Microtubule Regulation

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DESCRIPTION (provided by applicant): The essential function of mitosis is the delivery of a complete set of chromosomes to each daughter cell. This requires attachment of spindle microtubules to kinetochores, specialized structures formed at centromeres. Kinetochores are more than just structural elements that mediate faithful chromosome inheritance. The unattached kinetochore is also the signal generator for the mitotic checkpoint (also known as the spindle assembly checkpoint), the major cell cycle control mechanism in mitosis. Each unattached kinetochore generates a "wait anaphase" inhibitor that blocks destruction of cyclin B and securin, thereby blocking disjoining of duplicated sister chromosomes and advance into anaphase. We have previously identified a microtubule-dependent motor, CENPE, to be multifunctional these mitotic events. It directly tethers spindle microtubules to kinetochores and it functions as the cyclin-like activator of the essential mitotic checkpoint kinase BubR1. Microtubule capture by CENP-E in turn inactivates BubR1 kinase activity, thereby silencing mitotic checkpoint signaling and permitting anaphase onset. We now propose to determine the properties of CENP-E in microtubule capture at kinetochores (with electron microscopy), the motor properties of CENP-E (including processivity and residence time when microtubule bound), and how CENP-E acts to affect coupling to dynamic microtubules. The functional role of an unusual CENPE modification, farnesylation, will be determined. A major effort will use all purified components to reconstruct mitotic checkpoint signaling in vitro and to identify the "wait anaphase" inhibitor(s) produced by unattached kinetochores. Moreover, recognizing that most human solid tumors are aneuploid, that is, have other than the correct number of 46 chromosomes, we will exploit our discovery that reduced levels of CENP-E generate accelerated rates of such aneuploidy to enable a test of how whole chromosomal aneuploidy affects tumorigenesis initiated by loss of each of the major tumor suppressor genes. This will provide a test of the hypothesis posed more than 100 years ago by the great cytologist Boveri that aneuploidy drives tumorigenesis. Finally, recognizing that successful cancer therapeutics such as taxol yield chronic activation of the mitotic checkpoint, we will examine the mechanisms underlying cell death or adaptation after chrom0ic mitotic checkpoint arrest. PUBLIC HEALTH RELEVANCE: Unattached kinetochores control the cell cycle clock during cell division by generating a "wait" signal, the mitotic checkpoint, which delays cell cycle advance until all chromosomes have successfully attached to spindle microtubules. How this signaling works will be examined by determining how a kinetochore motor protein affects chromosome attachment to spindle microtubules and acts to activate and silence an essential checkpoint kinase. The overall signaling pathway will be determined by reconstructing it from individual components. Finally, since some successful anti-tumor drugs in humans chronically activate the mitotic checkpoint, the mechanisms tha determine cell fate, cell death or adaptation, when so arrested will be determined.