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The present article reports immunofluorescence-based analysis of the distribution of the actin-binding protein myosin-9 in the cytoplasm of human embryonic lung fibroblasts. Electrophoresis and Western blotting were used to demonstrate that myosin-9, actin, and high molecular weight tropomyosin isoforms are incorporated into cytoplasmic protein complexes not bound to cytoskeletal structures. Cross-immunoprecipitation was used to show that these complexes were rapidly disassembled when the cells were exposed to lysophosphatidic acid (LPA). Moreover, LPA induced proteolytic degradation of myosin-9 associated with the structures of the actin cytoskeleton. The results obtained point at the participation of multimolecular cytoplasmic protein complexes that contain myosin-9 and tropomyosin in the regulation of the cellular response to stimulation with LPA. [ABSTRACT FROM AUTHOR]

The presence of an actin-binding protein, tropomyosin, in particles or protein complexes not bound with actin structures were found during an assay of structural rearrangements of actin cytoskeleton. To study the composition and properties of these protein complexes, a novel method of their isolation without destroying cytoskeleton structures has been elaborated. The protein composition of isolated tropomyosin particles was assessed by gel filtration, electrophoresis, and Western blotting. It was demonstrated that they are about 700-kDa multimolecular complexes. In addition to tropomyosin and actin, these complexes contained Hsp70, Hsp90, and myosin-9 identified by mass spectrometry. It was found that the deacetylase inhibitor, trichostatin A, which induced actin cytoskeleton rearrangements, changed the number of tropomyosin particles and caused redistribution of tropomyosin between cytosol and cytoskeleton. These results demonstrate that these multimolecular complexes may participate in the process of reorganization of actin microfilaments. [ABSTRACT FROM AUTHOR]