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相等的分离。这发生在有丝分裂纺锤体上，这是一种细胞内的机器，它使用微管和基于微管的运动蛋白来协调染色体运动和细胞分裂。我们提出的研究目标是阐明染色体在纺锤体上定位的运动机制。我们的中心假设是，这涉及多个马达同时运作的合作活动，以产生互补和对抗力量的动态平衡。具体来说，位于着丝点上的马达产生指向纺锤极的力，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“极向”和“平向”的力精确平衡时，就会形成一个稳态结构，染色体保持稳定的位置，就像在中期一样。为了研究这一点，我们将利用果蝇早期胚胎作为我们的主要实验系统，实现以下具体目标：目标1)以高空间和时间分辨率表征染色体运动的速率和结构基础。目的2)验证着丝粒结合马达、动力蛋白/动力蛋白和KinI动力蛋白协同工作以在染色体上产生极性力的假设。目的3)验证染色体臂结合马达KLP38B和Nod协同工作在染色体上产生板向力的假设。目的4)检查两极和平板电机之间存在的功能相互关系，以确定染色体是否受到平衡电机产生的力。我们的总体实验策略是利用单个马达的分析结果来制定和测试更广泛的假设，即这些马达如何共同推动细胞增殖过程中必须发生的基因组的连贯和严格控制重组。由于这一过程中的缺陷导致许多人类疾病，包括出生缺陷和癌症，我们的发现应该提供对这些疾病的原因的见解，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.