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F-actin
Terms modified by F-actin Selected AbstractsA Polysaccharide-Based Container Transportation System Powered by Molecular Motors,ANGEWANDTE CHEMIE, Issue 4 2010Youichi Tsuchiya Dr. Molekularer Güterzug: Polysaccharide (,-1,3-Glucane) können verschiedene Nanomaterialien einschließen (z.,B. einwandige Kohlenstoffnanoröhren) und entlang einer Schiene (F-Actin) transportieren, wobei anhängende Myosin-Einheiten als Räder und molekulare Motoren wirken (siehe Bild). Dieses künstliche System hat ein Transportsystem zum Vorbild, das auf der Bewegung von Vesikeln in biologischen Zellen beruht. [source] Nucleotides and epidermal growth factor induce parallel cytoskeletal rearrangements and migration in cultured adult murine neural stem cellsACTA PHYSIOLOGICA, Issue 2 2010I. Grimm Abstract Aim:, The adult subventricular zone (SVZ) contains neural stem cells that generate neuroblasts migrating to the olfactory bulb (OB) and differentiating into interneurones. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, functional integration and cell survival are poorly understood. We have previously shown that cultured adult neural stem cells express a considerable variety of nucleotide receptors and that nucleotides and epidermal growth factor (EGF) induce converging intracellular signalling pathways that carry potential for synergism in the control of neural stem cell proliferation and cell survival. Here we investigate the role of EGF and the nucleotides ATP, ADP,S and UTP in neural stem cell migration. Methods:, Neural stem cells were prepared from adult mice and subjected to adherent culture. Labelling of F-actin was performed with tetramethylrhodamine isothiocyanate-phalloidin. Images were processed for quantitative evaluation of fluorescence labelling. Agonist-induced phosphorylation of AKT and focal adhesion kinase was analysed by quantitative Western blotting. Agonist-dependent cell migration was assayed using 48-well microchemotaxis chambers. Results:, Nucleotides and EGF induce the formation of stress fibres, an increase in the cortical actin cytoskeleton and in cell spreading. This is associated with increased phosphorylation of AKT and focal adhesion kinase. Using microchemotaxis chambers we demonstrate a parallel increase in cell migration. Conclusion:, Our results suggest that nucleotides and EGF acting as paracrine or autocrine signalling substances can be of relevance for structuring and maintaining the cytoarchitecture of the SVZ and the stream of neuroblasts migrating to the OB. [source] A FERM domain in a class XIV myosin interacts with actin and tubulin and localizes to the cytoskeleton, phagosomes, and nucleus in Tetrahymena thermophila,CYTOSKELETON, Issue 2 2010Michael Gotesman Abstract Previous studies have shown that Myo1(myosin class XIV) localizes to the cytoskeleton and is involved in amitosis of the macronucleus and trafficking of phagosomes. Myo1 contains a FERM domain that could be a site for interaction between Myo1 and the cytoskeleton. Here, we explore the function of FERM by investigating its cytoskeleton binding partners and involvement in localization of Myo1. Alignment of Myo1 FERM with a talin actin-binding sequence, a MAP-2 tubulin-binding sequence, the radixin FERM dimerization motif, and the SV40 nuclear localization sequence (NLS) revealed putative actin- and tubulin-binding sequences, a putative FERM dimerization motif, and NLS-like sequences in both the N-terminal and C-terminal regions of Myo1 FERM. Alignment of Myo1 with an ERM C-terminal motif revealed a similar sequence in the Myo1 motor domain. GFP-FERM and two truncated FERM domains were separately expressed in Tetrahymena. GFP-FERM contained the entire Myo1 FERM. Truncated Myo1 FERM domains contained either the N-terminal or the C-terminal region of FERM and one putative sequence for actin-binding, one for tubulin-binding, a putative dimerization motif, and a NLS-like sequence. Actin antibody coprecipitated GFP-fusion polypeptides and tubulin from lysate of cells expressing GFP-fusions. Cosedimentation assays performed with either whole cell extracts or anti-actin immunoprecipitation pellets revealed that F-actin (independent of ATP) and microtubules cosedimented with GFP-fusion polypeptides. GFP-FERM localized to the cytoskeleton, phagosomes, and nucleus. Truncated GFP-FERM domains localized to phagosomes but not to the cytoskeleton or nucleus. © 2009 Wiley-Liss, Inc. [source] AKAP-independent localization of type-II protein kinase A to dynamic actin microspikesCYTOSKELETON, Issue 9 2009Robert L. Rivard Abstract Regulation of the cyclic AMP-dependent protein kinase (PKA) in subcellular space is required for cytoskeletal dynamics and chemotaxis. Currently, spatial regulation of PKA is thought to require the association of PKA regulatory (R) subunits with A-kinase anchoring proteins (AKAPs). Here, we show that the regulatory RII, subunit of PKA associates with dynamic actin microspikes in an AKAP-independent manner. Both endogenous RII, and a GFP-RII, fusion protein co-localize with F-actin in microspikes within hippocampal neuron growth cones and the leading edge lamellae of NG108-15 cells. Live-cell imaging demonstrates that RII,-associated microspikes are highly dynamic and that the coupling of RII, to actin is tight, as the movement of both actin and RII, are immediately and coincidently stopped by low-dose cytochalasin D. Importantly, co-localization of RII, and actin in these structures is resistant to displacement by a cell-permeable disrupter of PKA-AKAP interactions. Biochemical fractionation confirms that a substantial pool of PKA RII, is associated with the detergent-insoluble cytoskeleton and is resistant to extraction by a peptide inhibitor of AKAP interactions. Finally, mutation of the AKAP-binding domain of RII, fails to disrupt its association with actin microspikes. These data provide the first demonstration of the physical association of a kinase with such dynamic actin structures, as well as the first demonstration of the ability of type-II PKA to localize to discrete subcellular structures independently of canonical AKAP function. This association is likely to be important for microfilament dynamics and cell migration and may prime the investigation of novel mechanisms for localizing PKA activity. