The ultimate objective of this research is to understand how mitosis works at a cellular and molecular level. This proposal is concerned with one aspect of mitosis, the elongation of spindles during anaphase chromosome separation. Experiments are described which utilize a functional, in vitro model system to gain biochemical, physiological and ultrastructural information on the mechanochemical enzymes responsible for spindle elongation. Preliminary results indocate that the enzymes are located in the zone of overlap, a region in the spindle where half spindle microtubules interdigitate. Large numbers of spindles will be isolated then the material in th overlap zone will be selectively extracted. The extracted material will be tested for ATPase activity and characterized by gel electrophoresis. Reconstitution experiments will be done to see if the material can restore function to extracted spindles. These extracts will be used to make monoclonal antibodies and the antibodies used to localize mechanochemical enzymes and as jamming agents in reactivation experiments. The ultrastructure of the overlap zone in physiologically treated spindles will be examined by cryo-EM techniques. Cryo-EM methods minimize the destruction inherent in EM preparation methods and improve the chances of directly visualizing the mechanochemical enzymes. The role of microtubule polymerization during spindle elongation will be investigated by video microscopy and EM immunocytochemistry. Because spindle elongation in vitro is regulated by protein phosphorylation, experiments will be done to determine which proteins are phosphorylated and which kinases are involved in regulating function. The regulation of mitosis is a topic of major medical interest since uncontrolled cell division is at the heart of the cancer problem and inaccurate chromosomal segregation (aneuploidy) is causal in several congenital malformations and a major cause of premature termination of pregnancy. An improved understanding of mitosis should eventually lead to new approaches to cancer chemotherapy and to control of abnormal chromosome segregation.
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