Mechanical activities ensuring accurate chromosome segregation

机械活动确保准确的染色体分离

• 批准号: 10458010
• 负责人: Matthew P Miller
• 金额: $ 38.13万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10458010
• 关键词: Address; Affect; Binding; Binding Sites; Biological; Biological Assay; Cell division; Cells; Chromosome Segregation; Chromosome abnormality; Chromosomes; Congenital Abnormality; Defect; Ensure; Genetic; Human; In Vitro; Kinetochores; Knowledge; Lead; Malignant Neoplasms; Measures; Mechanics; Mediating; Microtubules; Molecular; Monitor; Mutation; Pathway interactions; Process; Proteins; Side; Spontaneous abortion; Structure; Time; Tubulin; Work; base; biochemical tools; biophysical tools; fitness; human disease; innovation; insight; mechanical force; mechanotransduction; neoplastic cell; novel; protein complex; reconstitution; segregation; tool

PROJECT SUMMARY / ABSTRACT: A central aspect of cell division is the faithful segregation of chromosomes. Errors in chromosome segregation are the leading cause of miscarriages, and are associated with the vast majority of tumor cells. Despite this, we know very little about why this process is so defective in these circumstances, in large part because the molecules and mechanisms involved are not fully defined. A highly conserved protein complex, the kinetochore, physically attaches chromosomes to the spindle microtubules that are central to pulling chromosomes apart every time a cell divides. Doing this accurately requires kinetochores to remain persistently attached to a constantly changing substrate, the dynamically growing and shrinking tips of the spindle microtubules. They must also sense when they’re improperly attached and self-correct these errors. How kinetochores carry out these dynamic functions remains largely mysterious. We propose to investigate two key aspects of the kinetochore- microtubule interface. First, we know that the ability to sense tension (mechano-sensing) is vital for monitoring erroneous attachments of chromosomes to the spindle. Yet, how mechanical forces are sensed by and transmitted through this megadalton protein assembly is poorly understood. Second, the kinetochore’s ability to specifically recognize and bind to various microtubule tip structures is also crucial. How the microtubule side of this interface impacts kinetochore attachment fidelity is unknown, and has remained largely unstudied. Understanding these critical questions has proven challenging because of the lack of ways to measure and manipulate the kinetochore’s highly dynamic activities. Prior studies utilized mainly genetic and cell biological approaches, relying on downstream functional readouts that do not directly monitor protein activity. We use an innovative and fundamentally different strategy, combining cutting-edge biochemical and biophysical tools to reconstitute the activities of these protein machines in vitro. The ability to reconstitute these activities allows us to examine both sides of this interface, making our approach uniquely capable of addressing these fundamental questions in innovative ways. Capitalizing on my groundwork in which I identified the first factor, Stu2chTOG, required for a novel mechano-sensing pathway, we have now determined its exact kinetochore binding site via structural analysis and developed genetic tools allowing us to perturb and inducibly re-localize it to kinetochores. We will use these tools to expand insight into the mechanisms of mechano-sensing and extend our knowledge of the factors involved. We will also examine which kinetochore activities are affected by alterations to its microtubule substrate. We have identified numerous tubulin mutations that specifically disrupt chromosome segregation, and our reconstitution-based assays now give us the unique ability to understand this mechanistically. This approach constitutes the first direct examination of the microtubule side of this interface. I anticipate this work will greatly increase our understanding of how kinetochores work to segregate chromosomes and how defects arise in human disease.

项目摘要/摘要： 细胞分裂的一个核心方面是染色体的忠实分离。 是流产的主要原因，并且与大量肿瘤细胞有关。 我们对为什么这个过程在这些情况下如此有缺陷知之甚少，很大程度上是因为 所涉及的分子和机制尚未完全确定。 将染色体物理附着在纺锤体微管上，而纺锤体微管对于将染色体拉开至关重要 每次细胞分裂都需要着丝粒持续附着在细胞上。 不断变化的基质，纺锤体微管的动态生长和收缩的尖端。 动粒也能感知它们何时附着不当并自我纠正这些错误。 我们建议研究动粒的两个关键方面—— 首先，我们知道感知张力（机械传感）的能力对于监测至关重要。 然而，染色体与纺锤体的错误附着是如何被感知的。 其次，我们对动粒的传递能力知之甚少。 特异性识别并结合各种微管尖端结构也至关重要。 该界面对着丝粒附着保真度的影响尚不清楚，并且在很大程度上尚未得到研究。 事实证明，理解这些关键问题具有挑战性，因为缺乏衡量和解决问题的方法。 先前的研究主要利用遗传和细胞生物学来操纵动粒的高度动态活动。 方法，依赖于不直接监测蛋白质活性的下游功能读数。 创新且根本不同的策略，结合尖端的生化和生物物理工具 在体外重建这些蛋白质机器的活性使我们能够重建这些活性。 检查该界面的两侧，使我们的方法能够独特地解决这些基本问题 利用我确定第一个因素 Stu2chTOG 的基础工作， 一种新的机械传感途径所需的，我们现在已经通过以下方式确定了其确切的着丝粒结合位点 结构分析和开发的遗传工具使我们能够扰乱并诱导地将其重新定位到着丝粒。 我们将使用这些工具来扩展对机械传感机制的洞察并扩展我们的知识 我们还将研究哪些动粒活动受到其改变的影响。 我们已经鉴定出许多特异性破坏染色体的微管蛋白突变。 分离和基于重组的测定现在使我们具有理解这一点的独特能力 从机制上讲，这种方法构成了对该界面的微管侧的第一次直接检查。 预计这项工作将大大增加我们对动粒如何分离染色体的理解 以及人类疾病中缺陷是如何产生的。