Cell Cycle Control of the Cytoskeleton

细胞骨架的细胞周期控制

• 批准号: 10454822
• 负责人: DAVID S PELLMAN
• 金额: $ 34.71万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10454822
• 关键词: Address; Anaphase; Biochemical; Biological; Cell Cycle Regulation; Cell Nucleus; Cell division; Cells; Chromosome Segregation; Chromosome abnormality; Chromosomes; Cryoelectron Microscopy; Cytoskeleton; Data; Eukaryotic Cell; Event; Family; Fission Yeast; Frequencies; Funding; Genetic; Genome; Growth; Health; Human; Instruction; Kinesin; Lead; Length; Malignant Neoplasms; Mammalian Cell; Measures; Mediating; Microtubules; Mitosis; Mitotic; Modeling; Molecular; Molecular Conformation; Motor; Mutation; Nuclear Envelope; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nuclear Structure; Process; Regulation; Resolution; Series; Source; Structure; Subcellular structure; System; Testing; Thinking; Tubulin; Work; Yeasts; cancer genome; chromosome missegregation; chromothripsis; daughter cell; experimental study; insight; molecular imaging; recruit; single molecule; tool

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.

这一建议涉及控制微管长度的机制，微管的大小和功能， 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，微管长度的调节是一个主要的 机械控制整个主轴尺寸。纺锤体尺寸由浓度或活性全局控制 促进微管生长或分解的因素。此外，“测量”机制， 还描述了介导的长度依赖性微管组装或拆卸。最好的- 所研究的长度依赖性机制通过微管的驱动蛋白-8家族的活性发生 电动机.在哺乳动物细胞中受损的驱动蛋白8功能导致染色体的高频率 错误分离和异常核结构的形成，这在癌症中很常见，称为 微核我们最近发现微核可以引起“染色体断裂”，这是一个主要的突变过程 导致癌症中的染色体重排。 在上一个资助期间，我们确定了酵母驱动蛋白8选择性地修剪更长时间的机制， 微管与以前的建议相反，生物化学和单分子成像的组合 实验导致了一个新的构象转换模型，涉及驱动蛋白8识别弯曲的微管蛋白， 微管末端，触发微管解体。建立一种新的高分辨率冷冻EM结构 和其他数据，我们现在建议测试这个模型，并找出弯曲微管蛋白的分子机制 识别.此外，在上一个供资期，我们在了解 后期纺锤体功能如何与核被膜周围的重组协调， 染色体形成子细胞核。我们发现纺锤体微管阻止了 核被膜（NE）含有核孔蛋白，以解致密染色体，但允许其他方面的 发生NE组装。这导致落后染色体上不可逆的有缺陷的NE组装， 这解释了为什么微核周围的NE会发生自发破坏，这是产生微核的关键步骤。 色裂病这些发现改变了对后生动物细胞有丝分裂退出组织的看法。 我们的研究结果表明，染色体分离和NE，而不是精确的检查点控制， 装配只是通过后期主轴拆卸的时机松散地协调。没有 精确的调节控制可以解释为什么有丝分裂退出过程中的错误是频繁的，并代表了一个主要的 是灾难性基因组重排的源头提出了一系列细胞生物学实验， 解决关键的未回答的问题，如微管抑制NPC组装的机制。一 使用裂殖酵母S.它将使我们能够利用强大的遗传 理解NE组装及其与有丝分裂完成的协调的工具。 相关性（参见说明）： 这一提议解决了真核细胞分裂的核心问题： 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起作用。该项目具有广泛的相关性，因为它 解决了细胞内结构的大小如何按比例缩放到细胞和基因组大小。该项目还有 与人类健康的相关性，因为它提供了一个重要的突变过程， 癌症基因组中的染色体畸变。