Mitotic chromosome segregation is the process by which duplicated genomic information is transmitted faithfully to daughter cells during cell division. Although well characterized at the cytological level, many of the molecular mechanisms that underlie this complex process in eukaryotic cells remain obscure. Segregation errors during cell division have been determined to be the cause of various disorders including Down's syndrome and spontaneous fetal abortion. In addition, segregation errors can play an important role in the promotion of neoplasia. The long-term objective of the proposed project is to gain a molecular understanding of the molecules and mechanisms responsible for chromosome segregation in the yeast S. cerevisiae. Chromosomes are segregated in mitosis by the spindle, an extremely dynamic organelle. Previously, we demonstrated that bipolar spindle morphogenesis depends upon the actions of """"""""motor"""""""" proteins related to the mechanochemical enzyme kinesin. In the absence of the function of either Cin8p or Kip1p, spindle poles cannot separate and separated poles are rapidly drawn back together. In the simplest hypothesis, these motor proteins are exerting an outward force upon the spindle poles that counters an inwardly-directed force. We have demonstrated that kinesin- related Kar3p is contributing to the inward force. Therefore, it appears that normal spindle assembly requires the actions of numerous microtubule- based motors, some that act redundantly and some that act antagonistically. Aspects of this hypothesis and the functions of Cin8p, Kip1p and Kar3p will be tested in the proposed experiments. These include the development of in vitro assays for the functions of these proteins (i.e., microtubule binding and motility), in vivo assays of function, systematic mutagenesis studies to analyze protein structure as it relates to function, and a variety of genetic assays to identify interacting factors. In addition, the cell cycle regulation of Cin8p spindle assembly activity will be examined by both genetic and biochemical techniques. For mitotic motors, we have demonstrated that both cooperative and antagonistic relationships can be characterized by genetic analysis. We propose to extend our analysis with the goal of identifying by function all the molecular motors that operate within the yeast spindle. These studies will also identify and characterize many other important spindle components.
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