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Cell adhesion and spreading are regulated by complex interactions involving the cytoskeleton and extracellular matrix proteins. We examined the interaction of the intermediate filament protein vimentin with the actin cross-linking protein filamin A in regulation of spreading in HEK-293 and 3T3 cells. Filamin A and vimentin-expressing cells were well spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, cells treated with small interfering RNA (siRNA) to knock down filamin A or vimentin were poorly spread; both of these cell populations exhibited >50% reductions of cell adhesion, cell surface [31 integrin expression, and [beta]1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions, whereas knockdown of filamin and/or vimentin inhibited the formation of cell extensions. Reduced vimentin phosphorylation, cell spreading, and [beta]1 integrin surface expression, and activation were phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase C-[epsilon]. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins, we found an association between filamin A and vimentin. Filamin A also associated with protein kinase C-[epsilon], which was enriched in cell extensions. These data indicate that filamin A associates with vimentin and to protein kinase C-E, thereby enabling vimentin phosphorylation, which is important for [beta]1 integrin activation and cell spreading on collagen. actin; cell guidance; human embryonic kidney cells doi: 10.1152/ajpcell.00323.2009 more...

Cyclosporin A (CsA) inhibits collagen remodeling by interfering with the collagen-binding step of phagocytosis. In rapidly remodeling connective tissues such as human periodontium this interference manifests as marked tissue overgrowth and loss of function. Previous data have shown that CsA inhibits integrin-induced release of [Ca.sup.2+] from internal stores, which is required for the binding step of collagen phagocytosis. Because gelsolin is a [Ca.sup.2+]-dependent actin-severing protein that mediates collagen phagocytosis, we determined whether gelsolin is a CsA target. Compared with vehicle controls, CsA treatment of wild-type mice increased collagen accumulation by 60% in periodontal tissues; equivalent increases were seen in vehicle-treated gelsolin-null mice. Collagen degradation by phagocytosis in cultured gelsolin wild-type fibroblasts was blocked by CsA, comparable to levels of vehicletreated gelsolin-null fibroblasts. In wild-type cells treated with CsA, collagen binding was similar to that of gelsolin-null fibroblasts transfected with a gelsolin-severing mutant and treated with vehicle. CsA blocked collagen-induced [Ca.sup.2+] fluxes subjacent to bound collagen beads, gelsolin recruitment, and actin assembly at bead sites. CsA reduced gelsolin-dependent severing of actin in wild-type cells to levels similar to those in gelsolin-null fibroblasts. We conclude that CsA-induced accumulation of collagen in the extracellular matrix involves disruption of the actin-severing properties of gelsolin, thereby inhibiting the binding step of collagen phagocytosis. adhesion molecules; fibroblasts; knockout mice; actin more...

Many adult connective tissues undergo continuous remodeling to maintain matrix homeostasis. Physiological remodeling involves the degradation of collagen fibers by the intracellular cathepsin‐dependent phagocytic pathway. We considered that a multidomain, small GTPase activating protein, IQGAP1, which is involved in the generation of cell extensions, is required for collagen phagocytosis, possibly arising from its interactions with cdc42 and the actin‐binding protein Flightless I (FliI). We examined the role of IQGAP1 in collagen phagocytosis by human gingival fibroblasts (HGFs) and by IQGAP1+/+ and IQGAP1−/− mouse embryonic fibroblasts. IQGAP1 was strongly expressed by HGFs, localized to vinculin‐stained cell adhesions and sites where cell extensions are initiated, and colocalized with FliI. Immunoprecipitation showed that IQGAP1 associated with FliI. HGFs showed 10‐fold increases of collagen binding, 6‐fold higher internalization, and 3‐fold higher β1 integrin activation between 30 and 180 min after incubation with collagen. Compared with IQGAP1+/+ fibroblasts, deletion of IQGAP1 reduced collagen binding (1.4‐fold), collagen internalization (3‐fold), β1 integrin activation (2‐fold), and collagen degradation (1.8‐fold). We conclude that IQGAP1 affects collagen remodeling through its regulation of phagocytic degradation pathways, which may involve the interaction of IQGAP1 with FliI. [ABSTRACT FROM AUTHOR] more...

