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学过程。染色体分离的缺陷可能导致非整倍性，并且与癌症的进展相关。我们集中精力理解细胞如何调节微管的组装，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该组件。由于许多化学疗法是基于细胞分裂的放缓，因此对有丝分裂机制的更好理解将对人类健康产生相当大的影响。我们以前的研究集中在了解微管稳定器和微管稳定剂TOGP如何与微管相互作用以修改装配动力学的癌蛋白18如何与微管稳定剂TOGP相互作用。我们提出的研究将利用上一个赠款期间开发的工具，包括siRNA耗尽TOGP，以解决有丝分裂过程中微管组装的调节，以及该组装如何有助于形成有丝分裂纺锤体。我们的第一个目标是确定togp和oncoprotin 18如何调节纺锤体内的微管组件。我们还将确定是否需要TOGP来拮抗KCM1的活性，KCM1的活性是破坏微管的稳定性。接下来，我们将检查中心体处TOGP的功能，因为该蛋白质的耗竭会导致微管负端在纺锤形杆处的正常焦点的破坏。这些实验将确定TOGP是调节微管成核还是从中心体中释放，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具有缺陷。最后，我们将确定在形成主轴内是否存在微管稳定活性的梯度。为了解决这些问题，我们将应用许多基于荧光的成像方法与siRNA或注射功能阻断蛋白或抗体。