The accurate transmission of genetic information during cell division requires that chromosomes be segregated to daughter cells with extremely high fidelity. This fidelity is maintained not only by the intrinsic accuracy of the mitotic machinery, but also by the operation of mitotic checkpoints that link the exit from mitosis to the correct assembly of the mitotic spindle. The goal of this work is to probe the mechanisms of checkpoint action in animal cells through the study of two genes first cloned isolated in yeast, the Mitotic Arrest Defective 1 and 2 genes (Mad1, 2). Biochemical reconstitution experiments in vitro will be used to guide the design of specific Mad1 and Mad2 mutations. The status of mitotic checkpoint controls will then be monitored in cells carrying these mutations by real-time microscopy, by examining sensitivity to anti-microtubule drugs, and by monitoring checkpoint-specific phosphorylation events. The tumorgenic effects of mutating mitotic checkpoints and of increasing the rate of chromosome loss will be monitored in mice. Specifically: 1. Cells carrying a disruption of the Mad2 gene will be examined for mis-regulation of mitotic timing, for increased chromosome loss and for response to anti-microtubule drugs. Mice with Mad2 mutations will be examined for increased tumor formation. 2. Complexes containing Mad1-Mad2 and Cdc20 will be reconstituted in vitro and the domains responsible for protein-protein interactions will be mapped. Potential dominant negative and recessive loss-of-function mutations will be introduced into these domains, characterized in yeast and introduced into murine cells. 3. The effects of Mad1 and Mad2 mutations on other components of the checkpoint signaling pathway will be characterized using microscopy-based assays with genetically altered cell lines. 4. A chromosome marking method using GFP-tagged DNA binding proteins will be adapted from yeast for use in murine cells. Cells with marked chromosomes will then be used in the real-time analysis of chromosome segregation in cells carrying checkpoint mutations. The analysis of mitotic checkpoints will have an impact on two aspects of cancer biology. First, it will clarify the role of whole-chromosome genetic instability and the resulting aneuploidy in tumor development. Second, it will help to reveal the mechanism of action of the anti-microtubule chemotherapeutic such as taxol, and thereby assist in the development of drugs that modulate the action of these important therapies.
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