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] The roles of actin cytoskeleton and microtubules for membrane recycling of a food vacuole in Tetrahymena thermophilaCYTOSKELETON, Issue 7 2009Maki Sugita Abstract Phagocytosis is a fundamental cellular event for the uptake of nutrients from the environment in several kinds of eukaryote. Most ciliates egest waste and undigested materials in food vacuoles (FVs) through a cytoproct, which is a specific organelle for defecation. It is considered that FV egestion is initiated by fusion between the FV membrane and plasma membrane in a cytoproct and completed with retrieval of the membrane into a cytoplasmic space. In addition, electron microscopy indicated that microfilaments might be involved in the recycling process of the FV membrane in ciliates over 30 years ago; however, there is no conclusive evidence. Here we demonstrated actin organization on FV near a cytoproct in Tetrahymena thermophila by using a marker for a cytoproct. Moreover, it was revealed that cells treated with actin cytoskeletal inhibitor, Latrunculin B, might be suppressed for membrane retrieval in a cytoproct following FV egestion. On the other hand, the actin structures, likely to be the site of membrane retrieval, were frequently observed in the cells treated with cytoplasmic microtubules inhibitor, Nocodazole. We concluded that actin filaments were probably required for recycling of the FV membrane in a cytoproct although the role was not essential for FV egestion. In addition, it was possible that microtubules might be involved in transportation of recycling vesicles of FV coated with F-actin. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Inflammatory cytokines augments TGF-,1-induced epithelial-mesenchymal transition in A549 cells by up-regulating T,R-ICYTOSKELETON, Issue 12 2008Xiangde Liu Abstract Epithelial-mesenchymal transition (EMT) is believed to play an important role in fibrosis and tumor invasion. EMT can be induced in vitro cell culture by various stimuli including growth factors and matrix metalloproteinases. In this study, we report that cytomix (a mixture of IL-1,, TNF-, and IFN-,) significantly enhances TGF-,1-induced EMT in A549 cells as evidenced by acquisition of fibroblast-like cell shape, loss of E-cadherin, and reorganization of F-actin. IL-1, or TNF-, alone can also augment TGF-,1-induced EMT. However, a combination of IL-1, and TNF-, or the cytomix is more potent to induce EMT. Cytomix, but not individual cytokine of IL-1,, TNF-, or IFN-,, significantly up-regulates expression of TGF-, receptor type I (T,R-I). Suppression of T,R-I, Smad2 or Smad3 by siRNA partially blocks EMT induction by cytomix plus TGF-,1, indicating cytomix augments TGF-,1-induced EMT through enhancing T,R-I and Smad signaling. These results indicate that inflammatory cytokines together with TGF-,1 may play an important role in the development of fibrosis and tumor progress via the mechanism of epithelial-mesenchymal transition. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophinCYTOSKELETON, Issue 11 2007Brian M. Burkel Abstract Actin filaments (F-actin) are protein polymers that undergo rapid assembly and disassembly and control an enormous variety of cellular processes ranging from force production to regulation of signal transduction. Consequently, imaging of F-actin has become an increasingly important goal for biologists seeking to understand how cells and tissues function. However, most of the available means for imaging F-actin in living cells suffer from one or more biological or experimental shortcomings. Here we describe fluorescent F-actin probes based on the calponin homology domain of utrophin (Utr-CH), which binds F-actin without stabilizing it in vitro. We show that these probes faithfully report the distribution of F-actin in living and fixed cells, distinguish between stable and dynamic F-actin, and have no obvious effects on processes that depend critically on the balance of actin assembly and disassembly. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Severing of F-actin by yeast cofilin is pH-independentCYTOSKELETON, Issue 9 2006Dmitry Pavlov Abstract Cofilin plays an important role in actin turnover in cells by severing actin filaments and accelerating their depolymerization. The role of pH in the severing by cofilin was examined using fluorescence microscopy. To facilitate the imaging of actin filaments and to avoid the use of rhodamine phalloidin, which competes with cofilin, ,-actin was labeled with tetramethylrhodamine cadaverine (TRC) at Gln41. The TRC-labeling inhibited actin treadmilling strongly, as measured by ,ATP release. Cofilin binding, detected via an increase in light scattering, and the subsequent conformational change in filament structure, as detected by TRC fluorescence decay, occurred 2,3 times faster at pH 6.8 than at pH 8.0. In contrast, actin filaments severing by cofilin was pH-independent. The pH-independent severing by cofilin was confirmed using actin labeled at Cys374 with Oregon Green® 488 maleimide. The depolymerization of actin by cofilin was faster at high pH. