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Actin Filaments (actin + filament)
Terms modified by Actin Filaments Selected AbstractsActin 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] 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] Axon extension in the fast and slow lanes: Substratum-dependent engagement of myosin II functionsDEVELOPMENTAL NEUROBIOLOGY, Issue 10 2007Andrea R. Ketschek Abstract Axon extension involves the coordinated regulation of the neuronal cytoskeleton. Actin filaments drive protrusion of filopodia and lamellipodia while microtubules invade the growth cone, thereby providing structural support for the nascent axon. Furthermore, in order for axons to extend the growth cone must attach to the substratum. Previous work indicates that myosin II activity inhibits the advance of microtubules into the periphery of growth cones, and myosin II has also been implicated in mediating integrin-dependent cell attachment. However, it is not clear how the functions of myosin II in regulating substratum attachment and microtubule advance are integrated during axon extension. We report that inhibition of myosin II function decreases the rate of axon extension on laminin, but surprisingly promotes extension rate on polylysine. The differential effects of myosin II inhibition on axon extension rate are attributable to myosin II having the primary function of mediating substratum attachment on laminin, but not on polylysine. Conversely, on polylysine the primary function of myosin II is to inhibit microtubule advance into growth cones. Thus, the substratum determines the role of myosin II in axon extension by controlling the functions of myosin II that contribute to extension. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007. [source] Hepatocyte dynamics in a three-dimensional rotating bioreactorJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 11 2007Mitsuo Miyazawa Abstract Background and Aims:, The use of an artificial liver system with extracorporeal circulation or a three-dimensional bioreactor perfused with liquid culture medium inevitably exposes hepatocytes to fluid mechanical stress (MS). The expression of liver-specific hepatocyte functions seems to be modulated by the magnitude of MS. Nonetheless, few studies have focused on the direct effects of MS on hepatocytes. We subjected hepatocytes to MS using an MS loading device and investigated the effects on the cytoskeleton and hepatocyte dynamics inside three-dimensional scaffolds by monitoring the changes in actin fiber, one of the components of the cytoskeleton. We also assessed the influence of MS on specific hepatocyte functions. Methods:, We subjected hepatocytes to MS by a rotating radial flow bioreactor (RRFB) and examined the effects by comparing the MS-loaded culture cells with cells cultured under stationary conditions without MS loading. The hepatocytes (1 × 106/cm3) were seeded on gauze without collagen coating and examined to determine morphological changes after 60 h incubation. Actin filaments in samples from the MS-loaded hepatocyte culture were stained by fluorescein isothiocyanate-labeled phalloidin. Results:, Hepatocyte aggregation was observed in the MS-loaded culture, but not in the unloaded stationary culture. Better albumin products were observed in the MS-loaded group than in the stationary culture group at all measurement points. Actin filaments extended toward the scaffold after the start of MS loading incubation and polymerized around the hepatocytes. The hepatocyte aggregation eventually advanced to the formation of spheroids. Conclusion:, These results suggest that MS-induced polymerization of actin filaments stimulate hepatocyte aggregation and thereby improve hepatocyte-specific function. [source] The loose coupling mechanism in molecular machines of living cellsGENES TO CELLS, Issue 1 2000Fumio Oosawa Living cells have molecular machines for free energy conversion, for example, sliding machines in muscle and other cells, flagellar motors in bacteria, and various ion pumps in cell membranes. They are constructed from protein molecules and work in the nm (nanometer), pN (piconewton) and ms (millisecond) ranges, without inertia. In 1980s, a question was raised of whether the input,output or influx,efflux coupling in these molecular machines is tight or loose, and an idea of loose coupling was proposed. Recently, the long-distance multistep sliding of a single myosin head on an actin filament, coupled with the hydrolysis of one ATP molecule, was observed by Yanagida's group using highly developed techniques of optical microscopy and micromanipulation. This gave direct evidence for