Myosin has been shown to play an important role in several cellular processes, including cytokinesis, cell movement and phagocytosis. The goal of the proposed experimental program is to understand the role played by myosin, and in particular the two myosin light chains (MLC), in cell motility. cDNA clones which encode the Dictyostelium MLCs have recently been isolated. The DNA sequence of one of them has been completed, and the other is nearly done. This proposal continues the analysis of the MLC messages, and uses the cDNAs to isolate genomic clones corresponding to the Dictyostelium MLC genes. These studies will define the structure of the MLC mRNAs and the genes which encode them. The cloned cDNAs will be used to construct strains of E. coli which express the MLC polypeptides, both as beta- galactosidase fusion proteins and in their native form. The fusion proteins will be used to produce MLC specific antibodies. These antibodies as well as the cDNAs will be employed to study the regulation of MLC synthesis at both the polypeptide and mRNA level. The native protein will be reconstituted with heavy chains and the function of the reconstituted myosin assessed. In vitro techniques for site directed mutagenesis will be employed to modify the MLC coding sequences, and the mutant polypeptides produced in E. coli and subsequently reconstituted with heavy chains. The biochemical characteristics of myosin containing the mutant MLC will be analyzed in vitro to examine the effects of the mutations on filament formation, association of the MLCs with the heavy chain, actin binding, and ATPase activity. The effects of the mutations on cell motility will be assessed by reintroducing the modified MLC genes into Dictyostelium cells where expression of the modified genes will be assessed at both the mRNA and polypeptide level. The ability of the cells to undergo normal development, cytokinesis and chemotaxis will be directly assessed. Through these studies it should be possible to produce a """"""""function domain"""""""" map of the MLCs, and learn how the MLCs function in cell motility processes. This will contribute to our basic understanding of cell movement, a process critical for normal embryogenesis, morphogenesis and tissue development. Cell motility is also a critical step in the invasion of tissues by metastic tumors. A basic understanding of the process may suggest avenues for intervention.
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