The goals of experiments described in this proposal are to define mechanisms by which actomyosin-dependent contractile and motile functions are regulated in smooth and non-muscle cells. The proposed work will test hypotheses with regard to thick filament (myosin regulatory light chain (RLC) phosphorylation-dependent and independent activation) and thin filament (calponin) regulation. In smooth muscle tissues, RLC phosphorylation by Ca2+/CaM-dependent myosin light chain kinase (MLCK) is sufficient for force development or shortening; it has also been implicated in fibroblast contraction. Steady-state isometric force is linearly dependent upon the extent of RLC phosphorylation; however, the slope of this relation may change with different modes of stimulation. Regulation collateral to RLC phosphorylation may arise by activation of non-phosphorylated cross bridges or the action of the thin filament protein, calponin, which inhibits actin-activated myosin MgATPase in vitro in a Ca2+/CaM- and phosphorylation-reversible manner. FIRST, we will establish the dependence of isometric force on RLC phosphorylation in cultured aortic and tracheal smooth muscle cells and fibroblasts reconstituted in collagen gels. Conditions under which collateral regulation is maximal will be defined in these models. These experiments will indicate the relative extent of collateral regulation in smooth muscle as compared to fibroblast cells and will define optimal conditions for activation of non-phosphorylated cross bridges. SECOND, we will test the hypothesis that RLC phosphorylation is essential for contraction in smooth muscle cells and/or fibroblasts. Cultured smooth muscle cells and fibroblasts will be infected with adenovirus carrying cDNA sequences coding for 1) catalytically inactive MLCK (dominant negative), 2) antisense RNA to MLCK (enzyme depletion), 3) a non-phosphorylatable RLC (T18A/S19A), or 4) a combination of these. In addition, MLCK null mutant 3T3 fibroblasts will be generated by gene targeting through homologous recombination. After reconstituting treated cells into collagen gels, force, shortening and RLC phosphorylation will be measured in response to agents defined in Aim 1 as eliciting collateral regulation. These experiments will indicate whether non-phosphorylated cross bridges are capable of cycling. Comparison of results with overexpression of inactive enzyme to those with enzyme depletion will allow us to test the hypothesis that MLCK plays a structural role, in addition to its catalytic role. THIRD, MLCK-deficient cells generated as described in Specific Aim 2 will be evaluated for rates of cell migration (chemotaxis), rates of cellular proliferation (cytokinesis), and rates of cell attachment. These experiments will elucidate presently less well-defined functions for RLC phosphorylation in smooth muscle as well as non-muscle cells. FOURTH, the responses to calponin overexpression in smooth muscle cells and fibroblasts will be investigated in terms of force, cell migration, attachment, and proliferation. We will test the hypothesis that calponin depresses RLC-dependent motile responses.
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