the loose coupling between the chemical reaction and the mechanical event in the sliding machine. In this review, I will briefly describe a historical overview of the input,output problem in the molecular machines of living cells. [source] Cooperative expression of junctional adhesion molecule-C and -B supports growth and invasion of gliomaGLIA, Issue 5 2010Mirna Tenan Abstract Brain invasion is a biological hallmark of glioma that contributes to its aggressiveness and limits the potential of surgery and irradiation. Deregulated expression of adhesion molecules on glioma cells is thought to contribute to this process. Junctional adhesion molecules (JAMs) include several IgSF members involved in leukocyte trafficking, angiogenesis, and cell polarity. They are expressed mainly by endothelial cells, white blood cells, and platelets. Here, we report JAM-C expression by human gliomas, but not by their normal cellular counterpart. This expression correlates with the expression of genes involved in cytoskeleton remodeling and cell migration. These genes, identified by a transcriptomic approach, include poliovirus receptor and cystein-rich 61, both known to promote glioma invasion, as well as actin filament associated protein, a c-Src binding partner. Gliomas also aberrantly express JAM-B, a high affinity JAM-C ligand. Their interaction activates the c-Src proto-oncogene, a central upstream molecule in the pathways regulating cell migration and invasion. In the tumor microenvironment, this co-expression may thus promote glioma invasion through paracrine stimuli from both tumor cells and endothelial cells. Accordingly, JAM-C/B blocking antibodies impair in vivo glioma growth and invasion, highlighting the potential of JAM-C and JAM-B as new targets for the treatment of human gliomas. © 2009 Wiley-Liss, Inc. [source] The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filamentsNEW PHYTOLOGIST, Issue 3 2005Emmanuel Panteris Summary The morphogenesis of lobed plant cells has been considered to be controlled by microtubule (MT) and/or actin filament (AF) organization. In this article, a comprehensive mechanism is proposed, in which distinct roles are played by these cytoskeletal components. First, cortical MT bundles and, in the case of pavement cells, radial MT arrays combined with MT bundles determine the deposition of local cell wall thickenings, the cellulose microfibrils of which copy the orientation of underlying MTs. Cell growth is thus locally prevented and, consequently, lobes and constrictions are formed. Arch-like tangential expansion is locally imposed at the external periclinal wall of pavement cells by the radial arrangement of cellulose microfibrils at every wall thickening. Whenever further elongation of the original cell lobes occurs, AF patches assemble at the tips of growing lobes. Intercellular space formation is promoted or prevented by the opposite or alternate, respectively, arrangement of cortical MT arrays between neighboring cells. The genes that are possibly involved in the molecular regulation of the above morphogenetic procedure by MT and AF array organization are reviewed. [source] Multiple crystal structures of actin dimers and their implications for interactions in the actin filamentACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2008Michael R. Sawaya The structure of actin in its monomeric form is known at high resolution, while the structure of filamentous F-actin is only understood at considerably lower resolution. Knowing precisely how the monomers of actin fit together would lead to a deeper understanding of the dynamic behavior of the actin filament. Here, a series of crystal structures of actin dimers are reported which were prepared by cross-linking in either the longitudinal or the lateral direction in the filament state. Laterally cross-linked dimers, comprised of monomers belonging to different protofilaments, are found to adopt configurations in crystals that are not related to the native structure of filamentous actin. In contrast, multiple structures of longitudinal dimers consistently reveal the same interface between monomers within a single protofilament. The reappearance of the same longitudinal interface in multiple crystal structures adds weight to arguments that the interface visualized is similar to that in actin filaments. Highly conserved atomic interactions involving residues 199,205 and 287,291 are highlighted. [source] Actin on DNA,An ancient and dynamic relationship,CYTOSKELETON, Issue 8 2010Kari-Pekka Skarp Abstract In the cytoplasm of eukaryotic cells the coordinated assembly of actin filaments drives essential cell biological processes, such as