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  • Whitley Vistisen posted an update 4 months, 1 week ago

    To examine whether ROCK mediates LIMK activation in response to TGF, we performed in vitro kinase assays incubating LIMK immune complexes from cells pretreated with Y and then stimulated with TGF for various time points. . LIMK is required for actin reorganization induced by TGF.Infecting serumstarved cells with caALK adenovirus led to a robust actin reorganization that corresponded to the response seen after long treatment with TGF, because the exogenous receptor was active in the cells for over h.The present study has the unique feature of focusing on a fibroblastic model, instead of the more extensively studied epithelial cell types, such as mammary or prostate epithelial cells.Cells were fixed and stained with rhodamine phalloidin.Panels indicate serial optical sections from the top of the cell.B, cells were serumstarved for h, pretreated with M Y, then stimulated with ngml TGF for the indicated time points, and antiLIMK immune complexes were assayed for kinase activity in vitro.This study also demonstrates that two different mouse fibroblastic cell lines exhibit comparable cytoskeletal responses to TGF. When the complete signaling cascades regulating actin reorganization downstream of TGF become fully understood in several cell types, it will be of interest to compare the various effectors involved, as the current evidence suggests that cell type specificity is a prevalent factor that influences the cytoskeletal response.The role of the type I receptor was clearly demonstrated by ectopically expressing mutant receptors that mimic physiological activation by TGF. In all experiments, such a constitutively active receptor was always as efficient in eliciting actin reorganization events as TGF itself.This is in agreement with previous observations made in mammary epithelial cells. Cells were serumstarved for hand stimulated with ngml TGF for min.Immunoblots of the GSTRBD pulldown and tubulin antibody are shown.However, this response usually takes several hours and sometimes several days postTGF treatment to be fully elicited.Our current preliminary work suggests that this may be due to cell type specificity.The end point of the signaling cascade analyzed here is phosphorylation of cofilin by LIMK, which inhibits its function as a constitutive actindepolymerizing factor. However, despite the central role of cofilin in modulation of actin dynamics, the complexity of regulation of actin microfilament assembly by large numbers of signaling and regulatory proteins might suggest that additional factors could possibly contribute to the same phenotypic change of the cytoskeleton.This question can only be addressed by systematic screens for all actinregulatory factors.Thus, our study offers a deeper insight into the alternative signaling pathways initiated by the multifunctional polypeptide factor TGF aiming at the regulation of critical physiological responses such as actin cytoskeleton polymerization.jbc.M When a correction for this article is posted Click here to choose from all of JBCs email alerts This article cites references, of which can be accessed free at http:www.jbc.orgcontent.full.htmlreflist Downloadedfromhttp: http://www.jbc.orgbyguestonAugust, By fitting the Rapamycin fluorescence resonance energy transfer data to an isothermal binding equation, we observed a specific coclustering of raftassociated donor and acceptor probes that was sensitive to latrunculin B, which disrupts the actin cytoskeleton.Current models define cell membranes to be structurally heterogeneous in nature, composed of discrete domains with unique physical and biological properties.