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Actin filament binding by a monomeric IQGAP1 fragment with a single calponin homology domainCYTOSKELETON, Issue 4 2004Scott C. Mateer Abstract IQGAP1 is a homodimeric protein that reversibly associates with F-actin, calmodulin, activated Cdc42 and Rac1, CLIP-170, ,-catenin, and E-cadherin. Its F-actin binding site includes a calponin homology domain (CHD) located near the N-terminal of each subunit. Prior studies have implied that medium- to high-affinity F-actin binding (5,50 ,M Kd) requires multiple CHDs located either on an individual polypeptide or on distinct subunits of a multimeric protein. For IQGAP1, a series of six tandem IQGAP coiled-coil repeats (IRs) located past the C-terminal of the CHD of each subunit support protein dimerization and, by extension, the IRs or an undefined subset of them were thought to be essential for F-actin binding mediated by its CHDs. Here we describe efforts to determine the minimal region of IQGAP1 capable of binding F-actin. Several truncation mutants of IQGAP1, which contain progressive deletions of the IRs and CHD, were assayed for F-actin binding in vitro. Fragments that contain both the CHD and at least one IR could bind F-actin and, as expected, removal of all six IRs and the CHD abolished binding. Unexpectedly, a fragment called IQGAP12-210, which contains the CHD, but lacks IRs, could bind actin filaments. IQGAP12-210 was found to be monomeric, to bind F-actin with a Kd of ,47 ,M, to saturate F-actin at a molar ratio of one IQGAP12-210 per actin monomer, and to co-localize with cortical actin filaments when expressed by transfection in cultured cells. These collective results identify the first known example of high-affinity actin filament binding mediated by a single CHD. Cell Motil. Cytoskeleton 58:231,241, 2004. © 2004 Wiley-Liss, Inc. [source] Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation,CYTOSKELETON, Issue 4 2004Mike Lorenz Abstract Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-,-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured. Cell Motil. Cytoskeleton 57:207,217, 2004. © 2004 Wiley-Liss, Inc. [source] Activation of ADF/cofilin mediates attractive growth cone turning toward nerve growth factor and netrin-1DEVELOPMENTAL NEUROBIOLOGY, Issue 8 2010Bonnie M. Marsick Abstract Proper neural circuitry requires that growth cones, motile tips of extending axons, respond to molecular guidance cues expressed in the developing organism. However, it is unclear how guidance cues modify the cytoskeleton to guide growth cone pathfinding. Here, we show acute treatment with two attractive guidance cues, nerve growth factor (NGF) and netrin-1, for embryonic dorsal root ganglion and temporal retinal neurons, respectively, results in increased growth cone membrane protrusion, actin polymerization, and filamentous actin (F-actin). ADF/cofilin (AC) family proteins facilitate F-actin dynamics, and we found the inactive phosphorylated form of AC is decreased in NGF- or netrin-1-treated growth cones. Directly increasing AC activity mimics addition of NGF or netrin-1 to increase growth cone protrusion and F-actin levels. Extracellular gradients of NGF, netrin-1, and a cell-permeable AC elicit attractive growth cone turning and increased F-actin barbed ends, F-actin accumulation, and active AC in growth cone regions proximal to the gradient source. Reducing AC activity blunts turning responses to NGF and netrin. Our results suggest that gradients of NGF and netrin-1 locally activate AC to promote actin polymerization and subsequent growth cone turning toward the side containing higher AC activity. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 565,588, 2010 [source] A critical step for postsynaptic F-actin organization: Regulation of Baz/Par-3 localization by aPKC and PTENDEVELOPMENTAL NEUROBIOLOGY, Issue 9 2009Preethi Ramachandran Abstract Actin remodeling has emerged as a critical process during synapse development and plasticity. Thus, understanding the regulatory mechanisms controlling actin organization at synapses is exceedingly important. Here, we used the highly plastic Drosophila neuromuscular junction (NMJ) to understand mechanisms of actin remodeling at postsynaptic sites. Previous studies have suggested that the actin-binding proteins Spectrin and Coracle play a critical role in NMJ development and the anchoring of glutamate receptors most likely through actin regulation. Here, we show that an additional determinant of actin organization at the postsynaptic region is the PDZ protein Baz/Par-3. Decreasing Baz levels in postsynaptic muscles has dramatic consequences for the size of F-actin and spectrin domains at the postsynaptic region. In turn, proper localization of Baz at this site depends on both phosphorylation and dephosphorylation events. Baz phosphorylation by its binding partner, atypical protein kinase C (aPKC), is required for normal Baz targeting to the postsynaptic region. However, the retention of Baz at this site depends on its dephosphorylation mediated by the lipid and protein phosphatase PTEN. Misregulation of the phosphorylation state of Baz by genetic alterations in PTEN or aPKC activity has detrimental consequences for postsynaptic F-actin and spectrin localization, synaptic growth, and receptor localization. Our results provide a novel mechanism of postsynaptic actin regulation through Baz, governed by the antagonistic actions of aPKC and PTEN. Given the conservation of these proteins from worms to mammals, these results are likely to provide new insight into actin organization pathways. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009 [source] Disruption effects of monophthalate exposures on inter-Sertoli tight junction in a two-compartment culture modelENVIRONMENTAL TOXICOLOGY, Issue 3 2008Yun-Hui Zhang Abstract Phthalates are suspect environmental endocrine disruptors that may affect male reproduction and development by disturbing androgen synthesis and cell,cell interactions in the seminiferous epithelium. The in vivo metabolites, monophthalates, are thought to be the active agents, and toxicant effects including testicular damage and decreased sperm motility have been described previously. In this study, the aim was to investigate the effect of monophthalates on Sertoli cells using a two-compartment cell culture model, asking whether tight junction protein structures are affected, compromising the blood-testis barrier and contributing to male-mediated toxicity. Sertoli cells were isolated from Sprague Dawley rat testes and seeded onto the filters of two-compartment wells. A