TRPV4 mediates the Ca2+ influx required for the interaction between flightless-1 and non-muscle myosin, and collagen remodeling

Collagen remodeling by phagocytosis requires cell extension formation, which in turn involves interaction of the actin binding protein Flightless I (FliI) with non-muscle myosin IIA (NMMIIA) 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 extensions of fibroblasts. Western Blotting and qPCR 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. Blocking antibody to α2β1 integrin, the TRPV4-specific antagonist AB159908, or reduction of TRPV4 expression with siRNA, all blocked Ca2+ influx. These treatments also inhibited interaction of FliI with NMMIIA, reduced the number and length of cell extensions and blocked collagen remodeling. Pull-down assays showed that Ca2+ depletion inhibited interaction of purified FliI with NMMIIA filaments. Fluorescence resonance energy transfer experiments showed FliI-NMMIIA interactions require Ca2+ influx. We conclude that Ca2+ influx through TRPV4 channels regulates FliI-NMMIIA interactions, which in turn enable generation of the cell extensions essential for collagen remodeling.