cell migration. The discovery of prokaryotic actin homologues, as well as the appreciation of the existence of nuclear actin, have expanded the scope by which the actin family is utilized in different cell types. In bacteria, actin has been implicated in DNA movement tasks, while the connection with the RNA polymerase machinery appears to exist in both prokaryotes and eukaryotes. Within the nucleus, actin has further been shown to play a role in chromatin remodeling and RNA processing, possibly acting to link these to transcription, thereby facilitating the gene expression process. The molecular mechanism by which actin exerts these newly discovered functions is still unclear, because while polymer formation seems to be required in bacteria, these species lack conventional actin-binding proteins to regulate the process. Furthermore, although the nucleus contains a plethora of actin-regulating factors, the polymerization status of actin within this compartment still remains unclear. General theme, however, seems to be actin's ability to interact with numerous binding partners. A common feature to the novel modes of actin utilization is the connection between actin and DNA, and here we aim to review the recent literature to explore how this connection is exploited in different contexts. [source] An in vitro model system for cytoskeletal confinementCYTOSKELETON, Issue 10 2009Sarah Köster Abstract The motility, shape, and functionality of the cell depend sensitively on cytoskeletal mechanics which in turn is governed by the properties of filamentous proteins - mainly actin, microtubules, and intermediate filaments. These biopolymers are confined in the dense cytoplasm and therefore experience strong geometric constraints on their equilibrium thermal fluctuations. To obtain a better understanding of the influence of confinement on cytoskeletal filaments we study the thermal fluctuations of individual actin filaments in a microfluidic in vitro system by fluorescence microscopy and determine the persistence length of the filaments by analyzing the radial distribution function. A unique feature of this method is that we obtain the persistence length without detailed knowledge of the complete contour of the filament which makes the technique applicable to a broad range of biological polymers, including those with a persistence length smaller than the optical resolution. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] A cytoskeletal tropomyosin can compromise the structural integrity of skeletal muscleCYTOSKELETON, Issue 9 2009Anthony J. Kee Abstract We have identified a number of extra-sarcomeric actin filaments defined by cytoskeletal tropomyosin (Tm) isoforms. Expression of a cytoskeletal Tm (Tm3) not normally present in skeletal muscle in a transgenic mouse resulted in muscular dystrophy. In the present report we show that muscle pathology in this mouse is late onset (between 2 and 6 months of age) and is predominately in the back and paraspinal muscles. In the Tm3 mice, Evans blue dye uptake in muscle and serum levels of creatine kinase were markedly increased following downhill exercise, and the force drop following a series of lengthening contractions in isolated muscles (extensor digitorum longus) was also significantly increased in these mice. These results demonstrate that expression of an inappropriate Tm in skeletal muscle results in increased susceptibility to contraction-induced damage. The extra-sarcomeric actin cytoskeleton therefore may have an important role in protecting the muscle from contractile stress. 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] Compaction of cell shape occurs before decrease of elasticity in CHO-K1 cells treated with actin cytoskeleton disrupting drug cytochalasin DCYTOSKELETON, Issue 4 2009Christian Schulze Abstract The actin filaments of the cytoskeleton form a highly dynamic polymer scaffold which is actively involved in many essential mechanisms such as cell migration, transport, mitosis, and mechanosensitivity. We treated CHO-K1 cells with different concentrations of the actin cytoskeleton disrupting drug cytochalasin D. Then investigating the cells' elastic behaviour by scanning force microscopy-based rheology we confirmed for high cytochalasin D concentrations (,1.5 ,M) a significant decrease of mechanical stability. At lower concentrations we measured no significant softening, but flattening and a horizontal contraction was observable even at low concentrations (,0.3 ,M) of cytochalasin D. The observed changes in cell shape resulted in a lower cell volume, showing that there is compensation by volume for small decreases in cytoskeletal strength resulting from reduced numbers or lengths of actin filaments. These results suggest that the characteristic functions defining a cell's mechanical stability such as mechanosensitivity can be maintained via small changes in cell volume in order to counter fluctuations in cytoskeletal composition. