Molecular Analysis of Chromosome Segregation

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Project Summary Life depends on the accurate transmission of genetic material at each cell division. Errors in this process lead to aneuploidy, which is implicated in oncogenesis, birth defects and cell death. Duplicated chromosomes are captured and segregated by a microtubule-based molecular machine, the mitotic spindle. The spindle is bipolar and each spindle pole carries an exact complement of chromosomes to each daughter cell. During mitosis, microtubules nucleate from the poles and capture and organize the chromosomes. Kinetochores, large multiprotein organelles located at the centromeric DNA, bind the microtubules and anchor the chromosomes to the poles. Our work focuses on each end of the microtubule, the spindle poles and the kinetochores. Spindle morphogenesis requires spatially controlled microtubule nucleation. Using a combination of reconstitution and in vivo analysis, we will test hypotheses that address how microtubule nucleation is activated and spatially regulated. Kinetochores attach chromosomes to microtubules with a striking combination of strength and plasticity. The attachments are mobile and robust under tension, but can also rapidly destabilize in response to regulatory signals. As such, the kinetochore is at the center of an error correction mechanism that repairs incorrect attachments sensed by a lack of ‘proper’ tension. The identification of the proteins that are under tension, the measurement of the strength of the linkages and the requirements for the full strength of attachments are together the second focus of this project. We have found that individually no kinetochore protein binds the microtubule with strength or longevity. To reconstitute the full strength of microtubule attachment exhibited by native kinetochores requires synergy between proteins in contact with the microtubule with proteins within the interior of the kinetochore. We will use a reconstitution-based approach and in vivo analysis to test the contribution of affinity, avidity and geometry to this synergy. In this way we will understand how the whole achieves greater properties than the sum of the parts. In addition, by exploiting our reconstituted kinetochore, we will test hypotheses for how the tension signal that triggers error correction is transmitted from the kinetochore and received by the repair mechanisms.