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

果蝇有丝分裂的动力学和机制：后期 B 的机制

• 批准号: 8142310
• 负责人: Jonathan M. Scholey
• 金额: $ 5.58万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8142310
• 关键词: Anaphase; Antibodies; Antineoplastic Agents; Basic Science; Binding; Biochemical; Biochemistry; Biological Assay; C-terminal; Cell Cycle Regulation; Cells; Chromatids; Chromosomes; Congenital Abnormality; Coupled; Coupling; Crosslinker; Cyclin B; Defect; Diffuse; Disease; Down-Regulation; Drosophila genus; Electron Microscopy; Embryo; Equilibrium; Figs - dietary; Filament; Foundations; Genetic; Genetic Materials; Genomic Instability; Image; Invaded; Kinesin; Lead; Learning; Length; Life; Light; Malignant Neoplasms; Mechanics; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modeling; Molecular; Molecular Biology; Motor; Nature; Nucleotides; Phosphorylation; Polymerase; Polymers; Process; Prometaphase; Protein Biochemistry; Proteins; RNA Interference; Regulation; Research Project Grants; Resolution; Role; Sister; Slide; Tail; Techniques; Testing; Work; cell motility; crosslink; depolymerization; improved; in vivo; inhibitor/antagonist; insight; multidisciplinary; orientation selectivity; polymerization; preference; public health relevance; reconstitution; response; segregation; stem cell division; three-dimensional modeling

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.

描述（由申请人提供）：在有丝分裂期间，有丝分裂纺锤体使用微管（MT）加上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）。这里描述的工作的目的是为后期 B 提供全面的分子和定量解释。该提议的概念框架是纺锤体伸长取决于极间 (ip) MT 滑动丝机制，该机制由同四聚体驱动蛋白 5 马达产生，与极向 ipMT 通量协同作用，充当“开关”开关。我们将探索一种模型，其中通过纺锤体极点的 ipMT 滑动和 ipMT 解聚之间的平衡，使前期 B 纺锤体保持在稳态长度，产生极向通量。响应后期 A 结束时的细胞周期蛋白 B 降解； (i) MT突变梯度导致ipMT加端侵入发生向外ipMT滑动的重叠区域； (ii) ipMT负端解聚停止，因此通量被“关闭”，倾斜力的平衡以允许ipMT向外滑动以推开纺锤极。具体目标是： 1. 继续对纯化的驱动蛋白-5和MT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的滑动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合作创建 MT 灾难梯度并关闭极向通量以响应细胞周期蛋白 B 降解； 3. 分析与从前期 B 到后期 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测定、体内成像和电子显微镜、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大分子机器发挥作用，以拉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。 公共健康相关性：该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了解可能会阐明这一过程中的缺陷，这些缺陷会导致基因组不稳定、出生缺陷和癌症；并且还可以帮助我们了解干细胞分裂背后的不对称有丝分裂中发生的这一过程的变化。反过来，这可能会改善有丝分裂相关疾病的治疗，例如通过使用针对特定有丝分裂蛋白的抑制剂作为潜在的抗癌剂。