Cooperativity and forces in molecular interactions governing chromosome stability

活细胞不断地自我重组；这样它们就可以适应不断变化的环境，最终分裂并将其遗传物质转移到子细胞。这些过程都需要细胞以精确的方式对其内容物施加机械力。一些细胞内的运动是由微管推动的，微管是一种可以生长和缩短的蛋白质聚合物，它的末端可以拉住细胞的某些部分。微管提供的拉力对细胞分裂尤其重要，因为当遗传物质的两个副本--紧凑成染色体的DNA--需要在空间中物理分离时。染色体如何保持连接或连接到微管末端，微管末端随着染色体的缩短而解体，人们对此知之甚少。对这种“偶联”很重要的蛋白质成分在着丝粒中以多个副本的形式存在，着丝粒是一种将染色体与微管结合在一起的结构。保持动粒蛋白拷贝数的平衡可以防止染色体丢失，而这些蛋白的缺失和突变与癌症有关。我们有证据表明，动粒中相同的蛋白质相互作用，但这些相互作用的机制在活细胞中的研究是具有挑战性的。为了了解动粒蛋白质是如何联合起来正确地将染色体连接到微管上的，我们将在体外使用纯化的成分重建这些连接。我们将关注人类动粒的两个组成部分：连接动粒和微管末端的NDC80复合体和动态积累在NDC80-微管界面并稳定它的Ska复合体。Ska和Ndc80都是细胞存活所必需的，并且它们都以多个拷贝存在于着丝粒。利用光学和电子显微镜，我们将确定Ska复合体的结构域，这些结构域对于相邻的Ska分子之间的相互作用是重要的。通过突变这些结构域，我们将区分Ska：skA与Ska-微管和Ska：Ndc80的相互作用，从而更好地理解Ska是如何在正确形成的染色体-微管连接上具体积累的。一旦我们确定了控制Ska积累的相互作用，我们将研究Ska和Ndc80在微管拉动细胞时如何相互作用以及如何与自己相互作用。利用先进的光学显微镜技术，我们将研究Ska和Ndc80在力产生地点的积累。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-微管连接稳定的机制。在这项工作中，我们重点研究人类动粒的两个组成部分。然而，许多动粒-微管相互作用在其他物种中是保守的。由此得到的数据将使我们能够开始研究力是如何影响动心的组成和性能的。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扰乱细胞分裂的治疗方法，例如阻止癌细胞增殖的药物。

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.