Cell Cycle Control of the Cytoskeleton

本提案涉及控制微管长度、微管的大小和功能的机制 后期纺锤体，以及后期纺锤体功能与其他关键细胞事件的协调 有丝分裂退出。由于纺锤体是一种自组织结构，微管长度的调节是主要的 控制主轴整体尺寸的机构。磁盘轴大小由浓度或活动全局控制 促进微管生长或分解的因素。此外，“测量”机制 还描述了依赖于长度的中间微管组装或拆卸。最好的- 已研究的长度依赖机制是通过微管的kinesin-8家族的活动来实现的 摩托公司。哺乳动物细胞中激动素8功能受损导致染色体高频率 在癌症中常见的错误分离和异常核结构的形成，称为 微核。我们最近发现，微核可以引起“嗜铬症”，这是一种主要的突变过程。 导致癌症中的染色体重排。 在上一个资助期，我们确定了酵母激动素8选择性修剪更长时间的机制 微管。与之前的提议不同，生化和单分子成像相结合 实验导致了一种新的构象开关模型，涉及Kinesin 8对弯曲微管蛋白的识别 微管末端，触发微管解体。基于一种新的高分辨率低温电磁结构 和其他数据，我们现在建议测试这一模型，并找出弯曲微管蛋白的分子机制 承认。此外，在上一个资助期，我们在理解 后期纺锤体功能如何与核膜周围的重组相协调 染色体形成子代细胞核。我们发现纺锤形微管阻止了 核膜(NE)含有核孔蛋白使染色体解聚，但允许其他方面的 要发生的NE装配。这导致落后染色体上的NE组装出现不可逆转的缺陷， 解释微核周围的NE发生自发破坏的原因，这是产生 嗜铬线虫病。这些发现改变了后生动物细胞中有丝分裂退出的组织方式。 我们的发现不是精确的检查点控制，而是染色体分离和NE 装配只通过后期主轴拆卸的时机进行松散的协调。不存在 精确的监管控制可以解释为什么有丝分裂退出过程中的错误经常发生，并代表着一个主要的 造成灾难性基因组重排的根源。提出了一系列细胞生物学实验，以 解决尚未回答的关键问题，例如微管抑制NPC组装的机制。一个 描述了使用日本裂解酵母的易处理系统，这将使我们能够使用强大的基因 了解NE组装及其与有丝分裂完成的协调的工具。 相关性(请参阅说明)： 这一建议解决了真核细胞分裂中的核心问题：控制机制 微管长度、后期纺锤体的大小和功能以及后期纺锤体的整合 在有丝分裂退出期间与其他关键细胞事件一起发挥作用。该项目具有广泛的相关性，因为它 阐述了细胞内结构的大小是如何根据细胞和基因组的大小进行调整的。该项目还拥有 与人类健康相关，因为它提供了对一种重要的突变过程的洞察 癌症基因组中的染色体异常。

This proposal addresses the mechanisms controlling microtubule length, the size and function of the anaphase spindle, and the coordination of anaphase spindle function with other key cellular events during mitotic exit. Because the spindle is a self-organizing structure, the regulation of microtubule length is a major mechanism controlling overall spindle size. Spindle size is controlled globally by the concentration or activity of factors that promote microtubule growth or disassembly. Additionally, “measuring” mechanisms that mediate length-dependent microtubule assembly or disassembly have also been described. The best- studied length-dependent mechanism occurs through the activity of the kinesin-8 family of microtubule motors. Compromised kinesin 8 function in mammalian cells leads to high frequencies of chromosome missegregation and and the formation of abnormal nuclear structures, which are common in cancer, called micronuclei. We recently showed that micronuclei can cause “chromothripsis”, a major mutational process leading to chromosome rearrangement in cancer. In the last funding period, we defined the mechanism by which a yeast kinesin 8 selectively trims longer microtubules. In contrast to previous proposals, a combination of biochemical and single molecule imaging experiments lead to a new conformational switch model, involving kinesin 8 recognition of bent tubulin at the microtubule end, triggering microtubule disassembly. Building on a new high resolution cryo-EM structure and other data, we now propose to test this model and work out the molecular mechanism for bent tubulin recognition. Additionally, in the last funding period we have made a significant advance in understanding how anaphase spindle function is coordinated with the reassembly of the nuclear envelope around chromosomes to form daughter cell nuclei. We found that spindle microtubules block the recruitment of nuclear envelope (NE) containing nucleoporins to decondensing chromosomes, but allow other aspects of NE assembly to occur. This leads to irreversibly defective NE assembly on lagging chromosomes, explaining why the NE around micronuclei undergoes spontaneous disruption, a key step in generating chromothripsis. These findings alter the thinking on the organization of mitotic exit in metazoan cells. Rather than precise checkpoint controls, our findings indicate that chromosome segregation and NE assembly are only loosely coordinated through the timing of anaphase spindle disassembly. The absence of precise regulatory controls can explain why errors during mitotic exit are frequent, and represent a major source of catastrophic genome rearrangements. A series of cell biological experiments is proposed to address key unanswered questions, such as the mechanism by which microtubules inhibit NPC assembly. A tractable system using the fission yeast S. japonicus is described that will enable us to use powerful genetic tools for understanding NE assembly and its coordination with the completion of mitosis. RELEVANCE (See instructions): This proposal addresses centrals questions in eukaryotic cell division: the mechanisms controlling microtubule length, the size and function of the anaphase spindle, and the integration of anaphase spindle function with other key cellular events during mitotic exit. The project has broad relevance because it addresses how the size of an intracellular structure is scaled to cell and genome size. The project also has relevance to human health because it provides insight into an important mutational process generating chromosome aberrations in cancer genomes.