Kinetochores are complex assemblies of proteins bound to centromeric heterochromatin. They display two directions of microtubule (MT)- dependent motor activity, the ability to add and remove tubulin from MT plus ends to which they attach, and they signal in response to MT binding and/or tension to help the cell divide when to begin anaphase (the mitotic checkpoint). We propose to study the structure and function of mammalian kinetochores, both in vivo and in vitro, to help elucidate the mechanisms for these multiple functions and the relationships among them. We will characterize the structure of the MT-kinetochore interface by EM tomography of fast-frozen, freeze-substituted PtK cells, embedded in plastic, testing the hypothesis that MT end morphology is related to the polymerization of depolymerization of these polymers. We will use antibodies or other labeling methods to localize known kinetochore components rela6tive to fine structural markers, like the corona and the inner our outer kinetochore plates. This molecular anatomy will help to identify kinetochore components that may interact. We will extend our previous of kinetochore behavior in vitro, using laser tweezers to manipulate Mts relative to kinetochores on glass-bound chromosomes, testing the hypothesis that tension at the kinetochore promotes stability in the kinetochore-MT bond and that kinetochore-associated kinases to test the hypothesis that a MAP kinase serves as an up-stream regulator of the mitotic checkpoint, coupling MT binding and/or tension to the pathway that regulates the onset of anaphase.
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