Biochemical and functional characterization of intercellular adhesion and gap junctions in fibroblasts. Am J Physiol Cell Physiol 279: C147-C157, 2000.--Despite their significance in wound healing, little is known about the molecular determinants of cell-to-cell adhesion and gap junctional communication in fibroblasts. We characterized intercellular adherens junctions and gap junctions in human gingival fibroblasts (HGFs) using a novel model. Calcein-labeled donor cells in suspension were added onto an established, Texas red dextran (10 kDa)-labeled acceptor cell monolayer. Cell-to-cell adhesion required [Ca.sup.2+] and was [is greater than]30-fold stronger than cell-to-fibronectin adhesion at 15 min. Electron micrographs showed rapid formation of adherens junction-like structures at ~15 min that matured by ~2-3 h; distinct gap junctional complexes were evident by ~3 h. Immunoblotting showed that HGF expressed [Beta]-catenin and that cadherins and connexin43 were recruited to the Triton-insoluble cytoskeletal fraction in confluent cultures. Confocal microscopy localized the same molecules to intercellular contacts of acceptor and donor cells. There was extensive calcein dye transfer in a cohort of Texas red dextran-labeled cells, but this was almost completely abolished by the gap junction inhibitor [Beta]-glycyrrhetinic acid and the connexin43 mimetic peptide GAP 27. This donor-acceptor cell model allows large numbers ([is greater than][10.sup.5]) of cells to form synchronous cell-to-cell contacts, thereby enabling the simultaneous functional and molecular studies of adherens junctions and gap junctions. adherens junctions; fluorescence dye; cell adhesion more...

Gelsolin, a Ca2+-regulated actin filament severing, capping, and nucleating protein, is an ubiquitous, multifunctional regulator of cell structure and metabolism. More recent data show that gelsolin can act as a transcriptional cofactor in signal transduction and its own expression and function can be influenced by epigenetic changes. Here, we review the functions of the plasma and cytoplasmic forms of gelsolin, and their manifold impacts on cancer, apoptosis, infection and inflammation, cardiac injury, pulmonary diseases, and aging. An improved understanding of the functions and regulatory mechanisms of gelsolin may lead to new considerations of this protein as a potential biomarker and/or therapeutic target. [ABSTRACT FROM AUTHOR] more...

The spreading and migration of cells on adhesive substrates is regulated by the counterbalance of contractile and protrusive forces. Non-muscle myosin IIA, an ubiquitously expressed contractile protein and enzyme, is implicated in the regulation of cell spreading and directional migration in response to various stimuli. Here we show that discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor activated by type I collagen, associates with the non-muscle myosin IIA heavy chain (NMHC-IIA) upon ligand stimulation. An association was also indicated by coimmunoprecipitation of NMHC-IIA with full-length DDR1, but not with the truncated DDR1d-isoform lacking the kinase domain. DDR1 was important for assembly of NMHC-IIA into filaments on cells plated on collagen. DDR1 expression inhibited cell spreading over collagen but promoted cell migration. By contrast, blockade of non-muscle myosin II activity by blebbistatin enhanced cell spreading but inhibited migration over collagen. We propose that myosin and DDR1 impact cell spreading and migration by regulating adhesive contacts with collagen. [ABSTRACT FROM AUTHOR] more...

Cortactin regulates the strength of nascent N-cadherin-mediated intercellular adhesions through a tyrosine phosphorylation-dependent mechanism. Currently, the functional significance of cortactin phosphorylation and the kinases responsible for the regulation of adhesion strength are not defined. We show that the nonreceptor tyrosine kinase Fer phosphorylates cadherin-associated cortactin and that this process is involved in mediating intercellular adhesion strength. In wild-type fibroblasts N-cadherin ligation-induced transient phosphorylation of Fer, indicating that junction formation activates Fer kinase. Tyrosine phosphorylation of cortactin after N-cadherin ligation was strongly reduced in fibroblasts expressing only catalytically inactive Fer (D743R), compared with wild-type cells. In wild-type cells, N-cadherin-coated bead pull-off assays induced fourfold greater endogenous N-cadherin association than in D743R cells. Fluorescence recovery after photobleaching showed that GFP-N-cadherin mobility at nascent contacts was 50% faster in wild-type than D743R cells. In shear wash-off assays, nascent intercellular adhesion strength was twofold higher in wild-type than D743R cells. Cortactin recruitment to adhesions was independent of Fer kinase activity, but was impacted by N-cadherin ligation-provoked Rac activation. We conclude that N-cadherin ligation induces Rac-dependent cortactin recruitment and Fer-dependent cortactin phosphorylation, which in turn promotes enhanced mobilization and interaction of surface expressed N-cadherin in contacting cells. more...