Sertoli cell monolayer was allowed to form, whereupon the cultures were treated with 0, 10, 30, 150, and 600 ,mol/L monobutyl phthalate (MBP) or mono-2-ethylhexyl phthalate (MEHP) for 24 h. Effects on the tight junctions between adjacent Sertoli cells were studied by light and transmission electron microscopy, the transepithelial electrical resistance (TEER) assay, and immunofluorescence localization. Results showed that exposures to monophthalates destroyed tight junctional structure in Sertoli cell monolayers in a dose-depended manner, as evidenced by a loss of single-cell layer organization in the cultures, decline of TEER value, and decreased expression of proteins associated with tight junctions such as zonula occludens-1 (ZO-1), F-actin, and Occludin. The changes were observed at doses of 150 and 600 ,mol/L, which is 10,100 times higher relative to estimated human exposures from the environment. These results are consistent with monophthalate-induced damage to tight junctions between adjacent Sertoli cells, suggesting that damage to Sertoli cell tight junctions induced by monophthalates may be an underlying mechanism of their male-mediated reproductive toxicity. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source] Coronin 3 and its role in murine brain morphogenesisEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2005Andreas Hasse Abstract Coronins belong to the fundamental WD40-repeat proteins. They are mainly found at the submembraneous area, they bind F-actin in vitro, and most of the seven mammalian coronins have unclear roles. Coronin 3 is abundantly expressed in the adult CNS. All murine brain areas express coronin 3 during embryogenesis and the first postnatal stages. Expression in grey matter decreases postnatally, except for hippocampal pyramidal and dentate gyrus neurons, and cerebellar Purkinje cells, while levels in white matter increase in the course of myelination. Consistently, coronin 3 is abundant in differentiating neuro-2a and PC-12 cells and in primary oligodendrocytes. Treatment with PKC activator PMA reduced coronin 3 protein levels. To address its functions, neuro-2a and PC-12 cells were transfected with GFP-tagged coronin 3 versions. Full-length coronin 3 among other areas localized to outgrowing neurites, whereas truncated proteins efficiently suppressed neurite formation. Our results favour a role for coronin 3 in neuron morphogenesis and possibly migration. [source] Specific cleavage of the DNase-I binding loop dramatically decreases the thermal stability of actinFEBS JOURNAL, Issue 18 2010Anastasia V. Pivovarova Differential scanning calorimetry was used to investigate the thermal unfolding of actin specifically cleaved within the DNaseI-binding loop between residues Met47-Gly48 or Gly42-Val43 by two bacterial proteases, subtilisin or ECP32/grimelysin (ECP), respectively. The results obtained show that both cleavages strongly decreased the thermal stability of monomeric actin with either ATP or ADP as a bound nucleotide. An even more pronounced difference in the thermal stability between the cleaved and intact actin was observed when both actins were polymerized into filaments. Similar to intact F-actin, both cleaved F-actins were significantly stabilized by phalloidin and aluminum fluoride; however, in all cases, the thermal stability of the cleaved F-actins was much lower than that of intact F-actin, and the stability of ECP-cleaved F-actin was lower than that of subtilisin-cleaved F-actin. These results confirm that the DNaseI-binding loop is involved in the stabilization of the actin structure, both in monomers and in the filament subunits, and suggest that the thermal stability of actin depends, at least partially, on the conformation of the nucleotide-binding cleft. Moreover, an additional destabilization of the unstable cleaved actin upon ATP/ADP replacement provides experimental evidence for the highly dynamic actin structure that cannot be simply open or closed, but rather should be considered as being able to adopt multiple conformations. Structured digital abstract ,,MINT-7980274: Actin (uniprotkb:P68135) and Actin (uniprotkb:P68135) bind (MI:0407) by biophysical (MI:0013) [source] Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actinFEBS JOURNAL, Issue 22 2007Anastasia V. Pivovarova Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548,1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27,3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin,Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of ,,16 nm, a sedimentation coefficient of 17,20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions. [source] Deficiency of Myo18B in mice results in embryonic lethality with cardiac myofibrillar aberrationsGENES TO CELLS, Issue 10 2008Rieko Ajima Myo18B is an unconventional myosin family protein expressed predominantly in muscle cells. Although conventional myosins are known to be localized on the A-bands and function as a molecular motor for muscle contraction, Myo18B protein was localized on the Z-lines of myofibrils in striated muscles. Like Myo18A, another 18th class of myosin, the N-terminal unique domain of the protein and not the motor domain and the coiled-coil tail is critical for its localization to F-actin in myocytes. Myo18B expression was induced by myogenic differentiation through the binding of myocyte-specific enhancer factor-2 to its promoter. Deficiency of Myo18B caused an embryonic lethality in mice accompanied by disruption of myofibrillar structures in cardiac myocytes at embryonic day 10.5. Thus, Myo18B is a unique unconventional myosin that is predominantly expressed in myocytes and whose expression is essential for the development and/or maintenance of myofibrillar structure. [source] Actin filaments-stabilizing and -bundling activities of cofilin-phosphatase Slingshot-1GENES TO CELLS, Issue 5 2007Souichi Kurita Slingshot-1 (SSH1) is known to regulate actin filament dynamics by dephosphorylating and activating cofilin, an actin-depolymerizing factor. SSH1 binds