CYTOPLASMIC MYOSIN FUNCTION IN VIVO AND IN VITRO

Duke University, Durham NC

Investigators

Linked publications & trials

Abstract

We hypothesize that the classical genetic and molecular genetic approaches available in Drosophila provide a unique opportunity to investigate the molecular basis of nonmuscle myosin-II. Nonmuscle myosin-II is a highly conserved motor protein that contributes to cytokinesis, the distribution of cell surface receptors, post mitotic cell shape changes for morphogenesis, cellular locomotion and wound healing. Previously, we showed that in fly a single gene, zipper (zip) encodes the nonmuscle myosin-II heavy chain and a single gene, spaghetti squash (sqh) encodes the nonmuscle myosin regulatory light chain. These genes encode polypeptides that are very similar to their mammalian orthologs (the regulatory light chain protein is 81 percent identical and 93 percent similar). Thus, the fly proteins are considerably more similar to their mammalian orthologs than are myosin-IIs from other invertebrate model systems that are amenable to classical genetic or molecular genetic approaches. We have used a variety of methods to establish which cell shape changes require myosin-II function. Here we plan to investigate how myosin functions in living cells by using genetic, molecular, cell biological and protein biochemical approaches to identify proteins that are required for myosin function and then to characterize HOW they function. Our Specific Aims are as follows. 1) We will continue to use simple and powerful F1 screens to recover genes whose products interact with myosin-II to effect cytokinesis and morphogenesis. 2) We will characterize GENETICALLY new myosin alleles and putative interacting loci. 3) We will characterize MOLECULARLY new myosin alleles and putative interacting loci. Finally, 4) we will use a variety of CELL BIOLOGICAL approaches to investigate how these interacting proteins facilitate myosin function. We will evaluate links between a) the molecular defects that characterize myosin mutations; b) the molecular identity of interacting gene products and c) the molecular defects that characterize those alleles of interacting loci that actually modulate the ability of the myosin to perform its functions. Our strategy will be to investigate alterations in movements, the structure of the actin cytoskeleton and the localization of myosin in appropriate mutant animals. We will characterize these phenotypes of various myosin mutation homozygotes, interactor mutation homozygotes, myosin/interactor double homozygotes and if instructive, various other potential combinations. Our goal is to ascertain the basis of myosin function at the cellular and molecular level.