Mechanisms of Microtubule and Mitotic Spindle Assembly

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DESCRIPTION (provided by applicant): Accurate segregation of chromosomes during mitosis and meiosis is a critical biological process. Defects in chromosome segregation can lead to aneuploidy and are correlated with cancer progression. We have concentrated efforts on understanding how the cell regulates the assembly of microtubules, the major structural component of the mitotic spindle, and how several accessory proteins modify this assembly. Since many chemotherapies are based on slowing cell division, a better understanding of mitotic mechanisms will have considerable implications for human health. Our previous studies were focused on understanding how oncoprotein 18, a microtubule destabilizer, and TOGp, a microtubule stabilizer, interact with microtubules to modify assembly dynamics. Our proposed studies will take advantage of tools developed in the previous grant period, including depletion of TOGp by siRNA, to address regulation of microtubule assembly during mitosis and how this assembly contributes to formation of the mitotic spindle. Our first goal is to determine how TOGp and oncoprotein 18 regulate microtubule assembly within the spindle. We will also determine whether TOGp is necessary to antagonize the activity of KCM1, a kinesin that destabilizes microtubules. We will next examine the function of TOGp at centrosomes, since depletion of this protein results in disruption of the normal focus of microtubule minus ends at the spindle poles. These experiments will determine whether TOGp regulates microtubule nucleation or release from the centrosome, and whether TOGp stabilizes microtubule minus ends or protects microtubules from the action of severing proteins. We will also examine why TOGp-depleted cells have defects in bipolar spindle organization. Finally, we will determine whether gradients of microtubule stabilizing activity are present within the forming spindle. To address these issues, we will apply a number of fluorescence-based imaging methods combined with siRNA or injection of function-blocking proteins or antibodies.