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Cells in mechanically challenged environments cope with high-amplitude exogenous forces that can lead to cell death, but the mechanisms that mediate force-induced apoptosis and the identity of mechanoprotective cellular factors are not defined. We assessed apoptosis in NIH 3T3 and HEK (human embryonic kidney)-293 cells exposed to tensile forces applied through β1-integrins. Apoptosis was mediated by Rac-dependent activation of p38α. Depletion of Pak1 (p21-activated kinase 1), a downstream effector of Rac, prevented force-induced p38 activation and apoptosis. Rac was recruited to sites of force transfer by filamin A, which inhibited force-induced apoptosis mediated by Rac and p38α. We conclude that, in response to tensile force, filamin A regulates Rac-dependent signals, which induce apoptosis through Pak1 and p38.

Fibroblasts remodel extracellular matrix collagen, in part, through phagocytosis. This process requires formation of cell extensions, which in turn involves interaction of the actin-binding protein flightless-1 (FliI) with non-muscle myosin IIA (NMMIIA; heavy chain encoded by MYH9) at cell-matrix adhesion sites. As Ca2+ plays a central role in controlling actomyosin-dependent functions, we examined how Ca2+ controls the generation of cell extensions and collagen remodeling. Ratio fluorimetry demonstrated localized Ca2+ influx at the extensions of fibroblasts. Western blotting and quantitative (q)PCR showed high expression levels of the Ca2+-permeable transient receptor potential vanilloid-4 (TRPV4) channel, which co-immunoprecipitated with β1 integrin and localized to adhesions. Treatment with β2β1-integrin-blocking antibody or the TRPV4-specific antagonist AB159908, as well as reduction of TRPV4 expression through means of siRNA, blocked Ca2+ influx. These treatments also inhibited the interaction of FliI with NMMIIA, reduced the number and length of cell extensions, and blocked collagen remodeling. Pulldown assays showed that Ca2+ depletion inhibited the interaction of purified FliI with NMMIIA filaments. Fluorescence resonance energy transfer experiments showed that FliI-NMMIIA interactions require Ca2+ influx.We conclude that Ca2+ influx through the TRPV4 channel regulates FliI-NMMIIA interaction, which in turn enables generation of the cell extensions essential for collagen remodeling. [ABSTRACT FROM AUTHOR]

The role of adhesion-associated actin-binding proteins in cell migration is not well defined. In mouse fibroblasts we screened for focal adhesion-associated proteins that were isolated with collagen-coated beads and detected by tandem mass spectrometry. We identified flightless I (FliI) as an actin-binding protein in focal adhesion fractions, which was verified by immuno-blotting. By confocal microscopy most FliI was distributed throughout the cytosol and in focal adhesions. By sedimentation assays and in vitro binding assays, we found that FliI associates with actin filaments and actin monomers. Assays using purified proteins showed that FliI inhibits actin polymerization and caps but does not sever actin filaments. Cells with FliI knockdown or cells overexpress-ing FliI migrated more or less rapidly, respectively, than wild-type controls. Compared with controls, cells with Fill knockdown were less adherent than wild-type cells, exhibited reduced numbers of focal adhesions containing activated pi integrins and vinculin, and exhibited increased incorporation of actin monomers into nascent filaments at focal adhesions. These data indicate that FliI regulates cell migration through its localization to focal adhesions and its ability to cap actin filaments, which collectively affect focal adhesion maturation. [ABSTRACT FROM AUTHOR]