formation and re-attachment

    PubMed Central

    Chakrabarti, Kristi R.; Whipple, Rebecca A.; Boggs, Amanda E.; Hessler, Lindsay K.; Bhandary, Lekhana; Vitolo, Michele I.; Thompson, Keyata; Martin, Stuart S.

    2015-01-01

    The presence of tumor cells in the circulation is associated with a higher risk of metastasis in patients with breast cancer. Circulating breast tumor cells use tubulin-based structures known as microtentacles (McTNs) to re-attach to endothelial cells and arrest in distant organs. McTN formation is dependent on the opposing cytoskeletal forces of stable microtubules and the actin network. AMP-activated protein kinase (AMPK) is a cellular metabolic regulator that can alter actin and microtubule organization in epithelial cells. We report that AMPK can regulate the cytoskeleton of breast cancer cells in both attached and suspended conditions. We tested the effects of AMPK on microtubule stability and the actin-severing protein, cofilin. AMPK inhibition with compound c increased both microtubule stability and cofilin activation, which also resulted in higher McTN formation and re-attachment. Conversely, AMPK activation with A-769662 decreased microtubule stability and cofilin activation with concurrent decreases in McTN formation and cell re-attachment. This data shows for the first time that AMPK shifts the balance of cytoskeletal forces in suspended breast cancer cells, which affect their ability to form McTNs and re-attach. These results support a model where AMPK activators may be used therapeutically to reduce the metastatic efficiency of breast tumor cells. PMID:26431377

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

    PubMed Central

    Henty-Ridilla, Jessica L.; Shimono, Masaki; Li, Jiejie; Chang, Jeff H.; Day, Brad; Staiger, Christopher J.

    2013-01-01

    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 that 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. PMID:23593000

  11. Demonstration in vivo of the role of Arabidopsis PLIM2 actin-binding proteins during pollination.

    PubMed

    Sudo, Keisuke; Park, Jong-In; Sakazono, Satomi; Masuko-Suzuki, Hiromi; Osaka, Masaaki; Kawagishi, Mizuho; Fujita, Kotomi; Maruoka, Mayumi; Nanjo, Hikaru; Suzuki, Go; Suwabe, Keita; Watanabe, Masao

    2013-01-01

    In plant reproduction, pollination is the initial key process in bringing together the male and female gametophytes. When a pollen grain lands on the surface of the stigma, information is exchanged between the pollen and stigmatic cell to determine whether the pollen grain will be accepted or rejected. If it is accepted, the stigmatic papilla cell supplies water and other resources to the pollen for germination and pollen tube elongation. Cellular processes involving actin are essential for pollen germination and tube growth, and actin-binding proteins regulate these processes by interacting with actin filaments to assemble cytoskeletal structures and actin networks. LIM proteins, which belong to a subfamily of cysteine-rich proteins, are a family of actin-binding proteins in plants, and are considered to be important for formation of the actin cytoskeleton and maintenance of its dynamics. Although the physiological and biochemical characteristics of LIMs have been elucidated in vitro in a variety of cell types, their exact role in pollen germination and pollen tube growth during pollination remained unclear. In this manuscript, we focus on the pollen-specific LIM proteins, AtPLIM2a and AtPLIM2c, and define their biological function during pollination in Arabidopsis thaliana. The atplim2a/atplim2c double knockdown RNAi plants showed a reduced pollen germination, approximately one-fifth of wild type, and slower pollen tube growth in the pistil, that is 80.4 μm/hr compared to 140.8 μm/hr in wild type. These defects led to an occasional unfertilized ovule at the bottom of the silique in RNAi plants. Our data provide direct evidence of the biological function of LIM proteins during pollination as actin-binding proteins, modulating cytoskeletal structures and actin networks, and their consequent importance in seed production. PMID:24694391

  12. Changes in myocardial cytoskeletal intermediate filaments and myocyte contractile dysfunction in dilated cardiomyopathy: an in vivo study in humans

    PubMed Central

    Di, S; Marotta, M; Salvatore, G; Cudemo, G; Cuda, G; De Vivo, F; Di, B; Ciaramella, F; Caputo, G; de Divitiis, O

    2000-01-01

    AIM—To investigate in vivo the intermediate cytoskeletal filaments desmin and vimentin in myocardial tissues from patients with dilated cardiomyopathy, and to determine whether alterations in these proteins are associated with impaired contractility.
METHODS—Endomyocardial biopsies were performed in 12 patients with dilated cardiomyopathy and in 12 controls (six women with breast cancer before anthracycline chemotherapy and six male donors for heart transplantation). Biopsy specimens were analysed by light microscopy and immunochemistry (desmin, vimentin). Myocyte contractile protein function was evaluated by the actin-myosin in vitro motility assay. Left ventricular ejection fraction was assessed by echocardiography and radionuclide ventriculography.
RESULTS—Patients with dilated cardiomyopathy had a greater cardiomyocyte diameter than controls (p < 0.01). The increase in cell size was associated with a reduction in contractile function, as assessed by actin-myosin motility (r = −0.643; p < 0.01). Quantitative immunochemistry showed increased desmin and vimentin contents (p < 0.01), and the desmin distribution was disturbed in cardiomyopathy. There was a linear relation between desmin distribution and actin-myosin sliding in vitro (r = 0.853; p < 0.01) and an inverse correlation between desmin content and ejection fraction (r = −0.773; p < 0.02). Negative correlations were also found between myocardial vimentin content and the actin-myosin sliding rate (r = −0.74; p < 0.02) and left ventricular ejection fraction (r = −0.68; p < 0.01).
CONCLUSIONS—Compared with normal individuals, the myocardial tissue of patients with dilated cardiomyopathy shows alterations of cytoskeletal intermediate filament distribution and content associated with reduced myocyte contraction.


Keywords: dilated cardiomyopathy; desmin; vimentin; cardiac biopsy; actin-myosin PMID:11083750

  13. Intranuclear Actin Regulates Osteogenesis

    PubMed Central

    Sen, Buer; Xie, Zhihui; Uzer, Gunes; Thompson, William R.; Styner, Maya; Wu, Xin; Rubin, Janet

    2016-01-01

    Depolymerization of the actin cytoskeleton induces nuclear trafficking of regulatory proteins and global effects on gene transcription. We here show that in mesenchymal stem cells (MSCs), cytochalasin D treatment causes rapid cofilin-/importin-9-dependent transfer of G-actin into the nucleus. The continued presence of intranuclear actin, which forms rod-like structures that stain with phalloidin, is associated with induction of robust expression of the osteogenic genes osterix and osteocalcin in a Runx2-dependent manner, and leads to acquisition of osteogenic phenotype. Adipogenic differentiation also occurs, but to a lesser degree. Intranuclear actin leads to nuclear export of Yes-associated protein (YAP); maintenance of nuclear YAP inhibits Runx2 initiation of osteogenesis. Injection of cytochalasin into the tibial marrow space of live mice results in abundant bone formation within the space of 1 week. In sum, increased intranuclear actin forces MSC into osteogenic lineage through controlling Runx2 activity; this process may be useful for clinical objectives of forming bone. PMID:26140478

  14. Caldesmon and the Regulation of Cytoskeletal Functions

    PubMed Central

    Wang, C.-L. Albert

    2010-01-01

    Caldesmon (CaD) is an extraordinary actin-binding protein, because in addition to actin, it also binds myosin, calmodulin and tropomyosin. As a component of the smooth muscle and nonmuscle contractile apparatus CaD inhibits the actomyosin ATPase activity and its inhibitory action is modulated by both Ca2+ and phosphorylation. The multiplicity of binding partners and diverse biochemical properties suggest CaD is a potent and versatile regulatory protein both in contractility and cell motility. However, after decades of investigation in numerous laboratories, hard evidence is still lacking to unequivocally identify its in vivo functions, although indirect evidence is mounting to support an important role in connection with the actin cytoskeleton. This chapter reviews the highlights of the past findings and summarizes the current views on this protein, with emphasis of its interaction with tropomyosin. PMID:19209827

  15. Reversibility and Viscoelastic Properties of Micropillar Supported and Oriented Magnesium Bundled F-Actin

    PubMed Central

    Maier, Timo; Haraszti, Tamás

    2015-01-01

    Filamentous actin is one of the most important cytoskeletal elements. Not only is it responsible for the elastic properties of many cell types, but it also plays a vital role in cellular adhesion and motility. Understanding the bundling kinetics of actin filaments is important in the formation of various cytoskeletal structures, such as filopodia and stress fibers. Utilizing a unique pillar-structured microfluidic device, we investigated the time dependence of bundling kinetics of pillar supported free-standing actin filaments. Microparticles attached to the filaments allowed the measurement of thermal motion, and we found that bundling takes place at lower concentrations than previously found in 3-dimensional actin gels, i.e. actin filaments formed bundles in the presence of 5–12 mM of magnesium chloride in a time-dependent manner. The filaments also displayed long term stability for up to hours after removing the magnesium ions from the buffer, which suggests that there is an extensive hysteresis between cation induced crosslinking and decrosslinking. PMID:26322783

  16. Regulation of tubulin and actin synthesis and accumulation during Blastocladiella emersonii development.

    PubMed

    da Silva, A M; Juliani, M H

    1988-06-01

    Actin and alpha and beta-tubulin have been identified in Blastocladiella emersonii by two-dimensional gel electrophoresis and Western blotting. The kinetics of synthesis of these proteins were compared by pulse-labeling experiments with [35S]methionine and with the accumulation of their corresponding mRNAs, translated in a cell-free system. Large increases occur in the rates of actin and alpha- and beta-tubulin biosynthesis during sporulation and there is an accumulation of the corresponding mRNAs. In parallel to the increased synthesis, these cytoskeletal proteins accumulate during the late stage of sporulation. PMID:3409324

  17. Myosins, Actin and Autophagy.

    PubMed

    Kruppa, Antonina J; Kendrick-Jones, John; Buss, Folma

    2016-08-01

    Myosin motor proteins working together with the actin cytoskeleton drive a wide range of cellular processes. In this review, we focus on their roles in autophagy - the pathway the cell uses to ensure homeostasis by targeting pathogens, misfolded proteins and damaged organelles for degradation. The actin cytoskeleton regulated by a host of nucleating, anchoring and stabilizing proteins provides the filament network for the delivery of essential membrane vesicles from different cellular compartments to the autophagosome. Actin networks have also been implicated in structurally supporting the expanding phagophore, moving autophagosomes and enabling efficient fusion with the lysosome. Only a few myosins have so far been shown to play a role in autophagy. Non-muscle myosin IIA functions in the early stages delivering membrane for the initial formation of the autophagosome, whereas myosin IC and myosin VI are involved in the final stages providing specific membranes for autophagosome maturation and its fusion with the lysosome. PMID:27146966

  18. A multi-structural single cell model of force-induced interactions of cytoskeletal components.

    PubMed

    Barreto, Sara; Clausen, Casper H; Perrault, Cecile M; Fletcher, Daniel A; Lacroix, Damien

    2013-08-01

    Several computational models based on experimental techniques and theories have been proposed to describe cytoskeleton (CSK) mechanics. Tensegrity is a prominent model for force generation, but it cannot predict mechanics of individual CSK components, nor explain the discrepancies from the different single cell stimulating techniques studies combined with cytoskeleton-disruptors. A new numerical concept that defines a multi-structural 3D finite element (FE) model of a single-adherent cell is proposed to investigate the biophysical and biochemical differences of the mechanical role of each cytoskeleton component under loading. The model includes prestressed actin bundles and microtubule within cytoplasm and nucleus surrounded by the actin cortex. We performed numerical simulations of atomic force microscopy (AFM) experiments by subjecting the cell model to compressive loads. The numerical role of the CSK components was corroborated with AFM force measurements on U2OS-osteosarcoma cells and NIH-3T3 fibroblasts exposed to different cytoskeleton-disrupting drugs. Computational simulation showed that actin cortex and microtubules are the major components targeted in resisting compression. This is a new numerical tool that explains the specific role of the cortex and overcomes the difficulty of isolating this component from other networks in vitro. This illustrates that a combination of cytoskeletal structures with their own properties is necessary for a complete description of cellular mechanics. PMID:23702149

  19. Cytoskeletal architecture and immunocytochemical localization of fodrin in the terminal web of the ciliated epithelial cell.

    PubMed

    Kobayashi, N; Hirokawa, N

    1988-01-01

    In order to understand the cytoskeletal architecture at the terminal web of the ciliated cell, we examined chicken tracheal epithelium by quick-freeze deep-etch (QFDE) electron microscopy combined with immunocytochemistry of fodrin. At the terminal web, the cilia ended into the basal bodies and then to the rootlets. The rootlets were composed of several filaments and globular structures attached regularly to them. Decoration with myosin subfragment 1 (S1) revealed that some actin filaments ran parallel to the apical plasma membrane between the basal bodies, and other population traveled perpendicularly or obliquely, i.e., along the rootlets. Some actin filaments were connected to the surface of the basal bodies and the basal feet. Among the basal bodies and the rootlets there existed three kinds of fine crossbridges, which were not decorated with S1. In the deeper part of the terminal web, intermediate filaments were observed between the rootlets and were sometimes crosslinked with the rootlets. Immunocytochemistry combined with the QFDE method revealed that fodrin was a component of fine crossbridges associated with the basal bodies. We concluded that an extensive crosslinker system among the basal bodies and the rootlets along with networks of actin and intermediate filaments formed a structural basis for the effective beating of cilia. PMID:3208297

  20. Distinct Roles of Cytoskeletal Components in Immunological Synapse Formation and Directed Secretion

    PubMed Central

    Ueda, Hironori; Zhou, Jie; Xie, Jianming

    2015-01-01

    A hallmark of CD4+ T cell activation and immunological synapse (IS) formation is the migration of the microtubule organization center and associated organelles toward the APCs. In this study, we found that when murine CD4+ T cells were treated with a microtubule-destabilizing agent (vinblastine) after the formation of IS, the microtubule organization center dispersed and all of the major cellular organelles moved away from the IS. Cytokines were no longer directed toward the synapse but were randomly secreted in quantities similar to those seen in synaptic secretion. However, if the actin cytoskeleton was disrupted at the same time with cytochalasin D, the organelles did not shift away from the IS. These findings suggest that there is a complex interplay between the microtubules and actin cytoskeleton, where microtubules are important for directing particular cytokines into the synapse, but they are not involved in the amount of cytokines that are produced for at least 1 h after IS formation. In addition, we found that they play a critical role in mobilizing organelles to reorient toward the synapse during T cell activation and in stabilizing organelles against the force that is generated through actin polymerization so that they move toward the APCs. These findings show that there is a complex interplay between these major cytoskeletal components during synapse formation and maintenance. PMID:26392461

  1. Rapid Actin-Dependent Viral Motility in Live Cells

    PubMed Central

    Vaughan, Joshua C.; Brandenburg, Boerries; Hogle, James M.; Zhuang, Xiaowei

    2009-01-01

    During the course of an infection, viruses take advantage of a variety of mechanisms to travel in cells, ranging from diffusion within the cytosol to active transport along cytoskeletal filaments. To study viral motility within the intrinsically heterogeneous environment of the cell, we have developed a motility assay that allows for the global and unbiased analysis of tens of thousands of virus trajectories in live cells. Using this assay, we discovered that poliovirus exhibits anomalously rapid intracellular movement that was independent of microtubules, a common track for fast and directed cargo transport. Such rapid motion, with speeds of up to 5 μm/s, allows the virus particles to quickly explore all regions of the cell with the exception of the nucleus. The rapid, microtubule-independent movement of poliovirus was observed in multiple human-derived cell lines, but appeared to be cargo-specific. Other cargo, including a closely related picornavirus, did not exhibit similar motility. Furthermore, the motility is energy-dependent and requires an intact actin cytoskeleton, suggesting an active transport mechanism. The speed of this microtubule-independent but actin-dependent movement is nearly an order of magnitude faster than the fastest speeds reported for actin-dependent transport in animal cells, either by actin polymerization or by myosin motor proteins. PMID:19751669

  2. Increased beta-actin and tubulin polymerization in regrowing axons: relationship to the conditioning lesion effect.

    PubMed

    Lund, Linda M; Machado, Victor M; McQuarrie, Irvine G

    2002-12-01

    Spinal motor neurons of Sprague-Dawley rats were examined to determine which of the neuronal isoforms of actin (beta or gamma) upregulate following axon injury. In situ hybridization studies showed greater beta-actin mRNA levels but no change in gamma-actin mRNA levels-suggesting that axon regrowth utilizes beta-actin. We radiolabeled the newly synthesized actin and tubulin that are subsequently transported in the axon to the site of an axotomizing injury. This allowed us to evaluate changes in polymerization as new cytoskeletal elements approach the injury site. Previous studies had shown that the rate of the most rapid subcomponent of actin and tubulin transport (called SCb) accelerates following axotomy (J. Jacob and I. McQuarrie, J. Neurobiol. 22: 570-583, 1991). This rate increase is associated with an increased proportion of SCb tubulin and actin in polymer (vs monomer) form (J. Jacob and I. McQuarrie, J. Neurosci, Res. 43: 412-419, 1996). However, in that study newly synthesized proteins were radiolabeled at 7 days after axotomy-which is at the peak of increased protein synthesis. This time-course did not examine actin and tubulin that were already in transit in axons when the injury occurred. This actin and tubulin would enter the regrowing axons first. Here, we have radiolabeled newly synthesized proteins 3 days prior to axotomy. For beta-tubulin, the ratio of monomer to polymer was unaffected. For actin, the equilibrium shifted strongly toward polymerization. We conclude that the acceleration of axonal outgrowth seen after the second of two serial axotomies (the "conditioning lesion effect") is related to the ability of actin that is already in transit to polymerize in response to the first axotomy. PMID:12504890

  3. Structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum.

    PubMed

    Kim, Min-Kyu; Kim, Ji-Hye; Kim, Ji-Sun; Kang, Sa-Ouk

    2015-09-01

    The crystal structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum was solved by Ca(2+)/S-SAD phasing and refined at 1.89 Å resolution. ABP34 is a calcium-regulated actin-binding protein that cross-links actin filaments into bundles. Its in vitro F-actin-binding and F-actin-bundling activities were confirmed by a co-sedimentation assay and transmission electron microscopy. The co-localization of ABP34 with actin in cells was also verified. ABP34 adopts a two-domain structure with an EF-hand-containing N-domain and an actin-binding C-domain, but has no reported overall structural homologues. The EF-hand is occupied by a calcium ion with a pentagonal bipyramidal coordination as in the canonical EF-hand. The C-domain structure resembles a three-helical bundle and superposes well onto the rod-shaped helical structures of some cytoskeletal proteins. Residues 216-244 in the C-domain form part of the strongest actin-binding sites (193-254) and exhibit a conserved sequence with the actin-binding region of α-actinin and ABP120. Furthermore, the second helical region of the C-domain is kinked by a proline break, offering a convex surface towards the solvent area which is implicated in actin binding. The F-actin-binding model suggests that ABP34 binds to the side of the actin filament and residues 216-244 fit into a pocket between actin subdomains -1 and -2 through hydrophobic interactions. These studies provide insights into the calcium coordination in the EF-hand and F-actin-binding site in the C-domain of ABP34, which are associated through interdomain interactions. PMID:26327373

  4. Integrin β1 regulates leiomyoma cytoskeletal integrity and growth

    PubMed Central

    Malik, Minnie; Segars, James; Catherino, William H.

    2014-01-01

    Uterine leiomyomas are characterized by an excessive extracellular matrix, increased mechanical stress, and increased active RhoA. Previously, we observed that mechanical signaling was attenuated in leiomyoma, but the mechanisms responsible remain unclear. Integrins, especially integrin β1, are transmembrane adhesion receptors that couple extracellular matrix stresses to the intracellular cytoskeleton to influence cell proliferation and differentiation. Here we characterized integrin and laminin to signaling in leiomyoma cells. We observed a 2.25 ± 0.32 fold increased expression of integrin β1 in leiomyoma cells, compared to myometrial cells. Antibody-mediated inhibition of integrin β1 led to significant growth inhibition in leiomyoma cells and a loss of cytoskeletal integrity. Specifically, polymerization of actin filaments and formation of focal adhesions were reduced by inhibition of integrin p1. Inhibition of integrin β1 in leiomyoma cells led to 0.81 ± 0.02 fold decrease in active RhoA, and resembled levels found in serum-starved cells. Likewise, inhibition of integrin β1 was accompanied by a decrease in phospho-ERK. Compared to myometrial cells, leiomyoma cells demonstrated increased expression of integrin α6 subunit to laminin receptor (1.91 ± 0.11 fold), and increased expression of laminin 5α (1.52±0.02), laminin 5β (3.06±0.92), and laminin 5γ (1.66 ± 0.06). Of note, leiomyoma cells grown on laminin matrix appear to realign themselves. Taken together, the findings reveal that the attenuated mechanical signaling in leiomyoma cells is accompanied by an increased expression and a dependence on integrin β1 signaling in leiomyoma cells, compared to myometrial cells. PMID:23023061

  5. Computational Tension Mapping of Adherent Cells Based on Actin Imaging

    PubMed Central

    Manifacier, Ian; Milan, Jean-Louis; Jeanneau, Charlotte; Chmilewsky, Fanny; Chabrand, Patrick; About, Imad

    2016-01-01

    Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension. PMID:26812601

  6. Computational Tension Mapping of Adherent Cells Based on Actin Imaging.

    PubMed

    Manifacier, Ian; Milan, Jean-Louis; Jeanneau, Charlotte; Chmilewsky, Fanny; Chabrand, Patrick; About, Imad

    2016-01-01

    Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension. PMID:26812601

  7. Actin depolymerization mediated loss of SNTA1 phosphorylation and Rac1 activity has implications on ROS production, cell migration and apoptosis.

    PubMed

    Bhat, Sehar Saleem; Parray, Arif Ali; Mushtaq, Umar; Fazili, Khalid Majid; Khanday, Firdous Ahmad

    2016-06-01

    Alpha-1-syntrophin (SNTA1) and Rac1 are part of a signaling pathway via the dystrophin glycoprotein complex (DGC). Both SNTA1 and Rac1 proteins are over-expressed in various carcinomas. It is through the DGC signaling pathway that SNTA1 has been shown to act as a link between the extra cellular matrix, the internal cell signaling apparatus and the actin cytoskeleton. SNTA1 is involved in the modulation of the actin cytoskeleton and actin reorganization. Rac1 also controls actin cytoskeletal organization in the cell. In this study, we present the interplay between f-actin, SNTA1 and Rac1. We analyzed the effect of actin depolymerization on SNTA1 tyrosine phosphorylation and Rac1 activity using actin depolymerizing drugs, cytochalasin D and latrunculin A. Our results indicate a marked decrease in the tyrosine phosphorylation of SNTA1 upon actin depolymerization. Results suggest that actin depolymerization mediated loss of SNTA1 phosphorylation leads to loss of interaction between SNTA1 and Rac1, with a concomitant loss of Rac1 activation. The loss of SNTA1tyrosine phosphorylation and Rac1 activity by actin depolymerization results in increased apoptosis, decreased cell migration and decreased reactive oxygen species (ROS) levels in breast carcinoma cells. Collectively, our results present a possible role of f-actin in the SNTA1-Rac1 signaling pathway and implications of actin depolymerization on cell migration, ROS production and apoptosis. PMID:27048259

  8. Actin remodeling by Nck regulates endothelial lumen formation.

    PubMed

    Chaki, Sankar P; Barhoumi, Rola; Rivera, Gonzalo M

    2015-09-01

    Multiple angiogenic cues modulate phosphotyrosine signaling to promote vasculogenesis and angiogenesis. Despite its functional and clinical importance, how vascular cells integrate phosphotyrosine-dependent signaling to elicit cytoskeletal changes required for endothelial morphogenesis remains poorly understood. The family of Nck adaptors couples phosphotyrosine signals with actin dynamics and therefore is well positioned to orchestrate cellular processes required in vascular formation and remodeling. Culture of endothelial cells in three-dimensional collagen matrices in the presence of VEGF stimulation was combined with molecular genetics, optical imaging, and biochemistry to show that Nck-dependent actin remodeling promotes endothelial cell elongation and proper organization of VE-cadherin intercellular junctions. Major morphogenetic defects caused by abrogation of Nck signaling included loss of endothelial apical-basal polarity and impaired lumenization. Time-lapse imaging using a Förster resonance energy transfer biosensor, immunostaining with phospho-specific antibodies, and GST pull-down assays showed that Nck determines spatiotemporal patterns of Cdc42/aPKC activation during endothelial morphogenesis. Our results demonstrate that Nck acts as an important hub integrating angiogenic cues with cytoskeletal changes that enable endothelial apical-basal polarization and lumen formation. These findings point to Nck as an emergent target for effective antiangiogenic therapy. PMID:26157164

  9. Coupled actin-lamin biopolymer networks and protecting DNA

    NASA Astrophysics Data System (ADS)

    Zhang, Tao; Rocklin, D. Zeb; Mao, Xiaoming; Schwarz, J. M.

    The mechanical properties of cells are largely determined by networks of semiflexible biopolymers forming the cytoskeleton. Similarly, the mechanical properties of cell nuclei are also largely determined by networks of semiflexible biopolymers forming the nuclear cytoskeleton. In particular, a network of filamentous lamin sits just inside the inner nuclear membrane to presumably protect the heart of the cell nucleus--the DNA. It has been demonstrated over the past decade that the actin cytoskeletal biopolymer network and the lamin biopolymer network are coupled via a sequence of proteins bridging the outer and inner nuclear membranes, known as the LINC complex. We, therefore, probe the consequences of such a coupling in a model biopolymer network system via numerical simulations to understand the resulting deformations in the lamin network in response to perturbations in the actin cytoskeletal network. We find, for example, that the force transmission across the coupled system can depend sensitively on the concentration of LINC complexes. Such study could have implications for mechanical mechanisms of the regulation of transcription since DNA couples to lamin via lamin-binding domains so that deformations in the lamin network may result in deformations in the DNA.

  10. Actin remodeling by Nck regulates endothelial lumen formation

    PubMed Central

    Chaki, Sankar P.; Barhoumi, Rola; Rivera, Gonzalo M.

    2015-01-01

    Multiple angiogenic cues modulate phosphotyrosine signaling to promote vasculogenesis and angiogenesis. Despite its functional and clinical importance, how vascular cells integrate phosphotyrosine-dependent signaling to elicit cytoskeletal changes required for endothelial morphogenesis remains poorly understood. The family of Nck adaptors couples phosphotyrosine signals with actin dynamics and therefore is well positioned to orchestrate cellular processes required in vascular formation and remodeling. Culture of endothelial cells in three-dimensional collagen matrices in the presence of VEGF stimulation was combined with molecular genetics, optical imaging, and biochemistry to show that Nck-dependent actin remodeling promotes endothelial cell elongation and proper organization of VE-cadherin intercellular junctions. Major morphogenetic defects caused by abrogation of Nck signaling included loss of endothelial apical-basal polarity and impaired lumenization. Time-lapse imaging using a Förster resonance energy transfer biosensor, immunostaining with phospho-specific antibodies, and GST pull-down assays showed that Nck determines spatiotemporal patterns of Cdc42/aPKC activation during endothelial morphogenesis. Our results demonstrate that Nck acts as an important hub integrating angiogenic cues with cytoskeletal changes that enable endothelial apical-basal polarization and lumen formation. These findings point to Nck as an emergent target for effective antiangiogenic therapy. PMID:26157164

  11. Re-expression of ABP-120 rescues cytoskeletal, motility, and phagocytosis defects of ABP-120- Dictyostelium mutants.

    PubMed Central

    Cox, D; Wessels, D; Soll, D R; Hartwig, J; Condeelis, J

    1996-01-01

    The actin binding protein ABP-120 has been proposed to cross-link actin filaments in nascent pseudopods, in a step required for normal pseudopod extension in motile Dictyostelium amoebae. To test this hypothesis, cell lines that lack ABP-120 were created independently either by chemical mutagenesis or homologous recombination. Different phenotypes were reported in these two studies. The chemical mutant shows only a subtle defect in actin cross-linking, while the homologous recombinant mutants show profound defects in actin cross-linking, cytoskeletal structure, pseudopod number and size, cell motility and chemotaxis and, as shown here, phagocytosis. To resolve the controversy as to what the ABP-120- phenotype is, ABP-120 was re-expressed in an ABP-120- cell line created by homologous recombination. Two independently "rescued" cell lines that express wild-type levels of ABP-120 were analyzed. In both rescued cell lines, actin incorporation into the cytoskeleton, pseudopod formation, cell morphology, instantaneous velocity, phagocytosis, and chemotaxis were restored to wild-type levels. There is no alteration in the expression levels of several related actin binding proteins in either the original ABP-120- cell line or in the rescued cell lines, leading to the conclusion that neither the aberrant phenotype observed in ABP-120- cells nor the normal phenotype reasserted in rescued cells can be attributed to alterations in the levels of other abundant and related actin binding proteins. Re-expression of ABP-120 in ABP-120- cells reestablishes normal structural and behavioral parameters, demonstrating that the severity and properties of the structural and behavioral defects of ABP-120- cell lines produced by homologous recombination are the direct result of the absence of ABP-120. Images PMID:8744952

  12. Aggregation of Actin and Cofilin in Identical Twins with Juvenile-Onset Dystonia

    PubMed Central

    Gearing, Marla; Juncos, Jorge L.; Procaccio, Vincent; Gutekunst, Claire-Anne; Marino-Rodriguez, Elaine M.; Gyure, Kymberly A.; Ono, Shoichiro; Santoianni, Robert; Krawiecki, Nicolas S.; Wallace, Douglas C.; Wainer, Bruce H.