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unitCYTOSKELETON, Issue 1 2009Vijay S. Rao Abstract Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this "long-range" cooperative activation is lost and the filament behaves identically to bare actin filaments. However, these effects are not due to dissociation of dephosphorylated Tm from the reconstituted thin filament. The data suggest that end-to-end interactions of adjacent Tm molecules are strengthened when Tm is phosphorylated, and that phosphorylation is thus essential for long range cooperative activation along the thin filament. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] The involvement of phospholipases C and D in the asymmetric division of subsidiary cell mother cells of Zea maysCYTOSKELETON, Issue 11 2008Panagiotis Apostolakos Abstract In the present study, the involvement of phospholipase C and D (PLC and PLD) pathways in the asymmetric divisions that produce the stomatal complexes of Zea mays was investigated. In particular, the polar organization of microtubules (MTs) and actin filaments (AFs) and the process of asymmetric division were studied in subsidiary cell mother cells (SMCs) treated with PLC and PLD modulators. In SMCs treated with butanol-1 (but-1), which blocks phosphatidic acid (PA) production via PLDs, AF-patch formation laterally to the inducing guard cell mother cell (GMC) and the subsequent asymmetric division were inhibited. In these SMCs, cell division plane determination, as expressed by MT preprophase band (MT-PPB) formation, was not disturbed. Exogenously applied PA partially relieved the but-1 effects on SMCs. In contrast to SMCs, but-1 did not affect the symmetric GMC division. Inhibition of the PLC catalytic activity by neomycin or U73122 resulted in inhibition of asymmetric SMC division, while AF-patch and MT-PPB were organized as in control SMCs. These data show that the PLC and PLD signaling pathways are involved in the transduction and/or perception of the inductive stimulus that is emitted by the GMCs and induces the polar AF organization and asymmetric SMC division. In contrast, division plane determination in SMCs, as expressed by MT-PPB formation, does not depend on PLC and PLD signaling pathways. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] The translocation of signaling molecules in dark adapting mammalian rod photoreceptor cells is dependent on the cytoskeletonCYTOSKELETON, Issue 10 2008Boris Reidel Abstract In vertebrate rod photoreceptor cells, arrestin and the visual G-protein transducin move between the inner segment and outer segment in response to changes in light. This stimulus dependent translocation of signalling molecules is assumed to participate in long term light adaptation of photoreceptors. So far the cellular basis for the transport mechanisms underlying these intracellular movements remains largely elusive. Here we investigated the dependency of these movements on actin filaments and the microtubule cytoskeleton of photoreceptor cells. Co-cultures of mouse retina and retinal pigment epithelium were incubated with drugs stabilizing and destabilizing the cytoskeleton. The actin and microtubule cytoskeleton and the light dependent distribution of signaling molecules were subsequently analyzed by light and electron microscopy. The application of cytoskeletal drugs differentially affected the cytoskeleton in photoreceptor compartments. During dark adaptation the depolymerization of microtubules as well as actin filaments disrupted the translocation of arrestin and transducin in rod photoreceptor cells. During light adaptation only the delivery of arrestin within the outer segment was impaired after destabilization of microtubules. Movements of transducin and arrestin required intact cytoskeletal elements in dark adapting cells. However, diffusion might be sufficient for the fast molecular movements observed as cells adapt to light. These findings indicate that different molecular translocation mechanisms are responsible for the dark and light associated translocations of arrestin and transducin in rod photoreceptor cells. Cell Motil. Cytoskeleton 65: 785,800, 2008. © 2008 Wiley-Liss, Inc. [source] Dynamic compartmentalization of protein tyrosine phosphatase receptor Q at the proximal end of stereocilia: Implication of myosin VI-based transportCYTOSKELETON, Issue 7 2008Hirofumi