MCB 9507376 Margaret Titus The myosin family is rapidly expanding and currently consists of nine distinct classes, based on phylogenetic comparisons. This variety of myosins has raised interesting questions about the nature and extent of the actin-based motors utilized by the cell to generate movements. The best known classes of myosins, myosins I, II and V have been the subject of intense study, however information regarding the in vivo role of other types of myosins is lacking. The structural diversity in the myosin family suggests that each class of myosin generates a unique form of actin-based motility. A systematic approach to assessing the role of individual classes of myosins in generating actin-based movements is required to explore this possibility. The goal of our work is to explore the in vivo function of the different classes of myosin in a single cell by gene targeting. Dictyostelium is the subject of our investigations as undergoes a simple, yet elegant developmental program and exhibits chemotaxis and motility similar to that observed in higher eukaryotic cells. It is amenable to molecular genetic manipulation and cell biological investigations. Five unconventional myosins have been previously identified in this organism. A novel screen that combines physical mapping techniques with low stringency hybridization has identified at least seven additional myosin (myo) genes. Two of these, myoH and myoJ, appear to encode members of a new class of unconventional myosin, based on preliminary phylogenetic analyses. These myosins are expressed through the developmental cycle of Dictyostelium, suggesting that they play roles in morphogenesis and development. The in vivo role of the myo H and myoJ gene products, will be investigated using a combined approach that encompasses both cell biological and molecular genetic techniques. The genes will be fully cloned, sequenced, and used to generate fusion proteins. The fusion proteins will then be used to obtain specific antibodies for immunolocalization. Gene targeting studies will be carried out to generate strains that do not express either myoH or myoJ, and the mutants carefully analyzed for defects in various aspects of motility and development. Together, these experiments will provide insights into the role of this potentially new myosin family in generating different types of actin-based cellular movements that are carried out during the Dictyostelium life cycle. Understanding the role of myoH and myoJ gene products in movement carried out during development will provide important information about how cells use their actomyosin cytoskeleton to generate various movements. These potentially include, but are not limited to, organelle movement, the anchoring of organelles, or cellular translocation. %%% This project examine a class of molecules referred to as nonconventional myosins. These molecules have only recently been discovered, and they do a variety of types of work within the cell. They are essential for life and act in cell motility and the movement of components within cells. This project will result in a better understanding of these essential molecules. ***