    2005-01-01

    The neuropathology of the primary dystonias is not well understood. We examined brains from identical twins with DYT1-negative, dopa-unresponsive dystonia. The twins exhibited mild developmental delays until age 12 years when they began developing rapidly progressive generalized dystonia. Genetic, metabolic, and imaging studies ruled out known causes of dystonia. Cognition was subnormal but stable until the last few years. Death occurred at ages 21 and 22 years. The brains were macroscopically unremarkable. Microscopic examination showed unusual glial fibrillary acidic protein–immunoreactive astrocytes in multiple regions and iron accumulation in pallidal and nigral neurons. However, the most striking findings were 1) eosinophilic, rod-like cytoplasmic inclusions in neocortical and thalamic neurons that were actin depolymerizing factor/cofilin-immunoreactive but only rarely actin-positive; and 2) abundant eosinophilic spherical structures in the striatum that were strongly actin- and actin depolymerizing factor/cofilin-positive. Electron microscopy suggested that these structures represent degenerating neurons and processes; the accumulating filaments had the same dimensions as actin microfilaments. To our knowledge, aggregation of actin has not been reported previously as the predominant feature in any neurodegenerative disease. Thus, our findings may shed light on a novel neuropathological change associated with dystonia that may represent a new degenerative mechanism involving actin, a ubiquitous constituent of the cytoskeletal system. PMID:12325076

  13. Cortical instability drives periodic supracellular actin pattern formation in epithelial tubes.

    PubMed

    Hannezo, Edouard; Dong, Bo; Recho, Pierre; Joanny, Jean-François; Hayashi, Shigeo

    2015-07-14

    An essential question of morphogenesis is how patterns arise without preexisting positional information, as inspired by Turing. In the past few years, cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Theoretical and in vitro studies have suggested that biological polymers such as actomyosin gels have the property to self-organize, but the applicability of this concept in an in vivo setting remains unclear. Here, we report that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. We propose a simple biophysical model where pattern formation arises from the interplay of myosin contractility and actin turnover. We validate the hypotheses of the model using photobleaching experiments and report that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the actomyosin gel modify the spacing of the pattern, as the model predicted. In addition, our model posited a role of cortical friction in stabilizing the spacing pattern of actin rings. Consistently, genetic depletion of apical extracellular matrix caused strikingly dynamic movements of actin rings, mirroring our model prediction of a transition from steady to chaotic actin patterns at low cortical friction. Our results therefore demonstrate quantitatively that a hydrodynamical instability of the actin cortex can trigger regular pattern formation and drive morphogenesis in an in vivo setting. PMID:26077909

  14. Effect of tensile force on the mechanical behavior of actin filaments.

    PubMed

    Matsushita, Shinji; Inoue, Yasuhiro; Hojo, Masaki; Sokabe, Masahiro; Adachi, Taiji

    2011-06-01

    Actin filaments are the most abundant components of the cellular cytoskeleton, and play critical roles in various cellular functions such as migration, division and shape control. In these activities, mechanical tension causes structural changes in the double-helical structure of the actin filament, which is a key modulator of cytoskeletal reorganization. This study performed large-scale molecular dynamics (MD) and steered MD simulations to quantitatively analyze the effects of tensile force on the mechanical behavior of actin filaments. The results revealed that when a tensile force of 200pN was applied to a filament consisting of 14 actin subunits, the twist angle of the filament decreased by approximately 20°, corresponding to a rotation of approximately -2° per subunit, representing a critical structural change in actin filaments. Based on these structural changes, the variance in filament length and twist angle was found to decrease, leading to increases in extensional and torsional stiffness. Torsional stiffness increased significantly under the tensile condition, and the ratio of filament stiffness under tensile force to that under no external force increased significantly on longer temporal scales. The results obtained from this study contribute to the understanding of mechano-chemical interactions concerning actin dynamics, showing that increased tensile force in the filament prevents actin regulatory proteins from binding to the filament. PMID:21536289

  15. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    PubMed

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. PMID:25916847

  16. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria

    PubMed Central

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-01-01

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. PMID:25916847

  17. Cortical instability drives periodic supracellular actin pattern formation in epithelial tubes

    PubMed Central

    Hannezo, Edouard; Dong, Bo; Recho, Pierre; Joanny, Jean-François; Hayashi, Shigeo

    2015-01-01

    An essential question of morphogenesis is how patterns arise without preexisting positional information, as inspired by Turing. In the past few years, cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Theoretical and in vitro studies have suggested that biological polymers such as actomyosin gels have the property to self-organize, but the applicability of this concept in an in vivo setting remains unclear. Here, we report that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. We propose a simple biophysical model where pattern formation arises from the interplay of myosin contractility and actin turnover. We validate the hypotheses of the model using photobleaching experiments and report that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the actomyosin gel modify the spacing of the pattern, as the model predicted. In addition, our model posited a role of cortical friction in stabilizing the spacing pattern of actin rings. Consistently, genetic depletion of apical extracellular matrix caused strikingly dynamic movements of actin rings, mirroring our model prediction of a transition from steady to chaotic actin patterns at low cortical friction. Our results therefore demonstrate quantitatively that a hydrodynamical instability of the actin cortex can trigger regular pattern formation and drive morphogenesis in an in vivo setting. PMID:26077909

  18. TOR complex 2-Ypk1 signaling regulates actin polarization via reactive oxygen species.

    PubMed

    Niles, Brad J; Powers, Ted

    2014-12-01

    The evolutionarily conserved mTOR complex 2 (mTORC2) signaling pathway is an important regulator of actin cytoskeletal architecture and, as such, is a candidate target for preventing cancer cell motility and invasion. Remarkably, the precise mechanism(s) by which mTORC2 regulates the actin cytoskeleton have remained elusive. Here we show that in budding yeast, TORC2 and its downstream kinase Ypk1 regulate actin polarization by controlling reactive oxygen species (ROS) accumulation. Specifically, we find that TORC2-Ypk1 regulates actin polarization both by vacuole-related ROS, controlled by the phospholipid flippase kinase Fpk1 and sphingolipids, and by mitochondria-mediated ROS, controlled by the PKA subunit Tpk3. In addition, we find that the protein kinase C (Pkc1)/MAPK cascade, a well-established regulator of actin, acts downstream of Ypk1 to regulate ROS, in part by promoting degradation of the oxidative stress responsive repressor, cyclin C. Furthermore, we show that Ypk1 regulates Pkc1 activity through proper localization of Rom2 at the plasma membrane, which is also dependent on Fpk1 and sphingolipids. Together these findings demonstrate important links between TORC2/Ypk1 signaling, Fpk1, sphingolipids, Pkc1, and ROS as regulators of actin and suggest that ROS may play an important role in mTORC2-dependent dysregulation of the actin cytoskeleton in cancer cells. PMID:25253719

  19. Entropic forces drive contraction of cytoskeletal networks.

    PubMed

    Braun, Marcus; Lansky, Zdenek; Hilitski, Feodor; Dogic, Zvonimir; Diez, Stefan

    2016-05-01

    The cytoskeleton is a network of interconnected protein filaments, which provide a three-dimensional scaffold for cells. Remodeling of the cytoskeleton is important for key cellular processes, such as cell motility, division, or morphogenesis. This remodeling is traditionally considered to be driven exclusively by processes consuming chemical energy, such as the dynamics of the filaments or the action of molecular motors. Here, we review two mechanisms of cytoskeletal network remodeling that are independent of the consumption of chemical energy. In both cases directed motion of overlapping filaments is driven by entropic forces, which arise from harnessing thermal energy present in solution. Entropic forces are induced either by macromolecular crowding agents or by diffusible crosslinkers confined to the regions where filaments overlap. Both mechanisms increase filament overlap length and lead to the contraction of filament networks. These force-generating mechanisms, together with the chemical energy-dependent mechanisms, need to be considered for the comprehensive quantitative picture of the remodeling of cytoskeletal networks in cells. PMID:26996935

  20. Widespread cytoskeletal pathology characterizes corticobasal degeneration.

    PubMed Central

    Feany, M. B.; Dickson, D. W.

    1995-01-01

    Corticobasal degeneration (CBD) is a rare, progressive neurological disorder characterized by widespread neuronal and glial pathology. Using immunohistochemistry and laser confocal microscopy, we demonstrate that the nonamyloid cortical plaques of CBD are actually collections of abnormal tau in the distal processes of astrocytes. These glial cells express both vimentin and CD44, markers of astrocyte activation. Glial pathology also includes tau-positive cytoplasmic inclusions, here localized to Leu 7-expressing oligodendrocytes. In addition, a wide array of neuronal pathology is defined with tau-positive inclusions in multiple domains of a variety of cortical neurons. CBD thus exhibits widespread glial and neuronal cytoskeletal pathology, including a novel structure, the astrocytic plaque. CBD is a disease of generalized cytoskeletal disruption affecting several cell types and multiple domains of these cells. The further definition of CBD pathology refines the diagnosis and pathophysiological understanding of this unique disease and has important implications for other neurodegenerative diseases, like Alzheimer's disease, characterized by abnormal tau deposition. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7778678

  1. A Mutation of β-Actin That Alters Depolymerization Dynamics Is Associated with Autosomal Dominant Developmental Malformations, Deafness, and Dystonia

    PubMed Central

    Procaccio, Vincent; Salazar, Gloria; Ono, Shoichiro; Styers, Melanie L.; Gearing, Marla; Davila, Antonio; Jimenez, Richard; Juncos, Jorge; Gutekunst, Claire-Anne; Meroni, Germana; Fontanella, Bianca; Sontag, Estelle; Sontag, Jean Marie; Faundez, Victor; Wainer, Bruce H.

    2006-01-01

    Actin, one of the major filamentous cytoskeletal molecules, is involved in a variety of cellular functions. Whereas an association between muscle actin mutations and skeletal and cardiac myopathies has been well documented, reports of human disease arising from mutations of nonmuscle actin genes have been rare. We have identified a missense point mutation in the gene coding for β-actin that results in an arginine-to-tryptophan substitution at position 183. The disease phenotype includes developmental midline malformations, sensory hearing loss, and a delayed-onset generalized dystonia syndrome in monozygotic twins. Cellular studies of a lymphoblastoid cell line obtained from an affected patient demonstrated morphological abnormalities of the actin cytoskeleton and altered actin depolymerization dynamics in response to latrunculin A, an actin monomer–sequestering drug. Resistance to latrunculin A was also observed in NIH 3T3 cells expressing the mutant actin. These findings suggest that mutations in nonmuscle actins may be associated with a broad spectrum of developmental malformations and/or neurological abnormalities such as dystonia. PMID:16685646

  2. N-WASp is required for Schwann cell cytoskeletal dynamics, normal myelin gene expression and peripheral nerve myelination

    PubMed Central

    Jin, Fuzi; Dong, Baoxia; Georgiou, John; Jiang, Qiuhong; Zhang, Jinyi; Bharioke, Arjun; Qiu, Frank; Lommel, Silvia; Feltri, M. Laura; Wrabetz, Lawrence; Roder, John C.; Eyer, Joel; Chen, Xiequn; Peterson, Alan C.; Siminovitch, Katherine A.

    2011-01-01

    Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined. Here, we show that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin. Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the mutant animals achieving normal body weights and living to advanced age. Our observations demonstrate that N-WASp plays an essential role in Schwann cell maturation and myelin formation. PMID:21385763

  3. Thoracic Aortic Aneurysm (TAAD)-causing Mutation in Actin Affects Formin Regulation of Polymerization*

    PubMed Central

    Malloy, Lindsey E.; Wen, Kuo-Kuang; Pierick, Alyson R.; Wedemeyer, Elesa W.; Bergeron, Sarah E.; Vanderpool, Nicole D.; McKane, Melissa; Rubenstein, Peter A.; Bartlett, Heather L.

    2012-01-01

    More than 30 mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause autosomal dominant thoracic aortic aneurysm and dissection. The mutation R256H is of particular interest because it also causes patent ductus arteriosus and moyamoya disease. R256H is one of the more prevalent mutations and, based on its molecular location near the strand-strand interface in the actin filament, may affect F-actin stability. To understand the molecular ramifications of the R256H mutation, we generated Saccharomyces cerevisiae yeast cells expressing only R256H yeast actin as a model system. These cells displayed abnormal cytoskeletal morphology and increased sensitivity to latrunculin A. After cable disassembly induced by transient exposure to latrunculin A, mutant cells were delayed in reestablishing the actin cytoskeleton. In vitro, mutant actin exhibited a higher than normal critical concentration and a delayed nucleation. Consequently, we investigated regulation of mutant actin by formin, a potent facilitator of nucleation and a protein needed for normal vascular smooth muscle cell development. Mutant actin polymerization was inhibited by the FH1-FH2 fragment of the yeast formin, Bni1. This fragment strongly capped the filament rather than facilitating polymerization. Interestingly, phalloidin or the presence of wild type actin reversed the strong capping behavior of Bni1. Together, the data suggest that the R256H actin mutation alters filament conformation resulting in filament instability and misregulation by formin. These biochemical effects may contribute to abnormal histology identified in diseased arterial samples from affected patients. PMID:22753406

  4. Thoracic aortic aneurysm (TAAD)-causing mutation in actin affects formin regulation of polymerization.

    PubMed

    Malloy, Lindsey E; Wen, Kuo-Kuang; Pierick, Alyson R; Wedemeyer, Elesa W; Bergeron, Sarah E; Vanderpool, Nicole D; McKane, Melissa; Rubenstein, Peter A; Bartlett, Heather L

    2012-08-17

    More than 30 mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause autosomal dominant thoracic aortic aneurysm and dissection. The mutation R256H is of particular interest because it also causes patent ductus arteriosus and moyamoya disease. R256H is one of the more prevalent mutations and, based on its molecular location near the strand-strand interface in the actin filament, may affect F-actin stability. To understand the molecular ramifications of the R256H mutation, we generated Saccharomyces cerevisiae yeast cells expressing only R256H yeast actin as a model system. These cells displayed abnormal cytoskeletal morphology and increased sensitivity to latrunculin A. After cable disassembly induced by transient exposure to latrunculin A, mutant cells were delayed in reestablishing the actin cytoskeleton. In vitro, mutant actin exhibited a higher than normal critical concentration and a delayed nucleation. Consequently, we investigated regulation of mutant actin by formin, a potent facilitator of nucleation and a protein needed for normal vascular smooth muscle cell development. Mutant actin polymerization was inhibited by the FH1-FH2 fragment of the yeast formin, Bni1. This fragment strongly capped the filament rather than facilitating polymerization. Interestingly, phalloidin or the presence of wild type actin reversed the strong capping behavior of Bni1. Together, the data suggest that the R256H actin mutation alters filament conformation resulting in filament instability and misregulation by formin. These biochemical effects may contribute to abnormal histology identified in diseased arterial samples from affected patients. PMID:22753406

  5. Tumor metastatic promoter ABCE1 interacts with the cytoskeleton protein actin and increases cell motility.

    PubMed

    Han, Xu; Tian, Ye; Tian, Dali

    2016-06-01

    ABCE1, a member of the ATP-binding cassette (ABC) family, is a candidate tumor metastatic promoter in lung cancer. Overexpression of ABCE1 is correlated with aggressive growth and metastasis in lung cancer cells. However, the exact mechanism remains unclear. In the present study, GST pull-down assay provided evidence of the possible interaction between ABCE1 and β-actin using GST-ABCE1 as a bait protein. Co-immunoprecipitation manifested ABCE1 formed complexes with β-actin in vivo. ABCE1 overexpression significantly increased the migration of lung cancer cells which may be attributed to the promotion of F-actin rearrangements. Taken together, these data suggest that overexpression of ABCE1 produces an obvious effect on the motility of lung cancer cells through cytoskeleton rearrangement. PMID:27109616

  6. Immunohistochemical study of cytoskeletal and extracellular matrix components in the notochord and notochordal sheath of amphioxus

    PubMed Central

    Bočina, Ivana; Saraga-Babić, Mirna

    2006-01-01

    A major cytoskeletal and extracellular matrix proteins of the amphioxus notochordal cells and sheath were detected by immunohistochemical techniques. The three-layered amphioxus notochordal sheath strongly expressed fish collagen type I in its outer and middle layers, while in the innermost layer expression did not occur. The amphioxus notochordal sheath was reactive to applied anti-human antibodies for intermediate filament proteins such as cytokeratins, desmin and vimentin, as well as to microtubule components (ß-tubulin), particularly in the area close to the epipharyngeal groove. Alpha-smooth muscle actin was expressed in some notochordal cells and in the area of the notochordal attachment to the sheath. Thus muscular nature of notochordal cells was shown by immunohistochemistry in tissue section. Our results confirm that genes encoding intermediate filament proteins, microtubules and microfilaments are highly conserved during evolution. Collagen type I was proven to be the key extracellular matrix protein that forms the amphioxus notochordal sheath. PMID:16733537

  7. A vital role for myosin-9 in puromycin aminonucleoside-induced podocyte injury by affecting actin cytoskeleton.

    PubMed

    Yuan, Yanggang; Zhao, Chuanyan; An, Xiaofei; Wu, Lin; Wang, Hui; Zhao, Min; Bai, Mi; Duan, Suyan; Zhang, Bo; Zhang, Aihua; Xing, Changying

    2016-06-01

    Podocyte injury is an early pathological change of many kidney diseases. In particular, the actin cytoskeleton plays an important role in maintaining the normal function of podocytes. Disruption of the actin cytoskeleton is a feature of podocyte injury in proteinuric nephropathies. Recent studies showed that myosin-9 was localized in the podocyte foot processes and was necessary in maintaining podocyte structural homeostasis. However, it is unclear whether myosin-9 maintains podocyte structure by affecting actin cytoskleton. Here, the role of myosin-9 in puromycin aminonucleoside (PAN)-induced podocyte injury was explored both in vitro and in vivo. In cultured mouse podocytes (MPC5), it was determined that PAN downregulated myosin-9 expression, disrupted the actin cytoskeleton and reduced the adhesion ability. Reduced myosin-9 expression by siRNA precipitated podocyte cytoskeletal damage and accelerated PAN-induced podocyte detachment. Overexpression of myosin-9 protected against PAN-induced podocyte detachment. Furthermore, administration of an antioxidant Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) inhibited PAN-induced podocyte cytoskeletal damage and podocyte detachment by restoring the expression of myosin-9. In the rat PAN nephropathy model, MnTBAP could also attenuate PAN-induced reduction of myosin-9 and podocyte loss. Taken together, these findings pinpointed that oxidative stress contributed to PAN-induced podocyte injury through the repression of a cytoskeletal protein myosin-9, which provided novel insights into a potential target for the treatment of podocyte injury-associated glomerulopathies. PMID:26902808

  8. Molecular Mechanotransduction: how forces trigger cytoskeletal dynamics

    NASA Astrophysics Data System (ADS)

    Ehrlicher, Allen

    2012-02-01

    Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders, such as cardiac failure and pulmonary injury. Despite detailed knowledge of the cytoskeleton's structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein, filamin A (FLNa) as a central mechanotransduction element of the cytoskeleton by using Fluorescence Loss After photoConversion (FLAC), a novel high-speed alternative to FRAP. We reconstituted a minimal system consisting of actin filaments, FLNa and two FLNa-binding partners: the cytoplasmic tail of ß-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with ß integrin tails connecting to the actin cytoskeleton by binding directly to filamin. FilGAP is a FLNa-binding GTPase-activating protein specific for Rac, which in vivo regulates cell spreading and bleb formation. We demonstrate that both externally-imposed bulk shear and myosin II driven forces differentially regulate the binding of integrin and FilGAP to FLNa. Consistent with structural predictions, strain increases ß-integrin binding to FLNa, whereas it causes FilGAP to dissociate from FLNa, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify the first molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signaling molecules. Moreover, GAP activity has been shown to switch cell movement from mesenchymal to amoeboid motility, suggesting that mechanical forces directly impact the invasiveness of cancer.

  9. Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle-alpha-actin gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Smooth muscle a actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells(vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a ...

  10. Isoform-specific roles of the GTPase activating protein Nadrin in cytoskeletal reorganization of platelets.

    PubMed

    Beck, S; Fotinos, A; Lang, F; Gawaz, M; Elvers, M

    2013-01-01

    Cytoskeletal reorganization of activated platelets plays a crucial role in hemostasis and thrombosis and implies activation of Rho GTPases. Rho GTPases are important regulators of cytoskeletal dynamics and function as molecular switches that cycle between an inactive and an active state. They are regulated by GTPase activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signaling. The regulation of Rho GTPases in platelets is not explored. A detailed characterization of Rho regulation is necessary to understand activation and inactivation of Rho GTPases critical for platelet activation and aggregation. Nadrin is a RhoGAP regulating cytoplasmic protein explored in the central nervous system. Five Nadrin isoforms are known that share a unique GAP domain, a serine/threonine/proline-rich domain, a SH3-binding motif and an N-terminal BAR domain but differ in their C-terminus. Here we identified Nadrin in platelets where it co-localizes to actin-rich regions and Rho GTPases. Different Nadrin isoforms selectively regulate Rho GTPases (RhoA, Cdc42 and Rac1) and cytoskeletal reorganization suggesting that - beside the GAP domain - the C-terminus of Nadrin determines Rho specificity and influences cell physiology. Furthermore, Nadrin controls RhoA-mediated stress fibre and focal adhesion formation. Spreading experiments on fibrinogen revealed strongly reduced cell adhesion upon Nadrin overexpression. Unexpectedly, the Nadrin BAR domain controls Nadrin-GAP activity and acts as a guidance domain to direct this GAP to its substrate at the plasma membrane. Our results suggest a critical role for Nadrin in the regulation of RhoA, Cdc42 and Rac1 in platelets and thus for platelet adhesion and aggregation. PMID:22975681

  11. Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination

    PubMed Central

    Bonin, Sawyer R.; Gibeault, Sabrina; De Repentigny, Yves; Kothary, Rashmi

    2016-01-01

    Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dstdt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dstdt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dstdt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dstdt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dstdt-27J optic nerves was normal, as was myelin basic

  12. Cytoskeletal binding proteins distinguish cultured dental follicle cells and periodontal ligament cells.

    PubMed

    Li, Jie; Li, Hui; Tian, Ye; Yang, Yaling; Chen, Guoqing; Guo, Weihua; Tian, Weidong

    2016-07-01

    Human dental follicle cells (DFCs) and periodontal ligament cells (PDLCs) derived from the ectomesenchymal tissue, have been shown to exhibit stem/progenitor cell properties and the ability to induce tissue regeneration. Stem cells in dental follicle differentiate into cementoblasts, periodontal ligament fibroblasts and osteoblasts, these cells form cementum, periodontal ligament and alveolar bone, respectively. While stem cells in dental follicle are a precursor to periodontal ligament fibroblasts, the molecular changes that distinguish cultured DFCs from PDLCs are still unknown. In this study, we have compared the immunophenotypic features and cell cycle status of the two cell lines. The results suggest that DFCs and PDLCs displayed similar features related to immunophenotype and cell cycle. Then we employed an isobaric tag for relative and absolute quantitation (iTRAQ) proteomics strategy to reveal the molecular differences between the two cell types. A total of 2138 proteins were identified and 39 of these proteins were consistently differentially expressed between DFCs and PDLCs. Gene ontology analyses revealed that the protein subsets expressed higher in PDLCs were related to actin binding, cytoskeletal protein binding, and structural constituent of muscle. Upon validation by real-time PCR, western blotting, and immunofluorescence staining. Tropomyosin 1 (TPM1) and caldesmon 1 (CALD1) were expressed higher in PDLCs than in DFCs. Our results suggested that PDLCs display enhanced actin cytoskeletal dynamics relative to DFCs while DFCs may exhibit a more robust antioxidant defense ability relative to PDLCs. This study expands our knowledge of the cultured DFCs and PDLCs proteome and provides new insights into possible mechanisms responsible for the different biological features observed in each cell type. PMID:26708290

  13. Force-producing ADP state of myosin bound to actin

    PubMed Central

    Wulf, Sarah F.; Ropars, Virginie; Fujita-Becker, Setsuko; Oster, Marco; Hofhaus, Goetz; Trabuco, Leonardo G.; Pylypenko, Olena; Sweeney, H. Lee; Houdusse, Anne M.; Schröder, Rasmus R.

    2016-01-01

    Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V. PMID:26976594

  14. Force-producing ADP state of myosin bound to actin.

    PubMed

    Wulf, Sarah F; Ropars, Virginie; Fujita-Becker, Setsuko; Oster, Marco; Hofhaus, Goetz; Trabuco, Leonardo G; Pylypenko, Olena; Sweeney, H Lee; Houdusse, Anne M; Schröder, Rasmus R

    2016-03-29

    Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V. PMID:26976594

  15. Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension

    PubMed Central

    Johnson, Jennifer A.; Hemnes, Anna R.; Perrien, Daniel S.; Schuster, Manfred; Robinson, Linda J.; Gladson, Santhi; Loibner, Hans; Bai, Susan; Blackwell, Tom R.; Tada, Yuji; Harral, Julie W.; Talati, Megha; Lane, Kirk B.; Fagan, Karen A.

    2012-01-01

    The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2R899X). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2R899X transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2R899X mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease. PMID:22180660

  16. Integrin adhesion drives the emergent polarization of active cytoskeletal stresses to pattern cell delamination

    PubMed Central

    Meghana, C.; Ramdas, Nisha; Hameed, Feroz Meeran; Rao, Madan; Shivashankar, G. V.; Narasimha, Maithreyi

    2011-01-01

    Tissue patterning relies on cellular reorganization through the interplay between signaling pathways and mechanical stresses. Their integration and spatiotemporal coordination remain poorly understood. Here we investigate the mechanisms driving the dynamics of cell delamination, diversely deployed to extrude dead cells or specify distinct cell fates. We show that a local mechanical stimulus (subcellular laser perturbation) releases cellular prestress and triggers cell delamination in the amnioserosa during Drosophila dorsal closure, which, like spontaneous delamination, results in the rearrangement of nearest neighbors around the delaminating cell into a rosette. We demonstrate that a sequence of “emergent cytoskeletal polarities” in the nearest neighbors (directed myosin flows, lamellipodial growth, polarized actomyosin collars, microtubule asters), triggered by the mechanical stimulus and dependent on integrin adhesion, generate active stresses that drive delamination. We interpret these patterns in the language of active gels as asters formed by active force dipoles involving surface and body stresses generated by each cell and liken delamination to mechanical yielding that ensues when these stresses exceed a threshold. We suggest that differential contributions of adhesion, cytoskeletal, and external stresses must underlie differences in spatial pattern. PMID:21571643

  17. beta-Dystroglycan modulates the interplay between actin and microtubules in human-adhered platelets.

    PubMed

    Cerecedo, Doris; Cisneros, Bulmaro; Suárez-Sánchez, Rocío; Hernández-González, Enrique; Galván, Iván

    2008-05-01

    To maintain the continuity of an injured blood vessel, platelets change shape, secrete granule contents, adhere, aggregate, and retract in a haemostatic plug. Ordered arrays of microtubules, microfilaments, and associated proteins are responsible for these platelet responses. In full-spread platelets, microfilament bundles in association with other cytoskeleton proteins are anchored in focal contacts. Recent studies in migrating cells suggest that co-ordination and direct physical interaction of microtubules and actin network modulate adhesion development. In platelets, we have proposed a feasible association between these two cytoskeletal systems, as well as the participation of the dystrophin-associated protein complex, as part of the focal adhesion complex. The present study analysed the participation of microtubules and actin during the platelet adhesion process. Confocal microscopy, fluorescence resonance transfer energy and immunoprecipitation assays were used to provide evidence of a cross-talk between these two cytoskeletal systems. Interestingly, beta-dystroglycan was found to act as an interplay protein between actin and microtubules and an additional communication between these two cytoskeleton networks was maintained through proteins of focal adhesion complex. Altogether our data are indicative of a dynamic co-participation of actin filaments and microtubules in modulating focal contacts to achieve platelet function. PMID:18341635

  18. Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments.

