The Role of Motor Proteins in Astral and Spindle Forces

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9408249 Aist The objective of this project is to determine how forces that move the mitotic apparatus are generated. The organism of choice, the filamentous ascomycete Nectria haematococca, 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 to occur in vivo; both of these forces contribute to chromosome separation during anaphase B. Previous studies have also determined the time-course and ultrastructural details of mitosis in N. haematococca. The molecular targets of this investigation are kinesin-like proteins (KLPs), which are candidates for generating pushing force in the spindle, and dynein-like proteins (DLPs), which are thought to produce pulling force in the aster. KLPs are known to be necessary for normal mitotic progression in three other fungi (Saccharamyces cerevisiae, Schizosaccharomyces pombe, and Aspergillus nidulans), and a DLP has recently been shown to be necessary for mitosis to progress normally in S. cerevisiae. The superior cytological and optical properties of N. haematococca will be used to observe the roles of these proteins in mitosis in vivo. The strategy is to clone genes from N. haematococca encoding KLPs and DLPs, destroy the function of each cloned gene by site-specific mutation in the genome, and observe by video microscopy the cytological phenotype (if any) caused by each mutation. For each 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 proteins in mitotic force generation. Additionally, each protein will be localized in the mitotic apparatus in situ using immunofluorescence microscopy, and the polarity of force produced by each protein will be determined using in vitro motility assays. One KLP-encoding gene, Klp1, has already been cloned, the genomic copy has been disrupted, and the phenotypic effect of the loss of KLP1 has been shown to be abnormal behavior of the mitotic spindle. The role of KLP1 in mitosis will be investigated, and the analysis will be extended to additional KLPs and DLPs in the organism. The proposed work 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 microbean experiments on the mutants is entirely unique and, if different motor proteins are involved in different aspects of mitosis, the results may provide clear demonstrations as to which motor protein is responsible for which mitotic force. This would contribute substantially to our understanding of the mechanisms of mitosis, a process that is fundamental to growth and development of all eukaryotes. %%% Mitosis, the process of cell division, is fundamental to all eukaryotic life. The process ensures that the genetic material of the cell is equally distributed between the two daughter cells, by carefully regulating the movement of paired sister chromosomes to opposite ends of the cell prior to physical division of the cell into two. This is critical to the viability of the daughter cells. The process is accomplished by a complex, highly regulated and predictable series of microtubule-mediated movements of the chromosomes. The biochemical mechanisms involved in these movements and their regulation are not yet well understood, although there is evidence that two kinds of microtubule-based motor proteins (chemomechanical transducers), kinesin and dynein, are involved. With prior NSF support, this laboratory has used state-of-the-art laser microscopic techniques to study the forces exerted by the microtubular apparatus (known as the "spindle") on the chromosomes during the various stages of mitosis. Th e laboratory is now planning to apply state- of-the-art molecular genetic approaches to determine the biochemical basis for each stage of the mitotic process. The work has broad significance. The better we understand the process, the better we can manipulate it, either to disrupt unwanted mitotic events (e.g., pest or weed control in agriculture, novel treatments for cancer, etc.) or to understand and perhaps intervene in unwanted, pathological disruptions. Also, since these processes take place on very small size scales (microtubule diameters are in the nanometer range), a better understanding of the mechanism and regulation of these small but precise movements may serve as useful paradigms, or even as novel materials, in nanofabrication engineering. ***