1993;268:15958C15964. fibronectin, whereas cells expressing a GFP-fascin S39D mutant with constitutive bad charge spread more extensively than wild-type cells. In contrast, C2C12 cells coexpressing S39A fascin with endogenous fascin remained competent to form microspikes on thrombospondin-1, and cells that indicated fascin S39D attached to thrombospondin-1 but did not form microspikes. Blockade of PKC activity by TPA-induced down-regulation led to actin association of wild-type fascin in fibronectin-adherent C2C12 and LLC-PK1 cells but did not alter the distribution of S39A or S39D fascins. The association of fascin with actin in fibronectin-adherent cells was also obvious in the presence of an inhibitory antibody to integrin 5 subunit. These novel results set up matrix-initiated PKC-dependent rules of fascin phosphorylation at serine 39 like a mechanism whereby matrix adhesion is definitely coupled to the organization of cytoskeletal structure. Intro Cell adhesion to extracellular matrix macromolecules is definitely mediated by specific Croverin cell surface receptors, of which integrins and proteoglycans form major family members (examined by Hynes, 1987 , 1992 ; Ruoslahti, 1988 , 1989 ; Hardingham and Fosang, 1992 ). Relationships with individual matrix components lead to distinct outcomes in terms of subsequent cell behavior (examined by Adams and Watt, 1993 ). In cell types for which this phenomenon has been analyzed in depth, the association of individual integrins with cytoplasmic adaptor molecules has been demonstrated to provide linkage to specific intracellular signaling pathways (Wary [Palo Alto, CA] and Perkin Elmer-Cetus [Norwalk, CT]; detection on Hyperfilm ECL [Amersham, Arlington Heights, IL]). Cell Adhesion Assays and Croverin Immunofluorescence Cell adhesion assays were carried out as explained (Adams, 1995 ) for 1 h at 37C. Some experiments involved a altered protocol in which cells were treated with pharmacological inhibitors or activators of PKC, either before and during the adhesion assay or after cells experienced adhered to a specific matrix for 45 min. In pilot experiments, these inhibitors were tested at a range of concentrations for his or her effects on cell adhesion or cell viability. The concentrations used in the main experiments were 50 nM TPA, 100 nM calphostin C, 320 nM chelerythrine chloride, and 80 M myristoylated PKC peptide inhibitor. These ideals represent the lowest concentrations needed to accomplish clear effects on cell adhesion. Down-regulation of PKC was achieved by 24-h treatment with 100 nM TPA (LLC-PK1 cells) or 24-h treatment with 500 nM TPA (C2C12 cells) and was Croverin confirmed on Western blots of whole cell components using rabbit antibody specific to Smoc1 PKC. In some assays, antibody 5H10-27 to mouse 5 integrin subunit was added at 5 g/ml at the start of the adhesion period. Adherent cells were quantified, fixed and processed for fascin immunofluorescence, and costained with TRITC-phalloidin or monoclonal VIN 11.5 to vinculin (Sigma Chemical) as explained (Adams, 1995 ). Staining with antibody to -actin was carried out on methanol-fixed cells and visualized as double staining with GFP-fascin. For staining with PKC antibodies, cells were fixed in 3.7% formyl saline Croverin and then permeabilized for 10 min with 0.2% Triton X-100 in PBS. Main antibodies were detected with the use of appropriate varieties- and class-specific TRITC- or FITC-conjugated secondary antibodies (ICN Biomedical, Costa Mesa, CA). RESULTS Fibronectin Adhesion and TPA Treatment Have Similar Effects on Fascin Localization in Diverse Cell Types We Croverin used C2C12 myoblasts to examine whether adhesion to fibronectin or TPA treatment would have comparative effects on fascin localization in one cell type. As shown for additional cell types (Adams,.