Cooperativity and forces in molecular interactions governing chromosome stability

控制染色体稳定性的分子相互作用中的合作性和作用力

• 批准号: BB/X014975/1
• 负责人: Vladimir Volkov
• 金额: $ 91.41万
• 依托单位: Queen Mary University of London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX014975%2F1
• 关键词: Cooperativity; forces; molecular; interactions; governing

Living cells constantly rearrange themselves; this way they can adapt to the changing environment, and eventually divide and transfer their genetic material to daughter cells. These processes all require mechanical force to be applied by the cells to their contents in a precise manner. Some of the intracellular movements are fuelled by microtubules, protein polymers that can grow and shorten, and pull on parts of the cell with their ends. Pulling forces provided by microtubules are especially important for division of the cells, when two copies of the genetic material, DNA compacted into chromosomes, need to be physically separated in space. How chromosomes keep attached, or coupled, to microtubule ends which are falling apart as they shorten, is poorly understood. Protein components that are important for this 'coupling' are present in multiple copies in a kinetochore, a structure that binds chromosomes to microtubules. Keeping the copy number of kinetochore proteins in balance prevents chromosome loss, while deletions and mutations in these proteins are associated with cancer. We have evidence that identical proteins in the kinetochore interact with each other, but the mechanisms of these interactions are challenging to study in living cells.To understand how kinetochore proteins team up to properly attach chromosomes to microtubules, we will recreate these attachments using purified components in vitro. We will focus on two components of human kinetochore: Ndc80 complex, which cross-links kinetochores and microtubule ends, and Ska complex that dynamically accumulates at the Ndc80-microtubule interface and stabilizes it. Both Ska and Ndc80 are essential for cell viability, and both of them are present at kinetochore in multiple copies. Using light and electron microscopy, we will determine domains of the Ska complex that are important for interactions between neighbouring Ska molecules. By mutating these domains, we will distinguish Ska:Ska interactions from Ska-microtubule and Ska:Ndc80 interactions, leading to a better understanding how accumulation of Ska is specifically happening at properly formed chromosome-microtubule attachments. Once we have identified the interactions that control Ska accumulation, we will study how Ska and Ndc80 interact with each other and with themselves as microtubules pull on kinetochores. Using advanced light microscopy techniques, we will study accumulation of Ska and Ndc80 at the site of force generation. We will also study Ska with impaired self-interactions to further understand the mechanism of Ska-mediated stabilization of chromosome-microtubule attachments. In this work, we focus on two components of the human kinetochore. However, many kinetochore-microtubule interactions are conserved in other species. The resulting data will allow us to start investigating how force influences the composition and performance of kinetochores. Detailed understanding of the mechanism of cell division will help researchers to design more specific treatments that disrupt cell division, for example drugs that stop cancer cells from proliferating.

活细胞不断地重新排列自己;这样它们就可以适应不断变化的环境，并最终分裂并将其遗传物质转移到子细胞中。这些过程都需要细胞以精确的方式向其内容物施加机械力。一些细胞内的运动是由微管，蛋白质聚合物，可以生长和缩短，并拉在细胞的一部分与他们的结束。微管提供的拉力对于细胞分裂特别重要，当遗传物质的两个拷贝，DNA压缩成染色体时，需要在空间中物理分离。染色体如何保持附着或耦合到微管末端，这些末端在缩短时会分开，人们对此知之甚少。对于这种“耦合”很重要的蛋白质成分以多个拷贝存在于着丝粒中，着丝粒是一种将染色体与微管结合的结构。保持动粒蛋白质的拷贝数平衡可以防止染色体丢失，而这些蛋白质的缺失和突变与癌症有关。我们有证据表明，相同的蛋白质在动粒相互作用，但这些相互作用的机制是具有挑战性的活细胞中的研究。为了了解如何动粒蛋白团队正确地将染色体连接到微管，我们将重新创建这些附件使用纯化的成分在体外。我们将重点关注人类动粒的两个组成部分：Ndc 80复合物，其交联动粒和微管末端，以及Ska复合物，其在Ndc 80-微管界面动态积累并稳定它。Ska和Ndc 80都是细胞活力所必需的，并且它们都以多个拷贝存在于动粒。使用光学和电子显微镜，我们将确定域的斯卡复杂的是重要的相邻斯卡分子之间的相互作用。通过突变这些结构域，我们将区分Ska：Ska相互作用与Ska-微管和Ska：Ndc 80相互作用，从而更好地了解Ska的积累是如何在正确形成的染色体-微管附件中发生的。一旦我们确定了控制Ska积累的相互作用，我们将研究Ska和Ndc 80如何相互作用，以及微管如何拉动动粒。使用先进的光学显微镜技术，我们将研究积累的斯卡和Ndc 80的网站的力量产生。我们还将研究自身相互作用受损的Ska，以进一步了解Ska介导的染色体微管附着稳定化的机制。在这项工作中，我们专注于人类动粒的两个组成部分。然而，在其他物种中，许多激动剂微管相互作用是保守的。由此产生的数据将使我们能够开始研究力如何影响动粒的组成和性能。对细胞分裂机制的详细了解将有助于研究人员设计更具体的治疗方法来破坏细胞分裂，例如阻止癌细胞增殖的药物。