CYTOPLASMIC MYOSIN FUNCTION IN VITRO AND IN VIVO

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Our long range goals are to understand, on the molecular level, the mechanism of cytoplasmic myosin function in vitro and in vivo and to determine which cellular movements require myosin for force production. We focus on the function of cytoplasmic myosin in Drosophila for two reasons. First, a diverse array of interesting movements provide the structural basis for spatial differentiation of the embryo and are characteristic of various movements seen in other cells. They include nuclear divisions and migrations, pole cell formation and cellularization, and the complex cellular migrations and shape changes of gastrulation. Moreover, these movements are amenable to study by a range of powerful approaches. We used classical protein biochemical and immunological methods to purify and characterize cytoplasmic myosin from Drosophila cells in culture. This protein is myosin by five structural and three functional criteria. It is distinct from the muscle myosin heavy chain isoform by peptide mapping and immunochemical criteria. The real advantage of this organism is of course its accessibility to genetic, molecular biological and modern molecular genetic manipulation. We used antibodies to screen a library of Drosophila genomic DNA in the expression vector lambda gtll and have isolated a fragment of the gene that encodes cytoplasmic myosin. Its gene product shares epitopes with cytoplasmic but not muscle myosin heavy chain and hybridizes with a 7.4 kb message that is sufficiently large to encode this 205 kD polypeptide. Hybridization studies with restriction fragments of Drosophila DNA and clones that encode muscle myosin heavy chain confirm that it is not part of the muscle myosin gene. Moreover, preliminary mapping studies by in situ hybridization with polytene chromosomes show that this gene maps to 60E,F, distinct from the location of the muscle myosin heavy chain gene. Here we propose a multidisciplinary approach to the analysis of cytoplasmic myosin that includes further characterization of cytoplasmic myosin function in vitro, localization of this protein in embryos and cells, the use of site specific antibodies to evaluate myosin function in vitro and in vivo, characterization of the gene that encodes this peptide and its transcription unit, synthesis of cytoplasmic myosin heavy chain or its fragments in bacterial and eukaryotic expression vector systems for detailed analysis of protein structure/function relationships in vitro, and genetic manipulation of cytoplasmic myosin in living cells and embryos by cultured cell transformation and P-element mediated germ line transformation. Together these studies forge a comprehensive investigation of the mechanism of cytoplasmic myosin function and its role in cellular movements.