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Cytoskeletal Organization (cytoskeletal + organization)
Selected AbstractsThe heel and toe of the cell's foot: A multifaceted approach for understanding the structure and dynamics of focal adhesionsCYTOSKELETON, Issue 11 2009Haguy Wolfenson Abstract Focal adhesions (FAs) are large clusters of transmembrane receptors of the integrin family and a multitude of associated cytoplasmic "plaque" proteins, which connect the extracellular matrix-bound receptors with the actin cytoskeleton. The formation of nearly stationary FAs defines a boundary between the dense and highly dynamic actin network in lamellipodium and the sparser and more diverse cytoskeletal organization in the lamella proper, creating a template for the organization of the entire actin network. The major "mechanical" and "sensory" functions of FAs; namely, the nucleation and regulation of the contractile, myosin-II-containing stress fibers and the mechanosensing of external surfaces depend, to a major extent, on the dynamics of molecular components within FAs. A central element in FA regulation concerns the positive feedback loop, based on the most intriguing feature of FAs; that is, their dependence on mechanical tension developing by the growing stress fibers. FAs grow in response to such tension, and rapidly disassemble upon its relaxation. In this article, we address the mechanistic relationships between the process of FA development, maturation and dissociation and the dynamic molecular events, which take place in different regions of the FA, primarily in the distal end of this structure (the "toe") and the proximal "heel," and discuss the central role of local mechanical forces in orchestrating the complex interplay between FAs and the actin system. Cell Motil. Cytoskeleton 66: 1017,1029, 2009. © 2009 Wiley-Liss, Inc. [source] A decade of site- and phosphorylation state-specific antibodies: recent advances in studies of spatiotemporal protein phosphorylationGENES TO CELLS, Issue 8 2001Koh-ichi Nagata From 1990 to 2001, numerous site- and phosphorylation state-specific antibodies have been developed and many are now commercially available. These antibodies have facilitated understanding of the cytoskeletal organization, signal transduction and transcriptional mechanisms as well as clinical diseases. This review is an attempt to cover all these aspects. [source] ,IV tubulin is selectively expressed by oligodendrocytes in the central nervous systemGLIA, Issue 3 2005Nobuo Terada Abstract Oligodendrocyte differentiation and myelination involve dramatic changes in cell signaling pathways, gene expression patterns, cell shape, and cytoskeletal organization. In a pilot study investigating CNS angiogenesis, oligodendrocytes were intensely labeled by antisera directed against the C-terminal of Tie-2, a 140-kDa transmembrane receptor for angiopoietin. Immunoprecipitation of rat brain proteins with Tie-2 C-terminal antisera, however, produced a single spot of ,55-kDa pI ,5 by two-dimensional (2D) electrophoresis, which was identified as ,-tubulin by mass spectrometry. Isotype-specific antibodies for ,IV tubulin selectively labeled oligodendrocytes. First detected in premyelinating oligodendrocytes, ,IV tubulin was abundant in myelinating oligodendrocyte perinuclear cytoplasm and processes extending to and along developing myelin internodes. ,IV tubulin-positive MTs were diffusely distributed in oligodendrocyte perinuclear cytoplasm and not organized around the centrosome. ,IV tubulin may play a role in establishing the oligodendrocyte MT network, which is essential for the transport of myelin proteins, lipids, and RNA during myelination. © 2005 Wiley-Liss, Inc. [source] Annexin A1 subcellular expression in laryngeal squamous cell carcinomaHISTOPATHOLOGY, Issue 6 2008V A F Alves Aims:, Annexin A1 (ANXA1) is a soluble cytoplasmic protein, moving to membranes when calcium levels are elevated. ANXA1 has also been shown to move to the nucleus or outside the cells, depending on tyrosine-kinase signalling, thus interfering in cytoskeletal organization and cell differentiation, mostly in inflammatory and neoplastic processes. The aim was to investigate subcellular patterns of immunohistochemical expression of ANXA1 in neoplastic and non-neoplastic samples from patients with laryngeal squamous cell carcinomas (LSCC), to elucidate the role of ANXA1 in laryngeal carcinogenesis. Methods and results:, Serial analysis of gene expression experiments detected reduced expression of ANXA1 gene in LSCC compared with the corresponding non-neoplastic margins. Quantitative polymerase chain reaction confirmed ANXA1 low expression in 15 LSCC and eight matched normal samples. Thus, we investigated subcellular patterns of immunohistochemical expression of ANXA1 in 241 paraffin-embedded samples from 95 patients with LSCC. The results showed ANXA1 down-regulation in dysplastic, tumourous and metastatic lesions and provided evidence for the progressive migration of ANXA1 from the nucleus towards the membrane during laryngeal tumorigenesis. Conclusions:, ANXA1 dysregulation was observed early in laryngeal carcinogenesis, in intra-epithelial neoplasms; it was not found related to prognostic parameters, such