In Rous sarcoma virus (RSV)-transformed baby hamster kidney (BHK) cells, invadopodia can self-organize into rings and belts, similarly to podosome distribution during osteoclast differentiation. The composition of individual invadopodia is spatiotemporally regulated and depends on invadopodia localization along the ring section: the actin core assembly precedes the recruitment of surrounding integrins and integrin-linked proteins, whereas the loss of the actin core was a prerequisite to invadopodia disassembly. We have shown that invadopodia ring expansion is controlled by paxillin phosphorylations on tyrosine 31 and 118, which allows invadopodia disassembly. In BHK-RSV cells, ectopic expression of the paxillin mutant Y31F-Y118F induces a delay in invadopodia disassembly and impairs their self-organization. A similar mechanism is unraveled in osteoclasts by using paxillin knockdown. Lack of paxillin phosphorylation, calpain or extracellular signal-regulated kinase inhibition, resulted in similar phenotype, suggesting that these proteins belong to the same regulatory pathways. Indeed, we have shown that paxillin phosphorylation promotes Erk activation that in turn activates calpain. Finally, we observed that invadopodia/podosomes ring expansion is required for efficient extracellular matrix degradation both in BHK-RSV cells and primary osteoclasts, and for transmigration through a cell monolayer.
Cells from the myeloid lineage, namely macrophages, dendritic cells and osteoclasts develop podosomes instead of stress fibers and focal adhesions to adhere and migrate.Podosomes share many components with focal adhesions but differ in their molecular organization, with a dense core of polymerized actin surrounded by scaffolding proteins, kinases and integrins. Podosomes are found either isolated both in macrophages and dendritic cells or arranged into superstructures in osteoclasts. When osteoclasts resorb bone, they form an F-actin rich sealing zone, which is a dense array of connected podosomes that firmly anchors osteoclasts to bone. It delineates a compartment in which protons and proteases are secreted to dissolve and degrade the mineralized matrix. Since Rho GTPases have been shown to control F-actin stress fibers and focal adhesions in mesenchymal cells, the question of whether they could also control podosome formation and arrangement in cells from the myeloid lineage, and particularly in osteoclasts, rapidly emerged. This article considers recent advances made in our understanding of podosome arrangements in osteoclasts and how Rho GTPases may control it.
Organization of actin filaments is controlled by the Rho family of small GTPases, Rho, Rac, and Cdc42 (1). Among them, Rho regulates the assembly of actin stress fibers and focal contacts, through activation of the downstream effectors mDia and the closely related kinases ROCKI and ROCKII (2, 3). Activation of ROCK by Rho is implicated in stress fiber and focal contact formation (2, 4). ROCK increases the phosphorylation of myosin light chains by directly phosphorylating myosin light chains and negatively regulating myosin phosphatase and thus increases actomyosin-based contractility (5, 6). The resulting contractile forces are thought to contribute to the formation of stress fibers and focal contacts (7,8). ROCK also activates LIMK, 1 which subsequently phosphorylates cofilin and thereby inhibits its actin-depolymerizing activity, thus contributing to actin stress fiber stabilization (9 -11).In cells transformed by the product of the Rous sarcoma virus, the v-Src tyrosine kinase, the shape and cytoskeletal architecture are dramatically altered, with a disruption of actin stress fibers and focal contacts and the formation of dot-like actin-associated adhesions called podosomes. This disruption of the cytoskeleton is believed to contribute to several aspects of the transformed phenotype, including adhesion-independent cell growth and increased migration abilities (12). However, the mechanism by which v-Src disrupts actin stress fibers and focal contacts is not clear. Stimulation of a Rho-GAP (GTPase-activating protein) activity was reported upon v-Src activation in chicken embryo fibroblasts (13), suggesting that v-Src-induced cytoskeleton disruption is linked to inhibition of Rho activity. In addition, an active mutant form of Rho was shown to restore stress fibers and adhesion plaques in v-Src-transformed fibroblasts (14). Despite these experiments, the relationship between Rho-mediated stress fiber assembly and v-Src-induced morphological transformation, as well as the signaling pathways mediating v-Src effects on the cytoskeleton, have not been clearly identified. Expression of v-Src results in tyrosine phosphorylation of numerous substrates and simultaneous activation of several signaling pathways, including the Ras/MEK and PI3K pathways (15). Both pathways have been implicated in cytoskeleton regulation in normal cells. Activation of PI3K is necessary for Rac-and Rho-mediated rearrangements of the actin cytoskeleton induced by platelet-derived growth factor and other growth factors (16,17). ERK activity has been implicated in cell migration and was shown to directly phosphorylate myosin light chain kinase, suggesting a role for ERK in the regulation of cytoskeleton and focal contact dynamics (18,19). In v-Srctransformed chicken embryo fibroblasts, both ERK and PI3K are translocated to focal adhesions upon v-Src activation (20,21), raising the possibility that both pathways participate in cellular adhesion dynamic during transformation.We report here that activation of MEK, but not PI3K, is required for the ...
