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

描述(申请人提供)：在有丝分裂过程中，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点的运动(后期A)和纺锤体的伸长(后期B)。这项工作的目的是为后期B提供一个全面的分子和定量解释。该提议背后的概念框架是，纺锤体的伸长依赖于由同源四聚体Kinesin-5马达与向极方向的ipMT磁通协同作用而产生的极间(IP)MT滑动细丝机制，该机制起到“开-关”开关的作用。我们将探索一个模型，在该模型中，通过在纺锤体两极的ipMT滑动和ipMT解聚之间的平衡，使前后期B纺锤体保持稳定的长度，从而产生向极的通量。(I)MT突变梯度导致ipMT正端侵入发生向外ipMT滑动的重叠区；以及(Ii)ipMT减端解聚停止，因此通量被关闭，倾斜力平衡以允许向外ipMT滑动来推开纺锤体两极。具体目标是：1.继续我们的生化和结构分析纯化的Kinesin-5和MTS之间的相互作用，以提高我们对后期B的滑动细丝机制及其调控的理解；2.确定MT聚合酶、解聚酶、交联剂和滑动马达网络如何协同作用，创建MT突变梯度并关闭极向通量，以响应细胞周期蛋白B的降解；为了分析纺锤体MTS的动力学和结构重组，与从前期后期B到后期B的转变相关的马达和图谱。这个多学科的项目将利用蛋白质生物化学和运动性分析，活体成像和电子显微镜，活细胞的遗传和生化操作，以及定量建模。我们的目标是了解后期纺锤体如何作为一台大分子机器伸长自身并拉开姐妹染色体，从而为其功能缺陷如何导致基因组不稳定、出生缺陷和癌症提供见解。 与公共健康相关：这个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准确分离的机制，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更好的理解可能会揭示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的变化，这些变化发生在干细胞分裂的不对称有丝分裂中。这反过来可以改善有丝分裂相关疾病的治疗，例如通过使用以特定有丝分裂蛋白为靶点的抑制剂作为潜在的抗癌药物。

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.