as nodal metastases. [source] Disease Status in Autosomal Dominant Osteopetrosis Type 2 Is Determined by Osteoclastic Properties,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2006Kang Chu Abstract Asymptomatic gene carriers and clinically affected ADO2 subjects have the same ClCN7 mutation. We examined osteoclastic bone resorption in vitro as well as osteoclast formation, several markers, acid secretion, and cytoskeletal structure. We found that ADO2 expression results from osteoclast specific properties. Introduction: Autosomal dominant osteopetrosis type II (ADO2) is a heritable osteosclerotic disorder that results from heterozygous mutations in the ClCN7 gene. However, of those individuals with a ClCN7 mutation, one third are asymptomatic gene carriers who have no clinical, biochemical, or radiological manifestations. Disease severity in the remaining two thirds is highly variable. Materials and Methods: Human peripheral blood mononuclear cells were isolated and differentiated into osteoclasts by stimulation with hRANKL and human macrophage-colony stimulating factor (hM-CSF). Study subjects were clinically affected subjects, unaffected gene carriers, and normal controls (n = 6 in each group). Pit formation, TRACP staining, RANKL dose response, osteoclast markers, acid secretion, F-actin ring, and integrin ,v,3 expression and co-localization were studied. Results: Osteoclasts from clinically affected subjects had severely attenuated bone resorption compared with those from normal controls. However, osteoclasts from unaffected gene carriers displayed similar bone resorption to those from normal controls. In addition, the resorption lacunae from both unaffected gene carriers and normal controls appeared much earlier and spread much more rapidly than those from clinically affected subjects. As time progressed, the distinction between clinically affected subjects and the other two groups increased. No significant difference was found in acidic secretion or osteoclast formation between the three groups. Osteoclast cytoskeletal organization showed no difference between the three groups but there was low cellular motility in clinically affected subjects. Conclusions: Osteoclasts from the unaffected gene carriers, in contrast to those from the clinically affected subjects, functioned normally in cell culture. This finding supports the hypothesis that intrinsic osteoclast factors determine disease expression in ADO2. Further understanding of this mechanism is likely to lead to the development of new approaches to the treatment of clinically affected patients. [source] Embryonic undifferentiated cells show scattering activity on a surface coated with immobilized E-cadherinJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2008Masato Nagaoka Abstract Rearrangement of cell,cell adhesion is a critical event in embryonic development and tissue formation. We investigated the regulatory function of E-cadherin, a key adhesion protein, in the developmental process by using E-cadherin/IgG Fc fusion protein as an adhesion matrix in cell culture. F9 embryonal carcinoma cells usually form colonies when cultured on gelatin or fibronectin matrices. However, F9 cells cultured on the E-cadherin/IgG Fc fusion protein matrix formed a scattered distribution, with a different cytoskeletal organization and E-cadherin-rich protrusions that were regulated by Rac1 activity. The same scattering activity was observed in P19 embryonal carcinoma cells. In contrast, three types of differentiated cells, NMuMG mammary gland cells, MDCK kidney epithelial cells, and mouse primary isolated hepatocytes, did not show the scattering activity observed in F9 and P19 cells. These results suggest that migratory behavior on an E-cadherin-immobilized surface is only observed in embryonic cells, and that the regulatory mechanisms underlying E-cadherin-mediated cell adhesion vary with the state of differentiation. J. Cell. Biochem. 103: 296,310, 2008. © 2007 Wiley-Liss, Inc. [source] Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004L. Formigli We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca2+ mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca2+ -independent mechanisms of cell contraction have been the focus of numerous studies on Ca2+ sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca2+ -independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca2+, by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca2+ transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca2+ and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKC, and PKC,, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca2+ -independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction. J. Cell. Physiol. 