to filamentous (F-) actin through its multiple F-actin-binding sites and its cofilin-phosphatase activity is enhanced by binding to F-actin. In this study, we demonstrate that SSH1 has F-actin-stabilizing and -bundling activities. In vitro actin depolymerization assays revealed that SSH1 suppressed spontaneous and cofilin-induced actin depolymerization in a dose-dependent manner. SSH1 inhibited F-actin binding and severing activities of cofilin. Low-speed centrifugation assays combined with fluorescence and electron microscopic analysis revealed that SSH1 has F-actin-bundling activity, independently of its cofilin-phosphatase activity. Deletion of N- or C-terminal regions of SSH1 significantly reduced its F-actin-stabilizing and -bundling activities, indicating that both regions are critical for these functions. As SSH1 does not form a homodimer, it probably bundles F-actin through its multiple F-actin-binding sites. Knockdown of SSH1 expression by RNA interference significantly suppressed stress fiber formation in C2C12 myoblast cells, indicating a role for SSH1 in stress fiber formation or stabilization in cells. SSH1 thus has the potential to regulate actin filament dynamics and organization in cells via F-actin-stabilizing and -bundling activities, in addition to its ability to dephosphorylate cofilin. [source] Involvement of Cdc42 and Rac small G proteins in invadopodia formation of RPMI7951 cellsGENES TO CELLS, Issue 12 2003Hirokazu Nakahara Background:, Invadopodia are membrane protrusions into the extracellular matrix by aggressive tumour cells. These structures are associated with sites of matrix degradation and invasiveness of malignant tumour cells in an in vitro fibronectin degradation/invasion assay. The Rho family small G proteins, consisting of the Rho, Rac and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, adhesion, and motility, through reorganization of the actin cytoskeleton. We studied the roles of the Rho family small G proteins in invadopodia formation. Results:, We first demonstrated that invadopodia of RPMI7951 human melanoma cells extended into the matrix substratum on a vertical view using a laser scanning confocal microscope system. We confirmed that invadopodia were rich in actin filaments (F-actin) and visualized clearly with F-actin staining on a vertical view as well as on a horizontal view. We then studied the roles of Rho, Rac, and Cdc42 in invasiveness of the same cell line. In the in vitro fibronectin degradation/invasion assay, a dominant active mutant of Cdc42 enhanced dot-like degradation, whereas a dominant active mutant of Rac enhanced diffuse-type degradation. Furthermore, frabin, a GDP/GTP exchange protein for Cdc42 with F-actin-binding activity, enhanced both dot-like and diffuse-type degradation. However, a dominant active mutant of Rho did not affect the fibronectin degradation. Moreover, inhibition of phosphatidylinositol-3 kinase (PI3K) disrupted the Rac and Cdc42-dependent actin structures and blocked the fibronectin degradation. Conclusion:, These results suggest that Cdc42 and Rac play important roles in fibronectin degradation and invasiveness in a coordinate manner through the frabin-Cdc42/Rac-PI3K signalling pathway. [source] The cofilin activity cycle in lamellipodia and invadopodiaJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009Matthew Oser Abstract The actin severing protein cofilin is essential for directed cell migration and chemotaxis, in many cell types and is also important for tumor cell invasion during metastasis. Through its severing activity, cofilin increases the number of free barbed ends to initiate actin polymerization for actin-based protrusion in two distinct subcellular compartments in invasive tumor cells: lamellipodia and invadopodia. Cofilin severing activity is tightly regulated and multiple mechanisms are utilized to regulate cofilin activity. In this prospect, we have grouped the primary on/off regulation into two broad categories, both of which are important for inhibiting cofilin from binding to F-actin or G-actin: (1) Blocking cofilin activity by the binding of cofilin to either PI(4,5)P2 at lamellipodia, or cortactin at invadopodia. (2) Blocking cofilin's ability to bind to actin via serine phosphorylation. Although the literature suggests that these cofilin regulatory mechanisms may be cell-type dependent, we propose the existence of a common cofilin activity cycle in which both operate. In this common cycle, the mechanism used to initiate cofilin activity is determined by the starting point in the cycle in a given subcellular compartment. J. Cell. Biochem. 108: 1252,1262, 2009. © 2009 Wiley-Liss, Inc. [source] Vinculin, VASP, and profilin are coordinately regulated during actin remodeling in epithelial cells, which requires de novo protein synthesis and protein kinase signal transduction pathwaysJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2004Margaret P. Quinlan Transformation progression of epithelial cells involves alterations in their morphology, polarity, and adhesive characteristics, all of which are associated with the loss and/or reorganization of actin structures. To identify the underlying mechanism of formation of the adhesion-dependent, circumferential actin network, the expression and localization of the actin binding and regulating proteins (ABPs), vinculin, VASP, and profilin were evaluated. Experimental depolarization of epithelial cells results in the loss of normal F-actin structures and the transient upregulation of vinculin, VASP, and profilin. This response is due to the loss of cell,cell, and not cell,substrate interactions, since cells that no longer express focal adhesions or stress fibers are still sensitive to changes in adhesion and manifest this in the altered profile of expression of these ABPs. Transient upregulation is dependent upon de novo protein synthesis, and protein kinase-, but not phosphatase-sensitive signal transduction pathway(s). Inhibition of the synthesis of these proteins is accompanied by dephosphorylation of the ribosomal S6 protein, but does not involve inhibition of the PI3-kinase-Akt-mTOR pathway. Constitutive expression of VASP results in altered cell morphology and adhesion and F-actin and vinculin structures. V12rac1 expressing epithelial cells are constitutively nonadhesive, malignantly transformed, and constitutively express high levels of these ABPs, with altered subcellular localizations. Transformation suppression is accompanied by the restoration of normal levels of the three ABPs, actin structures, adhesion, and epithelial morphology. Thus, vinculin, VASP, and profilin are coordinately regulated by signal transduction pathways that effect a translational response. Additionally, their expression profile maybe indicative of the adhesion and transformation status of epithelial cells. J. Cell. Physiol. 