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Actin Stress Fibers (actin + stress_fiber)
Selected AbstractsActin stress fiber pre-extension in human aortic endothelial cellsCYTOSKELETON, Issue 4 2008Lan Lu Abstract Actin stress fibers (SFs) enable cells to sense and respond to mechanical stimuli and affect adhesion, motility and apoptosis. We and others have demonstrated that cultured human aortic endothelial cells (HAECs) are internally stressed so that SFs are pre-extended beyond their unloaded lengths. The present study explores factors affecting SF pre-extension. In HAECs cultured overnight the baseline pre-extension was 1.10 and independent of the amount of cell shortening. Decreasing contractility with 30 mM BDM or 10 ,M blebbistatin decreased pre-extension to 1.05 whereas increasing contractility with 2 nM calyculin A increased pre-extension to 1.26. Knockdown of ,-actinin-1 with an interfering RNA increased pre-extension to 1.28. None of these affected the wavelength of the buckled SFs. Pre-extension was the same in unperturbed cells as in those in which the actin cytoskeleton was disrupted by both chemical and mechanical means and then allowed to reassemble. Finally, disrupting MTs or IFs did not affect pre-extension but increased the wavelength. Taken together, these results suggest that pre-extension of SFs is determined primarily by intrinsic factors, i.e. the level of actin-myosin interaction. This intrinsic control of pre-extension is sufficiently robust that pre-extension is the same even after the actin cytoskeleton has been disrupted and reorganized. Unlike pre-extension, the morphology of the compressed SFs is partially determined by MTs and IFs which appear to support the SFs along their lengths. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Opposite effects of overexpressed myosin Va or heavy meromyosin Va on vesicle distribution, cytoskeleton organization, and cell motility in nonmuscle cellsCYTOSKELETON, Issue 3 2008Robbin D. Eppinga Abstract Myosin Va, an actin-based motor protein that transports intracellular cargos, can bundle actin in vitro. Whether myosin Va regulates cellular actin dynamics or cell migration remains unclear. To address this, we compared Chinese Hamster Ovary (CHO) cells that stably express GFP fused to either full length mouse myosin Va (GFP-M5) or heavy meromyosin Va (GFP-M5,). GFP-M5 and GFP-M5, co-immunoprecipitate with CHO myosin Va and serve as overexpression of wild-type and dominant negative mutants of myosin Va. Compared to non-expressing control cells, GFP-M5-overexpressing cells have peripheral endocytic vesicles, spread slowly after plating, as well as produce robust interior actin stress fibers, myosin II bundles, and focal adhesions. However, these cells display normal cell migration and lamellipodial dynamics. In contrast, GFP-M5,-expressing cells have perinuclear endocytic vesicles, produce thin interior actin and myosin bundles and contain no interior focal adhesions. In addition, these cells spread rapidly, migrate slowly and display reduced lamellipodial dynamics. Similarly, neurite outgrowth is compromised in neurons cultured from transgenic Drosophila that express M5,-dsRed and in neurons cultured from Drosophila that produce a tailless version of endogenous myosin V. Together, these data suggest that myosin Va overexpression induces actin bundles in vivo whereas the tailless version fails to bundle actin and disrupts cell motility. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [source] Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesionCYTOSKELETON, Issue 1 2005Tony Yeung Abstract The morphology and cytoskeletal structure of fibroblasts, endothelial cells, and neutrophils are documented for cells cultured on surfaces with stiffness ranging from 2 to 55,000 Pa that have been laminated with fibronectin or collagen as adhesive ligand. When grown in sparse culture with no cell-cell contacts, fibroblasts and endothelial cells show an abrupt change in spread area that occurs at a stiffness range around 3,000 Pa. No actin stress fibers are seen in fibroblasts on soft surfaces, and the appearance of stress fibers is abrupt and complete at a stiffness range coincident with that at which they spread. Upregulation of ,5 integrin also occurs in the same stiffness range, but exogenous expression of ,5 integrin is not sufficient to cause cell spreading on soft surfaces. Neutrophils, in contrast, show no dependence of either resting shape or ability to spread after activation when cultured on surfaces as soft as 2 Pa compared to glass. The shape and cytoskeletal differences evident in single cells on soft compared to hard substrates are eliminated when fibroblasts or endothelial cells make cell-cell contact. These results support the hypothesis that mechanical factors impact different cell types in fundamentally different ways, and can trigger specific changes similar to those stimulated by soluble ligands. Cell Motil. Cytoskeleton 60:24,34, 2005. © 2004 Wiley-Liss, Inc. [source] Dominant-negative Rac increases both inherent and ionizing radiation-induced cell migration in C6 rat glioma cellsINTERNATIONAL