James A. Spudich, Professor of Biochemistry |
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Our general research interest is the molecular basis of cell motility. Specific research interests include the molecular basis of energy transduction that leads to ATP-driven myosin movement on actin, the biochemical basis of the regulation of actin and myosin interaction and their assembly states, and the roles these proteins play in vivo, in cell movement and changes in cell shape. We work on two experimental systems: contraction of mammalian muscle and chemotaxis of Dictyostelium discoideum cells. Dictyostelium, the cell that commands most of our attention, exhibits all of the behavior of nonmuscle
mammalian cells and, unlike many |
| other eukaryotic cells, can be grown in large amounts for biochemical work. We have demonstrated efficient gene targeting by homologous recombination in the Dictyostelium myosin gene. The phenotype of cells that are lacking a functional myosin-II indicate that this motor participates in cytokinesis, development and cell motility. Mutagenized forms of this myosin have been constructed and characterized biochemically after expression in Dictyostelium as a host. In addition, we probe the effects of each altered myosin form on the phenotype of the cell. Our approaches include biochemical and structural studies of actin, myosin, and associated regulatory proteins. To better understand the mechano-chemical coupling of the myosin enzyme, we have designed and developed a number of in vitro assays for ATP-dependent movement of purified myosin on filaments reconstituted from purified actin. These assays also allow us to examine mutant myosin molecules for altered function. We are currently extending the in vitro motility assay system using laser optical trap technology in order to explore the production of force in vitro. Crystallographic approaches are also being used to examine these proteins structurally. We are thus examining the relationship between the enzyme's structure and its function, both at the atomic and the cellular levels, by combining detailed biochemical and sensitive in vitro movement assays with the in vivo analysis of mutant myosins and analysis of their structure. |