Sakaguchi Abstract Hair cell stereocilia are apical membrane protrusions filled with uniformly polarized actin filament bundles. Protein tyrosine phosphatase receptor Q (PTPRQ), a membrane protein with extracellular fibronectin repeats has been shown to localize at the stereocilia base and the apical hair cell surface, and to be essential for stereocilia integrity. We analyzed the distribution of PTPRQ and a possible mechanism for its compartmentalization. Using immunofluorescence we demonstrate that PTPRQ is compartmentalized at the stereocilia base with a decaying gradient from base to apex. This distribution can be explained by a model of transport directed toward the stereocilia base, which counteracts diffusion of the molecules. By mathematical analysis, we show that this counter transport is consistent with the minus end-directed movement of myosin VI along the stereocilia actin filaments. Myosin VI is localized at the stereocilia base, and exogenously expressed myosin VI and PTPRQ colocalize in the perinuclear endosomes in COS-7 cells. In myosin VI-deficient mice, PTPRQ is distributed along the entire stereocilia. PTPRQ-deficient mice show a pattern of stereocilia disruption that is similar to that reported in myosin VI-deficient mice, where the predominant features are loss of tapered base, and fusion of adjacent stereocilia. Thin section and freeze-etching electron microscopy showed that localization of PTPRQ coincides with the presence of a dense cell surface coat. Our results suggest that PTPRQ and myosin VI form a complex that dynamically maintains the organization of the cell surface coat at the stereocilia base and helps maintain the structure of the overall stereocilia bundle. Cell Motil. Cytoskeleton 2008. Published 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] Membrane associated nonmuscle myosin II functions as a motor for actin-based vesicle transport in clam oocyte extractsCYTOSKELETON, Issue 10 2007Ana S. DePina Abstract Nonmuscle myosin II (Myo2) has been shown to associate with membranes of the trans -Golgi network and to be involved in Golgi to ER retrograde protein transport. Here, we provide evidence that Myo2 not only associates with membranes but functions to transport vesicles on actin filaments (AFs). We used extracts from unactivated clam oocytes for these studies. AFs assembled spontaneously in these extracts and myosin-dependent vesicle transport was observed upon activation. In addition, actin bundles formed and moved relative to each other at an average speed of ,0.30 ,m/s. Motion analysis revealed that vesicles moved on the spontaneously assembled AFs at speeds greater than 1 ,m/s. The motor on these vesicles was identified as a member of the nonmuscle Myo2 family based on sequence determination by Edman chemistry. Vesicles in these extracts were purified by sucrose gradient centrifugation and movement was reconstituted in vitro using skeletal muscle actin coated coverslips. When peripheral membrane proteins of vesicles including Myo2 were removed by salt stripping or when extracts were treated with an antibody specific to clam oocyte nonmuscle Myo2, vesicle movement was inhibited. Blebbistatin, a Myo2 specific inhibitor, also blocked vesicle movement. Myo2 light chain kinase activity was found to be essential for vesicle movement and sliding of actin bundles. Together, our data provide direct evidence that nonmuscle Myo2 is involved in actin-dependent vesicle transport in clam oocytes. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] An actin-stabilizing peptide conjugate deduced from the major outer sheath protein of the bacterium Treponema denticolaCYTOSKELETON, Issue 9 2007Mohsen Amin Abstract A synthetic peptide conjugated to bovine serum albumin, P34BSA, based on a 10-mer in the deduced amino acid sequence of the major outer sheath protein of Treponema denticola, was found to stabilize actin filaments of fibroblasts. Pretreatment of cells with P34BSA inhibited the actin disruption induced by cytochalasin D and latrunculin B. P34BSA was taken up by the cells and localized among actin filaments. P34BSA bound actin from fibroblast lysates, and cell exposure to P34BSA led to the activation of RhoA, a key regulator of actin filament assembly in fibroblasts. Exposure of fibroblasts to P34BSA retarded their migration on a collagen substratum. P34BSA also inhibited chemotaxis of murine neutrophils. Our findings with a novel peptide conjugate imply that bacterial proteins known to perturb the cytoskeleton represent a rich source of molecular models upon which to design synthetic reagents for modulating actin-dependent cellular functions. