Cell Cycle Control of the Cytoskeleton

细胞骨架的细胞周期控制

• 批准号: 9980909
• 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/9980909
• 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.

该提案解决了控制微管长度，大小和功能的机制 后期纺锤，以及后期纺锤函数与其他关键细胞事件的协调 有丝分裂出口。由于主轴是自组织结构，因此微管长度的调节是主要的 控制整体主轴尺寸的机制。主轴尺寸通过浓度或活性在全球控制 促进微管生长或拆卸的因素。此外，“测量”机制 还描述了介导的长度依赖性微管组件或拆卸。最好的- 螺学的长度依赖性机制是通过微管的驱动蛋白-8家族的活性发生的 电动机。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 在癌症中常见的异常核结构的异常核分析和形成称为 微核。我们最近表明，微核可能会引起“ Chromothripsis”，这是一个主要的突变过程 导致癌症的染色体重排。 在最后一个资金期间，我们定义了酵母动力蛋白8选择性修剪更长的机制 微管。与以前的建议相反，生化和单分子成像的组合 实验导致了新的会议开关模型，涉及动机8识别弯曲微管蛋白 微管端，触发微管拆卸。建立新的高分辨率冷冻结构 和其他数据，我们现在建议测试该模型并确定弯曲微管蛋白的分子机制 认出。此外，在最后的资金期间，我们在理解方面取得了重大进步 后期的主轴函数如何与核包膜的重新组装一起协调 形成子细胞核的染色体。我们发现纺锤微管阻止了 核包络（NE）含有核oporins以对染色体进行反应，但允许其他方面 内组件发生。这导致在滞后染色体上不可逆转地有缺陷的NE组装， 解释为什么Micronuclei周围的NE会受到赞助破坏，这是生成的关键步骤 Chromothripsis。这些发现改变了对后生细胞中有丝分裂出口的组织的思维。 我们的发现不是精确检查点控件，而是表明染色体隔离和NE 组装仅通过后期纺锤体拆卸的时机松散地协调。没有 精确调节控制可以解释为什么有丝分裂出口期间的错误经常是主要的，并且代表主要 灾难性基因组重排的来源。提出了一系列细胞生物学实验 解决关键的未解决问题，例如微管抑制NPC组装的机制。一个 描述了使用裂变酵母S. japonicus的可进行的系统，这将使我们能够使用强大的通用 理解NE组装及其与有丝分裂的协调的工具。 相关性（请参阅说明）： 该提案解决了真核细胞部门的中心问题：控制机制 微管长度，后期主轴的大小和功能以及后期纺锤的整合 有丝分裂出口期间其他关键细胞事件的功能。该项目具有广泛的相关性，因为它 解决细胞内结构的大小如何缩放到细胞和基因组大小。该项目也有 与人类健康相关，因为它可以洞悉重要的突变过程产生 癌症基因组中的染色体畸变。