    PubMed

    Heaslip, Aoife T; Nelson, Shane R; Warshaw, David M

    2016-07-01

    The survival of Toxoplasma gondii within its host cell requires protein release from secretory vesicles, called dense granules, to maintain the parasite's intracellular replicative niche. Despite the importance of DGs, nothing is known about the mechanisms underlying their transport. In higher eukaryotes, secretory vesicles are transported to the plasma membrane by molecular motors moving on their respective cytoskeletal tracks (i.e., microtubules and actin). Because the organization of these cytoskeletal structures differs substantially in T. gondii, the molecular motor dependence of DG trafficking is far from certain. By imaging the motions of green fluorescent protein-tagged DGs in intracellular parasites with high temporal and spatial resolution, we show through a combination of molecular genetics and chemical perturbations that directed DG transport is independent of microtubules and presumably their kinesin/dynein motors. However, directed DG transport is dependent on filamentous actin and a unique class 27 myosin, TgMyoF, which has structural similarity to myosin V, the prototypical cargo transporter. Actomyosin DG transport was unexpected, since filamentous parasite actin has yet to be visualized in vivo due in part to the prevailing model that parasite actin forms short, unstable filaments. Thus our data uncover new critical roles for these essential proteins in the lytic cycle of this devastating pathogen. PMID:27146112

  19. Claisen thermally rearranged (CTR) polymers.

    PubMed

    Tena, Alberto; Rangou, Sofia; Shishatskiy, Sergey; Filiz, Volkan; Abetz, Volker

    2016-07-01

    Thermally rearranged (TR) polymers, which are considered the next-generation of membrane materials because of their excellent transport properties and high thermal and chemical stability, are proven to have significant drawbacks because of the high temperature required for the rearrangement and low degree of conversion during this process. We demonstrate that using a [3,3]-sigmatropic rearrangement, the temperature required for the rearrangement of a solid glassy polymer was reduced by 200°C. Conversions of functionalized polyimide to polybenzoxazole of more than 97% were achieved. These highly mechanically stable polymers were almost five times more permeable and had more than two times higher degrees of conversion than the reference polymer treated under the same conditions. Properties of these second-generation TR polymers provide the possibility of preparing efficient polymer membranes in a form of, for example, thin-film composite membranes for various gas and liquid membrane separation applications. PMID:27482538

  20. Claisen thermally rearranged (CTR) polymers

    PubMed Central

    Tena, Alberto; Rangou, Sofia; Shishatskiy, Sergey; Filiz, Volkan; Abetz, Volker

    2016-01-01

    Thermally rearranged (TR) polymers, which are considered the next-generation of membrane materials because of their excellent transport properties and high thermal and chemical stability, are proven to have significant drawbacks because of the high temperature required for the rearrangement and low degree of conversion during this process. We demonstrate that using a [3,3]-sigmatropic rearrangement, the temperature required for the rearrangement of a solid glassy polymer was reduced by 200°C. Conversions of functionalized polyimide to polybenzoxazole of more than 97% were achieved. These highly mechanically stable polymers were almost five times more permeable and had more than two times higher degrees of conversion than the reference polymer treated under the same conditions. Properties of these second-generation TR polymers provide the possibility of preparing efficient polymer membranes in a form of, for example, thin-film composite membranes for various gas and liquid membrane separation applications. PMID:27482538

  1. Radical Smiles Rearrangement: An Update.

    PubMed

    Allart-Simon, Ingrid; Gérard, Stéphane; Sapi, Janos

    2016-01-01

    Over the decades the Smiles rearrangement and its variants have become essential synthetic tools in modern synthetic organic chemistry. In this mini-review we summarized some very recent results of the radical version of these rearrangements. The selected examples illustrate the synthetic power of this approach, especially if it is incorporated into a domino process, for the preparation of polyfunctionalized complex molecules. PMID:27399654

  2. Dynamic simulations of membranes with cytoskeletal interactions

    NASA Astrophysics Data System (ADS)

    Lin, Lawrence C.-L.; Brown, Frank L. H.

    2005-07-01

    We describe a simulation algorithm for the dynamics of elastic membrane sheets over long length and time scales. Our model includes implicit hydrodynamic coupling between membrane and surrounding solvent and allows for arbitrary external forces acting on the membrane surface. In particular, the methodology is well suited to studying membranes in interaction with cytoskeletal filaments. We present results for the thermal undulations of a lipid bilayer attached to a regular network of spectrin filaments as a model for the red blood cell membrane. The dynamic fluctuations of the bilayer over the spectrin network are quantified and used to predict the macroscopic diffusion constant of band 3 on the surface of the red blood cell. We find that thermal undulations likely play a role in the mobility of band 3 in the plane of the erythrocyte membrane.

  3. Interplay of active processes modulates tension and drives phase transition in self-renewing, motor-driven cytoskeletal networks

    PubMed Central

    Mak, Michael; Zaman, Muhammad H.; Kamm, Roger D.; Kim, Taeyoon

    2016-01-01

    The actin cytoskeleton—a complex, nonequilibrium network consisting of filaments, actin-crosslinking proteins (ACPs) and motors—confers cell structure and functionality, from migration to morphogenesis. While the core components are recognized, much less is understood about the behaviour of the integrated, disordered and internally active system with interdependent mechano-chemical component properties. Here we use a Brownian dynamics model that incorporates key and realistic features—specifically actin turnover, ACP (un)binding and motor walking—to reveal the nature and underlying regulatory mechanisms of overarching cytoskeletal states. We generate multi-dimensional maps that show the ratio in activity of these microscopic elements determines diverse global stress profiles and the induction of nonequilibrium morphological phase transition from homogeneous to aggregated networks. In particular, actin turnover dynamics plays a prominent role in tuning stress levels and stabilizing homogeneous morphologies in crosslinked, motor-driven networks. The consequence is versatile functionality, from dynamic steady-state prestress to large, pulsed constrictions. PMID:26744226

  4. Actin evolution in ciliates (Protist, Alveolata) is characterized by high diversity and three duplication events.

    PubMed

    Yi, Zhenzhen; Huang, Lijuan; Yang, Ran; Lin, Xiaofeng; Song, Weibo

    2016-03-01

    Ciliates possess two distinct nuclear genomes and unique genomic features, including highly fragmented chromosomes and extensive chromosomal rearrangements. Recent transcriptomic surveys have revealed that ciliates have several multi-copy genes providing an ideal template to study gene family evolution. Nonetheless, this process remains little studied in ciliated protozoa and consequently, the evolutionary patterns that govern it are not well understood. In this study, we focused on obtaining fine-scale information relative to ciliate species divergence for the first time. A total of 230 actin gene sequences were derived from this study, among which 217 were from four closely related Pseudokeronopsis species and 13 from other hypotrichous ciliates. Our investigation shows that: (1) At least three duplication events occurred in ciliates: diversification of three actin genes (Actin I, II, III) happened after the divergence of ciliate classes but before that of subclasses. And several recent and genus-specific duplications were followed within Actin I (Sterkiella, Oxytricha, Uroleptus, etc.), Actin II (Sterkiella), respectively. (2) Within the genus Pseudokeronopsis, Actin I gene duplication events happened after P. carnea and P. erythrina diverged. In contrast, in the morphologically similar species P. flava and P. rubra, the duplication event preceded diversification of the two species. The Actin II gene duplication events preceded divergence of the genus Pseudokeronopsis. (3) Phylogenetic analyses revealed that actin is suitable for resolving ciliate classes, but may not be used to infer lower taxon relationships. PMID:26721556

  5. Interaction of myelin basic protein with cytoskeletal and signaling proteins in cultured primary oligodendrocytes and N19 oligodendroglial cells

    PubMed Central

    2014-01-01

    Background The classic myelin basic protein (MBP) isoforms are intrinsically-disordered proteins of 14–21.5 kDa in size arising from the Golli (Gene in the Oligodendrocyte Lineage) gene complex, and are responsible for formation of the multilayered myelin sheath in the central nervous system. The predominant membrane-associated isoform of MBP is not simply a structural component of compact myelin but is highly post-translationally modified and multi-functional, having interactions with numerous proteins such as Ca2+-calmodulin, and with actin, tubulin, and proteins with SH3-domains, which it can tether to a lipid membrane in vitro. It co-localizes with such proteins in primary oligodendrocytes (OLGs) and in early developmental N19-OLGs transfected with fluorescently-tagged MBP. Results To provide further evidence for MBP associations with these proteins in vivo, we show here that MBP isoforms are co-immunoprecipitated from detergent extracts of primary OLGs together with actin, tubulin, zonula occludens 1 (ZO-1), cortactin, and Fyn kinase. We also carry out live-cell imaging of N19-OLGs co-transfected with fluorescent MBP and actin, and show that when actin filaments re-assemble after recovery from cytochalasin D treatment, MBP and actin are rapidly enriched and co-localized at certain sites at the plasma membrane and in newly-formed membrane ruffles. The MBP and actin distributions change similarly with time, suggesting a specific and dynamic association. Conclusions These results provide more direct evidence for association of the predominant 18.5-kDa MBP isoform with these proteins in primary OLGs and in live cells than previously could be inferred from co-localization observations. This study supports further a role for classic MBP isoforms in protein-protein interactions during membrane and cytoskeletal extension and remodeling in OLGs. PMID:24956930

  6. Activated ADF/cofilin sequesters phosphorylated microtubule-associated-protein during the assembly of Alzheimer-like neuritic cytoskeletal striations

    PubMed Central

    Whiteman, Ineka T.; Gervasio, Othon L.; Cullen, Karen M.; Guillemin, Gilles J.; Jeong, Erica V.; Witting, Paul K.; Antao, Shane T.; Minamide, Laurie S.; Bamburg, James R.; Goldsbury, Claire

    2009-01-01

    In Alzheimer disease (AD), rod-like cofilin aggregates (cofilin-actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads) and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/tau and mimic neuropil threads. Fluorescence Resonance Energy Transfer (FRET) analysis revealed co-localization of cofilin-GFP and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin-actin rods was further investigated using actin-modifying drugs and siRNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin-actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous Aβ peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction or Aβ peptides, all suspected initiators of synaptic loss and neurodegeneration in AD. PMID:19828813

  7. Papaverine Prevents Vasospasm by Regulation of Myosin Light Chain Phosphorylation and Actin Polymerization in Human Saphenous Vein

    PubMed Central

    Hocking, Kyle M.; Putumbaka, Gowthami; Wise, Eric S.; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2016-01-01

    Objective Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. PMID:27136356

  8. Viscoelastic properties of actin networks influence material transport

    NASA Astrophysics Data System (ADS)

    Stam, Samantha; Weirich, Kimberly; Gardel, Margaret

    2015-03-01

    Directed flows of cytoplasmic material are important in a variety of biological processes including assembly of a mitotic spindle, retraction of the cell rear during migration, and asymmetric cell division. Networks of cytoskeletal polymers and molecular motors are known to be involved in these events, but how the network mechanical properties are tuned to perform such functions is not understood. Here, we construct networks of either semiflexible actin filaments or rigid bundles with varying connectivity. We find that solutions of rigid rods, where unimpeded sliding of filaments may enhance transport in comparison to unmoving tracks, are the fastest at transporting network components. Entangled solutions of semiflexible actin filaments also transport material, but the entanglements provide resistance. Increasing the elasticity of the actin networks with crosslinking proteins slows network deformation further. However, the length scale of correlated transport in these networks is increased. Our results reveal how the rigidity and connectivity of biopolymers allows material transport to occur over time and length scales required for physiological processes. This work was supported by the U. Chicago MRSEC

  9. Expression of transfected mutant beta-actin genes: transitions toward the stable tumorigenic state.

    PubMed Central

    Leavitt, J; Ng, S Y; Varma, M; Latter, G; Burbeck, S; Gunning, P; Kedes, L

    1987-01-01

    . Our results suggest that this mutant beta-actin contributes to the neoplastic phenotype of immortalized human fibroblasts by imposing a cytoarchitectural defect and inducing abnormal expression of cytoskeletal tropomyosins. Images PMID:3614199

  10. A theoretical model of cytokinesis implicates feedback between membrane curvature and cytoskeletal organization in asymmetric cytokinetic furrowing.

    PubMed

    Dorn, Jonas F; Zhang, Li; Phi, Tan-Trao; Lacroix, Benjamin; Maddox, Paul S; Liu, Jian; Maddox, Amy Shaub

    2016-04-15

    During cytokinesis, the cell undergoes a dramatic shape change as it divides into two daughter cells. Cell shape changes in cytokinesis are driven by a cortical ring rich in actin filaments and nonmuscle myosin II. The ring closes via actomyosin contraction coupled with actin depolymerization. Of interest, ring closure and hence the furrow ingression are nonconcentric (asymmetric) within the division plane across Metazoa. This nonconcentricity can occur and persist even without preexisting asymmetric cues, such as spindle placement or cellular adhesions. Cell-autonomous asymmetry is not explained by current models. We combined quantitative high-resolution live-cell microscopy with theoretical modeling to explore the mechanistic basis for asymmetric cytokinesis in theCaenorhabditis eleganszygote, with the goal of uncovering basic principles of ring closure. Our theoretical model suggests that feedback among membrane curvature, cytoskeletal alignment, and contractility is responsible for asymmetric cytokinetic furrowing. It also accurately predicts experimental perturbations of conserved ring proteins. The model further suggests that curvature-mediated filament alignment speeds up furrow closure while promoting energy efficiency. Collectively our work underscores the importance of membrane-cytoskeletal anchoring and suggests conserved molecular mechanisms for this activity. PMID:26912796

  11. A theoretical model of cytokinesis implicates feedback between membrane curvature and cytoskeletal organization in asymmetric cytokinetic furrowing

    PubMed Central

    Dorn, Jonas F.; Zhang, Li; Phi, Tan-Trao; Lacroix, Benjamin; Maddox, Paul S.; Liu, Jian; Maddox, Amy Shaub

    2016-01-01

    During cytokinesis, the cell undergoes a dramatic shape change as it divides into two daughter cells. Cell shape changes in cytokinesis are driven by a cortical ring rich in actin filaments and nonmuscle myosin II. The ring closes via actomyosin contraction coupled with actin depolymerization. Of interest, ring closure and hence the furrow ingression are nonconcentric (asymmetric) within the division plane across Metazoa. This nonconcentricity can occur and persist even without preexisting asymmetric cues, such as spindle placement or cellular adhesions. Cell-autonomous asymmetry is not explained by current models. We combined quantitative high-resolution live-cell microscopy with theoretical modeling to explore the mechanistic basis for asymmetric cytokinesis in the Caenorhabditis elegans zygote, with the goal of uncovering basic principles of ring closure. Our theoretical model suggests that feedback among membrane curvature, cytoskeletal alignment, and contractility is responsible for asymmetric cytokinetic furrowing. It also accurately predicts experimental perturbations of conserved ring proteins. The model further suggests that curvature-mediated filament alignment speeds up furrow closure while promoting energy efficiency. Collectively our work underscores the importance of membrane–cytoskeletal anchoring and suggests conserved molecular mechanisms for this activity. PMID:26912796

  12. Immunoglobulin λ Gene Rearrangement Can Precede κ Gene Rearrangement

    DOE PAGESBeta

    Berg, Jörg; Mcdowell, Mindy; Jäck, Hans-Martin; Wabl, Matthias

    1990-01-01

    Imore » mmunoglobulin genes are generated during differentiation of B lymphocytes by joining gene segments. A mouse pre-B cell contains a functional immunoglobulin heavy-chain gene, but no light-chain gene. Although there is only one heavy-chain locus, there are two lightchain loci: κ and λ .It has been reported that κ loci in the germ-line configuration are never (in man) or very rarely (in the mouse) present in cells with functionally rearranged λ -chain genes. Two explanations have been proposed to explain this: (a) the ordered rearrangement theory, which postulates that light-chain gene rearrangement in the pre-B cell is first attempted at the κ locus, and that only upon failure to produce a functional κ chain is there an attempt to rearrange the λ locus; and (b) the stochastic theory, which postulates that rearrangement at the λ locus proceeds at a rate that is intrinsically much slower than that at the κ locus. We show here that λ -chain genes are generated whether or not the κ locus has lost its germ-line arrangement, a result that is compatible only with the stochastic theory.« less

  13. Heterogeneous Porphyromonas gingivalis LPS modulates immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in human gingival fibroblasts

    PubMed Central

    Herath, Thanuja D. K.; Darveau, Richard P.; Seneviratne, Chaminda J.; Wang, Cun-Yu; Wang, Yu; Jin, Lijian

    2016-01-01

    Periodontal (gum) disease is a highly prevalent infection and inflammation accounting for the majority of tooth loss in adult population worldwide. Porphyromonas gingivalis is a keystone periodontal pathogen and its lipopolysaccharide (PgLPS) acts as a major virulence attribute to the disease. Herein, we deciphered the overall host response of human gingival fibroblasts (HGFs) to two featured isoforms of tetra-acylated PgLPS1435/1449 and penta-acylated PgLPS1690 with reference to E. coli LPS through quantitative proteomics. This study unraveled differentially expressed novel biomarkers of immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs. PgLPS1690 greatly upregulated inflammatory proteins (e.g. cyclophilin, inducible nitric oxide synthase, annexins, galectin, cathepsins and heat shock proteins), whereas the anti-inflammatory proteins (e.g. Annexin A2 and Annexin A6) were significantly upregulated by PgLPS1435/1449. Interestingly, the antioxidants proteins such as mitochondrial manganese-containing superoxide dismutase and peroxiredoxin 5 were only upregulated by PgLPS1690. The cytoskeletal rearrangement-related proteins like myosin were differentially regulated by these PgLPS isoforms. The present study gives new insight into the biological properties of P. gingivalis LPS lipid A moiety that could critically modulate immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs, and thereby enhances our understanding of periodontal pathogenesis. PMID:27538450

  14. Heterogeneous Porphyromonas gingivalis LPS modulates immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in human gingival fibroblasts.

    PubMed

    Herath, Thanuja D K; Darveau, Richard P; Seneviratne, Chaminda J; Wang, Cun-Yu; Wang, Yu; Jin, Lijian

    2016-01-01

    Periodontal (gum) disease is a highly prevalent infection and inflammation accounting for the majority of tooth loss in adult population worldwide. Porphyromonas gingivalis is a keystone periodontal pathogen and its lipopolysaccharide (PgLPS) acts as a major virulence attribute to the disease. Herein, we deciphered the overall host response of human gingival fibroblasts (HGFs) to two featured isoforms of tetra-acylated PgLPS1435/1449 and penta-acylated PgLPS1690 with reference to E. coli LPS through quantitative proteomics. This study unraveled differentially expressed novel biomarkers of immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs. PgLPS1690 greatly upregulated inflammatory proteins (e.g. cyclophilin, inducible nitric oxide synthase, annexins, galectin, cathepsins and heat shock proteins), whereas the anti-inflammatory proteins (e.g. Annexin A2 and Annexin A6) were significantly upregulated by PgLPS1435/1449. Interestingly, the antioxidants proteins such as mitochondrial manganese-containing superoxide dismutase and peroxiredoxin 5 were only upregulated by PgLPS1690. The cytoskeletal rearrangement-related proteins like myosin were differentially regulated by these PgLPS isoforms. The present study gives new insight into the biological properties of P. gingivalis LPS lipid A moiety that could critically modulate immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs, and thereby enhances our understanding of periodontal pathogenesis. PMID:27538450

  15. Polyphosphoinositides-dependent regulation of the osteoclast actin cytoskeleton and bone resorption

    PubMed Central

    Biswas, Rajat S; Baker, De Anna; Hruska, Keith A; Chellaiah, Meenakshi A

    2004-01-01

    Background Gelsolin, an actin capping protein of osteoclast podosomes, has a unique function in regulating assembly and disassembly of the podosome actin filament. Previously, we have reported that osteopontin (OPN) binding to integrin αvβ3 increased the levels of gelsolin-associated polyphosphoinositides, podosome assembly/disassembly, and actin filament formation. The present study was undertaken to identify the possible role of polyphosphoinositides and phosphoinositides binding domains (PBDs) of gelsolin in the osteoclast cytoskeletal structural organization and osteoclast function. Results Transduction of TAT/full-length gelsolin and PBDs containing gelsolin peptides into osteoclasts demonstrated: 1) F-actin enriched patches; 2) disruption of actin ring; 3) an increase in the association polyphosphoinositides (PPIs) with the transduced peptides containing PBDs. The above-mentioned effects were more pronounced with gelsolin peptide containing 2 tandem repeats of PBDs (PBD (2)). Binding of PPIs to the transduced peptides has resulted in reduced levels of PPIs association with the endogenous gelsolin, and thereby disrupted the actin remodeling processes in terms of podosome organization in the clear zone area and actin ring formation. These peptides also exhibited a dominant negative effect in the formation of WASP-Arp2/3 complex indicating the role of phosphoinositides in WASP activation. The TAT-PBD gelsolin peptides transduced osteoclasts are functionally defective in terms of motility and bone resorption. Conclusions Taken together, these data demonstrate that transduction of PBD gelsolin peptides into osteoclasts produced a dominant negative effect on actin assembly, motility, and bone resorption. These findings indicate that phosphoinositide-mediated signaling mechanisms regulate osteoclast cytoskeleton, podosome assembly/disassembly, actin ring formation and bone resorption activity of osteoclasts. PMID:15142256

  16. Berberine induces dedifferentiation by actin cytoskeleton reorganization via phosphoinositide 3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes.

    PubMed

    Yu, Seon-Mi; Cho, Hongsik; Kim, Gwang-Hoon; Chung, Ki-Wha; Seo, Sung-Yum; Kim, Song-Ja

    2016-04-01

    Osteoarthritis is a nonrheumatologic joint disease characterized by progressive degeneration of the cartilage extracellular matrix. Berberine (BBR) is an isoquinoline alkaloid used in traditional Chinese medicine, the majority of which is extracted from Huang Lian (Coptis chinensis). Although numerous studies have revealed the anticancer activity of BBR, its effects on normal cells, such as chondrocytes, and the molecular mechanisms underlying its actions remain elusive. Therefore, we examined the effects of BBR on rabbit articular chondrocytes, and the underlying molecular mechanisms, focusing on actin cytoskeletal reorganization. BBR induced dedifferentiation by inhibiting activation of phosphoinositide-3(PI3)-kinase/Akt and p38 kinase. Furthermore, inhibition of p38 kinase and PI3-kinase/Akt with SB203580 and LY294002, respectively, accelerated the BBR-induced dedifferentiation. BBR also caused actin cytoskeletal architecture reorganization and, therefore, we investigated if these effects were involved in the dedifferentiation. Disruption of the actin cytoskeleton by cytochalasin D reversed the BBR-induced dedifferentiation by activating PI3-kinase/Akt and p38 kinase. In contrast, the induction of actin filament aggregation by jasplakinolide accelerated the BBR-induced dedifferentiation via PI3-kinase/Akt inhibition and p38 kinase activation. Taken together, these data suggest that BBR strongly induces dedifferentiation, and actin cytoskeletal reorganization is a crucial requirement for this effect. Furthermore, the dedifferentiation activity of BBR appears to be mediated via PI3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes. PMID:26851252

  17. Short Stop provides an essential link between F-actin and microtubules during axon extension.

    PubMed

    Lee, Seungbok; Kolodziej, Peter A

    2002-03-01

    Coordination of F-actin and microtubule dynamics is important for cellular motility and morphogenesis, but little is known about underlying mechanisms. short stop (shot) encodes an evolutionarily conserved, neuronally expressed family of rod-like proteins required for sensory and motor axon extension in Drosophila melanogaster. We identify Shot isoforms that contain N-terminal F-actin and C-terminal microtubule-binding domains, and that crosslink F-actin and microtubules in cultured cells. The F-actin- and microtubule-binding domains of Shot are required in the same molecule for axon extension, though the length of the connecting rod domain can be dramatically reduced without affecting activity. Shot therefore functions as a cytoskeletal crosslinker in axon extension, rather than mediating independent interactions with F-actin and microtubules. A Ca(2+)-binding motif located adjacent to the microtubule-binding domain is also required for axon extension, suggesting that intracellular Ca(2+) release may regulate Shot activity. These results suggest that Shot coordinates regulated interactions between F-actin and microtubules that are crucial for neuronal morphogenesis. PMID:11874915

  18. The dynamin inhibitor dynasore inhibits bone resorption by rapidly disrupting actin rings of osteoclasts.

    PubMed

    Thirukonda, Gnanasagar J; Uehara, Shunsuke; Nakayama, Takahiro; Yamashita, Teruhito; Nakamura, Yukio; Mizoguchi, Toshihide; Takahashi, Naoyuki; Yagami, Kimitoshi; Udagawa, Nobuyuki; Kobayashi, Yasuhiro

    2016-07-01

    The cytoskeletal organization of osteoclasts is required for bone resorption. Binding of dynamin with guanosine triphosphate (GTP) was previously suggested to be required for the organization of the actin cytoskeleton. However, the role of the GTPase activity of dynamin in the organization of the actin cytoskeleton as well as in the bone-resorbing activity of osteoclasts remains unclear. This study investigated the effects of dynasore, an inhibitor of the GTPase activity of dynamin, on the bone-resorbing activity of and actin ring formation in mouse osteoclasts in vitro and in vivo. Dynasore inhibited the formation of resorption pits in osteoclast cultures by suppressing actin ring formation and rapidly disrupting actin rings in osteoclasts. A time-lapse image analysis showed that dynasore shrank actin rings in osteoclasts within 30 min. The intraperitoneal administration of dynasore inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced trabecular bone loss in mouse femurs. These in vitro and in vivo results suggest that the GTPase activity of dynamin is critical for the bone-resorbing activity of osteoclasts and that dynasore is a seed for the development of novel anti-resorbing agents. PMID:26063501

  19. Requirements for contractility in disordered cytoskeletal bundles

    NASA Astrophysics Data System (ADS)

    Lenz, Martin; Gardel, Margaret L.; Dinner, Aaron R.

    2012-03-01

    Actomyosin contractility is essential for biological force generation, and is well understood in highly organized structures such as striated muscle. Additionally, actomyosin bundles devoid of this organization are known to contract both in vivo and in vitro, which cannot be described by standard muscle models. To narrow down the search for possible contraction mechanisms in these systems, we investigate their microscopic symmetries. We show that contractile behavior requires non-identical motors that generate large-enough forces to probe the nonlinear elastic behavior of F-actin. This suggests a role for filament buckling in the contraction of these bundles, consistent with recent experimental results on reconstituted actomyosin bundles.

  20. CONSERVED ROLES FOR CYTOSKELETAL COMPONENTS IN DETERMINING LATERALITY

    PubMed Central

    McDowell, Gary S.; Lemire, Joan M.; Paré, Jean-Francois; Cammarata, Garrett; Lowery, Laura Anne; Levin, Michael

    2016-01-01

    SUMMARY Consistently-biased left-right (LR) patterning is required for the proper placement of organs including the heart and viscera. The LR axis is especially fascinating as an example of multi-scale pattern formation, since here chiral events at the subcellular level are integrated and amplified into asymmetric transcriptional cascades and ultimately into the anatomical patterning of the entire body. In contrast to the other two body axes, there is considerable controversy about the earliest mechanisms of embryonic laterality. Many molecular components of asymmetry have not been widely tested among phyla with diverse bodyplans, and it is unknown whether parallel (redundant) pathways may exist that could reverse abnormal asymmetry states at specific checkpoints in development. To address conservation of the early steps of LR patterning, we used the Xenopus laevis (frog) embryo to functionally test a number of protein targets known to direct asymmetry in plants, fruit fly, and rodent. Using the same reagents that randomize asymmetry in Arabidopsis, Drosophila, and mouse embryos, we show that manipulation of the microtubule and actin cytoskeleton immediately post-fertilization, but not later, results in laterality defects in Xenopus embryos. Moreover, we observed organ-specific randomization effects and a striking dissociation of organ situs from effects on the expression of left side control genes, which parallel data from Drosophila and mouse. Remarkably, some early manipulations that disrupt laterality of transcriptional asymmetry determinants can be subsequently “rescued” by the embryo, resulting in normal organ situs. These data reveal the existence of novel corrective mechanisms, demonstrate that asymmetric expression of Nodal is not a definitive marker of laterality, and suggest the existence of amplification pathways that connect early cytoskeletal processes to control of organ situs bypassing Nodal. Counter to alternative models of symmetry breaking

  1. Conserved roles for cytoskeletal components in determining laterality.

    PubMed

    McDowell, Gary S; Lemire, Joan M; Paré, Jean-Francois; Cammarata, Garrett; Lowery, Laura Anne; Levin, Michael

    2016-03-14

    Consistently-biased left-right (LR) patterning is required for the proper placement of organs including the heart and viscera. The LR axis is especially fascinating as an example of multi-scale pattern formation, since here chiral events at the subcellular level are integrated and amplified into asymmetric transcriptional cascades and ultimately into the anatomical patterning of the entire body. In contrast to the other two body axes, there is considerable controversy about the earliest mechanisms of embryonic laterality. Many molecular components of asymmetry have not been widely tested among phyla with diverse bodyplans, and it is unknown whether parallel (redundant) pathways may exist that could reverse abnormal asymmetry states at specific checkpoints in development. To address conservation of the early steps of LR patterning, we used the Xenopus laevis (frog) embryo to functionally test a number of protein targets known to direct asymmetry in plants, fruit fly, and rodent. Using the same reagents that randomize asymmetry in Arabidopsis, Drosophila, and mouse embryos, we show that manipulation of the microtubule and actin cytoskeleton immediately post-fertilization, but not later, results in laterality defects in Xenopus embryos. Moreover, we observed organ-specific randomization effects and a striking dissociation of organ situs from effects on the expression of left side control genes, which parallel data from Drosophila and mouse. Remarkably, some early manipulations that disrupt laterality of transcriptional asymmetry determinants can be subsequently "rescued" by the embryo, resulting in normal organ situs. These data reveal the existence of novel corrective mechanisms, demonstrate that asymmetric expression of Nodal is not a definitive marker of laterality, and suggest the existence of amplification pathways that connect early cytoskeletal processes to control of organ situs bypassing Nodal. Counter to alternative models of symmetry breaking during

  2. Effects of transforming growth factor type beta on expression of cytoskeletal proteins in endosteal mouse osteoblastic cells

    SciTech Connect

    Lomri, A.; Marie, P.J. )

    1990-01-01

    Transforming growth factor beta (TGF beta) has been shown to influence the growth and differentiation of many cell types in vitro. We have examined the effects of TGF beta on cell morphology and cytoskeletal organization in relation to parameters of cell proliferation and differentiation in endosteal osteoblastic cells isolated from mouse caudal vertebrae. Treatment of mouse osteoblastic cells cultured in serum free medium for 24 hours with TGF beta (1.5-30 ng/mL) slightly (-23%) inhibited alkaline phosphatase activity. In parallel, TGF beta (0.5-30 ng/mL, 24 hours) greatly increased cell replication as evaluated by (3H)-thymidine incorporation into DNA (157% to 325% of controls). At a median dose (1.5 ng/mL) that affected both alkaline phosphatase and DNA synthesis (235% of controls) TGF beta induced rapid (six hours) cell respreading of quiescent mouse osteoblastic cells. This effect was associated with increased polymerization of actin, alpha actinin, and tubulins, as evaluated by both biochemical and immunofluorescence methods. In addition, TGF beta (1.5 ng/mL) increased the de novo biosynthesis of actin, alpha actinin, vimentin, and tubulins, as determined by {sup 35}S methionine labeling and fractionation of cytoskeletal proteins using two-dimensional gel electrophoresis. These effects were rapid and transient, as they occurred at six hours and were reversed after 24 hours of TGF beta exposure. The results indicate that the stimulatory effect of TGF beta on DNA synthesis in endosteal mouse osteoblastic cells is associated with a transient increase in cell spreading associated with enhanced polymerization and synthesis of cytoskeletal proteins.