200: 277,290, 2004. © 2004 Wiley-Liss, Inc. [source] DOES THE CYTOSKELETON OF INTESTINAL EPITHHELIAL CELLS FUNCTION AS A CELLULAR ALARM TO IDENTIFY THE E. COLI INFECTIONJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 2001Zhe Li Intestinal epithelial cells play an important role in regulating host immunity in response to intestinal infection. Pathogenic bacteria (EPEC and EHEC) cause profound cytoskeletal rearrangement in intestinal epithelial cells during attachment or invasion. Rearrangement of cytoskeletal proteins could be a signal to up-regulate host defence response. Aims, To determine the role of actin cytoskeleton and microtubles in IL-8 mRNA response to E. coli infection. Methods, T84 cell monolayers in 6-well plates were infected with HB101, EPEC and EHEC (105 CFU/well) and compared with uninfected control at 3, 6 and 12 h post infection. Control and infected monolayers were treated with nocodazole (Noc, microtubule disrupter, 30 mm), taxol (Tax, microtubule stabiliser, 10 mm), cytochalasin D (CytoD, actin depolymeriser, 100 nm) and Jasplakinolide (Jasp, actin polymeriser, stabilise actin filaments, 1 mm) and studied 6 h post infection. IL-8 gene expression was measured by semiquantitative RT,PCR in control and uninfected monolayers with and without drug treatment and IL8 protein secretion by ELISA. The morphology of F-actin and ,-tubulin was examined by FITC-phaloidin staining (FAS), immunohistochemistry and confocal microscopy. Results, IL-8 mRNA and IL-8 were increased by infection with all bacterial strains at 3 and 6 h but both IL-8 mRNA and IL-8 in EHEC and EPEC infection were decreased compared with control and HB101 at 12 h. Disruption of microfilaments by Noc increased IL-8 (2.7 fold) while preservation of microfilaments by Tax inhibited IL8 response (0.5 fold) to HB101 infection only. CytoD decreased (0.1,0.5 fold) IL8 expression at all time points in all infections while stabilising actin by Jasp markedly increased the IL8 response (2,6 fold) in control, HB101, EHEC and EPEC at 3 and 6 h. CytoD inhibited Noc-induced IL8 gene expression. Confocal microscopy demonstrated that CytoD and Noc caused major morphological damage to the actin and ,-tubulin by 6 h. Similar changes were also observed in EPEC and EHEC infection at 12 h but not HB101. Jasp preserved actin stress filaments in both EPEC and EHEC. Conclusions, Disruption of microtubules and exogenous rearrangement of actin by pathogenic organism may be primary stimuli to up-regulate proinflammatory cytokine gene expression. Preservation of actin filaments is required for this response and may be necessary for signal transduction to the nucleus. [source] The Actin Cytoskeleton and Signaling Network during Pollen Tube Tip GrowthJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 2 2010Ying Fu The organization and dynamics of the actin cytoskeleton play key roles in many aspects of plant cell development. The actin cytoskeleton responds to internal developmental cues and environmental signals and is involved in cell division, subcellular organelle movement, cell polarity and polar cell growth. The tip-growing pollen tubes provide an ideal model system to investigate fundamental mechanisms of underlying polarized cell growth. In this system, most signaling cascades required for tip growth, such as Ca2+ -, small GTPases- and lipid-mediated signaling have been found to be involved in transmitting signals to a large group of actin-binding proteins. These actin-binding proteins subsequently regulate the structure of the actin network, as well as the rapid turnover of actin filaments (F-actin), thereby eventually controlling tip growth. The actin cytoskeleton acts as an integrator in which multiple signaling pathways converge, providing a general growth and regulatory mechanism that applies not only for tip growth but also for polarized diffuse growth in plants. [source] Phosphorylation of Microtubule-associated Protein SB401 from Solanum berthaultii Regulates Its Effect on MicrotubulesJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 3 2009Bao-Quan Liu Abstract We reported previously that the protein SB401 from Solanum berthaultii binds to and bundles both microtubules and F-actin. In the current study, we investigated the regulation of SB401 activity by its phosphorylation. Our experimental results showed that the phosphorylation of SB401 by casein kinase II (CKII) downregulates the activities of SB401, namely the bundling of microtubules and enhancement of the polymerization of tubulin. However, phosphorylation of SB401 had no observable effect on its bundling of F-actin. Further investigation using extract of potato pollen indicated that a CKII-like kinase may exist in potato pollen. Antibodies against CKII alpha recognized specifically a major band from the pollen extract and the pollen extract was able to phosphorylate the SB401 protein in vitro. The CKII-like kinase showed a similar ability to downregulate the bundling of microtubules. Our experiments demonstrated that phosphorylation plays an important role in the regulation of SB401 activity. We propose that this phosphorylation may regulate the effects of SB401 on microtubules and the actin