Roles of Microtubule-Associated Motor Proteins in Mitosis

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9807829 Aist Cell division, with its concomitant uniform distribution of genetic material between daughter cells, is absolutely fundamental to all life processes. The part of the cell division process in eukaryotes whereby the genetic material, in the form of chromosomes, becomes evenly distributed between the two daughter cells is called mitosis. During the mitotic process, the chromosomes become condensed and collect halfway between the poles of the spindle, a microtubular array so named because of its shape. The chromosomes become attached to the ends of specialized microtubules within the spindle. As a result of microtubule-based chemomechanical movements mediated by motor proteins known as dyneins, the chromosomes separate into two groups and move toward the spindle poles, from each of which another set of microtubules, termed asters, radiate. The phase of mitosis during which this migration toward the poles occurs is termed anaphase. The objective of this project is to determine how forces that move the mitotic apparatus and its attached chromosomes are generated. The filamentous ascomycete, Nectria haematococca (a.k.a. Fusarium solani), has been chosen for study because it is the most cytologically tractable fungus which is also amenable to both conventional and molecular genetic technologies. It is the only organism in which both astral pulling forces and spindle pushing forces have been demonstrated experimentally to occur in vivo; both of these forces contribute to chromosome separation during anaphase B. The time-course and ultrastructural details of mitosis have been determined. Four microtubule-associated motor protein genes have been cloned and sequenced, and one role of the cytoplasmic dynein heavy chain gene in mitosis has been demonstrated. The continuing goal is to use the superior cytological and optical properties of N. haematococca to further ascertain the roles of these motor proteins by direct observation of mitosis in vivo. The strategy is to destroy t he function of each cloned gene by site-specific mutation in the genome and then to observe the in vivo mitotic phenotypes caused by each mutation. With each motor protein, the hypothesis that elimination or overproduction will alter the rate of mitotic movements will be tested. Laser microbeam experiments will then be used as a confirmation of the suspected roles of the motor proteins in mitotic force generation. Additionally, each motor protein will be localized in the mitotic apparatus in vivo using green fluorescent protein - motor protein fusions, and the polarity of force produced by the kinesin-related proteins will be determined using in vitro motility assays. The project represents a rare opportunity to correlate specific motor proteins with in vivo demonstrated mitotic forces of known polarity and location. The strategy of testing functional inferences by performing laser microbeam experiments on mutants is unique to this project and should lead to unequivocal conclusions as to which motor protein is responsible for which mitotic force. Thus, the project has the potential to be the first to fully elucidate the molecular basis of both the astral pulling force and the spindle pushing and counter forces. In doing so, it would contribute substantially to our understanding of the mechanisms of mitosis, which is fundamental to all growth and development in eukaryotes.