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] The deaf mouse mutant whirler suggests a role for whirlin in actin filament dynamics and stereocilia developmentCYTOSKELETON, Issue 7 2007Mette M. Mogensen Abstract Stereocilia, finger-like projections forming the hair bundle on the apical surface of sensory hair cells in the cochlea, are responsible for mechanosensation and ultimately the perception of sound. The actin cytoskeleton of the stereocilia contains hundreds of tightly cross-linked parallel actin filaments in a paracrystalline array and it is vital for their function. Although several genes have been identified and associated with stereocilia development, the molecular mechanisms responsible for stereocilia growth, maintenance and organisation of the hair bundle have not been fully resolved. Here we provide further characterisation of the stereocilia of the whirler mouse mutant. We found that a lack of whirlin protein in whirler mutants results in short stereocilia with larger diameters without a corresponding increase in the number of actin filaments in inner hair cells. However, a decrease in the actin filament packing density was evident in the whirler mutant. The electron-density at the tip of each stereocilium was markedly patchy and irregular in the whirler mutants compared with a uniform band in controls. The outer hair cell stereocilia of the whirler homozygote also showed an increase in diameter and variable heights within bundles. The number of outer hair cell stereocilia was significantly reduced and the centre-to-centre spacing between the stereocilia was greater than in the wildtype. Our findings suggest that whirlin plays an important role in actin filament packing and dynamics during postnatal stereocilium elongation. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Expression of nebulette during early cardiac developmentCYTOSKELETON, Issue 4 2007Michael Esham Abstract Nebulette, a cardiac homologue of nebulin, colocalizes with ,-actinin in the pre-myofibrils of spreading cardiomyocytes and has been hypothesized to play a critical role in the formation of the thin-filament-Z-line complex early during myofibrillogenesis. Data from mesodermal explants or whole tissue mounts of developing hearts suggest that the pattern of myofibrillogenesis in situ may differ from observations of spreading cardiomyocytes. To evaluate the role of nebulette in myofibrillogenesis, we have analyzed the expression of nebulette in chicken heart rudiments by immunoblots and immunofluorescence. We detect the 110 kDa nebulette in heart rudiments derived from stage 9,10 using the anti-nebulin mAb, N114, or polyclonal anti-nebulette Abs by immunoblotting. Immunofluorescence analysis of explants stained with anti-nebulette and anti-,-actinin Abs demonstrates that both proteins localize along actin filaments in punctate to continuous manner at early stages of cardiac development and later give rise to striations. In both cases, the punctate staining had a periodicity of ,1.0 ,m indicating a pre-myofibrils distribution at the earliest time points examined. We demonstrate that nebulette is indeed associated with premyofibrils in very early stages of myofibrillogenesis and suggest that nebulette may play an important role in the formation of these structures. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] The effect of combined simulated microgravity and microgrooved surface topography on fibroblastsCYTOSKELETON, Issue 3 2007W. A. Loesberg Abstract This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and microgrooved substrata (groove depth: 0.5 ,m, width: 1, 2, 5, and 10 ,m), which were subjected to simulated microgravity. The aim of the study was to clarify which of these parameters was more dominant to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell alignment and area. Confocal laser scanning microscopy visualised distribution of actin filaments and focal adhesion points. Finally, expression of collagen type I, fibronectin, and ,1- and ,1-integrin were investigated by PCR. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata, cells had spread out in a random fashion. The alignment of cells cultured on grooved surfaces decreased under simulated microgravity, especially after 24 h of culturing. Cell surface area on grooved substrata were significantly smaller than on smooth substrata, but simulated microgravity on the grooved groups resulted in an enlargement of cell area. ANOVA was performed on all main parameters: topography, gravity force, and time. In this analysis, all parameters proved significant. In addition, gene levels were reduced by microgravity particularly those of ,1-integrin and fibronectin. From our data it is concluded that the fibroblasts primarily adjust their shape according to morphological environmental cues like substratum surface whilst a secondary, but significant, role is played by microgravity conditions. 