Previous studies of transformed rodent fibroblasts have suggested that specific isoforms of the actin-binding protein tropomyosin (TM) could function as suppressors of transformation, but an analysis of TM expression in patient tumor tissue is limited. The purpose of our study was to characterize expression of the different TM isoforms in human transitional cell carcinoma of the urinary bladder by immunohistochemistry and Western blot analysis. We found that TM1 and TM2 protein levels were markedly reduced and showed >60% reduction in 61% and 55% of tumor samples, respectively. TM5, which was expressed at very low levels in normal bladder mucosa, exhibited aberrant expression in 91% of tumor specimens. The Western blot findings were confirmed by immunohistochemical analysis in a number of tumors. We then investigated the mechanism underlying TM expression deregulation, in the T24 human bladder cancer cell line. We showed that levels of TM1, TM2 and TM3 are reduced in T24 cells, but significantly upregulated by inhibition of the mitogen-activated protein kinase-signaling pathway. In addition, inhibition of this pathway was accompanied by restoration of stress fibers. Overall, changes in TM expression levels seem to be an early event during bladder carcinogenesis. We conclude that alterations in TM isoform expression may provide further insight into malignant transformation in transitional cell carcinomas of the bladder and may be a useful target for early detection strategies. © 2004 Wiley-Liss, Inc. Key words: tropomyosin; cytoskeleton; bladder neoplasmsTransitional cell carcinoma (TCC) is the most common cancer type of the urinary bladder, representing approximately 90% of all cases. More than 57,000 people are diagnosed with TCC of the urinary bladder each year in the United States alone, and 12,500 of these patients are expected to die from the cancer. 1 Cytoscopy is the most valuable method to detect and monitor TCC, but it remains difficult to predict tumor progression, optimal therapy and finally clinical outcome. 2,3 There is a clear need for developing biomarkers for bladder cancer management.Alterations of the actin-based cytoskeleton are an established part of the neoplastic phenotype, and it is now demonstrated that such alteration is not a byproduct of cellular transformation but contributes to malignant transformation. 4,5 These alterations in actin remodeling are associated with downregulation of numerous actin-binding proteins and cell adhesion molecules, which are therefore candidate markers for early cancer detection and prognostic indication. 6 Tropomyosins (TMs) are a family of actin-binding proteins essential for the integrity of actin filaments. 7 Although TMs have been known to function in regulation of muscle contraction, the functional significance of the multiple TM isoforms present in non-muscle cells has remained largely unclear. Several studies have suggested that specific isoforms of TMs may possess tumor suppressor activity. Consistent with this hypothesis it was shown that (i) expr...
Transformation by oncogenic Ras profoundly alters actin cytoskeleton organization. We investigated Ras-dependent signaling pathways involved in cytoskeleton disruption by transfecting normal rat kidney (NRK) cells with different Ras mutants. RasV12S35, a mutant known to activate specifically the Raf/MAPK pathway, led to stress fiber and focal contact disruption, whereas the adherens junctions remained intact. Next, we found that pharmacological inhibition of MEK was sufficient to restore the cytoskeletal defects of ras-transformed NRK cells, including assembly of stress fibers and focal contacts, but it did not induce reorganization of the cell-cell junctions. Investigating the mechanism underlying this phenotypic reversion, we found that the sustained MAPK signaling resulting from Ras-transformation down-regulated the expression of ROCKI and Rho-kinase, two-Rho effectors required for stress fiber formation, at the post-transcriptional level. On MEK inhibition, ROCKI/Rho-kinase expression and cofilin phosphorylation were increased, demonstrating that the Rho-kinase/LIM-kinase/cofilin pathway was functionally restored. Finally, using dominant negative or constitutively active mutants, we demonstrated that expression of ROCKI/Rho-kinase was both necessary and sufficient to promote cytoskeleton reorganization in NRK/ras cells. These findings further establish the Ras/MAPK pathway as the critical pathway involved in cytoskeleton disruption during Ras-transformation, and they suggest a new mechanism, involving alteration in ROCKI/Rho-kinase expression, by which oncogenic Ras can specifically target the actin-based cytoskeleton and achieve morphological transformation of the cells.
Movement of individual cells and of cellular cohorts, chains or sheets requires physical forces that are established through interactions of cells with their environment. In vivo, migration occurs extensively during embryonic development and in adults during wound healing and tumorigenesis. In order to identify the molecular events involved in cell movement, in vitro systems have been developed. These have contributed to the definition of a number of molecular pathways put into play in the course of migratory behaviours, such as mesenchymal and amoeboid movement. More recently, our knowledge of migratory modes has been enriched by analyses of cells exploring and moving through three-dimensional (3D) matrices. While the cells' morphologies differ in 2D and 3D environments, the basic mechanisms that put a cellular body into motion are remarkably similar. Thus, in both 2D and 3D, the polarity of the migrating cell is initially defined by a specific subcellular localization of signalling molecules and components of molecular machines required for motion. While the polarization can be initiated either in response to extracellular signalling or be a chance occurrence, it is reinforced and sustained by positive feedback loops of signalling molecules. Second, adhesion to a substratum is necessary to generate forces that will propel the cell engaged in either mesenchymal or ameboid migration. For collective cell movement, intercellular coordination constitutes an additional requirement: a cell cohort remains stationary if individual cells pull in opposite directions. Finally, the availability of space to move into is a general requirement to set cells into motion. Lack of free space is probably the main obstacle for migration of most healthy cells in an adult multicellular organism. Thus, the requirements for cell movement are both intrinsic to the cell, involving coordinated signalling and interactions with molecular machines, and extrinsic, imposed by the physicochemical nature of the environment. In particular, the geometry and stiffness of the support act on a range of signalling pathways that induce specific cell migratory responses. These issues are discussed in the present review in the context of published work and our own data on collective migration of hepatocyte cohorts.
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