  3. Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin

    PubMed Central

    Wilhelmsen, Kevin; Litjens, Sandy H.M.; Kuikman, Ingrid; Tshimbalanga, Ntambua; Janssen, Hans; van den Bout, Iman; Raymond, Karine; Sonnenberg, Arnoud

    2005-01-01

    Despite their importance in cell biology, the mechanisms that maintain the nucleus in its proper position in the cell are not well understood. This is primarily the result of an incomplete knowledge of the proteins in the outer nuclear membrane (ONM) that are able to associate with the different cytoskeletal systems. Two related ONM proteins, nuclear envelope spectrin repeat (nesprin)–1 and –2, are known to make direct connections with the actin cytoskeleton through their NH2-terminal actin-binding domain (ABD). We have now isolated a third member of the nesprin family that lacks an ABD and instead binds to the plakin family member plectin, which can associate with the intermediate filament (IF) system. Overexpression of nesprin-3 results in a dramatic recruitment of plectin to the nuclear perimeter, which is where these two molecules are colocalized with both keratin-6 and -14. Importantly, plectin binds to the integrin α6β4 at the cell surface and to nesprin-3 at the ONM in keratinocytes, suggesting that there is a continuous connection between the nucleus and the extracellular matrix through the IF cytoskeleton. PMID:16330710

  4. Organization of cytoskeletal elements and organelles preceding growth cone emergence from an identified neuron in situ.

    PubMed

    Lefcort, F; Bentley, D

    1989-05-01

    The purpose of this study was to investigate the arrangement of cytoskeletal elements and organelles in an identified neuron in situ at the site of emergence of its growth cone just before and concurrent with the onset of axonogenesis. The Ti1 pioneer neurons are the first pair of afferent neurons to differentiate in embryonic grasshopper limbs. They arise at the distal tip of the limb bud epithelium, the daughter cells of a single precursor cell, the Pioneer Mother Cell (PMC). Using immunohistochemical markers, we characterized the organization of microtubules, centrosomes, Golgi apparatus, midbody, actin filaments, and chromatin from mitosis in the PMC through axonogenesis in the Tils. Just before and concurrent with the onset of axonogenesis, a characteristic arrangement of tubulin, actin filaments, and Golgi apparatus is localized at the proximal pole of the proximal pioneer neuron. The growth cone of the proximal cell stereotypically arises from this site. Although the distal cell's axon generally grows proximally, occasionally it arises from its distal pole; in such limbs, the axons from the sister cells extend from mirror symmetric locations on their somata. In the presence of cytochalasin D, the PMC undergoes nuclear division but not cytokinesis and although other neuronal phenotypes are expressed, axongenesis is inhibited. Our data suggest that intrinsic information determines the site of growth cone emergence of an identified neuron in situ. PMID:2654140

  5. Effects of fixation protocol and gravistimulation on cytoskeletal organization in Brassica rapa roots

    NASA Astrophysics Data System (ADS)

    Edge, Andrea; Hasenstein, Karl H.

    2012-07-01

    In preparation for a flight experiment we have studied the optimization of the staining protocols for microtubules and actin filaments in Brassica rapa seedlings. Microtubules (MT) were stained with monoclonal antibody (mAb) YOL 1/34. F-actin (FA) staining was achieved with C4 mAb antibody. Fixative prepared more than three weeks before use produces specimens that stained poorly. Storage in fixative for more than four weeks resulted in noticeably poorer staining. Staining was best in cortical cells but more difficult and less consistent in cap cells, especially for FA. In addition, the quality of staining of root cap cells was dependent on the age of the formaldehyde. The organization of the MTs corresponded with previously published descriptions; FA was prominent in the stele with thick and numerous parallel bundles; cortical cells showed less dense and less directional organization of mostly thinner filaments. FA organization was determined by tissue rather than by differential elongation. The organization of MTs in cortical cells of curving roots was uniformly circular and perpendicular to the long cell axis despite different cell length. The effect of clinorotation around the horizontal axis and centrifugation on the cytoskeletal organization was inconsistent. (Supported by NASA grant NNX10AP91G)

  6. Bacteroides fragilis enterotoxin induces cytoskeletal changes and surface blebbing in HT-29 cells.

    PubMed Central

    Donelli, G; Fabbri, A; Fiorentini, C

    1996-01-01

    Certain strains of the anaerobic bacterium Bacteroides fragilis are known to produce an enterotoxin of about 20 kDa which is able to induce a fluid response in ligated intestinal loops and a cytotoxic response in HT-29 cells. It presents protease activity, belonging to a family of metalloproteases termed metzincins. In order to investigate the mode of action of the enterotoxin in cultured cells, we performed a study with HT-29 cells, using both fluoresence and electron microscopy. Treated cells underwent morphological changes, mainly consisting of the retraction of the cell body and the formation of numerous blebs on the cell surface. The microfilament system was reorganized, the F-actin being condensed as a ring at the cell periphery, whereas other cell organelles appeared to be unaffected. All these changes, clearly visible after 3 h of exposure to the toxin, were reversed within 24 h of treatment. By inhibiting the protease activity of the toxin with specific metal chelators, the cytoskeletal effects were also prevented. Thus, B. fragilis enterotoxin appears to act on cells by reversibly modifying the actin cytoskeleton, an effect probably dependent on its proteolytic activity. PMID:8557328

  7. Quantitative analysis of cytoskeletal reorganization during epithelial tissue sealing by large-volume electron tomography.

    PubMed

    Eltsov, Mikhail; Dubé, Nadia; Yu, Zhou; Pasakarnis, Laurynas; Haselmann-Weiss, Uta; Brunner, Damian; Frangakis, Achilleas S

    2015-05-01

    The closure of epidermal openings is an essential biological process that causes major developmental problems such as spina bifida in humans if it goes awry. At present, the mechanism of closure remains elusive. Therefore, we reconstructed a model closure event, dorsal closure in fly embryos, by large-volume correlative electron tomography. We present a comprehensive, quantitative analysis of the cytoskeletal reorganization, enabling separated epidermal cells to seal the epithelium. After establishing contact through actin-driven exploratory filopodia, cells use a single lamella to generate 'roof tile'-like overlaps. These shorten to produce the force, 'zipping' the tissue closed. The shortening overlaps lack detectable actin filament ensembles but are crowded with microtubules. Cortical accumulation of shrinking microtubule ends suggests a force generation mechanism in which cortical motors pull on microtubule ends as for mitotic spindle positioning. In addition, microtubules orient filopodia and lamellae before zipping. Our 4D electron microscopy picture describes an entire developmental process and provides fundamental insight into epidermal closure. PMID:25893916

  8. Oxytocin Increases Neurite Length and Expression of Cytoskeletal Proteins Associated with Neuronal Growth.

    PubMed

    Lestanova, Z; Bacova, Z; Kiss, A; Havranek, T; Strbak, V; Bakos, J

    2016-06-01

    Neuropeptide oxytocin acts as a growth and differentiation factor; however, its effects on neurite growth are poorly understood. The aims of the present study were (1) to evaluate time effects of oxytocin on expression of nestin and MAP2; (2) to measure the effect of oxytocin on gene expression of β-actin, vimentin, cofilin, and drebrin; and (3) to measure changes in neurite length and number in response to oxytocin/oxytocin receptor antagonist L-371,257. Exposure of SH-SY5Y cells to 1 μM oxytocin resulted in a significant increase in gene expression and protein levels of nestin after 12, 24, and 48 h. Oxytocin treatment induced no changes in gene expression of MAP2; however, a decrease of protein levels was observed in all time intervals. Gene expression of β-actin, vimentin, and drebrin increased in response to oxytocin. Oxytocin induced significant elongation of neurites after 12, 24, and 48 h. No change in neurite length was observed in the presence of the combination of retinoic acid and oxytocin receptor antagonist L-371,257. Oxytocin treatment for 12 h increased the number of neurites. Overall, the present data suggest that oxytocin contributes to the regulation of expression of cytoskeletal proteins associated with growth of neuronal cones and induces neurite elongation mediated by oxytocin receptors at least in certain types of neuronal cells. PMID:26474566

  9. Simultaneous tracking of 3D actin and microtubule strains in individual MLO-Y4 osteocytes under oscillatory flow.

    PubMed

    Baik, Andrew D; Qiu, Jun; Hillman, Elizabeth M C; Dong, Cheng; Guo, X Edward

    2013-02-22

    Osteocytes in vivo experience complex fluid shear flow patterns to activate mechanotransduction pathways. The actin and microtubule (MT) cytoskeletons have been shown to play an important role in the osteocyte's biochemical response to fluid shear loading. The dynamic nature of physiologically relevant fluid flow profiles (i.e., 1Hz oscillatory flow) impedes the ability to image and study both actin and MT cytoskeletons simultaneously in the same cell with high spatiotemporal resolution. To overcome these limitations, a multi-channel quasi-3D microscopy technique was developed to track the actin and MT networks simultaneously under steady and oscillatory flow. Cells displayed high intercellular variability and intracellular cytoskeletal variability in strain profiles. Shear Exz was the predominant strain in both steady and oscillatory flows in the form of viscoelastic creep and elastic oscillations, respectively. Dramatic differences were seen in oscillatory flow, however. The actin strains displayed an oscillatory strain profile more often than the MT networks in all the strains tested and had a higher peak-to-trough strain magnitude. Taken together, the actin networks are the more responsive cytoskeletal networks in osteocytes under oscillatory flow and may play a bigger role in mechanotransduction pathway activation and regulation. PMID:23352617

  10. Keratinocyte cytoskeletal roles in cell sheet engineering

    PubMed Central

    2013-01-01

    Background There is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragile and, over time, will contract. While the role of the cytoskeleton in maintaining the structure and adhesion of cells cultured on a rigid substrate is well-characterized, a systematic examination of the role played by different components of the cytoskeleton in regulating cell sheet contraction and cohesion in the absence of a substrate has been lacking. Results In this study, keratinocytes were cultured until confluent and cell sheets were generated using dispase to remove the influence of the substrate. The effects of disrupting actin, microtubules or intermediate filaments on cell-cell interactions were assessed by measuring cell sheet cohesion and contraction. Keratin intermediate filament disruption caused comparable effects on cell sheet cohesion and contraction, when compared to actin or microtubule disruption. Interfering with actomyosin contraction demonstrated that interfering with cell contraction can also diminish cell cohesion. Conclusions All components of the cytoskeleton are involved in maintaining cell sheet cohesion and contraction, although not to the same extent. These findings demonstrate that substrate-free cell sheet biomechanical properties are dependent on the integrity of the cytoskeleton network. PMID:23442760

  11. Regulation of the Postsynaptic Compartment of Excitatory Synapses by the Actin Cytoskeleton in Health and Its Disruption in Disease

    PubMed Central

    Stefen, Holly; Chaichim, Chanchanok

    2016-01-01

    Disruption of synaptic function at excitatory synapses is one of the earliest pathological changes seen in wide range of neurological diseases. The proper control of the segregation of neurotransmitter receptors at these synapses is directly correlated with the intact regulation of the postsynaptic cytoskeleton. In this review, we are discussing key factors that regulate the structure and dynamics of the actin cytoskeleton, the major cytoskeletal building block that supports the postsynaptic compartment. Special attention is given to the complex interplay of actin-associated proteins that are found in the synaptic specialization. We then discuss our current understanding of how disruption of these cytoskeletal elements may contribute to the pathological events observed in the nervous system under disease conditions with a particular focus on Alzheimer's disease pathology. PMID:27127658

  12. Emergence of Large-Scale Cell Morphology and Movement from Local Actin Filament Growth Dynamics

    PubMed Central

    Lacayo, Catherine I; Pincus, Zachary; VanDuijn, Martijn M; Wilson, Cyrus A; Fletcher, Daniel A; Gertler, Frank B; Mogilner, Alex; Theriot, Julie A

    2007-01-01

    Variations in cell migration and morphology are consequences of changes in underlying cytoskeletal organization and dynamics. We investigated how these large-scale cellular events emerge as direct consequences of small-scale cytoskeletal molecular activities. Because the properties of the actin cytoskeleton can be modulated by actin-remodeling proteins, we quantitatively examined how one such family of proteins, enabled/vasodilator-stimulated phosphoprotein (Ena/VASP), affects the migration and morphology of epithelial fish keratocytes. Keratocytes generally migrate persistently while exhibiting a characteristic smooth-edged “canoe” shape, but may also exhibit less regular morphologies and less persistent movement. When we observed that the smooth-edged canoe keratocyte morphology correlated with enrichment of Ena/VASP at the leading edge, we mislocalized and overexpressed Ena/VASP proteins and found that this led to changes in the morphology and movement persistence of cells within a population. Thus, local changes in actin filament dynamics due to Ena/VASP activity directly caused changes in cell morphology, which is coupled to the motile behavior of keratocytes. We also characterized the range of natural cell-to-cell variation within a population by using measurable morphological and behavioral features—cell shape, leading-edge shape, filamentous actin (F-actin) distribution, cell speed, and directional persistence—that we have found to correlate with each other to describe a spectrum of coordinated phenotypes based on Ena/VASP enrichment at the leading edge. This spectrum stretched from smooth-edged, canoe-shaped keratocytes—which had VASP highly enriched at their leading edges and migrated fast with straight trajectories—to more irregular, rounder cells migrating slower with less directional persistence and low levels of VASP at their leading edges. We developed a mathematical model that accounts for these coordinated cell-shape and behavior

  13. Motion in partially and fully cross-linked F-actin networks

    NASA Astrophysics Data System (ADS)

    Morris, Eliza; Ehrlicher, Allen; Weitz, David

    2012-02-01

    Single molecule experiments have measured stall forces and procession rates of molecular motors on isolated cytoskeletal fibers in Newtonian fluids. But in the cell, these motors are transporting cargo through a highly complex cytoskeletal network. To compare these single molecule results to the forces exerted by motors within the cell, an evaluation of the response of the cytoskeletal network is needed. Using magnetic tweezers and fluorescence confocal microscopy we observe and quantify the relationship between bead motion and filament response in F-actin networks both partially and fully cross-linked with filamin We find that when the transition from full to partial cross-linking is brought about by a decrease in cross-linker concentration there is a simultaneous decline in the elasticity of the network, but the response of the bead remains qualitatively similar. However, when the cross-linking is reduced through a shortening of the F-actin filaments the bead response is completely altered. The characteristics of the altered bead response will be discussed here.

  14. A biosensor for the protease TACE reveals actin damage induced TACE activation

    PubMed Central

    Chapnick, Douglas A.; Bunker, Eric; Liu, Xuedong

    2016-01-01

    Ligand shedding has gained increased attention as a major posttranslational modification mechanism used by cells to respond to diverse environmental conditions. The TACEadam17 protease is a critical mediator of such ligand shedding, regulating the maturation and release of an impressive range of extracellular substrates that drive diverse cellular responses. Exactly how this protease is itself activated remains unclear, in part due to the lack of available tools to measure TACE activity with temporal and spatial resolution in live cells. We have developed a FRET based biosensor for TACE activity (TSen), which is capable of reporting TACE activation kinetics in live cells with a high degree of specificity. TSen was used in combination with chemical biology to probe the dependence of various means of TACE activation on p38 and Erk kinase activities, as well as to identify a novel connection between actin cytoskeletal disruption and TACE activation. Such cytoskeletal disruption leads to rapid and robust TACE activation in some cell types and accumulation of TACE at the plasma membrane, allowing for increased cleavage of endogenous substrates. Our study highlights both the versatility of TSen as a tool to understand the mechanisms of TACE activation in live cells and the importance of actin cytoskeletal integrity as a modulator of TACE activity. PMID:25714465

  15. Initiation of Chondrocyte Self-Assembly Requires an Intact Cytoskeletal Network.

    PubMed

    Lee, Jennifer K; Hu, Jerry C Y; Yamada, Soichiro; Athanasiou, Kyriacos A

    2016-02-01

    Self-assembly and self-organization have recently emerged as robust scaffold-free tissue engineering methodologies that can be used to generate various tissues, including cartilage, vessel, and liver. Self-assembly, in particular, is a scaffold-free platform for tissue engineering that does not require the input of exogenous energy to the system. Although self-assembly can generate functional tissues, most notably neocartilage, the mechanisms of self-assembly remain unclear. To study the self-assembling process, we used articular chondrocytes as a model to identify parameters that can affect this process. Specifically, the roles of cell-cell and cell-matrix adhesion molecules, surface-bound collagen, and the actin cytoskeletal network were investigated. Using time-lapse imaging, we analyzed the early stages of chondrocyte self-assembly. Within hours, chondrocytes rapidly coalesced into cell clusters before compacting to form tight cellular structures. Chondrocyte self-assembly was found to depend primarily on integrin function and secondarily on cadherin function. In addition, actin or myosin II inhibitors prevented chondrocyte self-assembly, suggesting that cell adhesion alone is not sufficient, but rather the active contractile actin cytoskeleton is essential for proper chondrocyte self-assembly and the formation of neocartilage. Better understanding of the self-assembly mechanisms allows for the rational modulation of this process toward generating neocartilages with improved properties. These findings are germane to understanding self-assembly, an emerging platform for tissue engineering of a plethora of tissues, especially as these neotissues are poised for translation. PMID:26729374

  16. Specific inhibition of skeletal alpha-actin gene transcription by applied mechanical forces through integrins and actin.

    PubMed Central

    Lew, A M; Glogauer, M; Mculloch, C A

    1999-01-01

    Skeletal alpha-actin (skA), a prominent fetal actin isoform that is re-expressed by adult cardiac myocytes after chronic overload in vivo, provides a model for studying cytoskeletal gene regulation by mechanical forces in vitro. We have determined the mechanisms by which perpendicular applied forces acting through integrins and the actin cytoskeleton regulate the expression of skA. Rat-2 fibroblasts were transiently transfected with plasmids containing 5'-regulatory regions of the skA gene fused to luciferase coding sequences. A constant, perpendicular force (0.2 pN/micrometer(2)) was applied by using a collagen-magnetic bead model; a 25% deformation was obtained on the dorsal cell surface. In this system, force is applied through focal adhesion integrins and strongly induces actin assembly [Glogauer, Arora, Yao, Sokholov, Ferrier and McCulloch (1997) J. Cell Sci. 110, 11-21]. skA promoter activity was inhibited by 68% in cells subjected to 4 h of applied force, whereas Rous sarcoma virus promoter activity was unaffected. In cells transiently transfected with a skA expression vector there was also a parallel 40% decrease in skA protein levels by force, as shown by Western blotting. In L8 cells, constitutive skA expression was decreased by more than 50%. Analyses of specific motifs in the skA promoter revealed that transcriptional enhancer factor 1 and Yin and Yang 1 sites, but not serum response factor and Sp1 sites, mediated inhibitory responses to force. In cells treated with cycloheximide the force-induced inhibition was abrogated, indicating a dependence on new protein synthesis. Inhibition of actin filament assembly with either cytochalasin D or Ca(2+)-depleted medium blocked the inhibitory effect induced by the applied force, suggesting that actin filaments are required for the regulation of skA promoter activity. Western blot analysis showed that p38 kinase, but not Jun N-terminal kinase or extracellular signal-regulated protein kinase 1/2, was activated by

  17. Methods for modeling cytoskeletal and DNA filaments

    NASA Astrophysics Data System (ADS)

    Andrews, Steven S.

    2014-02-01

    This review summarizes the models that researchers use to represent the conformations and dynamics of cytoskeletal and DNA filaments. It focuses on models that address individual filaments in continuous space. Conformation models include the freely jointed, Gaussian, angle-biased chain (ABC), and wormlike chain (WLC) models, of which the first three bend at discrete joints and the last bends continuously. Predictions from the WLC model generally agree well with experiment. Dynamics models include the Rouse, Zimm, stiff rod, dynamic WLC, and reptation models, of which the first four apply to isolated filaments and the last to entangled filaments. Experiments show that the dynamic WLC and reptation models are most accurate. They also show that biological filaments typically experience strong hydrodynamic coupling and/or constrained motion. Computer simulation methods that address filament dynamics typically compute filament segment velocities from local forces using the Langevin equation and then integrate these velocities with explicit or implicit methods; the former are more versatile and the latter are more efficient. Much remains to be discovered in biological filament modeling. In particular, filament dynamics in living cells are not well understood, and current computational methods are too slow and not sufficiently versatile. Although primarily a review, this paper also presents new statistical calculations for the ABC and WLC models. Additionally, it corrects several discrepancies in the literature about bending and torsional persistence length definitions, and their relations to flexural and torsional rigidities.

  18. Inflammation Drives Retraction, Stiffening, and Nodule Formation via Cytoskeletal Machinery in a Three-Dimensional Culture Model of Aortic Stenosis.

    PubMed

    Lim, Jina; Ehsanipour, Arshia; Hsu, Jeffrey J; Lu, Jinxiu; Pedego, Taylor; Wu, Alexander; Walthers, Chris M; Demer, Linda L; Seidlits, Stephanie K; Tintut, Yin

    2016-09-01

    In calcific aortic valve disease, the valve cusps undergo retraction, stiffening, and nodular calcification. The inflammatory cytokine, tumor necrosis factor (TNF)-α, contributes to valve disease progression; however, the mechanisms of its actions on cusp retraction and stiffening are unclear. We investigated effects of TNF-α on murine aortic valvular interstitial cells (VICs) within three-dimensional, free-floating, compliant, collagen hydrogels, simulating their natural substrate and biomechanics. TNF-α increased retraction (percentage of diameter), stiffness, and formation of macroscopic, nodular structures with calcification in the VIC-laden hydrogels. The effects of TNF-α were attenuated by blebbistatin inhibition of myosin II-mediated cytoskeletal contraction. Inhibition of actin polymerization with cytochalasin-D, but not inhibition of Rho kinase with Y27632, blocked TNF-α-induced retraction in three-dimensional VIC hydrogels, suggesting that actin stress fibers mediate TNF-α-induced effects. In the hydrogels, inhibitors of NF-κB blocked TNF-α-induced retraction, whereas simultaneous inhibition of c-Jun N-terminal kinase was required to block TNF-α-induced stiffness. TNF-α also significantly increased collagen deposition, as visualized by Masson's trichrome staining, and up-regulated mRNA expression of discoidin domain receptor tyrosine kinase 2, fibronectin, and α-smooth muscle actin. In human aortic valves, calcified cusps were stiffer and had more collagen deposition than noncalcified cusps. These findings suggest that inflammation, through stimulation of cytoskeletal contractile activity, may be responsible for valvular cusp retraction, stiffening, and formation of calcified nodules. PMID:27392969

  19. Changes in Ultrastructure and Cytoskeletal Aspects of Human Normal and Osteoarthritic Chondrocytes Exposed to Interleukin-1β and Cyclical Hydrostatic Pressure

    PubMed Central

    Pascarelli, Nicola Antonio; Collodel, Giulia; Moretti, Elena; Cheleschi, Sara; Fioravanti, Antonella

    2015-01-01

    The aim of this study was to examine the ultrastructure and cytoskeletal organization in human normal and Osteoarhritic (OA) chondrocytes, exposed to interleukin-1β (IL-1β) and cyclic hydrostatic pressure (HP). Morphological examination by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed differences between normal and OA chondrocytes at the nuclear and cytoplasmic level. IL-1β (5 ng/mL) induced a decrease of the number of mitochondria and Golgi bodies and a significant increase on the percentage of cells rich in vacuolization and in marginated chromatin. Cyclical HP (1–5 MPa, 0.25 Hz, for 3 h) did not change the morphology of normal chondrocytes, but had a beneficial effect on OA chondrocytes increasing the number of organelles. Normal and OA cells subjected to IL-1β and HP recovered cytoplasmic ultrastructure. Immunofluorescence (IF) examination of normal chondrocytes showed an actin signal polarized on the apical sides of the cytoplasm, tubulin and vimentin uniformly distributed throughout cytoplasm and vinculin revealed a punctuated pattern under the plasma membrane. In OA chondrocytes, these proteins partially lost their organization. Stimulation with IL-1β caused, in both type of cells, modification in the cytoskeletal organization; HP counteracted the negative effects of IL-1β. Our results showed structural differences at nuclear, cytoplasmic and cytoskeletal level between normal and OA chondrocytes. IL-1β induced ultrastructural and cytoskeletal modifications, counteracted by a cyclical low HP. PMID:26528971

  20. Pseudorabies virus US3 leads to filamentous actin disassembly and contributes to viral genome delivery to the nucleus.

    PubMed

    Jacob, Thary; Van den Broeke, Céline; Grauwet, Korneel; Baert, Kim; Claessen, Christophe; De Pelsmaeker, Steffi; Van Waesberghe, Cliff; Favoreel, Herman W

    2015-06-12

    The conserved alphaherpesvirus US3 tegument protein induces rearrangements of the actin cytoskeleton, consisting of protrusion formation and stress fiber breakdown. Although US3 does not affect levels of total actin protein, it remains unclear whether US3 modulates the total levels of filamentous (F) actin. In this report, we show that the pseudorabies virus (PRV) US3 protein, via its kinase activity, leads to disassembly of F-actin in porcine ST cells. F-actin disassembly has been reported before to contribute to host cell entry of HIV. In line with this, in the current study, we report that US3 has a previously uncharacterized role in viral genome delivery to the nucleus, since quantitative polymerase chain reaction (qPCR) assays on nuclear fractions demonstrated a reduced nuclear delivery of US3null PRV compared to wild type PRV genomes. Treatment of cells with the actin depolymerizing drug cytochalasin D enhanced virus genome delivery to the nucleus, particularly of US3null PRV, supporting a role for F-actin disassembly during certain aspects of viral entry. In conclusion, the US3 kinase of PRV leads to F-actin depolymerization, and US3 and F-actin disassembly contribute to viral genome delivery to the nucleus. PMID:25869795

  1. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation.

    PubMed

    Lee, Joomin; Ahn, Eunsook; Park, Wyun Kon; Park, Seyeon

    2016-01-01

    Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways. PMID:27611435

  2. Actin cross-link assembly and disassembly mechanics for alpha-Actinin and fascin.

    PubMed

    Courson, David S; Rock, Ronald S

    2010-08-20

    Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and alpha-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. alpha-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo. PMID:20551315

  3. VAMP-7 links granule exocytosis to actin reorganization during platelet activation.

    PubMed

    Koseoglu, Secil; Peters, Christian G; Fitch-Tewfik, Jennifer L; Aisiku, Omozuanvbo; Danglot, Lydia; Galli, Thierry; Flaumenhaft, Robert

    2015-07-30

    Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein (VAMP)-7 is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-) platelets compared to wild-type controls. Immunoprecipitation of VAMP-7 revealed an association with VPS9-domain ankyrin repeat protein (VARP), an adaptor protein that interacts with both membrane-bound and cytoskeleton proteins and with Arp2/3. VAMP-7, VARP, and Arp2/3 localized to the platelet periphery during spreading. These studies demonstrate that VAMP-7 participates in both platelet granule secretion and spreading and suggest a mechanism whereby VAMP-7 links granule exocytosis with actin reorganization. PMID:25999457

  4. Biotransformation and Rearrangement of Laromustine.

    PubMed

    Nassar, Alaa-Eldin F; Wisnewski, Adam V; King, Ivan

    2016-08-01

    This review highlights the recent research into the biotransformations and rearrangement of the sulfonylhydrazine-alkylating agent laromustine. Incubation of [(14)C]laromustine with rat, dog, monkey, and human liver microsomes produced eight radioactive components (C-1 to C-8). There was little difference in the metabolite profile among the species examined, partly because NADPH was not required for the formation of most components, which instead involved decomposition and/or hydrolysis. The exception was C-7, a hydroxylated metabolite, largely formed by CYP2B6 and CYP3A4/5. Liquid chromatography-multistage mass spectrometry (LC-MS(n)) studies determined that collision-induced dissociation, and not biotransformation or enzyme catalysis, produced the unique mass spectral rearrangement. Accurate mass measurements performed with a Fourier-transform ion cyclotron resonance mass spectrometer (FTICR-MS) significantly aided determination of the elemental compositions of the fragments and in the case of laromustine revealed the possibility of rearrangement. Further, collision-induced dissociation produced the loss of nitrogen (N2) and methylsulfonyl and methyl isocyanate moieties. The rearrangement, metabolite/decomposition products, and conjugation reactions were analyzed utilizing hydrogen-deuterium exchange, exact mass, (13)C-labeled laromustine, nuclear magnetic resonance spectroscopy (NMR), and LC-MS(n) experiments to assist with the assignments of these fragments and possible mechanistic rearrangement. Such techniques produced valuable insights into these functions: 1) Cytochrome P450 is involved in C-7 formation but plays little or no role in the conversion of [(14)C]laromustine to C-1 through C-6 and C-8; 2) the relative abundance of individual degradation/metabolite products was not species-dependent; and 3) laromustine produces several reactive intermediates that may produce the toxicities seen in the clinical trials. PMID:27278961

  5. Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells via depolymerizing F-actin to impede TAZ nuclear translocation

    PubMed Central

    Chen, Zhe; Luo, Qing; Lin, Chuanchuan; Kuang, Dongdong; Song, Guanbin

    2016-01-01

    Microgravity induces observed bone loss in space flight, and reduced osteogenesis of bone mesenchymal stem cells (BMSCs) partly contributes to this phenomenon. Abnormal regulation or functioning of the actin cytoskeleton induced by microgravity may cause the inhibited osteogenesis of BMSCs, but the underlying mechanism remains obscure. In this study, we demonstrated that actin cytoskeletal changes regulate nuclear aggregation of the transcriptional coactivator with PDZ-binding motif (TAZ), which is indispensable for osteogenesis of bone mesenchymal stem cells (BMSCs). Moreover, we utilized a clinostat to model simulated microgravity (SMG) and demonstrated that SMG obviously depolymerized F-actin and hindered TAZ nuclear translocation. Interestingly, stabilizing the actin cytoskeleton induced by Jasplakinolide (Jasp) significantly rescued TAZ nuclear translocation and recovered the osteogenic differentiation of BMSCs in SMG, independently of large tumor suppressor 1(LATS1, an upstream kinase of TAZ). Furthermore, lysophosphatidic acid (LPA) also significantly recovered the osteogenic differentiation of BMSCs in SMG through the F-actin-TAZ pathway. Taken together, we propose that the depolymerized actin cytoskeleton inhibits osteogenic differentiation of BMSCs through impeding nuclear aggregation of TAZ, which provides a novel connection between F-actin cytoskeleton and osteogenesis of BMSCs and has important implications in bone loss caused by microgravity. PMID:27444891

  6. Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells via depolymerizing F-actin to impede TAZ nuclear translocation.

