Dynamics and Mechanics of Mitosis in Drosophila.

翻译后摘要：有丝分裂纺锤体使用多个力发生器和调节分子组装本身， 分离姐妹染色单体。这里所描述的工作的广泛目标是提供一个全面的 有丝分裂蛋白的集合如何合作产生的分子和定量解释 主轴在装配、维护和伸长过程中的动态和机械性能。的 这一提议的基本概念框架是，有丝分裂运动取决于 稳态结构稳态结构，例如，保持恒定长度的中期纺锤体 由多个力发生器产生的平衡。有丝分裂运动（例如纺锤极 在主轴装配和后期主轴伸长期间分离）发生在天平倾斜时。 具体目标是1.为了验证和阐明我们的早期纺锤体模型的分子机制， 组装，这取决于由基于MT的马达、皮质马达和核马达产生的力平衡 弹性2.为了检验双极驱动蛋白-5马达交联MT并驱动滑动细丝的假设， 一种被驱动蛋白-14拮抗以维持前中期纺锤体的机制，使用运动性测定 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， 其中驱动蛋白-5驱动的极间（ip）MT滑动丝机制产生运动力， MT通量充当通断开关。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组与注射供体组和有丝分裂组的动态延时成像， 突变果蝇胚胎; RNAi后培养的S2细胞的高通量荧光显微镜 有丝分裂蛋白质的消耗;以及从天然或 杆状病毒感染的昆虫细胞。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方面的基本 分子。我们的目标是了解有丝分裂纺锤体如何作为一台机器工作，从而提供见解 它的功能缺陷如何导致基因组不稳定、癌症和出生缺陷。

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 offerees 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 elasticitry 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.