Mechanisms of Microtubule and Mitotic Spindle Assembly

微管和有丝分裂纺锤体组装机制

• 批准号: 6785325
• 负责人: LYNNE CASSIMERIS
• 金额: $ 35.04万
• 依托单位: LEHIGH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6785325
• 关键词: RNA interference; binding proteins; cell cycle; cell growth regulation; centromere; centrosome; gene induction /repression; gene targeting; kinesin; microtubule associated protein; microtubules; mitotic spindle apparatus; nucleic acid sequence; oncoproteins; protein structure function; small interfering RNA; tissue /cell culture; tubulin

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.

描述（由申请人提供）：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过程。染色体分离缺陷可能导致非整倍体，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组成部分，以及几种辅助蛋白如何修改这种组装。由于许多化疗都是基于减缓细胞分裂，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。我们之前的研究重点是了解癌蛋白 18（一种微管去稳定剂）和 TOGp（一种微管稳定剂）如何与微管相互作用以改变组装动力学。我们提出的研究将利用先前资助期间开发的工具，包括通过 siRNA 耗尽 TOGp，来解决有丝分裂期间微管组装的调节以及该组装如何有助于有丝分裂纺锤体的形成。我们的首要目标是确定 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（一种使微管不稳定的驱动蛋白）活性所必需的。接下来我们将检查 TOGp 在中心体的功能，因为这种蛋白质的消耗会导致纺锤体极处微管负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，我们将确定成形主轴内是否存在微管稳定活性的梯度。为了解决这些问题，我们将应用多种基于荧光的成像方法，结合 siRNA 或注射功能阻断蛋白或抗体。