198: 1,11, 2004© 2003 Wiley-Liss, Inc. [source] Developmental roles for Homer: more than just a pretty scaffoldJOURNAL OF NEUROCHEMISTRY, Issue 1 2009Lisa Foa Abstract Homer proteins are best known as scaffold proteins at the post-synaptic density where they facilitate synaptic signalling and are thought to be required for learning and memory. Evidence implicating Homer proteins in the development of the nervous system is also steadily accumulating. Homer is highly conserved and is expressed at key developmental time points in the nervous system of several species. Homer regulates intracellular calcium homeostasis, clustering and trafficking of receptors and proteins at the cytosolic surface of the plasma membrane, transcription and translation, and cytoskeletal organization. Each of these functions has obvious potential to regulate neuronal development, and indeed Homer is implicated in several pathologies associated with the developing nervous system. Current data justify more critical experimental approaches to the role of Homer in the developing nervous system and related neurological disorders. [source] Emerging functions of the calpain superfamily of cysteine proteases in neuroendocrine secretory pathwaysJOURNAL OF NEUROCHEMISTRY, Issue 3 2007Joanne S. Evans Abstract The first calpain protease was discovered over 40 years ago now, yet despite the vast amount of literature that has subsequently emerged detailing their involvement in the pathophysiology of a variety of human diseases, it is only in the last decade that calpain-mediated actions along the secretory pathway have begun to emerge. However, the number of secretory pathway substrates identified and their diversity of function continues to grow. This review summarizes our current knowledge of calpain-mediated mechanisms of action that are pertinent to synaptic vesicle assembly and budding, cytoskeletal organization, endosomal recycling, and exocytotic membrane fusion. [source] Characterization of fibronectin assembly by platelets adherent to adsorbed laminin-111JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 5 2006J. CHO Summary.,Background: Various types of laminin (LN) are ubiquitous components of basement membrane and exposed to blood upon localized damage of vascular endothelial cells. Fibronectin is a plasma protein that is insolubilized into fibrils in a regulated fashion by, for example, lysophosphatidic acid (LPA)-stimulated fibroblasts or platelets spread on supportive adhesive ligands. Objective: To study assembly of plasma fibronectin by LPA-activated platelets adherent to LN-111 via ,6,1 integrin. Results: Platelets adherent to LN-111-bound plasma fibronectin or its N-terminal 70 kD fragment in fibrillar arrays at the periphery of spread platelets under static but not shear conditions. Formation of fibronectin arrays under static conditions was inhibited by co-incubation with the N-terminal 70 kD fragment or with a 49-amino acid peptide that binds to the N-terminal region of fibronectin. Approximately 7000 fibronectin dimers bound per adherent platelet with a Kd of 50 nm. Bound 70 kD fragment was readily solubilized with deoxycholate (DOC), whereas bound fibronectin became progressively insoluble. Bound 70 kD fragment became resistant to DOC extraction after treatment with a cell-impermeable, reducible crosslinker. Crosslinked 70 kD fragment was found in a high molecular weight complex. As with fibroblasts, signaling molecules modulating actin cytoskeletal organization controlled expression of binding sites for the N-terminal 70 kD region of fibronectin on adherent platelets. Conclusions: These results indicate that platelets adherent to LN-111 via ,6,1 support subsequent assembly of fibronectin, but possibly only under conditions of intermittent or stagnant blood flow. [source] CSRP2, TIMP-1, and SM22, promoter fragments direct hepatic stellate cell-specific transgene expression in vitro, but not in vivoLIVER INTERNATIONAL, Issue 1 2004Jens Herrmann Abstract: Background/Aims: The activation of hepatic stellate cells (HSC) and their transdifferentiation into myofibroblasts (MFB) is the key step for development of liver fibrosis. Over the past several years, significant progress has been made in the understanding of the critical pathways involved incells undergoing activation. Cellular activation in the course of transdifferentiation involves, among other biochemical modifications, functionally relevant changes in the control of gene expression. These include the up-regulation of transcription factors, different extracellular matrix proteins, cell adhesion molecules, smooth muscle specific genes, and proteins involved in matrix remodelling, or cytoskeletal organization. The corresponding regulatory elements of these genes have afforded us the opportunity to express transgenes with antifibrotic potential in a cell type- and/or transdifferentiation-dependent manner. Methods: In the present study, we have tested several promoters for their ability to mediate cell-specific expression, including those for CSRP2, SM22,, and TIMP-1 (CSRP2, gene encoding the LIM domain protein CRP2; SM22,, smooth muscle-specific gene encoding a 22-kDa protein; TIMP-1, gene encoding the tissue inhibitor of metalloproteinases-1), which in liver are specifically expressed in HSC or become strongly activated during the acute remodelling into MFB. We constructed adenoviral reporter vectors in which relevant portions of the promoters were fused to the green fluorescent protein. Results and Conclusion: Our experiments demonstrate that each of these promoters is sufficient to achieve strong or partially selective expression in vitro but none is able to direct a specific or inducible expression of transgenes in HSC/MFB in