Microtubule Motor-Mechanisms of Chromosome Movements

描述(由申请人提供)：成功的有丝分裂需要从母细胞到子细胞的基因组平等分离。这发生在有丝分裂纺锤体上，这是一种细胞内机器，使用微管(MT)和基于MT的马达蛋白来协调染色体运动和细胞分裂。我们提出的研究的目标是阐明将染色体定位在纺锤体上的马达机制。我们的中心假设是，这涉及同时工作的多个马达的合作活动，以产生互补和对抗力量的动态平衡。具体地说，位于动轴上的马达产生指向纺锤体极的力，而位于染色体臂上的马达产生指向中期板的力。我们假设，当这些“极地”和“平台”力量精确平衡时，就会形成稳定的结构，染色体保持稳定的位置，就像在中期一样。为了研究这一点，我们将使用果蝇早期胚胎作为我们的主要实验系统：目的1)以高空间和高时间分辨率表征染色体运动的速率和结构基础。目的2)验证动粒结合马达、动力蛋白/动力蛋白和激动素协同作用在染色体上产生向极力的假设。目的3)验证KLP38B和NOD这两个染色体臂结合马达协同工作对染色体产生平台向力的假设。目的4)研究极向马达和北向马达之间存在的功能相互关系，以确定染色体是否受到平衡马达产生的力的影响。我们的总体实验策略是利用对单个马达的分析结果来制定和测试关于这些马达如何共同工作以驱动在细胞增殖过程中必须发生的连贯和严格控制的基因组重组的更广泛的假设。由于这一过程中的缺陷会导致许多人类疾病，包括出生缺陷和癌症，我们的发现应该会为这些疾病的原因提供见解，并提出潜在的治疗方法。

DESCRIPTION (provided by applicant): Successful mitosis requires the equal segregation of the genome from mother to daughter cells. This occurs on the mitotic spindle, an intracellular machine that uses microtubules (MTs) and MT-based motor proteins to coordinate chromosome movements with cell division. The goal of our proposed studies is to elucidate the motor-mechanisms that position chromosomes on the spindle. Our central hypothesis is that this involves the cooperative activity of multiple motors functioning simultaneously to generate a dynamic balance of complementary and antagonistic forces. Specifically, motors positioned on kinetochores generate forces directed toward the spindle poles while motors positioned on chromosome arms generate forces directed toward the metaphase plate. We posit that when these 'poleward' and 'plateward' forces precisely balance, a steady-state structure forms and chromosomes maintain a stable position, as during metaphase. Tipping this balance, via the up- or down-regulation of a subset of motors, results in specific chromosome movements such as chromatid-to-pole motion during anaphase A. To study this, we will carry out the following specific aims using Drosophila early embryos as our primary experimental system: Aim 1) Characterize the rates and structural basis of chromosome motility with high spatial and temporal resolution. Aim 2) Test the hypothesis that the kinetochore binding motors, dynein/dynactin and KinI kinesins, work cooperatively to generate poleward forces on chromosomes. Aim 3) Test the hypothesis that the chromosome-arm binding motors, KLP38B and Nod, work cooperatively to generate plateward forces on chromosomes. Aim 4) Examine the functional inter-relationships that exist between sets of poleward and plateward motors to determine whether chromosomes are subjected to counterbalancing motor-generated forces. Our overall experimental strategy is to utilize the results of analyses of individual motors to formulate and test broader hypotheses regarding how these motors work collectively to drive the coherent and tightly controlled reorganization of the genome that must occur during cell proliferation. Because defects in this process lead to numerous human maladies, including birth defects and cancer, our findings should provide insights into the causes of these diseases and suggest potential therapeutic approaches to their treatment.