cytoskeleton. [source] Major muscle systems in the larval caenogastropod, Ilyanassa obsoleta, display different patterns of developmentJOURNAL OF MORPHOLOGY, Issue 10 2009Carol C.E. Evans Abstract This study describes the anatomical and developmental aspects of muscular development from the early embryo to competent larval stage in the gastropod Ilyanassa obsoleta. Staining of F-actin revealed differential spatial and temporal patterns of several muscles. In particular, two major muscles, the larval retractor and pedal retractor muscles originate independently and display distinct developmental patterns similar to observations in other gastropod species. Additionally, together with the larval retractor muscle, the accessory larval muscle developed in the embryo at the trochophore stage. Therefore, both these muscles develop prior to ontogenetic torsion. The pedal retractor muscle marked the most abundant growth in the mid veliger stage. Also during the middle stage, the metapodial retractor muscle and opercular retractor muscle grew concurrently with development of the foot. We show evidence that juvenile muscles, such as the buccal mass muscle and siphon muscle develop initially during the late veliger stage. Collectively, these findings substantiate that larval myogenesis involves a complex sequence of events that appear evolutionary conserved within the gastropods, and set the stage for future studies using this model species to address issues concerning the evolution and eventual fates of larval musculature in molluscs. J. Morphol., 2009. © 2009 Wiley-Liss, Inc. [source] Areca nut extract represses migration and differentiation while activating matrix metalloproteinase-9 of normal gingival epithelial cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2008Y-H Tseng Background and Objective:, Areca (betel) chewing is associated with an increase in the incidence of periodontal diseases. Aberrations in matrix metalloproteinase (MMP) expression have been reported to be associated with periodontal disease. This study investigated the effects of areca nut extract on MMP activity and the phenotype of human gingival epithelial cells. Material and Methods:, Reverse transcription-polymerase chain reaction, western blotting and gelatin zymography were used to assay MMPs. Cell viability, mobility and detachment assays were performed to characterize the phenotypic impact. Confocal microscopy was employed to evaluate cell aggregation and the distribution of E-cadherin and F-actin. Results:, Treatment of gingival epithelial cells with 10 µg/mL of areca nut extract reduced its cell viability. Treatment with 5 and 10 µg/mL of areca nut extract for 24 h activated MMP-9 but not MMP-2 in gingival epithelial cells. This activation could be nuclear factor-,B dependent and was abrogated by 10 µm curcumin. Areca nut extract also reduced the migration and detachment of gingival epithelial cells. The differentiated cell,cell contact of gingival epithelial cells was markedly impaired by areca nut extract. This was accompanied by a disruption of distribution of E-cadherin and F-actin. Conclusion:, The areca nut extract-mediated activation of MMP-9 in gingival epithelial cells could signify a potential periodontal pathogenesis in areca chewers. The areca nut extract-mediated inhibition of cell viability and migration, together with the changed aggregation in gingival epithelial cells, suggests that impairment of the re-epithelization underlies the process and this, in turn, might exacerbate gingival inflammation. [source] Differential localization of laminin ,2 and integrin ,4 in primary cultures of the rat gingival epitheliumJOURNAL OF PERIODONTAL RESEARCH, Issue 1 2006Michie Tanno Objectives:, The aim of this study was to investigate the differential immunolocalization of laminin ,2 and integrin ,4 in primary cultures of the rat gingival epithelium. Methods:, The gingival epithelium was obtained from Sprague-Dawley rats and was cultured in serum-free keratinocyte growth medium (DK-SFM). Western blotting analysis, immunofluorescence, confocal laser scanning microscopy (CLSM), and immuno-gold labeling for laminin ,2 and integrin ,4 were employed. CLSM images for laminin and integrin were analyzed in horizontal (x,y axis) and in vertical (x,z axis) sections. Results:, Both laminin ,2 and integrin ,4 were detected by Western blot analysis in the gingival epithelium. Immunolocalization of laminin ,2 was distinct in the cytoplasm to form one or two irregular rings in gingival epithelial cells. By contrast, integrin ,4 was localized diffusely in the cytoplasm. F-actin (indicating actin filaments) was clearly discernible at the periphery of the cytoplasm to form a cellular fringe. In x,z axis images obtained by CLSM, laminin ,2 was recognized as large foci in the most inner portion just above the basal plasma membrane. Integrin ,4 existed in the area where F-actin was labeled surrounding the membrane. Immuno-electron microscopy showed that 10nm colloidal gold particles indicating laminin ,2 were mainly localized at the extracellular portion and in the peripheral cytoplasm, whereas integrin ,4 was distributed in the cytoplasm close to the basal plasma membrane but not in extracellular regions. Conclusions:, In primary cultures of the rat gingival epithelium, both laminin ,2 and integrin ,4 may be produced by the epithelium, and irregular rings of laminin ,2 are formed in areas where gingival cells adhere to the extracellular matrix. [source] Evaluation of human nasal RPMI 2650 cells grown at an air,liquid interface as a model for nasal drug transport studiesJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 3 2008Shuhua Bai Abstract This study tests the hypothesis that human nasal RPMI 2650 cells grown at an air,liquid interface is a feasible model for drug transport studies via the nasal route. RPMI 2650 cells were cultured in Eagle's minimal essential medium (MEM) at both air,liquid and liquid,liquid interfaces. For each culture regimen, monolayer integrity was tested