Mechanisms of Microtubule and Mitotic Spindle Assembly

描述（申请人提供）：有丝分裂和减数分裂过程中染色体的准确分离是一个关键的生物学过程。染色体分离缺陷可导致非整倍体，并与癌症进展相关。我们集中精力了解细胞如何调节微管的组装，微管是有丝分裂纺锤体的主要结构成分，以及几种辅助蛋白如何修饰这种组装。由于许多化疗是以减缓细胞分裂为基础的，因此更好地了解有丝分裂机制将对人类健康产生重大影响。我们之前的研究主要集中在了解癌蛋白18（一种微管不稳定剂）和TOGp（一种微管稳定剂）如何与微管相互作用以改变装配动力学。我们提出的研究将利用之前资助期开发的工具，包括siRNA耗尽TOGp，来解决有丝分裂过程中微管组装的调节以及这种组装如何促进有丝分裂纺锤体的形成。我们的第一个目标是确定TOGp和癌蛋白18如何调节纺锤体内的微管组装。我们还将确定TOGp是否需要拮抗KCM1的活性，KCM1是一种破坏微管稳定性的激酶。接下来，我们将研究TOGp在中心体上的功能，因为这种蛋白质的消耗会导致纺锤体极点微管负端正常聚焦的破坏。这些实验将确定TOGp是否调节微管成核或从中心体释放，以及TOGp是否稳定微管负端或保护微管免受切断蛋白质的作用。我们还将研究为什么togp耗尽的细胞在双极纺锤体组织中存在缺陷。最后，我们将确定微管稳定活动的梯度是否存在于成形主轴内。为了解决这些问题，我们将应用一些基于荧光的成像方法，结合siRNA或注射功能阻断蛋白或抗体。

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.