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Lysophosphatidic acid (LPA) is a bioactive lipid mediator that acts via specific G-protein-coupled receptors and activates Rho/ROCK pathway resulting in actin cytoskeletal rearrangements. LPA stimulation enables fibroblasts to promote tissue remodeling, inflammation, angiogenesis, wound healing and tumor progression. This study is directed to elucidation of mechanisms by which LPA induces modifications of the microfilament system. Myosin-9 is a non-muscle motor protein associated with the wide range of cellular functions, including cell shape and motility. Previously we have demonstrated that myosin-9 forms multimolecular complexes with high molecular weight tropomyosin isoforms and heat shock proteins in cytoplasm of cultured cells. It was found that LPA induced redistribution of tropomyosin between cytosol and cytomatrix in dose-dependent manner. Here we explored the levels of myosin-9 and tropomyosin in cytosol and cytomatrix extracts at different time points after LPA addition (20 ng/ml) to culture medium. Cytosole extracts from human embryonic lung fibroblasts were subjected to co-immunoprecipitation assay with polyclonal antibodies specific to C-terminal peptide of human myosin-9, or with monoclonal antibodies recognizing high molecular weight tropomyosin isoforms. The cross-immunoprecipitation method revealed changes in the composition of myosin-9/tropomyosin complexes under the action of LPA. We have observed the significant decrease in myosin-9 co-immunoprecipitated tropomyosin level immediately (1 min) after LPA addition. Gel-filtration chromatography confirmed the myosin-9/tropomyosin complex disassemble after LPA treatment. Additionally, we have found that LPA treatment during 1 h induces myosin-9 degradation with accumulation of 130 kDa and 40 kDa fragments in cytomatrix fraction. Further studies are needed to elucidate the molecular mechanisms responsible for these effects of LPA treatment. This work was supported by a grant (No. 14-50-00068) from the Russian Science Foundation.