    PubMed

    Chen, Zhe; Luo, Qing; Lin, Chuanchuan; Kuang, Dongdong; Song, Guanbin

    2016-01-01

    Microgravity induces observed bone loss in space flight, and reduced osteogenesis of bone mesenchymal stem cells (BMSCs) partly contributes to this phenomenon. Abnormal regulation or functioning of the actin cytoskeleton induced by microgravity may cause the inhibited osteogenesis of BMSCs, but the underlying mechanism remains obscure. In this study, we demonstrated that actin cytoskeletal changes regulate nuclear aggregation of the transcriptional coactivator with PDZ-binding motif (TAZ), which is indispensable for osteogenesis of bone mesenchymal stem cells (BMSCs). Moreover, we utilized a clinostat to model simulated microgravity (SMG) and demonstrated that SMG obviously depolymerized F-actin and hindered TAZ nuclear translocation. Interestingly, stabilizing the actin cytoskeleton induced by Jasplakinolide (Jasp) significantly rescued TAZ nuclear translocation and recovered the osteogenic differentiation of BMSCs in SMG, independently of large tumor suppressor 1(LATS1, an upstream kinase of TAZ). Furthermore, lysophosphatidic acid (LPA) also significantly recovered the osteogenic differentiation of BMSCs in SMG through the F-actin-TAZ pathway. Taken together, we propose that the depolymerized actin cytoskeleton inhibits osteogenic differentiation of BMSCs through impeding nuclear aggregation of TAZ, which provides a novel connection between F-actin cytoskeleton and osteogenesis of BMSCs and has important implications in bone loss caused by microgravity. PMID:27444891

  7. CRP2, a new invadopodia actin bundling factor critically promotes breast cancer cell invasion and metastasis

    PubMed Central

    Dieterle, Monika; Moreau, Flora; Al Absi, Antoun; Steinmetz, André; Oudin, Anaïs; Berchem, Guy; Janji, Bassam; Thomas, Clément

    2016-01-01

    A critical process underlying cancer metastasis is the acquisition by tumor cells of an invasive phenotype. At the subcellular level, invasion is facilitated by actin-rich protrusions termed invadopodia, which direct extracellular matrix (ECM) degradation. Here, we report the identification of a new cytoskeletal component of breast cancer cell invadopodia, namely cysteine-rich protein 2 (CRP2). We found that CRP2 was not or only weakly expressed in epithelial breast cancer cells whereas it was up-regulated in mesenchymal/invasive breast cancer cells. In addition, high expression of the CRP2 encoding gene CSRP2 was associated with significantly increased risk of metastasis in basal-like breast cancer patients. CRP2 knockdown significantly reduced the invasive potential of aggressive breast cancer cells, whereas it did not impair 2D cell migration. In keeping with this, CRP2-depleted breast cancer cells exhibited a reduced capacity to promote ECM degradation, and to secrete and express MMP-9, a matrix metalloproteinase repeatedly associated with cancer progression and metastasis. In turn, ectopic expression of CRP2 in weakly invasive cells was sufficient to stimulate cell invasion. Both GFP-fused and endogenous CRP2 localized to the extended actin core of invadopodia, a structure primarily made of actin bundles. Purified recombinant CRP2 autonomously crosslinked actin filaments into thick bundles, suggesting that CRP2 contributes to the formation/maintenance of the actin core. Finally, CRP2 depletion significantly reduced the incidence of lung metastatic lesions in two xenograft mouse models of breast cancer. Collectively, our data identify CRP2 as a new cytoskeletal component of invadopodia that critically promotes breast cancer cell invasion and metastasis. PMID:26883198

  8. Experimental and computational assessment of F-actin influence in regulating cellular stiffness and relaxation behaviour of fibroblasts.

    PubMed

    Fallqvist, Björn; Fielden, Matthew L; Pettersson, Torbjörn; Nordgren, Niklas; Kroon, Martin; Gad, Annica K B

    2016-06-01

    In biomechanics, a complete understanding of the structures and mechanisms that regulate cellular stiffness at a molecular level remain elusive. In this paper, we have elucidated the role of filamentous actin (F-actin) in regulating elastic and viscous properties of the cytoplasm and the nucleus. Specifically, we performed colloidal-probe atomic force microscopy (AFM) on BjhTERT fibroblast cells incubated with Latrunculin B (LatB), which results in depolymerisation of F-actin, or DMSO control. We found that the treatment with LatB not only reduced cellular stiffness, but also greatly increased the relaxation rate for the cytoplasm in the peripheral region and in the vicinity of the nucleus. We thus conclude that F-actin is a major determinant in not only providing elastic stiffness to the cell, but also in regulating its viscous behaviour. To further investigate the interdependence of different cytoskeletal networks and cell shape, we provided a computational model in a finite element framework. The computational model is based on a split strain energy function of separate cellular constituents, here assumed to be cytoskeletal components, for which a composite strain energy function was defined. We found a significant influence of cell geometry on the predicted mechanical response. Importantly, the relaxation behaviour of the cell can be characterised by a material model with two time constants that have previously been found to predict mechanical behaviour of actin and intermediate filament networks. By merely tuning two effective stiffness parameters, the model predicts experimental results in cells with a partly depolymerised actin cytoskeleton as well as in untreated control. This indicates that actin and intermediate filament networks are instrumental in providing elastic stiffness in response to applied forces, as well as governing the relaxation behaviour over shorter and longer time-scales, respectively. PMID:26766328

  9. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    SciTech Connect

    Woloschak, G.E. |; Felcher, P.; Chang-Liu, Chin-Mei

    1993-12-31

    Experiments were designed to examine the effects of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements ({gamma}- and {beta}-actin and {alpha}-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Results demonstrated little effect of dose-rate for JANUS fission-spectrum neutrons when comparing expression of either a-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Cycloheximide, however, repressed accumulation of {alpha}-tubulin following exposure to high dose-rate neutrons or {gamma} rays; this did not occur following similar low dose-rate exposures. Cycloheximide did not affect accumulation of mRNA for actin genes. Cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin mRNA accumulation following exposure to ionizing radiation and that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons.

  10. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    SciTech Connect

    Woloschak, G.E. |; Felcher, P.; Chang-Liu, Chin-Mei

    1992-12-31

    Experiments were designed to examine the effects of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements ({gamma}- and {beta}-actin and {alpha}-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either {alpha}-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide, however, revealed several interesting and novel findings: (1) Cycloheximide repressed accumulation of {alpha}-tubulin following exposure to high dose-rate neutrons or {gamma} rays; this did not occur following similar low dose-rate exposure (2) Cycloheximide did not affect accumulation of mRNA for actin genes. Cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin mRNA accumulation following exposure to ionizing radiation. In addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons.

  11. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    SciTech Connect

    Woloschak, G.E. |; Felcher, P.; Chang-Liu, Chin-Mei

    1994-05-01

    Experiments were designed to examine the effects Of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements ({gamma}- and {beta}-actin and {alpha}-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either {alpha}-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide revealed that cycloheximide repressed accumulation of {alpha}-tubulin following exposure to high dose-rate neutrons or {gamma} rays; this did not occur following similar low dose-rate exposure. (2) Cycloheximide did not affect accumulation of MRNA for actin genes; and that cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin MRNA accumulation following exposure to ionizing radiation. in addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons.

  12. Actin-based phagosome motility.

    PubMed

    Zhang, Fangliang; Southwick, Frederick S; Purich, Daniel L

    2002-10-01

    Despite abundant evidence of actin's involvement at the particle internalization stage of phagocytosis, little is known about whether phagosomes undergo the same type of actin-based motility as observed with endocytic vesicles or such intracellular pathogens as Listeria and Shigella. By employing video microscopy to follow the fate of latex bead-containing phagosomes within the cytoplasm of bone marrow macrophages, we have made the novel observation of actin-based phagosome motility. Immunofluorescence microscopy confirmed that phagosomes containing IgG-opsonized, bovine serum albumin (or BSA) -coated or uncoated latex beads all formed actin-rich rocket tails that persisted only during a brief, 1-2 min period of actin-based motility. Average speeds of actin-based phagosome motility were 0.13 +/- 0.06 microm/s for IgG-coated beads, 0.14 +/- 0.04 microm/s for BSA-coated beads, and 0.11+/- 0.03 microm/s for uncoated beads. Moreover, the speeds and motile-phase duration of each type of phagosome were comparable to the behavior of pinosomes [Merrifield et al., 1999: Nat. Cell Biol. 1:72-74.]. Determination of optimal conditions for observing and analyzing actin-based phagosome motility should facilitate future investigations of phagocytosis and phagosome maturation. PMID:12211106

  13. Antibodies covalently immobilized on actin filaments for fast myosin driven analyte transport.

    PubMed

    Kumar, Saroj; ten Siethoff, Lasse; Persson, Malin; Lard, Mercy; te Kronnie, Geertruy; Linke, Heiner; Månsson, Alf

    2012-01-01

    Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm(-1)). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments. PMID:23056279

  14. Study of the influence of actin-binding proteins using linear analyses of cell deformability.

    PubMed

    Plaza, Gustavo R; Uyeda, Taro Q P; Mirzaei, Zahra; Simmons, Craig A

    2015-07-21

    The actin cytoskeleton plays a key role in the deformability of the cell and in mechanosensing. Here we analyze the contributions of three major actin cross-linking proteins, myosin II, α-actinin and filamin, to cell deformability, by using micropipette aspiration of Dictyostelium cells. We examine the applicability of three simple mechanical models: for small deformation, linear viscoelasticity and drop of liquid with a tense cortex; and for large deformation, a Newtonian viscous fluid. For these models, we have derived linearized equations and we provide a novel, straightforward methodology to analyze the experiments. This methodology allowed us to differentiate the effects of the cross-linking proteins in the different regimes of deformation. Our results confirm some previous observations and suggest important relations between the molecular characteristics of the actin-binding proteins and the cell behavior: the effect of myosin is explained in terms of the relation between the lifetime of the bond to actin and the resistive force; the presence of α-actinin obstructs the deformation of the cytoskeleton, presumably mainly due to the higher molecular stiffness and to the lower dissociation rate constants; and filamin contributes critically to the global connectivity of the network, possibly by rapidly turning over cross-links during the remodeling of the cytoskeletal network, thanks to the higher rate constants, flexibility and larger size. The results suggest a sophisticated relationship between the expression levels of actin-binding proteins, deformability and mechanosensing. PMID:26059185

  15. Syndapin promotes pseudocleavage furrow formation by actin organization in the syncytial Drosophila embryo

    PubMed Central

    Sherlekar, Aparna; Rikhy, Richa

    2016-01-01

    Coordinated membrane and cytoskeletal remodeling activities are required for membrane extension in processes such as cytokinesis and syncytial nuclear division cycles in Drosophila. Pseudocleavage furrow membranes in the syncytial Drosophila blastoderm embryo show rapid extension and retraction regulated by actin-remodeling proteins. The F-BAR domain protein Syndapin (Synd) is involved in membrane tubulation, endocytosis, and, uniquely, in F-actin stability. Here we report a role for Synd in actin-regulated pseudocleavage furrow formation. Synd localized to these furrows, and its loss resulted in short, disorganized furrows. Synd presence was important for the recruitment of the septin Peanut and distribution of Diaphanous and F-actin at furrows. Synd and Peanut were both absent in furrow-initiation mutants of RhoGEF2 and Diaphanous and in furrow-progression mutants of Anillin. Synd overexpression in rhogef2 mutants reversed its furrow-extension phenotypes, Peanut and Diaphanous recruitment, and F-actin organization. We conclude that Synd plays an important role in pseudocleavage furrow extension, and this role is also likely to be crucial in cleavage furrow formation during cell division. PMID:27146115

  16. Actin marker lines in grapevine reveal a gatekeeper function of guard cells.

    PubMed

    Guan, Xin; Buchholz, Günther; Nick, Peter

    2014-08-15

    Resistance to abiotic and biotic stress is a central topic for sustainable agriculture, especially in grapevine, one of the field crops with the highest economic output per acreage. As early cellular factors for plant defense, actin microfilaments (AF) are of high relevance. We therefore generated a transgenic actin marker line for grapevine by expressing a fusion protein between green fluorescent protein and the second actin-binding domain of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. Based on this first cytoskeletal-marker line in grapevine, the response of AFs to phytopathogenic microorganisms could be followed in vivo. Upon inoculation with fluorescently labeled strains of phytopathogenic bacteria, actin responses were confined to the guard cells. In contrast, upon contact with zoospores of Plasmopara viticola, not only the guard cells, but also epidermal pavement cells, where no zoospores had attached responded with the formation of a perinuclear actin basket. Our data support the hypothesis that guard cells act as pacemakers of defense, dominating the responses of the remaining epidermal cells. PMID:24973589

  17. Syndapin promotes pseudocleavage furrow formation by actin organization in the syncytial Drosophila embryo.

    PubMed

    Sherlekar, Aparna; Rikhy, Richa

    2016-07-01

    Coordinated membrane and cytoskeletal remodeling activities are required for membrane extension in processes such as cytokinesis and syncytial nuclear division cycles in Drosophila Pseudocleavage furrow membranes in the syncytial Drosophila blastoderm embryo show rapid extension and retraction regulated by actin-remodeling proteins. The F-BAR domain protein Syndapin (Synd) is involved in membrane tubulation, endocytosis, and, uniquely, in F-actin stability. Here we report a role for Synd in actin-regulated pseudocleavage furrow formation. Synd localized to these furrows, and its loss resulted in short, disorganized furrows. Synd presence was important for the recruitment of the septin Peanut and distribution of Diaphanous and F-actin at furrows. Synd and Peanut were both absent in furrow-initiation mutants of RhoGEF2 and Diaphanous and in furrow-progression mutants of Anillin. Synd overexpression in rhogef2 mutants reversed its furrow-extension phenotypes, Peanut and Diaphanous recruitment, and F-actin organization. We conclude that Synd plays an important role in pseudocleavage furrow extension, and this role is also likely to be crucial in cleavage furrow formation during cell division. PMID:27146115

  18. Actin branches out to link pathogen perception and host gene regulation

    PubMed Central

    Porter, Katie; Day, Brad

    2013-01-01

    Cellular functions of actin, and associated actin binding proteins (ABPs), have been well characterized with respect to their dynamic cytosolic role as components of the complex cytoskeletal network. In this regard, the collective research in this field has vastly expanded our knowledge of the role of actin to more recently identify a key role within the nucleus as an integral part gene organization and expression. Herein, we describe the requirement of the ABP actin depolymerizing factor-4 (ADF4) as a regulator of resistance to Pseudomonas syringae DC3000 expressing the effector AvrPphB via ADF4’s cytosolic and nuclear functions. In total, our work has identified significant alterations in the expression of the resistance protein RPS5 in an ADF4 phosphorylation dependent manner. In this mini-review, we provide compelling evidence in support of both a nuclear function for ADF4, as well as potential targeting of the actin cytoskeleton bythe bacterial effector AvrPphB. PMID:23333960

  19. Antibodies Covalently Immobilized on Actin Filaments for Fast Myosin Driven Analyte Transport

    PubMed Central

    Kumar, Saroj; ten Siethoff, Lasse; Persson, Malin; Lard, Mercy; te Kronnie, Geertruy; Linke, Heiner; Månsson, Alf

    2012-01-01

    Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm−1). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments. PMID:23056279

  20. Actin Disorganization Plays a Vital Role in Impaired Embryonic Development of In Vitro-Produced Mouse Preimplantation Embryos

    PubMed Central

    Tan, Kun; An, Lei; Wang, Shu-Min; Wang, Xiao-Dong; Zhang, Zhen-Ni; Miao, Kai; Sui, Lin-Lin; He, Shu-Zhi; Nie, Jing-Zhou; Wu, Zhong-Hong; Tian, Jian-Hui

    2015-01-01

    Assisted reproductive technology (ART) is being increasingly applied to overcome infertility. However, the in vitro production process, the main procedure of ART, can lead to aberrant embryonic development and health-related problems in offspring. Understanding the mechanisms underlying the ART-induced side effects is important to improve the ART process. In this study, we carried out comparative transcriptome profiling between in vivo- (IVO) and in vitro- produced (IVP) mouse blastocysts. Our results suggested that aberrant actin organization might be a major factor contributing to the impaired development of IVP embryos. To test this, we examined the effect of actin disorganization on the development of IVP preimplantation embryos. Specific disruption of actin organization by cytochalasin B (CB) indicated that well-organized actin is essential for in vitro embryonic development. Supplementing the culture medium with 10–9 M melatonin, a cytoskeletal modulator in adult somatic cells, significantly reversed the disrupted expression patterns of genes related to actin organization, including Arhgef2, Bcl2, Coro2b, Flnc, and Palld. Immunofluorescence analysis showed that melatonin treatment of IVP embryos significantly improved the distribution and organization of actin filaments (F-actin) from the 8-cell stage onwards. More importantly, we found that melatonin alleviated the CB-mediated aberrant F-actin distribution and organization and rescued CB-induced impaired embryonic development. This is the first study to indicate that actin disorganization is implicated in impaired development of IVP embryos during the preimplantation stage. We also demonstrated that improving actin organization is a promising strategy to optimize existing IVP systems. PMID:26076347

  1. Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells

    PubMed Central

    Shiraishi, Takumi; Verdone, James E.; Huang, Jessie; Kahlert, Ulf D.; Hernandez, James R.; Torga, Gonzalo; Zarif, Jelani C.; Epstein, Tamir; Gatenby, Robert; McCartney, Annemarie; Elisseeff, Jennifer H.; Mooney, Steven M.; An, Steven S.; Pienta, Kenneth J.

    2015-01-01

    The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease. PMID:25426557

  2. Formin' actin in the nucleus.

    PubMed

    Baarlink, Christian; Grosse, Robert

    2014-01-01

    Many if not most proteins can, under certain conditions, change cellular compartments, such as, for example, shuttling from the cytoplasm to the nucleus. Thus, many proteins may exert functions in various and very different subcellular locations, depending on the signaling context. A large amount of actin regulatory proteins has been detected in the mammalian cell nucleus, although their potential roles are much debated and are just beginning to emerge. Recently, members of the formin family of actin nucleators were also reported to dynamically localize to the nuclear environment. Here we discuss our findings that specific diaphanous-related formins can promote nuclear actin assembly in a signal-dependent manner. PMID:24637338

  3. The cytogenetics of mammalian autosomal rearrangements

    SciTech Connect

    Daniel, A.

    1988-01-01

    Combining data from animal and clinical studies with classical cytogenetic observations, the volume provides information on various aspects of mammalian autosomal rearrangements. Topics range from the reproductive consequences to carriers of autosomal rearrangements to the application of structural rearrangements and DNA probes to gene mapping. In addition, the book presents an overview of new perspectives and future directions for research.

  4. Podocytic cytoskeletal disaggregation and basement-membrane detachment in puromycin aminonucleoside nephrosis.

    PubMed Central

    Whiteside, C. I.; Cameron, R.; Munk, S.; Levy, J.

    1993-01-01

    Puromycin aminonucleoside--(PAN) treated rats develop acute nephrotic syndrome, mimicking human minimal lesion disease. In PAN nephrosis, podocyte detachment from the glomerular basement membrane (GBM) is the most likely cause of massive proteinuria in this model. To elucidate further the mechanisms of PAN-induced cellular dysfunction, new methods were employed to visualize podocyte cytoskeletal aggregation and to measure fibrillar attachment to the GBM. Adult Sprague-Dawley rats (n = 4/group) received a single tail-vein injection of PAN (75 mg/kg). On days 1, 2, 3, and 5 following injection, 24-hour urine collections were obtained for creatinine clearance, albuminuria, and total proteinuria. Then kidneys from each group were fixed by perfusion. Podocytic cytoskeleton was visualized by scanning electron microscopy. Subepithelial GBM staining and attachment fiber number, observed on digitized images of transmission electron micrographs, were quantitated with computer-based density analysis. A significant reduction in attachment fiber number in the GBM lamina rara externa occurred by day 5. On scanning electron micrographs, the secondary and tertiary podocytic processes were observed to be formed by highly aggregated cytoskeleton, which became partially disaggregated by day 3, was totally absent by day 5, and normalized by day 20. Immunogold staining revealed that actin and vinculin localized to the tertiary podocytic processes in the normal state were dispersed into the cell body following PAN. Podocyte cytoskeletal disaggregation precedes, and detachment from the GBM occurs simultaneously with, the onset of massive proteinuria in the PAN model. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:8494056

  5. Neurobehavioral deficits in mice lacking the erythrocyte membrane cytoskeletal protein 4.1.

    PubMed

    Walensky, L D; Shi, Z T; Blackshaw, S; DeVries, A C; Demas, G E; Gascard, P; Nelson, R J; Conboy, J G; Rubin, E M; Snyder, S H; Mohandas, N

    1998-11-19

    The erythrocyte membrane cytoskeletal protein 4.1 (4.1R) is a structural protein that confers stability and flexibility to erythrocytes via interactions with the cytoskeletal proteins spectrin and F-actin and with the band 3 and glycophorin C membrane proteins. Mutations in 4.1R can cause hereditary elliptocytosis, a disease characterized by a loss of the normal discoid morphology of erythrocytes, resulting in hemolytic anemia [1]. Different isoforms of the 4.1 protein have been identified in a wide variety of nonerythroid tissues by immunological methods [2-5]. The variation in molecular weight of these different 4.1 isoforms, which range from 30 to 210 kDa [6], has been attributed to complex alternative splicing of the 4.1R gene [7]. We recently identified two new 4.1 genes: one is generally expressed throughout the body (4. 1G) [8] and the other is expressed in central and peripheral neurons (4.1N) [9]. Here, we examined 4.1R expression by in situ hybridization analysis and found that 4.1R was selectively expressed in hematopoietic tissues and in specific neuronal populations. In the brain, high levels of 4.1R were discretely localized to granule cells in the cerebellum and dentate gyrus. We generated mice that lacked 4.1R expression; these mice had deficits in movement, coordination, balance and learning, in addition to the predicted hematological abnormalities. The neurobehavioral findings are consistent with the distribution of 4.1R in the brain, suggesting that 4.1R performs specific functions in the central nervous system. PMID:9822582

  6. Conditioning nerve crush accelerates cytoskeletal protein transport in sprouts that form after a subsequent crush

    SciTech Connect

    McQuarrie, I.G.; Jacob, J.M. )

    1991-03-01

    To examine the relationship between axonal outgrowth and the delivery of cytoskeletal proteins to the growing axon tip, outgrowth was accelerated by using a conditioning nerve crush. Because slow component b (SCb) of axonal transport is the most rapid vehicle for carrying cytoskeletal proteins to the axon tip, the rate of SCb was measured in conditioned vs. sham-conditioned sprouts. In young Sprague-Dawley rats, the conditioning crush was made to sciatic nerve branches at the knee; 14 days later, the test crush was made where the L4 and L5 spinal nerves join to form the sciatic nerve in the flank. Newly synthesized proteins were labeled in motor neurons by injecting {sup 35}S-methionine into the lumbar spinal cord 7 days before the test crush. The wave of pulse-labeled SCb proteins reached the crush by the time it was made and subsequently entered sprouts. The nerve was removed and sectioned for SDS-PAGE and fluorography 4-12 days after the crush. Tubulins, neurofilament proteins, and representative 'cytomatrix' proteins (actin, calmodulin, and putative microtubule-associated proteins) were removed from gels for liquid scintillation counting. Labeled SCb proteins entered sprouts without first accumulating in parent axon stumps, presumably because sprouts begin to grow within hours after axotomy. The peak of SCb moved 11% faster in conditioned than in sham-conditioned sprouts: 3.0 vs. 2.7 mm/d (p less than 0.05). To confirm that sprouts elongate more rapidly when a test crush is preceded by a conditioning crush, outgrowth distances were measured in a separate group of rats by labeling fast axonal transport with {sup 3}H-proline 24 hours before nerve retrieval.

  7. Interplay of cytoskeletal activity and lipid phase stability in dynamic protein recruitment and clustering.

    PubMed

    Gómez-Llobregat, Jordi; Buceta, Javier; Reigada, Ramon

    2013-01-01

    Recent experiments have revealed that some membrane proteins aggregate to form clusters. This type of process has been proven to be dynamic and to be actively maintained by external kinetics. Additionally, this dynamic recruiting is cholesterol- and actin-dependent, suggesting that raft organization and cytoskeleton rearrangement play a crucial role. In the present study, we propose a simple model that provides a general framework to describe the dynamical behavior of lipid-protein assemblies. Our results suggest that lipid-mediated interactions and cytoskeleton-anchored proteins contribute to the modulation of such behavior. In particular, we find a resonant condition between the membrane protein and cytoskeleton dynamics that results in the invariance of the ratio of clustered proteins that is found in in vivo experimental observations. PMID:24018870

  8. Mutations in the Drosophila orthologs of the F-actin capping protein alpha- and beta-subunits cause actin accumulation and subsequent retinal degeneration.

    PubMed

    Delalle, Ivana; Pfleger, Cathie M; Buff, Eugene; Lueras, Paula; Hariharan, Iswar K

    2005-12-01

    The progression of several human neurodegenerative diseases is characterized by the appearance of intracellular inclusions or cytoskeletal abnormalities. An important question is whether these abnormalities actually contribute to the degenerative process or whether they are merely manifestations of cells that are already destined for degeneration. We have conducted a large screen in Drosophila for mutations that alter the growth or differentiation of cells during eye development. We have used mitotic recombination to generate patches of homozygous mutant cells. In our entire screen, mutations in only two different loci, burned (bnd) and scorched (scrd), resulted in eyes in which the mutant patches appeared black and the mutant tissue appeared to have undergone degeneration. In larval imaginal discs, growth and cell fate specification occur normally in mutant cells, but there is an accumulation of F-actin. Mutant cells degenerate much later during the pupal phase of development. burned mutations are allelic to mutations in the previously described cpb locus that encodes the beta-subunit of the F-actin capping protein, while scorched mutations disrupt the gene encoding its alpha-subunit (cpa). The alpha/beta-heterodimer caps the barbed ends of an actin filament and restricts its growth. In its absence, cells progressively accumulate actin filaments and eventually die. A possible role for their human orthologs in neurodegenerative disease merits further investigation. PMID:16143599

  9. ACD toxin-produced actin oligomers poison formin-controlled actin polymerization

    PubMed Central

    Heisler, David B.; Kudryashova, Elena; Grinevich, Dmitry O.; Suarez, Cristian; Winkelman, Jonathan D.; Birukov, Konstantin G.; Kotha, Sainath R.; Parinandi, Narasimham L.; Vavylonis, Dimitrios; Kovar, David R.; Kudryashov, Dmitri S.

    2015-01-01

    The actin crosslinking domain (ACD) is an actin-specific toxin produced by several pathogens, including life-threatening spp. of Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Actin crosslinking by ACD is thought to lead to slow cytoskeleton failure owing to a gradual sequestration of actin in the form of nonfunctional oligomers. Here we found that ACD converted cytoplasmic actin into highly toxic oligomers that potently “poisoned” the ability of major actin assembly proteins, formins, to sustain actin polymerization. Thus, ACD can target the most abundant cellular protein by employing actin oligomers as secondary toxins to efficiently subvert cellular functions of actin while functioning at very low doses. PMID:26228148

  10. Epidemiology of actinic keratoses.

    PubMed

    Green, Adèle C

    2015-01-01

    The epidemiology of actinic keratoses (AKs) reflects their causation by cumulative sun exposure, with the highest prevalence seen in pale-skinned people living at low latitudes and on the most sun-exposed body sites, namely the hands, forearms and face. AKs are markers of increased risk of basal cell carcinoma, squamous cell carcinoma and melanoma, especially when they are numerous and have coalesced into an area of 'field cancerisation'. The major risk factors are male sex, advanced age, sun-sensitive complexion, high lifetime sun exposure and prolonged immunosuppression. Clinical counts of AKs enable the assessment and monitoring of AK burden, but accurate counting is notoriously difficult, especially when skin is severely sun damaged. AK counting has been repeatedly shown to be unreliable, even among expert dermatologists. Notwithstanding these challenges, qualitative assessment of the natural history of AKs shows a high turnover, with new lesions developing and with other lesions regressing. A very small proportion of AKs undergo malignant transformation, but the precise rate of transformation is unknown due to the inaccuracies in monitoring AK lesions over time. Primary prevention of AKs is achieved by limiting intense sun exposure through sun-protective behaviour, including seeking deep shade, wearing sun-protective clothing and applying sunscreen regularly to exposed skin, from an early age. PMID:25561199

  11. Model of cellular mechanotransduction via actin stress fibers.

    PubMed

    Gouget, Cecile L M; Hwang, Yongyun; Barakat, Abdul I

    2016-04-01

    Mechanical stresses due to blood flow regulate vascular endothelial cell structure and function and play a key role in arterial physiology and pathology. In particular, the development of atherosclerosis has been shown to correlate with regions of disturbed blood flow where endothelial cells are round and have a randomly organized cytoskeleton. Thus, deciphering the relation between the mechanical environment, cell structure, and cell function is a key step toward understanding the early development of atherosclerosis. Recent experiments have demonstrated very rapid ([Formula: see text]100 ms) and long-distance ([Formula: see text]10 [Formula: see text]m) cellular mechanotransduction in which prestressed actin stress fibers play a critical role. Here, we develop a model of mechanical signal transmission within a cell by describing strains in a network of prestressed viscoelastic stress fibers following the application of a force to the cell surface. We find force transmission dynamics that are consistent with experimental results. We also show that the extent of stress fiber alignment and the direction of the applied force relative to this alignment are key determinants of the efficiency of mechanical signal transmission. These results are consistent with the link observed experimentally between cytoskeletal organization, mechanical stress, and cellular responsiveness to stress. Based on these results, we suggest that mechanical strain of actin stress fibers under force constitutes a key link in the mechanotransduction chain. PMID:26081725

  12. Actin in Action: Imaging Approaches to Study Cytoskeleton Structure and Function

    PubMed Central

    McKayed, Katey K.; Simpson, Jeremy C.