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] The effect of combined hypergravity and microgrooved surface topography on the behaviour of fibroblastsCYTOSKELETON, Issue 7 2006W. A. Loesberg Abstract This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and microgrooved substrata (groove depth: 1 ,m, width: 1, 2, 5, 10 ,m), which undergo artificial hypergravity by centrifugation (10, 24 and 50 g; or 1 g control). The aim of the study was to clarify which of these parameters was more important to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell spreading and alignment. Confocal laser scanning microscopy visualised distribution of actin filaments and vinculin anchoring points through immunostaining. Finally, expression of collagen type I, fibronectin, and ,1 - and ,1 -integrin were investigated by PCR. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata (control), cells spread out in a random fashion. The alignment of cells cultured on grooved surfaces increased with higher g-forces until a peak value at 25 g. An ANOVA was performed on the data, for all main parameters: topography, gravity force, and time. In this analysis, all parameters proved significant. In addition, most gene levels were reduced by hypergravity. Still, collagen type 1 and fibronectin are seemingly unaffected by time or force. From our data it is concluded that the fibroblasts primarily adjust their shape according to morphological environmental cues like substratum surface whilst a secondary, but significant, role is played by hypergravity forces. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Morphological irregularities and features of resistance to apoptosis in the dcp-1/pita double mutated egg chambers during Drosophila oogenesisCYTOSKELETON, Issue 1 2005Ioannis P. Nezis Abstract In the present study, we demonstrate the most novel characteristic morphological features of Drosophila egg chambers lacking both dcp-1 and pita functions in the germline cells. Dcp-1 is an effector caspase and it has been previously shown to play an important role during Drosophila oogenesis [McCall and Steller, 1998 : Science 279 : 230,234; Laundrie et al., 2003 : Genetics 165 : 1881,1888; Peterson et al., 2003 : Dev Biol 260 : 113,123]. The completion of sequencing and annotation of the Drosophila genome has revealed that the dcp-1 gene is nested within an intron of another distinct gene, called pita, a member of the C2H2 zinc finger protein family that regulates transcriptional initiation. The dcp-1,/,/pita,/, nurse cells exhibit euchromatic nuclei (delay of apoptosis) during the late stages of oogenesis, as revealed by conventional light and electron microscopy. The phalloidin-FITC staining discloses significant defects in actin cytoskeleton arrangement. The actin bundles fail to organize properly and the distribution of actin filaments in the ring canals is changed compared to the wild type. The oocyte and the chorion structures have been also modified. The oocyte nucleus is out of position and the chorion appears to contain irregular foldings, while the respiratory filaments obtain an altered morphology. The dcp-1,/,/pita,/, egg chambers do not exhibit the rare events of spontaneously induced apoptosis, observed for the wild type flies, during mid-oogenesis. Interestingly, the mutated egg chambers are protected by staurosporine-induced apoptosis in a percentage of 40%, strongly suggesting the essential role of dcp-1 and/or pita during mid-oogenesis. Cell Motil. Cytoskeleton 60:14,23, 2005. © 2004 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] Transient concentration of a ,-tubulin-related protein with a pericentrin-related protein in the formation of basal bodies and flagella during the differentiation of Naegleria gruberiCYTOSKELETON, Issue 2 2002Mi Ra Suh Abstract The distribution of two proteins in Naegleria gruberi, N-,TRP (Naegleria ,-tubulin-related protein) and N-PRP (Naegleria pericentrin-related protein), was examined during the de novo formation of basal bodies and flagella that occurs during the differentiation of N. gruberi. After the initiation of differentiation, N-,TRP and N-PRP began to concentrate at the same site within cells. The percentage of cells with a concentrated region of N-,TRP and N-PRP was maximal (68%) at 40 min when the synthesis of tubulin had just started but no assembled microtubules were visible. When concentrated tubulin became visible (60 min), the