MYOSIN I FUNCTION IN VIVO

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A novel small myosin gene, abmA, has been identified in Dictyostelium. The abmA gene encodes a protein that has a myosin head attached to a positively charged tail region that is similar to the Acanthamoeba myosin I membrane binding domain. Cells devoid of the abmA gene product were examined at the single cell level and preliminary results revealed that these mutants have significantly reduced rates of intracellular particle movement. The experiments described in this grant proposal are directed at further dissecting the role of abmA in cellular motility, specifically organelle transport, and biochemically characterizing the abmA protein. Dictyostelium is the organism of choice for such studies as it allows the investigator to employ a multi-faceted approach, combining molecular genetic experiments with traditional protein biochemistry and cell biology to dissect the role of abms in organelle transport or other forms of cellular motility. The first step will be the generation of abmA-specific antibodies and localization of the abmA protein in Dictyostelium using immunofluorescence. Mutant and wild-type abmA genes will be introduced into abmA- cells and the recovery of intracellular particle movement assessed. The mutations will include alteration of the regulatory phosphorylation site (which would affect the function of the molecule) or changes in the tail region (which would influence the intracellular localization of the protein). Secondly, a protocol will be developed to purify the abmA protein from Dictyostelium and the myosin-like properties of the abmA protein determined. A cell line that overexpresses the wild-type abmA protein will be generated to assist in the purification of the abmA protein. The final series of experiments will address the interaction of abmA with intercellular particles or other membranous elements. Vesicles will be isolated from Dictyostelium and their ability to be translocated along oriented Nitella actin cables will be examined. The identity of the actin-based motor attached to such vesicles or membranous elements will be determined by a combination of immunoblotting experiments and purification of the vesicle motor. Additional experiments will focus on how the identified abm is attached to the vesicle or membranous element, whether it is by direct association of the motor via electrostatic interaction or if there is an abmA receptor protein present in the membrane The approach described above is specifically directed at understanding the in vivo role of the abmA protein, but it is also broadly applicable to the study of other actin-based motors involved in organelle transport.