Dynamics and Mechanics of Mitosis in Drosophila

摘要：有丝分裂纺锤体使用多个力发生器和调节分子进行自身组装和分离姐妹染色单体。这里描述的工作的广泛目标是提供一个全面的分子和定量的解释，有丝分裂蛋白的集合如何在其组装，维护和延伸过程中合作产生纺锤体的动态和机械特性。该建议的概念框架是有丝分裂运动依赖于稳态结构之间的转换。恒定长度的中期纺锤体等稳态结构是由多个力发生器产生的力的平衡来维持的。有丝分裂运动（如主轴装配过程中的主轴杆分离和后期主轴伸长）发生在天平倾斜时。具体目标是：1。为了测试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。为了验证双极性激酶-5电机与MTs交联并驱动由激酶-14拮抗的滑动丝机制以维持前期纺锤体的假设，使用运动测定和低温电子显微镜。3. 为了测试和阐明我们的后期B模型的分子细节，在后期B中，一个由激酶5驱动的极性间（ip） MT滑动丝机制产生运动力，而向极性的MT通量充当开关。4. 开始对有丝分裂网络进行高通量的系统级分析。我们的实验将利用对照与抑制剂注射和有丝分裂突变果蝇胚胎的动态延时成像；RNAi去除有丝分裂蛋白后培养S2细胞的高通量荧光显微镜观察；从原生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。互补的定量建模将利用力平衡系统和速率方程来解释有丝分裂的特定阶段的动力学，根据潜在的分子。我们的目标是了解有丝分裂纺锤体是如何像机器一样工作的，从而为其行为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。

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.