region of concentrated N-,TRP and N-PRP was co-localized with the tubulin spot and then flagella began to elongate from the region of concentrated tubulin. When cells had elongated flagella, the concentrated N-,TRP and N-PRP were translocated to the opposite end of the flagellated cells and disappeared. The transient concentration of N-,TRP coincided with the transient formation of an F-actin spot at which N-,TRP and ,-tubulin mRNA were co-localized. The concentration of N-,TRP and formation of the F-actin spot occurred without the formation of microtubules but were inhibited by cytochalasin D. These observations suggest that the regional concentration of N-,TRP and N-PRP is mediated by actin filaments and might provide a site of microtubule nucleation for the assembly of newly synthesized tubulins into basal bodies and flagella. Cell Motil. Cytoskeleton 52:66,81, 2002. © 2002 Wiley-Liss, Inc. [source] Quantification of Shigella IcsA required for bacterial actin polymerizationCYTOSKELETON, Issue 4 2002Juana Magdalena Abstract Shigella move through the cytoplasm of host cells by active polymerization of host actin to form an "actin tail." Actin tail assembly is mediated by the Shigella protein IcsA. The process of Shigella actin assembly has been studied extensively using IcsA-expressing Escherichia coli in cytoplasmic extracts of Xenopus eggs. However, for reasons that have been unclear, wild type Shigella does not assemble actin in these extracts. We show that the defect in actin assembly in Xenopus extracts by Shigella can be rescued by increasing IcsA expression by approximately 3-fold. We calculate that the number of IcsA molecules required on an individual bacterium to assemble actin filaments in extracts is approximately 1,500,2,100 molecules, and the number of IcsA molecules required to assemble an actin tail is approximately 4,000 molecules. The majority of wild type Shigella do not express these levels of IcsA when grown in vitro. However, in infected host cells, IcsA expression is increased 3.2-fold, such that the number of IcsA molecules on a significant percentage of intracellular wild type Shigella would exceed that required for actin assembly in extracts. Thus, the number of IcsA molecules estimated from our studies in extracts as being required on an individual bacterium to assemble actin filaments or an actin tail is a reasonable prediction of the numbers required for these functions in Shigella -infected cells. Cell Motil. Cytoskeleton 51:187,196, 2002. © 2002 Wiley-Liss, Inc. [source] Remodeling of the actin cytoskeleton of target hepatocytes and NK cells during induction of apoptosisCYTOSKELETON, Issue 2 2001W. Marty Blom Abstract Natural Killer cells are immune cells that recognize and eliminate altered and non-self cells from the circulation. To study the interaction between NK cells and target cells, we set up an experimental system consisting of rat Interleukin-2 activated Natural Killer cells (A-NK cells) and rat hepatocytes with a masked Major Histocompatibility Complex (MHC). The masking of the MHC induces recognition of the hepatocytes by the NK cells as non-self. We showed that in vitro apoptosis is rapidly induced in the hepatocytes [Blom et al., 1999] after co-incubation with A-NK cells. Now we describe the morphological changes that occur during and after interaction of A-NK cells with hepatocytes. Confocal laser scanning microscopy showed that the actin cytoskeleton of the NK cells was remodeled during attack of hepatocytes. Some NK cells were in close contact with the hepatocytes while others had formed actin-containing dendrites of varying length that made contact with the hepatocytes. However, dendrite formation is not obligatory for induction of apoptosis because cells that were unable to form these did induce FAS-dependent apoptosis in hepatocytes. Apparently both direct as well as distant contact resulted in apoptosis. Formation of the dendrites was calcium-dependent as EGTA largely prevented it. Importantly, chelation of the calcium also suppressed killing of the hepatocytes. Within 1 h after addition of the A-NK cells, morphological changes in hepatocytes that are characteristic of apoptosis, such as the formation of apoptotic bodies and fragmented nuclei, became apparent. Specifically, the actin cytoskeleton of the hepatocytes was remodeled resulting in the formation of the apoptotic bodies. Inhibition of caspase activity by z-Val-Ala-DL-Asp-fluoromethylketone (100 ,M) partly protected against the rearrangement of the actin filaments in the hepatocytes. Cell Motil. Cytoskeleton 49:78,92, 2001. © 2001 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||||||||