vivo. [source] Emerging mechanisms in morphogen-mediated axon guidanceBIOESSAYS, Issue 10 2009Cristina Sánchez-Camacho Abstract Early in animal development, gradients of secreted morphogenic molecules, such as Sonic hedgehog (Shh), Wnt and TGF,/Bmp family members, regulate cell proliferation and determine the fate and phenotype of the target cells by activating well-characterized signalling pathways, which ultimately control gene transcription. Shh, Wnt and TGF,/Bmp signalling also play an important and evolutionary conserved role in neural circuit assembly. They regulate neuronal polarization, axon and dendrite development and synaptogenesis, processes that require rapid and local changes in cytoskeletal organization and plasma membrane components. A key question then is whether morphogen signalling at the growth cone uses similar mechanisms and intracellular pathway components to those described for morphogen-mediated cell specification. This review discusses recent advances towards the understanding of this problem, showing how Shh, Wnt and TGF,/Bmp have adapted their ,classical' signalling pathways or adopted alternative and novel molecular mechanisms to influence different aspects of neuronal circuit formation. [source] Effect of spatial architecture on cellular colonizationBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Yan Huang Abstract The spatial cell-material interaction remains vital issue in forming biodegradable scaffolds in Tissue Engineering. In this study, to understand the influence of spatial architecture on cellular behavior, 2D and 3D chitosan scaffolds of 50,190 kD and >310 kD MW were synthesized through air drying and controlled rate freezing/lypohilization technique, respectively. In addition, chitosan was emulsified with 19, 76, and 160 kD 50:50 poly lactide-co-glycolide (PLGA) using 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) as stabilizer. 2D and 3D scaffolds were formed by air drying and lyophilization as before. Tensile and compressive properties of films and scaffolds were analyzed in wet conditions at 37°C. Alterations in the cell spreading, proliferation, and cytoskeletal organization of human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblasts (MEFs) were studied. These results showed that the formed 3D chitosan scaffolds had interconnected open pore architecture (50,200 µm size). HUVECs and MEFs had reduced spreading areas and circular morphology on 2D chitosan membranes compared with 3D chitosan scaffolds. The fluorescence photomicrographs for actin (using Alexa Fluor 488 phalloidin) and cytoplasm staining (using carboxyfluorescein diacetate-succinimidyl ester) demonstrated that the cells spread within 3D chitosan matrix. 2D and 3D emulsified chitosan and chitosan/PLGA scaffolds reduced the spreading of HUVECs and MEFs even further. Proliferation results, analyzed via MTT-Formazan assay and BrdU uptake assay, correlated with the spreading characteristics. The reductions in cell spreading area on emulsified surfaces were not detrimental to the viability and endocytic activity but to proliferation. The observed alterations in cellular colonization are in part due to the substrate stiffness and surface topography. In summary, these results suggest a significant influence of spatial architecture on cellular colonization. © 2005 Wiley Periodicals, Inc. [source] RhoC is essential for angiogenesis induced by hepatocellular carcinoma cells via regulation of endothelial cell organizationCANCER SCIENCE, Issue 10 2008Wei Wang The angiogenesis induced by tumor cells is essential for metastasis of hepatocellular carcinoma. Available information suggests that RhoC participates in angiogenesis through regulation of vascular endothelial growth factor expression in tumor cells. For its broad functions in cell migration and cytoskeletal organization, we hypothesized that RhoC regulating angiogenesis does not exclusively depend on regulation of vascular endothelial growth factor expression. To address this question, in the present study, we used a retroviral small interfering RNA approach to selectively knockdown the expression of RhoC in a neovascularization model in vivo and in vitro. Our present results indicate that RhoC is the downstream regulator of vascular endothelial growth factor in endothelial cells and is essential for angiogenesis induced by vascular endothelial growth factor, notwithstanding that RhoC regulates the expression of vascular endothelial growth factor in tumor cells. Furthermore, we show that knockdown of RhoC is associated with the inhibition of invasion and migration but not apoptosis of endothelial cells. Knockdown of RhoC results in inhibition of endothelial cell organization through restraining the reorganization of F-actin filaments, which represses endothelial cell network and sprout formation. In conclusion, our results demonstrate that knockdown of RhoC inhibits angiogenesis induced by tumor cells not only through effecting the release of vascular endothelial growth factor, but also through inhibiting endothelial cell migration and organization, which implies that it blocks tumor metastasis by specifically inhibiting RhoC in endothelial cells. (Cancer Sci 2008; 99: 2012,2018) [source] | |||||||||||||||||||||||||