    2013-01-01

    The cytoskeleton plays several fundamental roles in the cell, including organizing the spatial arrangement of subcellular organelles, regulating cell dynamics and motility, providing a platform for interaction with neighboring cells, and ultimately defining overall cell shape. Fluorescence imaging has proved to be vital in furthering our understanding of the cytoskeleton, and is now a mainstay technique used widely by cell biologists. In this review we provide an introduction to various imaging modalities used to study focal adhesions and the actin cytoskeleton, and using specific examples we highlight a number of recent studies in animal cells that have advanced our knowledge of cytoskeletal behavior. PMID:24709877

  13. Molecular mechanisms and functional implications of polarized actin remodeling at the T cell immunological synapse

    PubMed Central

    Le Floc’h, Audrey; Huse, Morgan

    2014-01-01

    Transient, specialized cell-cell interactions play a central role in leukocyte function by enabling specific intercellular communication in the context of a highly dynamic systems level response. The dramatic structural changes required for the formation of these contacts are driven by rapid and precise cytoskeletal remodeling events. In recent years, the immunological synapse that forms between a T lymphocyte and its antigen-presenting target cell has emerged as an important model system for understanding immune cell interactions. In this review, we discuss how regulators of the cortical actin cytoskeleton control synaptic architecture and in this way specify T cell function. PMID:25355055

  14. Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

    SciTech Connect

    Muthukumaran, Padmalosini; Lim, Chwee Teck; Lee, Taeyong

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Estradiol induced stiffness changes of osteoblasts were quantified using AFM. Black-Right-Pointing-Pointer Estradiol causes significant decrease in the stiffness of osteoblasts. Black-Right-Pointing-Pointer Decreased stiffness was caused by decreased density of f-actin network. Black-Right-Pointing-Pointer Stiffness changes were not associated with mineralized matrix of osteoblasts. Black-Right-Pointing-Pointer Estradiol increases inherent alkaline phosphatase activity of osteoblasts. -- Abstract: Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E{sup Asterisk-Operator }) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with {beta}-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E{sup Asterisk-Operator }. The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E{sup Asterisk-Operator} of osteoblasts significantly decreased by 43-46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness

  15. 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

  16. A 133Cs nuclear magnetic resonance study of endothelial Na(+)-K(+)-ATPase activity: can actin regulate its activity?

    PubMed Central

    Gruwel, M L; Culíc, O; Schrader, J

    1997-01-01

    Using (133)Cs+ NMR, we developed a technique to repetitively measure, in vivo, Na(+)-K(+)-ATPase activity in endothelial cells. The measurements were made without the use of an exogenous shift reagent, because of the large chemical shift of 1.36 +/- 0.13 ppm between intra- and extracellular Cs+. Intracellularly we obtained a spin lattice relaxation time (T1) of 2.0 +/- 0.3 s, and extracellular T1 was 7.9 +/- 0.4 s. Na(+)-K+ pump activity in endothelial cells was determined at 12 +/- 3 nmol Cs+ x min(-1) x (mg Prot)[-1] under control conditions. When intracellular ATP was depleted by the addition of 5 mM 2-deoxy-D-glucose (DOG) and NaCN to about 5% of control, the pump rate decreased by 33%. After 80 min of perfusion with 5 mM DOG and NaCN, reperfusion with control medium rapidly reestablished the endothelial membrane Cs+ gradient. Using (133)Cs+ NMR as a convenient tool, we further addressed the proposed role of actin as a regulator of Na(+)-K+ pump activity in intact cells. Two models of actin rearrangement were tested. DOG caused a rearrangement of F-actin and an increase in G-actin, with a simultaneous decrease in ATP concentration. Cytochalasin D, however, caused an F-actin rearrangement different from that observed for DOG and an increase in G-actin, and cellular ATP levels remained unchanged. In both models, the Na(+)-K(+)-pump activity remained unchanged, as measured with (133)Cs NMR. Our results demonstrate that (133)Cs NMR can be used to repetitively measure Na(+)-K(+)-ATPase activity in endothelial cells. No evidence for a regulatory role of actin on Na(+)-K(+)-ATPase was found. Images FIGURE 6 PMID:9168052

  17. Design Principles of Length Control of Cytoskeletal Structures.

    PubMed

    Mohapatra, Lishibanya; Goode, Bruce L; Jelenkovic, Predrag; Phillips, Rob; Kondev, Jane

    2016-07-01

    Cells contain elaborate and interconnected networks of protein polymers, which make up the cytoskeleton. The cytoskeleton governs the internal positioning and movement of vesicles and organelles and controls dynamic changes in cell polarity, shape, and movement. Many of these processes require tight control of the size and shape of cytoskeletal structures, which is achieved despite rapid turnover of their molecular components. Here we review mechanisms by which cells control the size of filamentous cytoskeletal structures, from the point of view of simple quantitative models that take into account stochastic dynamics of their assembly and disassembly. Significantly, these models make experimentally testable predictions that distinguish different mechanisms of length control. Although the primary focus of this review is on cytoskeletal structures, we believe that the broader principles and mechanisms discussed herein will apply to a range of other subcellular structures whose sizes are tightly controlled and are linked to their functions. PMID:27145876

  18. CASK and protein 4.1 support F-actin nucleation on neurexins.

    PubMed

    Biederer, T; Sudhof, T C

    2001-12-21

    Rearrangements of the actin cytoskeleton are involved in a variety of cellular processes from locomotion of cells to morphological alterations of the cell surface. One important question is how local interactions of cells with the extracellular space are translated into alterations of their membrane organization. To address this problem, we studied CASK, a member of the membrane-associated guanylate kinase homologues family of adaptor proteins. CASK has been shown to bind the erythrocyte isoform of protein 4.1, a class of proteins that promote formation of actin/spectrin microfilaments. In neurons, CASK also interacts via its PDZ domain with the cytosolic C termini of neurexins, neuron-specific cell-surface proteins. We now show that CASK binds a brain-enriched isoform of protein 4.1, and nucleates local assembly of actin/spectrin filaments. These interactions can be reconstituted on the cytosolic tail of neurexins. Furthermore, CASK can be recovered with actin filaments prepared from rat brain extracts, and neurexins are recruited together with CASK and protein 4.1 into these actin filaments. Thus, analogous to the PDZ-domain protein p55 and glycophorin C at the erythrocyte membrane, a similar complex comprising CASK and neurexins exists in neurons. Our data suggest that intercellular junctions formed by neurexins, such as junctions initiated by beta-neurexins with neuroligins, are at least partially coupled to the actin cytoskeleton via an interaction with CASK and protein 4.1. PMID:11604393

  19. Myosin di-phosphorylation and peripheral actin bundle formation as initial events during endothelial barrier disruption

    PubMed Central

    Hirano, Mayumi; Hirano, Katsuya

    2016-01-01

    The phosphorylation of the 20-kD myosin light chain (MLC) and actin filament formation play a key role in endothelial barrier disruption. MLC is either mono- or di-phosphorylated (pMLC and ppMLC) at T18 or S19. The present study investigated whether there are any distinct roles of pMLC and ppMLC in barrier disruption induced by thrombin. Thrombin induced a modest bi-phasic increase in pMLC and a robust mono-phasic increase in ppMLC. pMLC localized in the perinuclear cytoplasm during the initial phase, while ppMLC localized in the cell periphery, where actin bundles were formed. Later, the actin bundles were rearranged into stress fibers, where pMLC co-localized. Rho-kinase inhibitors inhibited thrombin-induced barrier disruption and peripheral localization of ppMLC and actin bundles. The double, but not single, mutation of phosphorylation sites abolished the formation of peripheral actin bundles and the barrier disruption, indicating that mono-phosphorylation of MLC at either T18 or S19 is functionally sufficient for barrier disruption. Namely, the peripheral localization, but not the degree of phosphorylation, is suggested to be essential for the functional effect of ppMLC. These results suggest that MLC phosphorylation and actin bundle formation in cell periphery are initial events during barrier disruption. PMID:26863988

  20. The Strip-Hippo Pathway Regulates Synaptic Terminal Formation by Modulating Actin Organization at the Drosophila Neuromuscular Synapses.

    PubMed

    Sakuma, Chisako; Saito, Yoshie; Umehara, Tomoki; Kamimura, Keisuke; Maeda, Nobuaki; Mosca, Timothy J; Miura, Masayuki; Chihara, Takahiro

    2016-08-30

    Synapse formation requires the precise coordination of axon elongation, cytoskeletal stability, and diverse modes of cell signaling. The underlying mechanisms of this interplay, however, remain unclear. Here, we demonstrate that Strip, a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex that regulates these processes, is required to ensure the proper development of synaptic boutons at the Drosophila neuromuscular junction. In doing so, Strip negatively regulates the activity of the Hippo (Hpo) pathway, an evolutionarily conserved regulator of organ size whose role in synapse formation is currently unappreciated. Strip functions genetically with Enabled, an actin assembly/elongation factor and the presumptive downstream target of Hpo signaling, to modulate local actin organization at synaptic termini. This regulation occurs independently of the transcriptional co-activator Yorkie, the canonical downstream target of the Hpo pathway. Our study identifies a previously unanticipated role of the Strip-Hippo pathway in synaptic development, linking cell signaling to actin organization. PMID:27545887

  1. Exploitation of host signal transduction pathways and cytoskeletal functions by invasive bacteria.

    PubMed

    Rosenshine, I; Finlay, B B

    1993-01-01

    Many bacteria that cause disease have the capacity to enter into and live within eukaryotic cells such as epithelial cells and macrophages. The mechanisms used by these organisms to achieve and maintain this intracellular lifestyle vary considerably, but most mechanisms involve subversion and exploitation of host cell functions. Entry into non-phagocytic cells involves triggering host signal transduction mechanisms to induce rearrangement of the host cytoskeleton, thereby facilitating bacterial uptake. Once inside the host cell, intracellular pathogens either remain within membrane bound inclusions or escape to the cytoplasm. Those living in the cytoplasm can further pirate the host actin system, using actin as a mechanism to facilitate movement within and between host cells. Organisms remaining within the vacuole have specialized mechanisms for intracellular survival and growth which involve additional communication with the host cell. Some of the processes involved in the various steps of facultative intracellular parasitism are discussed in the context of subverting the host cell cytoskeleton and signal transduction pathways for bacterial benefit. PMID:8466472

  2. The pathogen-actin connection: A platform for defense signaling in plants

    SciTech Connect

    Day, B; Henty, Jessica L; Porter, K J; Staiger, Chris J

    2011-09-08

    The cytoskeleton, a dynamic network of cytoplasmic polymers, plays a central role in numerous fundamental processes, such as development, reproduction, and cellular responses to biotic and abiotic stimuli. As a platform for innate immune responses in mammalian cells, the actin cytoskeleton is a central component in the organization and activation of host defenses, including signaling and cellular repair. In plants, our understanding of the genetic and biochemical responses in both pathogen and host that are required for virulence and resistance has grown enormously. Additional advances in live-cell imaging of cytoskeletal dynamics have markedly altered our view of actin turnover in plants. In this review, we outline current knowledge of host resistance following pathogen perception, both in terms of the genetic interactions that mediate defense signaling, as well as the biochemical and cellular processes that are required for defense signaling.

  3. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    SciTech Connect

    Woloschak, G.E. |; Felcher, P.; Chin-Mei Chang-Liu

    1995-06-01

    Experiments examined the effects of radiation dose-rate and protein synthesis inhibition expression of cytoskeletal and matrix elements in Syrian hamster embryo cells. Results demonstrated little effect of dose-rate for neutrons when comparing expression of {alpha}-tubulin and fibronectin genes. Cycloheximide repressed accumulation of {alpha}-tubulin-mRNA following exposure to high dose-rate neutrons or {gamma} rays. Cycloheximide did not affect accumulation of actin mRNA. Cycloheximide abrogated induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin mRNA accumulation following exposure to radiation. 24 refs., 3 tabs.

  4. Freezing tolerance of sea urchin embryonic cells: Differentiation commitment and cytoskeletal disturbances in culture.

    PubMed

    Odintsova, Nelly A; Ageenko, Natalya V; Kipryushina, Yulia O; Maiorova, Mariia A; Boroda, Andrey V

    2015-08-01

    This study focuses on the freezing tolerance of sea urchin embryonic cells. To significantly reduce the loss of physiological activity of these cells that occurs after cryopreservation and to study the effects of ultra-low temperatures on sea urchin embryonic cells, we tested the ability of the cells to differentiate into spiculogenic or pigment directions in culture, including an evaluation of the expression of some genes involved in pigment differentiation. A morphological analysis of cytoskeletal disturbances after freezing in a combination of penetrating (dimethyl sulfoxide and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants revealed that the distribution pattern of filamentous actin and tubulin was similar to that in the control cultures. In contrast, very rare spreading cells and a small number of cells with filamentous actin and tubulin were detected after freezing in the presence of only non-penetrating cryoprotectants. The largest number of pigment cells was found in cultures frozen with trehalose or trehalose and dimethyl sulfoxide. The ability to induce the spicule formation was lost in the cells frozen only with non-penetrating cryoprotectants, while it was maximal in cultures frozen in a cryoprotective mixture containing both non-penetrating and penetrating cryoprotectants (particularly, when ethylene glycol was present). Using different markers for cell state assessment, an effective cryopreservation protocol for sea urchin cells was developed: three-step freezing with a low cooling rate (1-2°C/min) and a combination of non-penetrating and penetrating cryoprotectants made it possible to obtain a high level of cell viability (up to 65-80%). PMID:26049089

  5. A novel neuron-enriched homolog of the erythrocyte membrane cytoskeletal protein 4.1.

    PubMed

    Walensky, L D; Blackshaw, S; Liao, D; Watkins, C C; Weier, H U; Parra, M; Huganir, R L; Conboy, J G; Mohandas, N; Snyder, S H

    1999-08-01

    We report the molecular cloning and characterization of 4.1N, a novel neuronal homolog of the erythrocyte membrane cytoskeletal protein 4.1 (4.1R). The 879 amino acid protein shares 70, 36, and 46% identity with 4.1R in the defined membrane-binding, spectrin-actin-binding, and C-terminal domains, respectively. 4.1N is expressed in almost all central and peripheral neurons of the body and is detected in embryonic neurons at the earliest stage of postmitotic differentiation. Like 4.1R, 4.1N has multiple splice forms as evidenced by PCR and Western analysis. Whereas the predominant 4.1N isoform identified in brain is approximately 135 kDa, a smaller 100 kDa isoform is enriched in peripheral tissues. Immunohistochemical studies using a polyclonal 4.1N antibody revealed several patterns of neuronal staining, with localizations in the neuronal cell body, dendrites, and axons. In certain neuronal locations, including the granule cell layers of the cerebellum and dentate gyrus, a distinct punctate-staining pattern was observed consistent with a synaptic localization. In primary hippocampal cultures, mouse 4.1N is enriched at the discrete sites of synaptic contact, colocalizing with the postsynaptic density protein of 95 kDa (a postsynaptic marker) and glutamate receptor type 1 (an excitatory postsynaptic marker). By analogy with the roles of 4.1R in red blood cells, 4.1N may function to confer stability and plasticity to the neuronal membrane via interactions with multiple binding partners, including the spectrin-actin-based cytoskeleton, integral membrane channels and receptors, and membrane-associated guanylate kinases. PMID:10414974

  6. Overexpression of p49/STRAP alters cellular cytoskeletal structure and gross anatomy in mice

    PubMed Central

    2014-01-01

    Background The protein p49/STRAP (SRFBP1) is a transcription cofactor of serum response factor (SRF) which regulates cytoskeletal and muscle-specific genes. Results Two conserved domains were found in the p49/STRAP protein. The SRF-binding domain was at its N-terminus and was highly conserved among mammalian species, xenopus and zebrafish. A BUD22 domain was found at its C-terminus in three sequence databases. The BUD22 domain was conserved among mammalian p49/STRAP proteins, and yeast cellular morphogenesis proteins, which is involved in ribosome biogenesis that affects growth rate and cell size. The endogenous p49/SRAP protein was localized mainly in the nucleus but also widely distributed in the cytoplasm, and was in close proximity to the actin. Transfected GFP-p49/STRAP protein co-localized with nucleolin within the nucleolus. Overexpression of p49/STRAP reduced actin content in cultured cells and resulted in smaller cell size versus control cells. Increased expression of p49/STRAP in transgenic mice resulted in newborns with malformations, which included asymmetric abdominal and thoracic cavities, and substantial changes in cardiac morphology. p49/STRAP altered the expression of certain muscle-specific genes, including that of the SRF gene, which is a key regulator of cardiac genes at the developmental, structural and maintenance level and has two SRE binding sites. Conclusions Since p49/STRAP is a co-factor of SRF, our data suggest that p49/STRAP likely regulates cell size and morphology through SRF target genes. The function of its BUD22 domain warrants further investigation. The observed increase in p49/STRAP expression during cellular aging may contribute to observed morphological changes in senescence. PMID:25183317

  7. Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones.

    PubMed

    Munnamalai, Vidhya; Weaver, Cory J; Weisheit, Corinne E; Venkatraman, Prahatha; Agim, Zeynep Sena; Quinn, Mark T; Suter, Daniel M

    2014-08-01

    NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)-type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F-actin content, retrograde F-actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91(phox) localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40(phox) . p40(phox) itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91(phox) and p40(phox) with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40(phox) with NOX2/gp91(phox) at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth. We have previously shown that reactive oxygen species (ROS) are critical for actin organization and dynamics in neuronal growth cones as well as neurite outgrowth. Here, we report that the cytosolic subunit p40(phox) of the NOX2-type NADPH oxidase complex is partially associated with F-actin in neuronal growth cones, while ROS produced by this complex regulates F-actin dynamics and neurite growth. These findings provide evidence for a bidirectional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones. PMID:24702317

  8. Microcephaly-dystonia due to mutated PLEKHG2 with impaired actin polymerization.

    PubMed

    Edvardson, Simon; Wang, Haibo; Dor, Talya; Atawneh, Osamah; Yaacov, Barak; Gartner, Jutta; Cinnamon, Yuval; Chen, Songhai; Elpeleg, Orly

    2016-01-01

    Rearrangement of the actin cytoskeleton is controlled by RhoGTPases which are activated by RhoGEFs. We identified homozygosity for Arg204Trp mutation in the Rho guanidine exchange factor (RhoGEF) PLEKHG2 gene in five patients with profound mental retardation, dystonia, postnatal microcephaly, and distinct neuroimaging pattern. The activity of the mutant PLEKHG2 was significantly decreased, both in basal state and when Gβγ- or lysophosphatidic acid (LPA)-stimulated. SDF1a-stimulated actin polymerization was significantly impaired in patient cells, and this abnormality was duplicated in control cells when PLEKHG2 expression was downregulated. These results underscore the role of PLEKHG2 in actin polymerization and delineate the clinical and radiological findings in PLEKHG2 deficiency. PMID:26573021

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

    SciTech Connect

    Vieira da Silva, Claudio; Alves da Silva, Erika; Costa Cruz, Mario; Chavrier, Philippe; Arruda Mortara, Renato

    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 RH 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.

  10. Regulation of water flow by actin-binding protein-induced actin gelatin.

    PubMed Central

    Ito, T; Suzuki, A; Stossel, T P

    1992-01-01

    Actin filaments inhibit osmotically driven water flow (Ito, T., K.S. Zaner, and T.P. Stossel. 1987. Biophys. J. 51: 745-753). Here we show that the actin gelation protein, actin-binding protein (ABP), impedes both osmotic shrinkage and swelling of an actin filament solution and reduces markedly the concentration of actin filaments required for this inhibition. These effects depend on actin filament immobilization, because the ABP concentration that causes initial impairment of water flow by actin filaments corresponds to the gel point measured viscometrically and because gelsolin, which noncovalently severs actin filaments, solates actin gels and restores water flow in a solution of actin cross-linked by ABP. Since ABP gels actin filaments in the periphery of many eukaryotic cells, such actin networks may contribute to physiological cell volume regulation. PMID:1318095

  11. 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.

  12. Cdc42 and Actin Control Polarized Expression of TI-VAMP Vesicles to Neuronal Growth Cones and Their Fusion with the Plasma MembraneV⃞

    PubMed Central

    Alberts, Philipp; Rudge, Rachel; Irinopoulou, Theano; Danglot, Lydia; Gauthier-Rouvière, Cécile; Galli, Thierry

    2006-01-01

    Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth. PMID:16381811

  13. Functional Cooperation between the Proteins Nck and ADAP Is Fundamental for Actin Reorganization▿†

    PubMed Central

    Pauker, Maor H.; Reicher, Barak; Fried, Sophie; Perl, Orly; Barda-Saad, Mira

    2011-01-01

    T cell antigen receptor (TCR) activation triggers profound changes in the actin cytoskeleton. In addition to controlling cellular shape and polarity, this process regulates vital T cell responses, such as T cell adhesion, motility, and proliferation. These depend on the recruitment of the signaling proteins Nck and Wiskott-Aldrich syndrome protein (WASp) to the site of TCR activation and on the functional properties of the adapter proteins linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kDa (SLP76). We now demonstrate that Nck is necessary but insufficient for the recruitment of WASp. We show that two pathways lead to SLP76-dependent actin rearrangement. One requires the SLP76 acidic domain, crucial to association with the Nck SH2 domain, and another requires the SLP76 SH2 domain, essential for interaction with the adhesion- and degranulation-promoting adapter protein ADAP. Functional cooperation between Nck and ADAP mediates SLP76-WASp interactions and actin rearrangement. We also reveal the molecular mechanism linking ADAP to actin reorganization. PMID:21536650

  14. Cytoskeletal modulation and tyrosine phosphorylation of tight junction proteins are associated with mainstream cigarette smoke-induced permeability of airway epithelium.

    PubMed

    Olivera, Dorian; Knall, Cindy; Boggs, Susan; Seagrave, JeanClare

    2010-03-01

    Cigarette smoke increases the permeability of the lung epithelium. Consequences of increased permeability include increased access of toxins and pathogens from the air spaces to the interstitium and even the blood stream, and leakage of fluids into the air spaces. The mechanisms for permeability alterations have not been elucidated for airway epithelia. By analogy with other types of epithelia, we hypothesized that changes in the phosphorylation status and function of tight junction (TJ) or cytoskeletal proteins might mediate the smoke-induced permeability changes. We investigated the effects of exposure to mainstream cigarette smoke (MS) on cultures of Calu-3 cells, an airway epithelial cell line. Specifically, MS exposure caused increases in phosphorylation of the myosin-binding subunit (MBS) of myosin phosphatase and myosin light chain (MLC), proteins involved in the regulation of actin polymerization. These results implicate activation of Rho kinase (ROCK), consistent with previously reported data indicating that inhibition of ROCK activation suppressed MS-induced increases in permeability. MS exposure also increased polymerized (filamentous) actin (f-actin) content and caused redistribution of the TJ proteins from the normal apical circumferential band to a more basal location. The translocation of the TJ proteins was spatially associated with local increases in both f-actin and macromolecular permeability. Finally, MS exposure increased tyrosine phosphorylation of occludin but not ZO-1 and decreased association between the two TJ proteins. These results indicate that MS exposure causes alterations in cytoskeletal and TJ structure and function, resulting in increased macromolecular permeability that may contribute to the adverse health effects of MS. PMID:19376691

  15. The secretion-stimulated 80K phosphoprotein of parietal cells is ezrin, and has properties of a membrane cytoskeletal linker in the induced apical microvilli.

    PubMed Central

    Hanzel, D; Reggio, H; Bretscher, A; Forte, J G; Mangeat, P

    1991-01-01

    Stimulation of gastric acid secretion in parietal cells involves the translocation of the proton pump (H,K-ATPase) from cytoplasmic tubulovesicles to the apical membrane to form long, F-actin-containing, microvilli. Following secretion, the pump is endocytosed back into tubulovesicles. The parietal cell therefore offers a system for the study of regulated membrane recycling, with temporally separated endocytic and exocytic steps. During cAMP-mediated stimulation, an 80 kDa peripheral membrane protein becomes phosphorylated on serine residues. This protein is a major component, together with actin and the pump, of the isolated apical membrane from stimulated cells, but not the resting tubulovesicular membrane. Here we show that the gastric 80 kDa phosphoprotein is closely related or identical to ezrin, a protein whose phosphorylation on serine and tyrosine residues was recently implicated in the induction by growth factors of cell surface structures on cultured cells [Bretscher, A. (1989) J. Cell Biol., 108, 921-930]. Light and electron microscopy reveal that ezrin is associated with the actin filaments of the microvilli of stimulated cells, but not with the filaments in the terminal web. In addition, a significant amount of ezrin is present in the basolateral membrane infoldings of both resting and stimulated cells. Extraction studies show that ezrin is a cytoskeletal protein in unstimulated and stimulated cells, and its association with the cytoskeleton is more stable in stimulated cells. These studies indicate that ezrin is a membrane cytoskeletal linker that may play a key role in the control of the assembly of secretory apical microvilli in parietal cells and ultimately in the regulation of acid secretion. Taken together with the earlier studies, we suggest that ezrin might be a general substrate for kinases involved in the regulation of actin-containing cell surface structures. Images PMID:1831124

  16. Programmed Rearrangement in Ciliates: Paramecium.

    PubMed

    Betermier, Mireille; Duharcourt, Sandra

    2014-12-01

    Programmed genome rearrangements in the ciliate Paramecium provide a nice illustration of the impact of transposons on genome evolution and plasticity. During the sexual cycle, development of the somatic macronucleus involves elimination of ∼30% of the germline genome, including repeated DNA (e.g., transposons) and ∼45,000 single-copy internal eliminated sequences (IES). IES excision is a precise cut-and-close process, in which double-stranded DNA cleavage at IES ends depends on PiggyMac, a domesticated piggyBac transposase. Genome-wide analysis has revealed that at least a fraction of IESs originate from Tc/mariner transposons unrelated to piggyBac. Moreover, genomic sequences with no transposon origin, such as gene promoters, can be excised reproducibly as IESs, indicating that genome rearrangements contribute to the control of gene expression. How the system has evolved to allow elimination of DNA sequences with no recognizable conserved motif has been the subject of extensive research during the past two decades. Increasing evidence has accumulated for the participation of noncoding RNAs in epigenetic control of elimination for a subset of IESs, and in trans-generational inheritance of alternative rearrangement patterns. This chapter summarizes our current knowledge of the structure of the germline and somatic genomes for the model species Paramecium tetraurelia, and describes the DNA cleavage and repair factors that constitute the IES excision machinery. We present an overview of the role of specialized RNA interference machineries and their associated noncoding RNAs in the control of DNA elimination. Finally, we discuss how RNA-dependent modification and/or remodeling of chromatin may guide PiggyMac to its cognate cleavage sites. PMID:26104450

  17. Studies on the structure of actin gels using time correlation spectroscopy of fluorescent beads.

    PubMed Central

    Qian, H; Elson, E L; Frieden, C

    1992-01-01

    Fluorescence correlation spectroscopy (FCS) has been used to measure the diffusion of fluorescently labeled beads in solutions of polymerized actin or buffer. The results, obtained at actin concentrations of 1 mg/ml, show that small beads (0.09 micron in diameter) diffuse nearly as rapidly in the actin gel as in buffer, whereas the largest beads tested (0.5 micron in diameter) are immobilized. Measured autocorrelation times for motions of beads with intermediate sizes show that the diffusion is retarded (relative to buffer) and that the time behavior cannot be represented as a single diffusive process. In addition to the retarded diffusion observed over distances > 1 micron, 0.23-micron beads also show a faster motion over smaller distances. Based on the measured rate of this faster motion, we estimate that the beads may be constrained within a cage approximately 0.67 micron on a side, equal to a filament length of approximately 250 subunits. Fluorescence correlation spectroscopy measurements made in the same small spot (radius of 1.4 microns) of the gel vary over time. From the variations of both the autocorrelation functions and the mean fluorescence, we conclude that, corresponding to a spatial scale of 1.4 microns, the actin gel is a dynamic structure with slow rearrangement of the gel occurring over periods of 20-50 s at 21-22 degrees C. This rearrangement may result from local reorganization of the actin matrix. Data for the retardation of beads by the actin gel are consistent with a detailed theory of the diffusion of particles through solutions of rigid rods that have longitudinal diffusion coefficients much less than that of the particles (Ogston, A. G., B. N. Preston, and J. D. Wells. 1973. Proc. R. Soc. Lond. A. 333:297-316). PMID:1420920

  18. Regulation of the actin cytoskeleton in Helicobacter pylori-induced migration and invasive growth of gastric epithelial cells

    PubMed Central

    2011-01-01

    Dynamic rearrangement of the actin cytoskeleton is a significant hallmark of Helicobacter pylori (H. pylori) infected gastric epithelial cells leading to cell migration and invasive growth. Considering the cellular mechanisms, the type IV secretion system (T4SS) and the effector protein cytotoxin-associated gene A (CagA) of H. pylori are well-studied initiators of distinct signal transduction pathways in host cells targeting kinases, adaptor proteins, GTPases, actin binding and other proteins involved in the regulation of the actin lattice. In this review, we summarize recent findings of how H. pylori functionally interacts with the complex signaling network that controls the actin cytoskeleton of motile and invasive gastric epithelial cells. PMID:22044652

  19. 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.