by measuring the transepithelial resistance (TEER) as well as the transport of paracellular and transcellular markers across the monolayer. The expression of tight junction proteins,differentiation markers,in cells of the different monolayers was studied by western blot analysis and confocal microscopy. The highest TEER values (192,±,3 ,,·,cm2) were observed for RPMI 2650 cells seeded onto collagen-coated permeable polytetrafluoroethylene inserts and grown at an air,liquid interface for 10 days; a seeding density of 4,×,105/cm2 generated and maintained a cell monolayer with suitable barrier properties at days 9,12. Microscopic examination showed that RPMI 2650 cells grown on filter inserts formed a fully confluent monolayer. The apparent permeability coefficients of the paracellular marker, [14C] mannitol, and the transcellular marker, [3H] propranolol, were 5.07,±,0.01,×,10,6 cm/s and 16.1,±,0.1,×,10,6 cm/s, respectively. Western blot analysis indicated the presence of four tight junction proteins: ZO-1, occludin, claudin-1 and E-cadherin; and the quantities of ZO-1, occludin, and E-cadherin were significantly higher in cells grown at an air,liquid interface than in cells grown at a liquid,liquid interface. Confocal microscopic studies showed ZO-1, F-actin, occludin and claudin-1 proteins at cell-cell contacts and revealed significant differences in the distributions and densities of ZO-1 protein in cells grown at the two types of interface. The data indicate that RPMI 2650 cells grown at an air,liquid interface form polarized monolayers with the cells interconnected by tight junction proteins. This human nasal cell line model could provide a useful tool for in vitro screening of nasal drug candidates. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1165,1178, 2008 [source] Recent progress on the molecular organization of myelinated axonsJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2002Steven S. Scherer Abstract The structure of myelinated axons was well described 100 years ago by Ramón y Cajal, and now their molecular organization is being revealed. The basal lamina of myelinating Schwann cells contains laminin-2, and their abaxonal/outer membrane contains two laminin-2 receptors, ,6,4 integrin and dystroglycan. Dystroglycan binds utrophin, a short dystrophin isoform (Dp116), and dystroglycan-related protein 2 (DRP2), all of which are part of a macromolecular complex. Utrophin is linked to the actin cytoskeleton, and DRP2 binds to periaxin, a PDZ domain protein associated with the cell membrane. Non-compact myelin,found at incisures and paranodes,contains adherens junctions, tight junctions, and gap junctions. Nodal microvilli contain F-actin, ERM proteins, and cell adhesion molecules that may govern the clustering of voltage-gated Na+ channels in the nodal axolemma. Nav1.6 is the predominant voltage-gated Na+ channel in mature nerves, and is linked to the spectrin cytoskeleton by ankyrinG. The paranodal glial loops contain neurofascin 155, which likely interacts with heterodimers composed of contactin and Caspr/paranodin to form septate-like junctions. The juxtaparanodal axonal membrane contains the potassium channels Kv1.1 and Kv1.2, their associated ,2 subunit, as well as Caspr2. Kv1.1, Kv1.2, and Caspr2 all have PDZ binding sites and likely interact with the same PDZ binding protein. Like Caspr, Caspr2 has a band 4.1 binding domain, and both Caspr and Caspr2 probably bind to the band 4.1B isoform that is specifically found associated with the paranodal and juxtaparanodal axolemma. When the paranode is disrupted by mutations (in cgt -, contactin -, and Caspr -null mice), the localization of these paranodal and juxtaparanodal proteins is altered: Kv1.1, Kv1.2, and Caspr2 are juxtaposed to the nodal axolemma, and this reorganization is associated with altered conduction of myelinated fibers. Understanding how axon-Schwann interactions create the molecular architecture of myelinated axons is fundamental and almost certainly involved in the pathogenesis of peripheral neuropathies. [source] What is vinculin needed for in platelets?JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2010J. V. MITSIOS Summary.,Background: Vinculin links integrins to the cell cytoskeleton by virtue of its binding to proteins such as talin and F-actin. It has been implicated in the transmission of mechanical forces from the extracellular matrix to the cytoskeleton of migrating cells. Vinculin's function in platelets is unknown. Objective: To determine whether vinculin is required for the functions of platelets and their major integrin, ,IIb,3. Methods: The murine vinculin gene (Vcl) was deleted in the megakaryocyte/platelet lineage by breeding Vcl fl/fl mice with Pf4,Cre mice. Platelet and integrin functions were studied in vivo and ex vivo. Results: Vinculin was undetectable in platelets from Vcl fl/fl Cre+ mice, as determined by immunoblotting and fluorescence microscopy. Vinculin-deficient megakaryocytes exhibited increased membrane tethers in response to mechanical pulling on ,IIb,3 with laser tweezers, suggesting that vinculin helps to maintain membrane cytoskeleton integrity. Surprisingly, vinculin-deficient platelets displayed normal agonist-induced fibrinogen binding to ,IIb,3, aggregation, spreading, actin polymerization/organization, clot retraction and the ability to form a procoagulant surface. Furthermore, vinculin-deficient platelets adhered to immobilized fibrinogen or collagen normally, under both static and flow conditions. Tail bleeding times were prolonged in 59% of vinculin-deficient mice. However, these mice exhibited no spontaneous bleeding and they formed occlusive platelet thrombi comparable to those in wild-type littermates in response to carotid artery injury with FeCl3. Conclusion: Despite promoting membrane cytoskeleton integrity when mechanical force is applied to ,IIb,3, vinculin is not required for the traditional functions of ,IIb,3 or the platelet actin cytoskeleton. [source] | ||||||||||||||||||||||||||||||||||||||||