JOURNAL OF CANCER, Issue 8 2006So-Young Hwang Abstract Rho-like GTPases, including Cdc42, Rac1 and RhoA, regulate distinct actin cytoskeleton changes required for cell adhesion, migration and invasion. In the present study, we examined the role of Rac signaling in inherent migration, as well as radiation-induced migration, of rat glioma cells. Stable overexpression of dominant-negative Rac1N17 in a C6 rat glioma cell line (C6-RacN17) promoted cell migration, and ionizing radiation further increased this migration. Migration was accompanied by decreased expression of the focal adhesion molecules FAK and paxillin. Focal contacts and actin stress fibers were also reduced in C6-RacN17 cells. Downstream effectors of Rac include JNK and p38 MAP kinases. Irradiation transiently activated p38, JNK and ERK1/2 MAP kinases in C6-RacN17 cells, while p38 and JNK were constitutively activated in C6 control cells. Blocking JNK activity with JNK inhibitor SP600125 inhibited migration, suggesting that the JNK pathway may regulate radiation-induced, as well as inherent, migration of C6-RacN17 cells. Additionally, the radiation-induced migration increase was also inhibited by SB203580, a specific inhibitor of p38 MAP kinase. However, PD98059, a MEK kinase 1 inhibitor, failed to influence migration. This is the first evidence that suppression of Rac signaling may be involved in invasion or metastasis of glioma cells before and/or after radiotherapy. These data further suggest that radiotherapy for malignant glioma needs to be used with caution because of the potential for therapy-induced cell migration or invasion and that pharmacological inhibition of cell migration and invasion through targeting the Rac signaling pathway may represent a new approach for improving the therapeutic efficacy of radiotherapy for malignant glioma. © 2005 Wiley-Liss, Inc. [source] Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathwaysJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2007M. HASMIM Summary.,Background: Recent evidence indicates that zoledronate, a nitrogen-containing bisphosphonate used to treat conditions of increased bone resorption, may have anti-angiogenic activity. The endothelial cells signaling events modulated by zoledronate remain largely elusive. Objectives: The aim of this work was to identify signaling events suppressed by zoledronate in endothelial cells and responsible for some of its biological effects. Methods: Human umbilical vein endothelial cells (HUVEC) were exposed to zoledronate, isoprenoid analogs (i.e. farnesol and geranylgeraniol) and various inhibitors of signaling, and the effect on adhesion, survival, migration, actin cytoskeleton and signaling events characterized. Results: Zoledronate reduced Ras prenylation, Ras and RhoA translocation to the membrane, and sustained ERK1/2 phosphorylation and tumor necrosis factor (TNF) induced JNK phosphorylation. Isoprenoid analogs attenuated zoledronate effects on HUVEC adhesion, actin stress fibers and focal adhesions, migration and survival. Isoprenoid analogs also restored Ras prenylation, RhoA translocation to the membrane, sustained FAK and ERK1/2 phosphorylation and prevented suppression of protein kinase B (PKB) and JNK phosphorylation in HUVEC exposed to TNF in the presence of zoledronate. Pharmacological inhibition of Rock, a RhoA target mediating actin fiber formation, phosphatidylinositol 3-kinase, an activator of PKB, MEK1/2, an activator of ERK1/2, and JNK, recapitulated individual zoledronate effects, consistent with the involvement of these molecules and pathways and their inhibition in the zoledronate effects. Conclusions: This work has demonstrated that zoledronate inhibits HUVEC adhesion, survival, migration and actin stress fiber formation by interfering with protein prenylation and has identified ERK1/2, JNK, Rock, FAK and PKB as kinases affected by zoledronate in a prenylation-dependent manner. [source] Extracellular and intracellular mechanisms that mediate the metastatic activity of exogenous osteopontinCANCER, Issue 8 2009Jami Mandelin PhD Abstract BACKGROUND: Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. In this study, the authors used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS: Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo that were designed to test the extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS: In the extracellular environment, the results confirmed that soluble osteopontin is required for its prometastatic effects; this phenomenon is specific, arginine-glycine-aspartic acid (RGD)-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induced rapid tyrosine 418 (Tyr-418) dephosphorylation of the cellular homolog of the Rous sarcoma virus (c-Src), with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation was followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSIONS: The results of this study revealed a complex molecular interaction as well as a dual role for osteopontin in metastasis that depends on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to antimetastasis strategies. Cancer 2009. © 2009 American Cancer Society. [source] | ||||||||||