Since the term """"""""mitosis"""""""" was introduced over 100 years ago by Fleming, a challenge has been to understand how cells divide and faithfully transmit chromosomes at each cell division. Accurate sister chromatid segregation during anaphase is pivotal for the faithful transmission of genetic information during each cell division. Errors in this process cause aneuploidy, which early in development lead to lethal developmental defects and later are hallmarks of human tumor progression. Protection from aneuploidy is provided by the mitotic (or spindle assembly) checkpoint in which the centromere/kinetochores of chromosomes that have not yet attached to spindle microtubules generate an at least partially diffusible """"""""stop anaphase""""""""signal that arrests mitosis. Using frog egg extracts, siRNA, purified proteins in vitro, and gene targeting in mice, we intend to identify the signaling cascade that activates and silences this essential cell cycle control pathway. We have already shown that a kinetochore-associated motor, CENP-E, is essential for spindle microtubule capture ant by binding at kinetochores to its partner, BubR1, it activates BubR1 kinase activity, itself essential for the """"""""stop anaphase"""""""" signaling. With purified components (including Bubl, BubR1, Bub3, CENP-E, Cdc20, chromosomes, and the anaphase promoting complex APC/C), we will now reconstruct the signaling pathway in vitro. We will directly test whether CENP-E is the signal transducing linker whose tethering of kinetochores to spindle microtubules serves to silence the BubRl-dependent checkpoint signal. We will identify substrates for BubR1 and Bubl kinases and how such phosphoryation activates the signaling cascade. Using similar approaches, we will also examine how two other kinetochore components ZW10 and Rod participate in silencing signaling from unattached kinetochores. We will examine how the earliest transient kinetochore component, CENP-F, contributes to cell cycle advance into and through mitosis. Lastly, as an extension to our early findings that NuMA, in combination with cytoplasmic dynein, is essential for tethering spindle microtubules to the poles, we will use gene replacement in mice to examine how NuMA contributes to nuclear architecture.
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