Mechanisms of Microtubule and Mitotic Spindle Assembly

描述（由申请人提供）：有丝分裂和减数分裂期间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，有丝分裂纺锤体的主要结构组成部分，以及几个辅助蛋白质如何修改此组件。由于许多化疗是基于减缓细胞分裂，更好地了解有丝分裂机制将对人类健康产生重大影响。我们以前的研究集中在了解癌蛋白18，微管不稳定剂，和TOGp，微管稳定剂，如何与微管相互作用，以修改组装动力学。我们提出的研究将利用在前一个资助期开发的工具，包括通过siRNA耗尽TOGp，以解决有丝分裂期间微管组装的调节以及这种组装如何有助于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是否是拮抗KCM1活性所必需的，KCM1是一种使微管不稳定的驱动蛋白。我们接下来将研究TOGp在中心体的功能，因为这种蛋白质的缺失会导致纺锤体两极微管负末端的正常焦点被破坏。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是否稳定微管负末端或保护微管免受切断蛋白的作用。我们还将研究为什么TOGp缺失的细胞在双极纺锤体组织中有缺陷。最后，我们将确定微管稳定活性的梯度是否存在于形成的纺锤体中。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.