Dynamics and Mechanics of Mitosis in Drosophila.

果蝇有丝分裂的动力学和机制。

• 批准号: 7931676
• 负责人: Jonathan M. Scholey
• 金额: $ 8.73万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931676
• 关键词: Actins; Anaphase; Baculoviruses; Biochemical; Biochemistry; Biological Assay; Cell division; Cells; Cellular biology; Confocal Microscopy; Congenital Abnormality; Cryoelectron Microscopy; Daughter; Defect; Development; Drosophila genus; Dynein ATPase; Elasticity; Embryo; Equation; Equilibrium; Event; Filament; Fluorescence Microscopy; Genome; Genomic Instability; Image; Insecta; Isometric Exercise; Kinesin; Learning; Length; Life; Maintenance; Malignant Neoplasms; Mechanics; Metaphase; Microscopy; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modeling; Molecular; Motor; Movement; Myosin ATPase; Nuclear; Phase; Polymers; Prometaphase; Property; Proteins; RNA Interference; Role; Sister Chromatid; Slide; Staging; Structure; System; Testing; Time; Work; abstracting; base; cell motility; crosslink; in vivo; inhibitor/antagonist; insight; mutant; protein complex; research study; time use

DESCRIPTION (provided by applicant): Abstract: The mitotic spindle uses multiple force-generators and regulatory molecules to assemble itself and segregate sister chromatids. The broad aim of the work described here is to provide a comprehensive molecular and quantitative explanation of how ensembles of mitotic proteins cooperate to produce the dynamic and mechanical properties of the spindle during its assembly, maintenance and elongation. The conceptual framework underlying the proposal is that mitotic movements depend on transitions between steady state structures. Steady state structures e.g. metaphase spindles at constant length are maintained by a balance of forces, generated by multiple force generators. Mitotic movements (e.g. spindle pole separation during spindle assembly and anaphase spindle elongation) occur when the balance is tipped. The specific aims are 1. To test and elucidate the molecular mechanisms of our model for early spindle assembly which depends on a force-balance generated by MT-based motors, cortical motors and nuclear elasticity 2. To test the hypothesis that bipolar kinesin-5 motors crosslink MTs and drive a sliding filament mechanism that is antagonized by kinesin-14 to maintain the prometaphase spindle, using motility assays and cryo-electron microscopy. 3. To test and elucidate the molecular details of our model for anaphase B in which a kinesin-5-driven interpolar (ip) MT sliding filament mechanism generates motile force and poleward MT flux acts as an on-off switch. 4. To begin a high-throughput, system-level analysis of the mitotic network. Our experiments will utilize dynamic time-lapse imaging of control versus inhibitor-injected and mitotic mutant Drosophila embryos; high-throughput fluorescence microscopy of cultured S2 cells following RNAi depletion of mitotic proteins; and biochemical analysis of mitotic proteins prepared from native or baculovirus-infected insect cells. Complementary quantitative modeling will exploit systems of force-balance and rate equations to explain the dynamics of specific phases of mitosis in terms of the underlying molecules. We aim to understand how the mitotic spindle works as a machine and thus to provide insights into how defects in its action can give rise to genomic instability, cancer and birth defects.

描述(申请人提供)：摘要：有丝分裂纺锤体使用多个力发生器和调节分子来组装自己并分离姐妹染色单体。这里描述的工作的主要目的是提供一个全面的分子和定量的解释，说明有丝分裂蛋白集合如何在纺锤体的组装、维护和延伸过程中相互协作，产生纺锤体的动态和机械特性。这一提议背后的概念框架是有丝分裂运动依赖于稳态结构之间的转变。恒定长度的中期纺锤体等稳态结构由多个力发生器产生的力平衡来维持。有丝分裂运动(例如，纺锤体组装过程中的纺锤体极分离和后期纺锤体的伸长)发生在平衡倾斜时。其具体目的是：1.检验和阐明我们的早期纺锤体组装模型的分子机制，该模型依赖于基于MT的马达、皮质马达和核弹性产生的力平衡。2.利用运动学分析和冷冻电子显微镜，验证双极Kinesin-5马达与MTS交织并驱动被Kinesin-14拮抗的滑动细丝机制维持早期纺锤体的假说。3.为了验证和解释我们的后期B模型的分子细节，在该模型中，一个由kinesin-5驱动的极间(IP)MT滑动细丝机制产生动力，向极面的MT通量充当开关。4.开始对有丝分裂网络进行高通量、系统水平的分析。我们的实验将使用对照与抑制剂注射和有丝分裂突变果蝇胚胎的动态时间推移成像；高通量荧光显微镜，在RNAi耗尽有丝分裂蛋白后培养的S2细胞；以及从本地或杆状病毒感染的昆虫细胞制备的有丝分裂蛋白的生化分析。互补的定量模型将利用力平衡和速率方程系统来解释根据潜在分子的特定有丝分裂阶段的动力学。我们的目标是了解有丝分裂纺锤体是如何作为机器工作的，从而为其活动中的缺陷如何导致基因组不稳定、癌症和出生缺陷提供见解。