Dynamics and Mechanics of Mitosis in Drosophila: Mechanisms of Anaphase B

描述（由申请方提供）：在有丝分裂期间，有丝分裂纺锤体使用微管（MT）和有丝分裂马达来协调染色单体到极的运动（后期A）和纺锤体伸长（后期B）。本文的目的是对后期B提供一个全面的分子和定量的解释。该提议的概念框架是，纺锤体伸长取决于由同四聚体驱动蛋白-5马达与向极ipMT通量协同作用产生的极间（ip）MT滑动丝机制，其充当“开-关”开关。我们将探索一种模型，其中前后期B纺锤体通过纺锤体极点处ipMT滑动和ipMT解聚之间的平衡而保持在稳态长度，从而产生向极通量。响应于细胞周期蛋白B在后期A结束时的降解;（i）MT突变梯度导致ipMT正末端侵入发生向外ipMT滑动的重叠区;和（ii）ipMT负末端解聚停止，因此通量被“关闭”，使力的平衡倾斜以允许向外ipMT滑动以推开纺锤体极。具体目标是：1.继续对纯化的驱动蛋白-5与MTs之间的相互作用进行生化和结构分析，以提高我们对后期B的滑丝机制及其调控的理解; 2.确定MT聚合酶、解聚酶、交联剂和滑动马达的网络如何合作以产生MT突变梯度并响应于细胞周期蛋白B降解而关闭向极通量;以及3.分析与前后期B向后期B转变相关的纺锤体MT、马达和MAP的动力学和结构重组。这个多学科项目将利用蛋白质生物化学和运动分析，体内成像和电子显微镜，活细胞的遗传和生物化学操作，以及定量建模。我们的目标是了解后期纺锤体如何作为一个大分子机器来延长自身并拉开姐妹染色体，从而深入了解其功能缺陷如何导致基因组不稳定，出生缺陷和癌症。 公共卫生相关性：这一基础科学研究项目旨在了解有丝分裂纺锤体协调遗传物质精确分离的机制，这是地球上所有生命繁殖的基本过程。对有丝分裂正常机制的进一步理解可能会阐明这一过程中导致基因组不稳定、出生缺陷和癌症的缺陷;也可能有助于我们理解这一过程中发生在干细胞分裂基础的不对称有丝分裂中的变化。反过来，这可能导致有丝分裂相关疾病治疗的改善，例如通过使用靶向特定有丝分裂蛋白的抑制剂作为潜在的抗癌剂。

DESCRIPTION (provided by applicant): During mitosis, the mitotic spindle uses microtubules (MT) plus mitotic motors to coordinate chromatid-to-pole motility (anaphase A) and spindle elongation (anaphase B). The aim of the work described here is to provide a comprehensive molecular and quantitative explanation of anaphase B. The conceptual framework underlying the proposal is that spindle elongation depends on an interpolar (ip) MT sliding filament mechanism generated by homotetrameric kinesin-5 motors acting in concert with poleward ipMT flux, which acts as an "on-off" switch. We will explore a model in which the pre-anaphase B spindle is maintained at a steady state length by the balance between ipMT sliding and ipMT depolymerization at spindle poles, producing poleward flux. In response to cyclin B degradation at the end of anaphase A; (i) a MT catastrophe gradient causes ipMT plus ends to invade the overlap zone where outward ipMT sliding occurs; and (ii) ipMT minus end depolymerization ceases so flux is turned "off", tipping the balance of forces to allow outward ipMT sliding to push apart the spindle poles. The specific aims are: 1. To continue our biochemical and structural analysis of the interactions between purified kinesin-5 and MTs, in order to improve our understanding of the sliding filament mechanism underlying anaphase B and its regulation; 2. To determine how the network of MT polymerases, depolymerases, crosslinkers and sliding motors cooperate to create the MT catastrophe gradient and turn off poleward flux in response to cyclin B degradation; And 3. To analyze the dynamics and structural reorganization of spindle MTs, motors and MAPs associated with the transition from pre-anaphase B to anaphase B. This multidisciplinary project will utilize protein biochemistry and motility assays, in vivo imaging and electron microscopy, the genetic and biochemical manipulation of living cells, together with quantitative modeling. We aim to learn how the anaphase spindle functions as a macromolecular machine to elongate itself and pull apart sister chromosomes, and thus to provide insights into how defects in its function can give rise to genomic instability, birth defects and cancer. PUBLIC HEALTH RELEVANCE: This basic science research project is aimed at understanding the mechanism by which the mitotic spindle coordinates the accurate segregation of the genetic material, a fundamental process that underlies the propagation of all life on Earth. An improved understanding of the normal mechanisms of mitosis may illuminate defects in this process that lead to genomic instability, birth defects and cancer; and also may help us understand the changes in this process that occur in the asymmetric mitoses that underlie stem cell divisions. This, in turn, could lead to improvements in the treatment of mitosis-related diseases, e.g. through the use of inhibitors that target specific mitotic proteins as potential anti-cancer agents.