  20. Transposable elements as catalysts for chromosome rearrangements.

    PubMed

    Zhang, Jianbo; Yu, Chuanhe; Krishnaswamy, Lakshminarasimhan; Peterson, Thomas

    2011-01-01

    Barbara McClintock first showed that transposable elements in maize can induce major chromosomal rearrangements, including duplications, deletions, inversions, and translocations. More recently, researchers have made significant progress in elucidating the mechanisms by which transposons can induce genome rearrangements. For the Ac/Ds transposable element system, rearrangements are generated when the termini of different elements are used as substrates for transposition. The resulting alternative transposition reaction directly generates a variety of rearrangements. The size and type of rearrangements produced depend on the location and orientation of transposon insertion. A single locus containing a pair of alternative transposition-competent elements can produce a virtually unlimited number of genome rearrangements. With a basic understanding of the mechanisms involved, researchers are beginning to utilize both naturally occurring and in vitro-generated configurations of transposable elements in order to manipulate chromosome structure. PMID:21181539

  1. 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.

  2. Fliih, a Gelsolin-Related Cytoskeletal Regulator Essential for Early Mammalian Embryonic Development

    PubMed Central

    Campbell, Hugh D.; Fountain, Shelley; McLennan, Ian S.; Berven, Leise A.; Crouch, Michael F.; Davy, Deborah A.; Hooper, Jane A.; Waterford, Kynan; Chen, Ken-Shiung; Lupski, James R.; Ledermann, Birgit; Young, Ian G.; Matthaei, Klaus I.

    2002-01-01

    The Drosophila melanogaster flightless I gene is required for normal cellularization of the syncytial blastoderm. Highly conserved homologues of flightless I are present in Caenorhabditis elegans, mouse, and human. We have disrupted the mouse homologue Fliih by homologous recombination in embryonic stem cells. Heterozygous Fliih mutant mice develop normally, although the level of Fliih protein is reduced. Cultured homozygous Fliih mutant blastocysts hatch, attach, and form an outgrowing trophoblast cell layer, but egg cylinder formation fails and the embryos degenerate. Similarly, Fliih mutant embryos initiate implantation in vivo but then rapidly degenerate. We have constructed a transgenic mouse carrying the complete human FLII gene and shown that the FLII transgene is capable of rescuing the embryonic lethality of the homozygous targeted Fliih mutation. These results confirm the specific inactivation of the Fliih gene and establish that the human FLII gene and its gene product are functional in the mouse. The Fliih mouse mutant phenotype is much more severe than in the case of the related gelsolin family members gelsolin, villin, and CapG, where the homozygous mutant mice are viable and fertile but display alterations in cytoskeletal actin regulation. PMID:11971982

  3. TRPV4 regulates calcium homeostasis, cytoskeletal remodeling, conventional outflow and intraocular pressure in the mammalian eye.

    PubMed

    Ryskamp, Daniel A; Frye, Amber M; Phuong, Tam T T; Yarishkin, Oleg; Jo, Andrew O; Xu, Yong; Lakk, Monika; Iuso, Anthony; Redmon, Sarah N; Ambati, Balamurali; Hageman, Gregory; Prestwich, Glenn D; Torrejon, Karen Y; Križaj, David

    2016-01-01

    An intractable challenge in glaucoma treatment has been to identify druggable targets within the conventional aqueous humor outflow pathway, which is thought to be regulated/dysregulated by elusive mechanosensitive protein(s). Here, biochemical and functional analyses localized the putative mechanosensitive cation channel TRPV4 to the plasma membrane of primary and immortalized human TM (hTM) cells, and to human and mouse TM tissue. Selective TRPV4 agonists and substrate stretch evoked TRPV4-dependent cation/Ca(2+) influx, thickening of F-actin stress fibers and reinforcement of focal adhesion contacts. TRPV4 inhibition enhanced the outflow facility and lowered perfusate pressure in biomimetic TM scaffolds populated with primary hTM cells. Systemic delivery, intraocular injection or topical application of putative TRPV4 antagonist prodrug analogs lowered IOP in glaucomatous mouse eyes and protected retinal neurons from IOP-induced death. Together, these findings indicate that TRPV4 channels function as a critical component of mechanosensitive, Ca(2+)-signaling machinery within the TM, and that TRPV4-dependent cytoskeletal remodeling regulates TM stiffness and outflow. Thus, TRPV4 is a potential IOP sensor within the conventional outflow pathway and a novel target for treating ocular hypertension. PMID:27510430

  4. TRPV4 regulates calcium homeostasis, cytoskeletal remodeling, conventional outflow and intraocular pressure in the mammalian eye

    PubMed Central

    Ryskamp, Daniel A.; Frye, Amber M.; Phuong, Tam T. T.; Yarishkin, Oleg; Jo, Andrew O.; Xu, Yong; Lakk, Monika; Iuso, Anthony; Redmon, Sarah N.; Ambati, Balamurali; Hageman, Gregory; Prestwich, Glenn D.; Torrejon, Karen Y.; Križaj, David

    2016-01-01

    An intractable challenge in glaucoma treatment has been to identify druggable targets within the conventional aqueous humor outflow pathway, which is thought to be regulated/dysregulated by elusive mechanosensitive protein(s). Here, biochemical and functional analyses localized the putative mechanosensitive cation channel TRPV4 to the plasma membrane of primary and immortalized human TM (hTM) cells, and to human and mouse TM tissue. Selective TRPV4 agonists and substrate stretch evoked TRPV4-dependent cation/Ca2+ influx, thickening of F-actin stress fibers and reinforcement of focal adhesion contacts. TRPV4 inhibition enhanced the outflow facility and lowered perfusate pressure in biomimetic TM scaffolds populated with primary hTM cells. Systemic delivery, intraocular injection or topical application of putative TRPV4 antagonist prodrug analogs lowered IOP in glaucomatous mouse eyes and protected retinal neurons from IOP-induced death. Together, these findings indicate that TRPV4 channels function as a critical component of mechanosensitive, Ca2+-signaling machinery within the TM, and that TRPV4-dependent cytoskeletal remodeling regulates TM stiffness and outflow. Thus, TRPV4 is a potential IOP sensor within the conventional outflow pathway and a novel target for treating ocular hypertension. PMID:27510430

  5. Force-Induced Dynamical Properties of Multiple Cytoskeletal Filaments Are Distinct from that of Single Filaments

    PubMed Central

    Das, Dipjyoti; Das, Dibyendu; Padinhateeri, Ranjith

    2014-01-01

    How cytoskeletal filaments collectively undergo growth and shrinkage is an intriguing question. Collective properties of multiple bio-filaments (actin or microtubules) undergoing hydrolysis have not been studied extensively earlier within simple theoretical frameworks. In this paper, we study the collective dynamical properties of multiple filaments under force, and demonstrate the distinct properties of a multi-filament system in comparison to a single filament. Comparing stochastic simulation results with recent experimental data, we show that multi-filament collective catastrophes are slower than catastrophes of single filaments. Our study also shows further distinctions as follows: (i) force-dependence of the cap-size distribution of multiple filaments are quantitatively different from that of single filaments, (ii) the diffusion constant associated with the system length fluctuations is distinct for multiple filaments, and (iii) switching dynamics of multiple filaments between capped and uncapped states and the fluctuations therein are also distinct. We build a unified picture by establishing interconnections among all these collective phenomena. Additionally, we show that the collapse times during catastrophes can be sharp indicators of collective stall forces exceeding the additive contributions of single filaments. PMID:25531397

  6. Artificial cytoskeletal structures within enzymatically active bio-inorganic protocells.

    PubMed

    Kumar, Ravinash Krishna; Li, Mei; Olof, Sam N; Patil, Avinash J; Mann, Stephen

    2013-02-11

    The fabrication of enzymatically active, semi-permeable bio-inorganic protocells capable of self-assembling a cytoskeletal-like interior and undergoing small-molecule dephosphorylation reactions is described. Reversible disassembly of an amino acid-derived supramolecular hydrogel within the internalized reaction space is used to tune the enzymatic activity of the nanoparticle-bounded inorganic compartments. PMID:23027575

  7. FIBROBLAST CYTOSKELETAL REMODELING CONTRIBUTES TO CONNECTIVE TISSUE TENSION

    PubMed Central

    Langevin, Helene M.; Bouffard, Nicole A.; Fox, James R.; Palmer, Bradley M.; Wu, Junru; Iatridis, James C.; Barnes, William D.; Badger, Gary J.; Howe, Alan K.

    2011-01-01

    The viscoelastic behavior of connective tissue is generally attributed to the material properties of the extracellular matrix rather than cellular activity. We have previously shown that fibroblasts within areolar connective tissue exhibit dynamic cytoskeletal remodeling within minutes in response to tissue stretch ex vivo and in vivo. Here, we tested the hypothesis that fibroblasts, through this cytoskeletal remodeling, actively contribute to the viscoelastic behavior of the whole tissue. We measured significantly increased tissue tension when cellular function was broadly inhibited by sodium azide and when cytoskeletal dynamics were compromised by disrupting microtubules (with colchicine) or actomyosin contractility (via Rho kinase inhibition). These treatments led to a decrease in cell body cross-sectional area and cell field perimeter (obtained by joining the end of all of a fibroblast’s processes). Suppressing lamellipodia formation by inhibiting Rac-1 decreased cell body cross-sectional area but did not affect cell field perimeter or tissue tension. Thus, by changing shape, fibroblasts can dynamically modulate the viscoelastic behavior of areolar connective tissue through Rho-dependent cytoskeletal mechanisms. These results have broad implications for our understanding of the dynamic interplay of forces between fibroblasts and their surrounding matrix, as well as for the neural, vascular and immune cell populations residing within connective tissue. PMID:20945345

  8. Cytoskeletal prestress regulates nuclear shape and stiffness in cardiac myocytes.

    PubMed

    Lee, Hyungsuk; Adams, William J; Alford, Patrick W; McCain, Megan L; Feinberg, Adam W; Sheehy, Sean P; Goss, Josue A; Parker, Kevin Kit

    2015-11-01

    Mechanical stresses on the myocyte nucleus have been associated with several diseases and potentially transduce mechanical stimuli into cellular responses. Although a number of physical links between the nuclear envelope and cytoplasmic filaments have been identified, previous studies have focused on the mechanical properties of individual components of the nucleus, such as the nuclear envelope and lamin network. The mechanical interaction between the cytoskeleton and chromatin on nuclear deformability remains elusive. Here, we investigated how cytoskeletal and chromatin structures influence nuclear mechanics in cardiac myocytes. Rapid decondensation of chromatin and rupture of the nuclear membrane caused a sudden expansion of DNA, a consequence of prestress exerted on the nucleus. To characterize the prestress exerted on the nucleus, we measured the shape and the stiffness of isolated nuclei and nuclei in living myocytes during disruption of cytoskeletal, myofibrillar, and chromatin structure. We found that the nucleus in myocytes is subject to both tensional and compressional prestress and its deformability is determined by a balance of those opposing forces. By developing a computational model of the prestressed nucleus, we showed that cytoskeletal and chromatin prestresses create vulnerability in the nuclear envelope. Our studies suggest the cytoskeletal-nuclear-chromatin interconnectivity may play an important role in mechanics of myocyte contraction and in the development of laminopathies by lamin mutations. PMID:25908635

  9. Actin-associated protein palladin promotes tumor cell invasion by linking extracellular matrix degradation to cell cytoskeleton

    PubMed Central

    von Nandelstadh, Pernilla; Gucciardo, Erika; Lohi, Jouko; Li, Rui; Sugiyama, Nami; Carpen, Olli; Lehti, Kaisa

    2014-01-01

    Basal-like breast carcinomas, characterized by unfavorable prognosis and frequent metastases, are associated with epithelial-to-mesenchymal transition. During this process, cancer cells undergo cytoskeletal reorganization and up-regulate membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14), which functions in actin-based pseudopods to drive invasion by extracellular matrix degradation. However, the mechanisms that couple matrix proteolysis to the actin cytoskeleton in cell invasion have remained unclear. On the basis of a yeast two-hybrid screen for the MT1-MMP cytoplasmic tail-binding proteins, we identify here a novel Src-regulated protein interaction between the dynamic cytoskeletal scaffold protein palladin and MT1-MMP. These proteins were coexpressed in invasive human basal-like breast carcinomas and corresponding cell lines, where they were associated in the same matrix contacting and degrading membrane complexes. The silencing and overexpression of the 90-kDa palladin isoform revealed the functional importance of the interaction with MT1-MMP in pericellular matrix degradation and mesenchymal tumor cell invasion, whereas in MT1-MMP–negative cells, palladin overexpression was insufficient for invasion. Moreover, this invasion was inhibited in a dominant-negative manner by an immunoglobulin domain–containing palladin fragment lacking the dynamic scaffold and Src-binding domains. These results identify a novel protein interaction that links matrix degradation to cytoskeletal dynamics and migration signaling in mesenchymal cell invasion. PMID:24989798

  10. Regulation of immunoglobulin gene rearrangement and expression.

    PubMed

    Taussig, M J; Sims, M J; Krawinkel, U

    1989-05-01

    The molecular genetic events leading to Ig expression and their control formed the topic of a recent EMBO workshop. This report by Michael Taussig, Martin Sims and Ulrich Krawinkel discusses contributions dealing with genes expressed in early pre-B cells, the mechanism of rearrangement, aberrant rearrangements seen in B cells of SCID mice, the feedback control of rearrangement as studied in transgenic mice, the control of Ig expression at the transcriptional and post-transcriptional levels, and class switching. PMID:2787158

  11. 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.

  12. Complex relationship between TCTP, microtubules and actin microfilaments regulates cell shape in normal and cancer cells

    PubMed Central

    Bazile, Franck; Pascal, Aude; Arnal, Isabelle; Le Clainche, Christophe; Chesnel, Franck; Kubiak, Jacek Z.

    2009-01-01

    Translationally Controlled Tumor-associated Protein (TCTP) is a ubiquitous and highly conserved protein implicated in cancers. Here we demonstrate that interactions of TCTP with microtubules (MTs) are functionally important but indirect, and we reveal novel interaction of TCTP with the actin cytoskeleton. Firstly, immunofluorescence in Xenopus XL2 cells revealed cytoplasmic fibers stained with TCTP but not with tubulin antibodies, as well as MT-free of TCTP. Furthermore, TCTP localized to a subset of actin-rich fibers in migrating cells. Secondly XlTCTP did not affect in vitro assembly/disassembly of MTs, and lacked MT binding affinity both in pull-down assays and in cell-free extracts. Although TCTP also failed to bind to purified F-actin, it associated with microfilaments in cell-free extracts. Thirdly, TCTP concentrated in mitotic spindle did not colocalize with MTs, and was easily dissociated from these structures except at the poles. Finally, RNAi knockdown of TCTP in XL2 and HeLa cells provoked drastic, MT-dependent, shape change. These data show that although TCTP interacts with MTs it does not behave as classic MT Associated Protein (MAP). Our evidence for an association of TCTP with F-actin structures, and for an involvement in cell shape regulation, implicates this protein in integrating cytoskeletal interations both in interphase and mitosis providing a new avenue to fully understand the role of TCTP. PMID:19168579

  13. Regulatory elements mediating transcription from the Drosophila melanogaster actin 5C proximal promoter.

    PubMed Central

    Chung, Y T; Keller, E B

    1990-01-01

    The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the proximal one of which controls constitutive synthesis of actin in all growing tissues. To locate regulatory elements required for constitutive activity of the proximal promoter, mutants of this promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. An essential regulatory element has been located 313 base pairs upstream from the cap site. Deletion of this element lowered expression to one-third of the wild-type level. The element has the sequence AAGTTGTAGTTG, as shown by protein-binding footprinting with the reagent methidiumpropyl-EDTA-Fe(II). This element is probably not a general one, since it was not detected in a search of the published 5'-flanking sequences of 27 Drosophila genes. In addition to this regulatory element, there are five GAGA elements in the actin 5C proximal promoter, some or all of which are essential for the promoter activity as shown by an in vivo competition assay. Although this promoter has no classical TATA element, there is an essential promoter region about 35 base pairs upstream from the cap site that could be a TATA surrogate. The promoter also shows sequences homologous to the alcohol dehydrogenase factor 1-binding site and to the core of the vertebrate serum response element, but mutations of these sites did not affect promoter activity in transient expression assays. Images PMID:2104658

  14. Modulation of Prion Formation, Aggregation, and Toxicity by the Actin Cytoskeleton in Yeast

    PubMed Central

    Ganusova, Elena E.; Ozolins, Laura N.; Bhagat, Srishti; Newnam, Gary P.; Wegrzyn, Renee D.; Sherman, Michael Y.; Chernoff, Yury O.

    2006-01-01

    Self-perpetuating protein aggregates transmit prion diseases in mammals and heritable traits in yeast. De novo prion formation can be induced by transient overproduction of the corresponding prion-forming protein or its prion domain. Here, we demonstrate that the yeast prion protein Sup35 interacts with various proteins of the actin cortical cytoskeleton that are involved in endocytosis. Sup35-derived aggregates, generated in the process of prion induction, are associated with the components of the endocytic/vacuolar pathway. Mutational alterations of the cortical actin cytoskeleton decrease aggregation of overproduced Sup35 and de novo prion induction and increase prion-related toxicity in yeast. Deletion of the gene coding for the actin assembly protein Sla2 is lethal in cells containing the prion isoforms of both Sup35 and Rnq1 proteins simultaneously. Our data are consistent with a model in which cytoskeletal structures provide a scaffold for generation of large aggregates, resembling mammalian aggresomes. These aggregates promote prion formation. Moreover, it appears that the actin cytoskeleton also plays a certain role in counteracting the toxicity of the overproduced potentially aggregating proteins. PMID:16382152

  15. Surface adsorption and hopping cause probe-size-dependent microrheology of actin networks

    NASA Astrophysics Data System (ADS)

    He, Jun; Tang, Jay X.

    2011-04-01

    A network of filaments formed primarily by the abundant cytoskeletal protein actin gives animal cells their shape and elasticity. The rheological properties of reconstituted actin networks have been studied by tracking micron-sized probe beads embedded within the networks. We investigate how microrheology depends on surface properties of probe particles by varying the stickiness of their surface. For this purpose, we chose carboxylate polystyrene (PS) beads, silica beads, bovine serum albumin (BSA) -coated PS beads, and polyethylene glycol (PEG) -grafted PS beads, which show descending stickiness to actin filaments, characterized by confocal imaging and microrheology. Probe size dependence of microrheology is observed for all four types of beads. For the slippery PEG beads, particle-tracking microrheology detects weaker networks using smaller beads, which tend to diffuse through the network by hopping from one confinement “cage” to another. This trend is reversed for the other three types of beads, for which microrheology measures stiffer networks for smaller beads due to physisorption of nearby filaments to the bead surface. We explain the probe size dependence with two simple models. We also evaluate depletion effect near nonadsorption bead surface using quantitative image analysis and discuss the possible impact of depletion on microrheology. Analysis of these effects is necessary in order to accurately define the actin network rheology both in vitro and in vivo.

  16. Regional orientation of actin filaments in the pericanalicular cytoplasm of rat hepatocytes.

    PubMed

    Ishii, M; Washioka, H; Tonosaki, A; Toyota, T

    1991-12-01

    To elucidate how actin filaments participate in bile formation, polarity of actin filaments in the pericanalicular cytoplasm was determined with myosin subfragment 1 by transmission electron microscopy of ultrathin sections and deep-etching replicas. Densely concentrated actin filaments were identified around the bile canaliculi in the forms of microvillous core filaments, pericanalicular web filaments, and filaments on the junctional complex. They bound subfragment 1 to form double-helical strands on the deep-etching replica or typical arrowheads on the ultrathin section. All microvillous core filaments showed their arrowheads pointing basally, suggesting the molecular growth occurring at their apical ends. In contrast, filaments of the pericanalicular web, running in parallel to the cell surface, showed unfixed polarities as indicated by their arrowheads. Furthermore, neighboring filament pairs often showed opposite polarities, an alignment necessary for filament sliding. The junctional complex had filaments with arrowheads pointed mostly at the cell center with a small number in opposite direction. In addition, a group of sporadic filaments appeared to be installed to link to both the canalicular membrane and coated vesicles. Such regionally specialized actin filaments are considered inclusively to form a cytoskeletal system that is in charge of (a) maintenance of length of the microvilli, (b) contraction of the canalicular walls, and (c) translocation of coated vesicles in the pericanalicular cytoplasm. PMID:1955131

  17. Actin polymerization is stimulated by actin cross-linking protein palladin.

    PubMed

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G; Orlova, Albina; Egelman, Edward H; Beck, Moriah R

    2016-02-15

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the co-ordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. In the present study, we show that the actin-binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro cross-linking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of globular or monomeric actin (G-actin), akin to metal ions, either through charge neutralization or through conformational changes. PMID:26607837

  18. Cytoskeletal proteins from human skin fibroblasts, peripheral blood leukocytes, and a lymphoblastoid cell line compared by two-dimensional gel electrophoresis

    SciTech Connect

    Giometti, C.S.; Willard, K.E.; Anderson, N.L.

    1982-04-01

    Differences in proteins between cells grown as suspension cultures and those grown as attached cultures were studied by comparing the proteins of detergent-resistant cytoskeletons prepared from peripheral blood leukocytes and a lymphoblastoid cell line (GM607) (both grown as suspension cultures) and those of human skin fibroblasts (grown as attached cultures) by two-dimensional gel electrophoresis. The major cytoskeletal proteins of the leukocytes were also present in the protein pattern of GM607 cytoskeletons. In contrast, the fibroblast cytoskeletal protein pattern contained four groups of proteins that differed from the patterns of the leukocytes and GM607. In addition, surface labeling of GM607 and human fibroblasts with /sup 125/I demonstrated that substantial amounts of vimentin and actin are exposed at the surface of the attached fibroblasts, but there is little evidence of similar exposure at the surface of the suspension-grown GM607. These results demonstrate some differences in cytoskeletal protein composition between different types of cells could be related to their ability or lack of ability to grow as attached cells in tissue culture.

  19. Activation of a muscle-specific actin gene promoter in serum-stimulated fibroblasts.

    PubMed Central

    Stoflet, E S; Schmidt, L J; Elder, P K; Korf, G M; Foster, D N; Strauch, A R; Getz, M J

    1992-01-01

    Treatment of AKR-2B mouse fibroblasts with serum growth factors or inhibitors of protein synthesis, such as cycloheximide, results in a stimulation of cytoskeletal beta-actin transcription but has no effect on transcription of muscle-specific isotypes, such as the vascular smooth muscle (VSM) alpha-actin gene. Deletion mapping and site-specific mutagenesis studies demonstrated that a single "CArG" element of the general form CC(A/T)6GG was necessary and possibly sufficient to impart serum and cycloheximide-inducibility to the beta-actin promoter. Although the VSM alpha-actin promoter exhibits at least three similar sequence elements, it remained refractory to serum and cycloheximide induction. However, deletion of a 33 base pair sequence between -191 and -224 relative to the transcription start site resulted in the transcriptional activation of this muscle-specific promoter in rapidly growing or serum-stimulated fibroblasts. Although the activity of this truncated promoter was potentiated by cycloheximide in a manner indistinguishable from that of the beta-actin promoter, this was dependent on a more complex array of interacting elements. These included at least one CArG box and a putative upstream activating element closely associated with the -191 to -224 inhibitory sequences. These results demonstrate that the expression of a muscle-specific actin gene in fibroblasts is suppressed by a cis-acting negative control element and that in the absence of this element, the promoter is responsive to growth factor-induced signal transduction pathways. Images PMID:1421567

  20. A LIM domain protein from tobacco involved in actin-bundling and histone gene transcription.

    PubMed

    Moes, Danièle; Gatti, Sabrina; Hoffmann, Céline; Dieterle, Monika; Moreau, Flora; Neumann, Katrin; Schumacher, Marc; Diederich, Marc; Grill, Erwin; Shen, Wen-Hui; Steinmetz, André; Thomas, Clément

    2013-03-01

    The two LIM domain-containing proteins from plants (LIMs) typically exhibit a dual cytoplasmic-nuclear distribution, suggesting that, in addition to their previously described roles in actin cytoskeleton organization, they participate in nuclear processes. Using a south-western blot-based screen aimed at identifying factors that bind to plant histone gene promoters, we isolated a positive clone containing the tobacco LIM protein WLIM2 (NtWLIM2) cDNA. Using both green fluorescent protein (GFP) fusion- and immunology-based strategies, we provide clear evidence that NtWLIM2 localizes to the actin cytoskeleton, the nucleus, and the nucleolus. Interestingly, the disruption of the actin cytoskeleton by latrunculin B significantly increases NtWLIM2 nuclear fraction, pinpointing a possible novel cytoskeletal-nuclear crosstalk. Biochemical and electron microscopy experiments reveal the ability of NtWLIM2 to directly bind to actin filaments and to crosslink the latter into thick actin bundles. Electrophoretic mobility shift assays show that NtWLIM2 specifically binds to the conserved octameric cis-elements (Oct) of the Arabidopsis histone H4A748 gene promoter and that this binding largely relies on both LIM domains. Importantly, reporter-based experiments conducted in Arabidopsis and tobacco protoplasts confirm the ability of NtWLIM2 to bind to and activate the H4A748 gene promoter in live cells. Expression studies indicate the constitutive presence of NtWLIM2 mRNA and NtWLIM2 protein during tobacco BY-2 cell proliferation and cell cycle progression, suggesting a role of NtWLIM2 in the activation of basal histone gene expression. Interestingly, both live cell and in vitro data support NtWLIM2 di/oligomerization. We propose that NtWLIM2 functions as an actin-stabilizing protein, which, upon cytoskeleton remodeling, shuttles to the nucleus in order to modify gene expression. PMID:22930731

  1. Estrogen Regulates Protein Synthesis and Actin Polymerization in Hippocampal Neurons through Different Molecular Mechanisms

    PubMed Central

    Briz, Victor; Baudry, Michel

    2014-01-01

    Estrogen rapidly modulates hippocampal synaptic plasticity by activating selective membrane-associated receptors. Reorganization of the actin cytoskeleton and stimulation of mammalian target of rapamycin (mTOR)-mediated protein synthesis are two major events required for the consolidation of hippocampal long-term potentiation and memory. Estradiol regulates synaptic plasticity by interacting with both processes, but the underlying molecular mechanisms are not yet fully understood. Here, we used acute rat hippocampal slices to analyze the mechanisms underlying rapid changes in mTOR activity and actin polymerization elicited by estradiol. Estradiol-induced mTOR phosphorylation was preceded by rapid and transient activation of both extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) and by phosphatase and tensin homolog (PTEN) degradation. These effects were prevented by calpain and ERK inhibitors. Estradiol-induced mTOR stimulation did not require activation of classical estrogen receptors (ER), as specific ERα and ERβ agonists (PPT and DPN, respectively) failed to mimic this effect, and ER antagonists could not block it. Estradiol rapidly activated both RhoA and p21-activated kinase (PAK). Furthermore, a specific inhibitor of RhoA kinase (ROCK), H1152, and a potent and specific PAK inhibitor, PF-3758309, blocked estradiol-induced cofilin phosphorylation and actin polymerization. ER antagonists also blocked these effects of estrogen. Consistently, both PPT and DPN stimulated PAK and cofilin phosphorylation as well as actin polymerization. Finally, the effects of estradiol on actin polymerization were insensitive to protein synthesis inhibitors, but its stimulation of mTOR activity was impaired by latrunculin A, a drug that disrupts actin filaments. Taken together, our results indicate that estradiol regulates local protein synthesis and cytoskeletal reorganization via different molecular mechanisms and signaling pathways. PMID:24611062

  2. Synthesis of a tricyclic lactam via Beckmann rearrangement and ring-rearrangement metathesis as key steps

    PubMed Central

    Ravikumar, Ongolu; Majhi, Jadab

    2015-01-01

    Summary A tricyclic lactam is reported in a four step synthesis sequence via Beckmann rearrangement and ring-rearrangement metathesis as key steps. Here, we used a simple starting material such as dicyclopentadiene. PMID:26425207

  3. Genome-Wide siRNA Screen Identifies Complementary Signaling Pathways Involved in Listeria Infection and Reveals Different Actin Nucleation Mechanisms during Listeria Cell Invasion and Actin Comet Tail Formation

    PubMed Central

    Kühbacher, Andreas; Emmenlauer, Mario; Rämo, Pauli; Kafai, Natasha; Dehio, Christoph

    2015-01-01

    ABSTRACT Listeria monocytogenes enters nonphagocytic cells by a receptor-mediated mechanism that is dependent on a clathrin-based molecular machinery and actin rearrangements. Bacterial intra- and intercellular movements are also actin dependent and rely on the actin nucleating Arp2/3 complex, which is activated by host-derived nucleation-promoting factors downstream of the cell receptor Met during entry and by the bacterial nucleation-promoting factor ActA during comet tail formation. By genome-wide small interfering RNA (siRNA) screening for host factors involved in bacterial infection, we identified diverse cellular signaling networks and protein complexes that support or limit these processes. In addition, we could precise previously described molecular pathways involved in Listeria invasion. In particular our results show that the requirements for actin nucleators during Listeria entry and actin comet tail formation are different. Knockdown of several actin nucleators, including SPIRE2, reduced bacterial invasion while not affecting the generation of comet tails. Most interestingly, we observed that in contrast to our expectations, not all of the seven subunits of the Arp2/3 complex are required for Listeria entry into cells or actin tail formation and that the subunit requirements for each of these processes differ, highlighting a previously unsuspected versatility in Arp2/3 complex composition and function. PMID:25991686

  4. Oxidative stress and alterations in actin cytoskeleton trigger glutathione efflux in Saccharomyces cerevisiae.

    PubMed

    Bradamante, Silvia; Villa, Alessandro; Versari, Silvia; Barenghi, Livia; Orlandi, Ivan; Vai, Marina

    2010-12-01

    A marked deficiency in glutathione (GSH), the most abundant antioxidant in living systems, plays a major role in aging and the pathogenesis of diseases ranging from neurological disorders to early atherosclerosis and the impairment of various immunological functions. In an attempt to shed light on GSH homeostasis, we carried out the space experiment SCORE (Saccharomyces cerevisiae oxidative stress response evaluation) during the FOTON-M3 mission. Microgravity and hyperoxic conditions induced an enormous extracellular release of GSH from S. cerevisiae cells (≈40% w/dw), changed the distribution of the buds, and activated the high osmolarity glycerol (HOG) and cell integrity/PKC pathways, as well as protein carbonylation. The results from the single spaceflight experiment were validated by a complete set of experiments under conditions of simulated microgravity and indicate that cytoskeletal alterations are mainly responsible for the observed effects. The results of ground experiments in which we induced cytoskeletal modifications by means of treatment with dihydrocytochalasin B (DHCB), a potent inhibitor of actin polymerisation, or (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), a selective ROCK (Rho-associated coiled-coil forming protein serine/threonine kinase) inhibitor, confirmed the role of actin in GSH efflux. We also found that the GSH release can be inhibited using the potent chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). PMID:20708643

  5. Lung Epithelial Injury by B. Anthracis Lethal Toxin Is Caused by MKK-Dependent Loss of Cytoskeletal Integrity

    PubMed Central

    Lehmann, Mandy; Noack, Deborah; Wood, Malcolm; Perego, Marta; Knaus, Ulla G.

    2009-01-01

    Bacillus anthracis lethal toxin (LT) is a key virulence factor of anthrax and contributes significantly to the in vivo pathology. The enzymatically active component is a Zn2+-dependent metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MKKs). Using ex vivo differentiated human lung epithelium we report that LT destroys lung epithelial barrier function and wound healing responses by immobilizing the actin and microtubule network. Long-term exposure to the toxin generated a unique cellular phenotype characterized by increased actin filament assembly, microtubule stabilization, and changes in junction complexes and focal adhesions. LT-exposed cells displayed randomly oriented, highly dynamic protrusions, polarization defects and impaired cell migration. Reconstitution of MAPK pathways revealed that this LT-induced phenotype was primarily dependent on the coordinated loss of MKK1 and MKK2 signaling. Thus, MKKs control fundamental aspects of cytoskeletal dynamics and cell motility. Even though LT disabled repair mechanisms, agents such as keratinocyte growth factor or dexamethasone improved epithelial barrier integrity by reducing cell death. These results suggest that co-administration of anti-cytotoxic drugs may be of benefit when treating inhalational anthrax. PMID:19270742

  6. Slit2N Inhibits Transmission of HIV-1 from Dendritic Cells to T-cells by Modulating Novel Cytoskeletal Elements

    PubMed Central

    Shrivastava, Ashutosh; Prasad, Anil; Kuzontkoski, Paula M.; Yu, Jinlong; Groopman, Jerome E.

    2015-01-01

    Dendritic cells are among the first cells to encounter sexually acquired human immunodeficiency virus (HIV-1), in the mucosa, and they can transmit HIV-1 to CD4+ T-cells via an infectious synapse. Recent studies reveal that actin-rich membrane extensions establish direct contact between cells at this synapse and facilitate virus transmission. Genesis of these contacts involves signaling through c-Src and Cdc42, which modulate actin polymerization and filopodia formation via the Arp2/3 complex and Diaphanous 2 (Diaph2). We found that Slit2N, a ligand for the Roundabout (Robo) receptors, blocked HIV-1-induced signaling through Arp2/3 and Diaph2, decreased filopodial extensions on dendritic cells, and inhibited cell-to-cell transmission of HIV-1 in a Robo1-dependent manner. Employing proteomic analysis, we identified Flightless-1 as a novel, Robo1-interacting protein. Treatment with shRNAs reduced levels of Flightless-1 and demonstrated its role in efficient cell-to-cell transfer of HIV-1. These results suggest a novel strategy to limit viral infection in the host by targeting the Slit/Robo pathway with modulation of cytoskeletal elements previously unrecognized in HIV-1 transmission. PMID:26582347

  7. Induction of Membrane Ceramides: A Novel Strategy to Interfere with T Lymphocyte Cytoskeletal Reorganisation in Viral Immunosuppression

    PubMed Central

    Harms, Harry; Krohne, Georg; Gulbins, Erich; Schneider-Schaulies, Sibylle

    2009-01-01

    Silencing of T cell activation and function is a highly efficient strategy of immunosuppression induced by pathogens. By promoting formation of membrane microdomains essential for clustering of receptors and signalling platforms in the plasma membrane, ceramides accumulating as a result of membrane sphingomyelin breakdown are not only essential for assembly of signalling complexes and pathogen entry, but also act as signalling modulators, e. g. by regulating relay of phosphatidyl-inositol-3-kinase (PI3K) signalling. Their role in T lymphocyte functions has not been addressed as yet. We now show that measles virus (MV), which interacts with the surface of T cells and thereby efficiently interferes with stimulated dynamic reorganisation of their actin cytoskeleton, causes ceramide accumulation in human T cells in a neutral (NSM) and acid (ASM) sphingomyelinase–dependent manner. Ceramides induced by MV, but also bacterial sphingomyelinase, efficiently interfered with formation of membrane protrusions and T cell spreading and front/rear polarisation in response to β1 integrin ligation or αCD3/CD28 activation, and this was rescued upon pharmacological or genetic ablation of ASM/NSM activity. Moreover, membrane ceramide accumulation downmodulated chemokine-induced T cell motility on fibronectin. Altogether, these findings highlight an as yet unrecognised concept of pathogens able to cause membrane ceramide accumulation to target essential processes in T cell activation and function by preventing stimulated actin cytoskeletal dynamics. PMID:19834551

  8. Slit2N Inhibits Transmission of HIV-1 from Dendritic Cells to T-cells by Modulating Novel Cytoskeletal Elements.

    PubMed

    Shrivastava, Ashutosh; Prasad, Anil; Kuzontkoski, Paula M; Yu, Jinlong; Groopman, Jerome E

    2015-01-01

    Dendritic cells are among the first cells to encounter sexually acquired human immunodeficiency virus (HIV-1), in the mucosa, and they can transmit HIV-1 to CD4(+) T-cells via an infectious synapse. Recent studies reveal that actin-rich membrane extensions establish direct contact between cells at this synapse and facilitate virus transmission. Genesis of these contacts involves signaling through c-Src and Cdc42, which modulate actin polymerization and filopodia formation via the Arp2/3 complex and Diaphanous 2 (Diaph2). We found that Slit2N, a ligand for the Roundabout (Robo) receptors, blocked HIV-1-induced signaling through Arp2/3 and Diaph2, decreased filopodial extensions on dendritic cells, and inhibited cell-to-cell transmission of HIV-1 in a Robo1-dependent manner. Employing proteomic analysis, we identified Flightless-1 as a novel, Robo1-interacting protein. Treatment with shRNAs reduced levels of Flightless-1 and demonstrated its role in efficient cell-to-cell transfer of HIV-1. These results suggest a novel strategy to limit viral infection in the host by targeting the Slit/Robo pathway with modulation of cytoskeletal elements previously unrecognized in HIV-1 transmission. PMID:26582347

  9. Cytoskeletal proteins in gastric H/sup +/ secretion: cAMP dependent phosphorylation, immunolocalization, and protein blotting

    SciTech Connect

    Cuppoletti, J.; Sachs, G.; Malinowska, D.H.

    1986-05-01

    The rabbit gastric parietal cell is an excellent model for the study of regulation of secretion and the role of cytoskeleton in secretion. Changes in morphology (appearance of expanded secretory canaliculi lined with microvilli) accompany H/sup +/ secretion stimulated by histamine (cAMP mediated). Parietal cells contain immunoreactive tubulin and are highly enriched in F-actin at secretory canaliculi, detected with fluorescently labelled phallacidin. They have previously shown increased protein phosphorylation in histamine-stimulated purified parietal cells concommitant with increases in H/sup +/ secretion. They report here possible functions of the phosphoproteins. Four of these proteins of apparent size on SDS PAGE of 24, 30, 48 and 130 Kd were membrane associated. /sup 125/I-actin binding to three proteins (24, 30 and 48 Kd) was shown using overlays. A 130 Kd protein reacted with anti-vinculin monoclonal antibody on immunoblots, and was immunolocalized at secretory canaliculi. As a working hypothesis, parietal cells possess membrane-associated proteins which change their state of phosphorylation upon stimulation of H/sup +/. These proteins may be cytoskeletal elements involved in regulation of H/sup +/ secretion. The 130 Kd vinculin-like protein may serve a microfilament-membrane linking role.

  10. Association of actin with alpha crystallins

    NASA Technical Reports Server (NTRS)

    Gopalakrishnan, S.; Boyle, D.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The alpha crystallins are cytosolic proteins that co-localize and co-purify with actin-containing microfilaments. Affinity column chromatography employing both covalently-coupled actin or alpha crystallin was used to demonstrate specific and saturable binding of actin with alpha crystallin. This conclusion was confirmed by direct visualization of alpha aggregates bound to actin polymerized in vitro. The significance of this interaction in relation to the functional properties of these two polypeptides will be discussed.

  11. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

    PubMed Central

    Paredez, Alexander R.; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C.; Wang, Chung-Ju Rachel; Cande, W. Z.

    2011-01-01

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host. PMID:21444821

  12. Fractionation, rearrangement and subgenome dominance

    PubMed Central

    Sankoff, David; Zheng, Chunfang

    2012-01-01

    Motivation: Fractionation is arguably the greatest cause of gene order disruption following whole genome duplication, causing severe biases in chromosome rearrangement-based estimates of evolutionary divergence. Results: We show how to correct for this bias almost entirely by means of a ‘consolidation’ algorithm for detecting and suitably transforming identifiable regions of fractionation. We characterize the process of fractionation and the performance of the algorithm through realistic simulations. We apply our method to a number of core eudicot genomes, we and by studying the fractionation regions detected, are able to address topical issues in polyploid evolution. Availability and implementation: Code for the consolidation algorithm, and sample data, is available at: http://137.122.149.195/Software/Fractionation/fractionation.html Contact: sankoff@uottawa.ca PMID:22962459

  13. Interpolation schemes for peptide rearrangements

    NASA Astrophysics Data System (ADS)

    Bauer, Marianne S.; Strodel, Birgit; Fejer, Szilard N.; Koslover, Elena F.; Wales, David J.

    2010-02-01

    A variety of methods (in total seven) comprising different combinations of internal and Cartesian coordinates are tested for interpolation and alignment in connection attempts for polypeptide rearrangements. We consider Cartesian coordinates, the internal coordinates used in CHARMM, and natural internal coordinates, each of which has been interfaced to the OPTIM code and compared with the corresponding results for united-atom force fields. We show that aligning the methylene hydrogens to preserve the sign of a local dihedral angle, rather than minimizing a distance metric, provides significant improvements with respect to connection times and failures. We also demonstrate the superiority of natural coordinate methods in conjunction with internal alignment. Checking the potential energy of the interpolated structures can act as a criterion for the choice of the interpolation coordinate system, which reduces failures and connection times significantly.

  14. Interpolation schemes for peptide rearrangements.

    PubMed

    Bauer, Marianne S; Strodel, Birgit; Fejer, Szilard N; Koslover, Elena F; Wales, David J

    2010-02-01

    A variety of methods (in total seven) comprising different combinations of internal and Cartesian coordinates are tested for interpolation and alignment in connection attempts for polypeptide rearrangements. We consider Cartesian coordinates, the internal coordinates used in CHARMM, and natural internal coordinates, each of which has been interfaced to the OPTIM code and compared with the corresponding results for united-atom force fields. We show that aligning the methylene hydrogens to preserve the sign of a local dihedral angle, rather than minimizing a distance metric, provides significant improvements with respect to connection times and failures. We also demonstrate the superiority of natural coordinate methods in conjunction with internal alignment. Checking the potential energy of the interpolated structures can act as a criterion for the choice of the interpolation coordinate system, which reduces failures and connection times significantly. PMID:20136299

  15. Steady-state nuclear actin levels are determined by export competent actin pool.

    PubMed

    Skarp, Kari-Pekka; Huet, Guillaume; Vartiainen, Maria K

    2013-10-01

    A number of studies in the last decade have irrevocably promoted actin into a fully fledged member of the nuclear compartment, where it, among other crucial tasks, facilitates transcription and chromatin remodeling. Changes in nuclear actin levels have been linked to different cellular processes: decreased nuclear actin to quiescence and increased nuclear actin to differentiation. Importin 9 and exportin 6 transport factors are responsible for the continuous nucleocytoplasmic shuttling of actin, but the mechanisms, which result in modulated actin levels, have not been characterized. We find that in cells growing under normal growth conditions, the levels of nuclear actin vary considerably from cell to cell. To understand the basis for this, we have extensively quantified several cellular parameters while at the same time recording the import and export rates of green fluorescent protein (GFP)-tagged actin. Surprisingly, our dataset shows that the ratio of nuclear to cytoplasmic fluorescence intensity, but not nuclear shape, size, cytoplasm size, or their ratio, correlates negatively with both import and export rate of actin. This suggests that high-nuclear actin content is maintained by both diminished import and export. The high nuclear actin containing cells still show high mobility of actin, but it is not export competent, suggesting increased binding of actin to nuclear complexes. Creation of such export incompetent actin pool would ensure enough actin is retained in the nucleus and make it available for the various nuclear functions described for actin. PMID:23749625

  16. Role of Adducin-like (hu-li tai shao) mRNA and protein localization in regulating cytoskeletal structure and function during Drosophila Oogenesis and early embryogenesis.

    PubMed

    Zaccai, M; Lipshitz, H D

    1996-01-01

    Adducin is a cytoskeletal protein that can function in vitro to bundle F-actin and to control the assembly of the F-actin/spectrin cytoskeletal network. We previously reported cloning of the Drosophila Adducin-like (Add) locus [Ding et al., 1993] also referred to as hu-li tai shao (hts) [Yue and Spradling, 1992], and identification of two adducin-related protein isoforms: a 95 x 10(3) Mr form (ADD-95) and an 87 x 10(3) Mr form (ADD-87) [Zaccai and Lipshitz, 1996]. ADD-87 protein is present throughout the oocyte cortex at stages 9 and 10 of oogenesis but is restricted to its anterior pole from stage 11 onward. This ADD-87 protein localization is preceded by localization of Add-hts mRNA first to the cortex and then to the anterior pole of the oocyte. Mutation of the swallow gene results in delocalization of Add-hts mRNA and ADD-87 protein from the cortex of stage 9 and 10 oocytes, and from the anterior pole of later stage oocytes. Early embryos produced by swallow or Add-hts mutant females have severe defects in the distribution of F-actin and spectrin as well as abnormalities in nuclear division, nuclear migration, and cellularization. In addition to their cytoskeletal defects, embryos produced by swallow females have an abnormal anterior pattern because bicoid mRNA is delocalized from the anterior pole. In contrast, bicoid mRNA is still found at the anterior of embryos produced by Add-hts mothers. Thus swallow functions to restrict bicoid mRNA and Add-hts mRNA to the cortex of the oocyte. Cortical restriction of Add-hts mRNA and protein is required for the normal structure and function of the early embryonic F-actin/spectrin cytoskeleton. A defective embryonic cytoskeleton can be induced in either of two ways: (1) by delocalization of functional ADD from the oocyte cortex (as in swallow mutants), or (2) by reduction of ADD function while retaining its normal cortical localization during oogenesis (as in Add-hts mutants). PMID:8952067

  17. Dynamin-Actin Cross Talk Contributes to Phagosome Formation and Closure.

    PubMed

    Marie-Anaïs, Florence; Mazzolini, Julie; Herit, Floriane; Niedergang, Florence

    2016-05-01

    Phagocytosis is a mechanism used by macrophages to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force allowing plasma membrane extension around the particle. The closure step of phagocytosis, however, remains poorly defined. We used a dedicated experimental setup with Total Internal Reflection Fluorescence Microscopy (TIRFM) to monitor phagosome formation and closure in three dimensions in living cells. We show that dynamin-2, which mediates the scission of endocytic vesicles, was recruited early and concomitantly with actin during phagosome formation. Dynamin-2 accumulated at the site of phagosome closure in living macrophages. Inhibition of its activity with dominant negative mutants or drugs demonstrated that dynamin-2 is implicated in actin dynamics and pseudopod extension. Depolymerization of actin led to impaired dynamin-2 recruitment or activity. Finally, we show that dynamin-2 plays a critical role in the effective scission of the phagosome from the plasma membrane. Thus, we establish that a cross talk between actin and dynamin takes place for phagosome formation and closure before dynamin functions for scission. PMID:26847957

  18. Actin dynamics: from nanoscale to microscale.

    PubMed

    Carlsson, Anders E

    2010-01-01

    The dynamic nature of actin in cells manifests itself constantly. Polymerization near the cell edge is balanced by depolymerization in the interior, externally induced actin polymerization is followed by depolymerization, and spontaneous oscillations of actin at the cell periphery are frequently seen. I discuss how mathematical modeling relates quantitative measures of actin dynamics to the rates of underlying molecular level processes. The dynamic properties addressed include the rate of actin assembly at the leading edge of a moving cell, the disassembly rates of intracellular actin networks, the polymerization time course in externally stimulated cells, and spontaneous spatiotemporal patterns formed by actin. Although several aspects of actin assembly have been clarified by increasingly sophisticated models, our understanding of rapid actin disassembly is limited, and the origins of nonmonotonic features in externally stimulated actin polymerization remain unclear. Theory has generated several concrete, testable hypotheses for the origins of spontaneous actin waves and cell-edge oscillations. The development and use of more biomimetic systems applicable to the geometry of a cell will be key to obtaining a quantitative understanding of actin dynamics in cells. PMID:20462375

  19. Rearrangement of sulfonamidyl radicals with hydrogen migration

    SciTech Connect

    Troyanskii, E.I.; Lazareva, M.I.; Nikishin, G.I.

    1987-01-20

    One-step outlying oxidative chlorination of alkanesulfonamides by the action of the Na/sub 2/S/sub 2/O/sub 8/-CuCl/sub 2/ system via intermediate sulfonamidyl radicals gives 3- and 4-chloroalkanesulfonamides. Rearrangements of sulfonamidyl radicals with H atom migration from the sulfonyl segment predominates over rearrangement with H atom migration from the amide segment.

  20. Inspiring Examples in Rearrangements of Infinite Products

    ERIC Educational Resources Information Center

    Ramasinghe, W.

    2007-01-01

    It is well known that simple examples are really encouraging in the understanding of rearrangements of infinite series. In this paper a similar role is played by simple examples in the case of infinite products. Iterated products of double products seem to have a similar spirit of rearrangements of products, although they are not the same.…

  1. Systematic mutational analysis of the amino-terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin-based motility.

    PubMed

    Lauer, P; Theriot, J A; Skoble, J; Welch, M D; Portnoy, D A

    2001-12-01

    The Listeria monocytogenes ActA protein acts as a scaffold to assemble and activate host cell actin cytoskeletal factors at the bacterial surface, resulting in directional actin polymerization and propulsion of the bacterium through the cytoplasm. We have constructed 20 clustered charged-to-alanine mutations in the NH2-terminal domain of ActA and replaced the endogenous actA gene with these molecular variants. These 20 clones were evaluated in several biological assays for phenotypes associated with particular amino acid changes. Additionally, each protein variant was purified and tested for stimulation of the Arp2/3 complex, and a subset was tested for actin monomer binding. These specific mutations refined the two regions involved in Arp2/3 activation and suggest that the actin-binding sequence of ActA spans 40 amino acids. We also identified a 'motility rate and cloud-to-tail transition' region in which nine contiguous mutations spanning amino acids 165-260 caused motility rate defects and changed the ratio of intracellular bacteria associated with actin clouds and comet tails without affecting Arp2/3 activation. Several unusual motility phenotypes were associated with amino acid changes in this region, including altered paths through the cytoplasm, discontinuous actin tails in host cells and the tendency to 'skid' or dramatically change direction while moving. These unusual phenotypes illustrate the complexity of ActA functions that control the actin-based motility of L. monocytogenes. PMID:11886549

  2. Association of thrombospondin-1 with the actin cytoskeleton of human thrombin-activated platelets through an alphaIIbbeta3- or CD36-independent mechanism.

    PubMed Central

    Saumet, Anne; Jesus, Nando de; Legrand, Chantal; Dubernard, Véronique

    2002-01-01

    Thrombospondin-1 (TSP-1) is an adhesive glycoprotein which, when secreted from alpha-granules of activated platelets, can bind to the cell surface and participate in platelet aggregate formation. In this study, we show that thrombin activation leads to the rapid and specific association of a large amount of secreted alpha-granular TSP-1 with the actin cytoskeleton. This cytoskeletal association of TSP-1 was correlated with platelet secretion, but not aggregation, and was inhibited by cytochalasin D, an inhibitor of actin polymerization. Association of TSP-1 with the actin cytoskeleton was mediated by membrane receptors, as shown by using MAII, a TSP-1-specific monoclonal antibody that inhibited both TSP-1 surface binding to activated platelets and cytoskeletal association. TSP-1 and its potential membrane receptors, e.g. alphaIIbbeta3 integrin, CD36 and CD47, concomitantly associated with the actin cytoskeleton. However, studies on platelets from a patient with type I Glanzmann's thrombasthenia lacking alphaIIbbeta3 and another with barely detectable CD36 showed normal TSP-1 surface expression and association with the actin cytoskeleton. Likewise, no involvement of CD47 in TSP-1 association with the actin cytoskeleton could be inferred from experiments with control platelets using the function-blocking anti-CD47 antibody B6H12. Finally, assembly of signalling complexes, as observed through translocation of tyrosine-phosphorylated proteins and kinases to the actin cytoskeleton, was found to occur in concert with cytoskeletal association of TSP-1, in control platelets as well as in thrombasthenic and CD36-deficient platelets. Our results imply a role for the actin cytoskeleton in the membrane-surface expression process of TSP-1 molecules and suggest a possible coupling of TSP-1 receptors to signalling events occurring independently of alphaIIbbeta3 or CD36. These results provide new insights into the link between surface-bound TSP-1 and the contractile actin

  3. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization

    PubMed Central

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2016-01-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis. PMID:25664724

  4. Lack of feedback inhibition of V kappa gene rearrangement by productively rearranged alleles.

    PubMed

    Harada, K; Yamagishi, H

    1991-02-01

    Circular DNAs excised by immunoglobulin kappa chain gene rearrangements were cloned and characterized. 16 of 17 clones examined were double recombination products containing a V kappa-J kappa rearrangement (coding joint) as well as the reciprocal element (signal joint) of another V kappa-J kappa rearrangement. These products suggested multiple recombination, primary inversion, and secondary excision. In primary events, 5 of 16 translational reading frames were in-phase. Thus, V kappa gene rearrangement may not be inhibited by the presence of a productively rearranged allele. An unusually large trinucleotide (P) insertion forming a palindrome of 12 nucleotides was also observed in one of the coding joints. PMID:1988542

  5. Rearrangements of organic peroxides and related processes

    PubMed Central

    Yaremenko, Ivan A; Vil’, Vera A; Demchuk, Dmitry V

    2016-01-01

    Summary This review is the first to collate and summarize main data on named and unnamed rearrangement reactions of peroxides. It should be noted, that in the chemistry of peroxides two types of processes are considered under the term rearrangements. These are conventional rearrangements occurring with the retention of the molecular weight and transformations of one of the peroxide moieties after O–O-bond cleavage. Detailed information about the Baeyer−Villiger, Criegee, Hock, Kornblum−DeLaMare, Dakin, Elbs, Schenck, Smith, Wieland, and Story reactions is given. Unnamed rearrangements of organic peroxides and related processes are also analyzed. The rearrangements and related processes of important natural and synthetic peroxides are discussed separately. PMID:27559418

  6. 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

  7. Vibration Induced Osteogenic Commitment of Mesenchymal Stem Cells is Enhanced by Cytoskeletal Remodeling but not Fluid Shear

    PubMed Central

    Uzer, Gunes; Pongkitwitoon, Suphannee; Chan, M Ete; Judex, Stefan

    2013-01-01

    Consistent across studies in humans, animals and cells, the application of vibrations can be anabolic and/or anti-catabolic to bone. The physical mechanisms modulating the vibration-induced response have not been identified. Recently, we developed an in vitro model in which candidate parameters including acceleration magnitude and fluid shear can be controlled independently during vibrations. Here, we hypothesized that vibration induced fluid shear does not modulate mesenchymal stem cell (MSC) proliferation and mineralization and that cell’s sensitivity to vibrations can be promoted via actin stress fiber formation. Adipose derived human MSCs were subjected to vibration frequencies and acceleration magnitudes that induced fluid shear stress ranging from 0.04Pa to 5Pa. Vibrations were applied at magnitudes of 0.15g, 1g, and 2g using frequencies of both 100Hz and 30Hz. After 14d and under low fluid shear conditions associated with 100Hz oscillations, mineralization was greater in all vibrated groups than in controls. Greater levels of fluid shear produced by 30Hz vibrations enhanced mineralization only in the 2g group. Over 3d, vibrations led to the greatest increase in total cell number with the frequency/acceleration combination that induced the smallest level of fluid shear. Acute experiments showed that actin remodeling was necessary for early mechanical up-regulation of RUNX-2 mRNA levels. During osteogenic differentiation, mechanically induced up-regulation of actin remodeling genes including Wiskott-Aldrich syndrome (WAS) protein, a critical regulator of Arp2/3 complex, was related to the magnitude of the applied acceleration but not to fluid shear. These data demonstrate that fluid shear does not regulate vibration induced proliferation and mineralization and that cytoskeletal remodeling activity may play a role in MSC mechanosensitivity. PMID:23870506

  8. Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

    PubMed Central

    Harder, T; Kellner, R; Parton, R G; Gruenberg, J

    1997-01-01

    Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin. Our biochemical and immunological observations indicate that these proteins are part of a complex containing annexin II and that stability of the complex is sensitive to cholesterol sequestering agents. Since annexin II is tightly membrane-associated in a cholesterol-dependent manner, and since it seems to interact physically with elements of the cortical actin cytoskeleton, we propose that the protein serves as interface between membranes containing high amounts of cholesterol and the actin cytoskeleton. Images PMID:9188103

  9. Rho-kinase-dependent actin turnover and actomyosin disassembly are necessary for mouse spinal neural tube closure

    PubMed Central

    Escuin, Sarah; Vernay, Bertrand; Savery, Dawn; Gurniak, Christine B.; Witke, Walter; Greene, Nicholas D. E.; Copp, Andrew J.

    2015-01-01

    ABSTRACT The cytoskeleton is widely considered essential for neurulation, yet the mouse spinal neural tube can close despite genetic and non-genetic disruption of the cytoskeleton. To investigate this apparent contradiction, we applied cytoskeletal inhibitors to mouse embryos in culture. Preventing actomyosin cross-linking, F-actin assembly or myosin II contractile activity did not disrupt spinal closure. In contrast, inhibiting Rho kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) or blocking F-actin disassembly prevented closure, with apical F-actin accumulation and adherens junction disturbance in the neuroepithelium. Cofilin-1-null embryos yielded a similar phenotype, supporting the hypothesis that there is a key role for actin turnover. Co-exposure to Blebbistatin rescued the neurulation defects caused by RhoA inhibition, whereas an inhibitor of myosin light chain kinase, ML-7, had no such effect. We conclude that regulation of RhoA, Rho kinase, LIM kinase and cofilin signalling is necessary for spinal neural tube closure through precise control of neuroepithelial actin turnover and actomyosin disassembly. In contrast, actomyosin assembly and myosin ATPase activity are not limiting for closure. PMID:26040287

  10. Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

    PubMed Central

    Kumari, Sudha; Depoil, David; Martinelli, Roberta; Judokusumo, Edward; Carmona, Guillaume; Gertler, Frank B; Kam, Lance C; Carman, Christopher V; Burkhardt, Janis K; Irvine, Darrell J; Dustin, Michael L

    2015-01-01

    Wiscott Aldrich Syndrome protein (WASP) deficiency results in defects in calcium ion signaling, cytoskeletal regulation, gene transcription and overall T cell activation. The activation of WASP constitutes a key pathway for actin filament nucleation. Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling. Here, we identify a fraction of total synaptic F-actin selectively generated by WASP in the form of distinct F-actin ‘foci’. These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation. We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response. DOI: http://dx.doi.org/10.7554/eLife.04953.001 PMID:25758716

  11. 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. PMID:26590149

  12. Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones

    PubMed Central

    Munnamalai, Vidhya; Weaver, Cory J.; Weisheit, Corinne E.; Venkatraman, Prahatha; Agim, Zeynep Sena; Quinn, Mark T.; Suter, Daniel M.

    2014-01-01

    NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NOX2-type NADPH oxidase complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F-actin content, retrograde F-actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91phox localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40phox.