How are mono-oriented chromosome-microtubule attachments protected to prevent errors in mitosis and associated cellular ageing?

当母细胞一分为二时，它的染色体被绳状微管拉成相等的两组。染色体-微管连接的错误会导致染色体的丢失或获得，导致细胞中染色体数目不规则-这是动物和人类不育和几种过早衰老综合征的标志。为了精确地找出某些疾病中染色体数目不正确的原因，对微管如何捕获和拉开染色体的分子理解是必不可少的。染色体-微管的附着是由近100种蛋白质组成的大分子结构介导的。Draviam小组报告了一种蛋白质复合物Astrin-SKAP，它在正确的染色体微管附着形成后不久就被招募到着丝粒中，并且该复合物是维持正确附着所必需的。我们还不知道阿斯特林是如何感知依恋状态以及它是如何稳定依恋状态的。通过结合结构生物学（Pickersgill实验室）和进化生物学（Martin-Duran实验室）的方法，Draviam小组表明，Astrin与外动粒蛋白HEC 1相互作用，而Astrin提供了一种酶PP1-磷酸酶。仔细安排收缩素介导的PP1递送以选择性地稳定正确的附着。换句话说，Astrin就像一个“信使”，有选择地到达正确连接的动粒，提供稳定附件所需的“工具”。这意味着确定Astrin-HEC 1和Astrin-PP 1相互作用是如何控制的，将揭示细胞如何确保正确的染色体-微管附着并防止染色体错误分离。这个项目是及时的，因为它利用了超分辨率显微镜（由BBSRC资助）以尽可能高的空间分辨率跟踪外动粒的动态变化。首先，Astrin到达动粒将与外动粒的纳米级结构变化相关，以了解正确附着的特定变化。第二，确定HEC 1或PP 1相互作用所必需的Astrin区域，并在细胞中表达不能与HEC 1或PP 1结合的Astrin突变体，以研究Astrin如何感知附着以及细胞如何安排Astrin-PP 1相互作用，以确保染色体的准确分离。为了进一步加强研究计划，计划开展两项合作：（i）使用Owenia胚胎裂解物的下拉，以从进化上保守的Astrin相互作用中获得线索。(ii)蛋白质结构的计算建模，从HEC 1和PP 1晶体结构中获取线索，用于设计Astrin相互作用突变体。将研究破坏染色体微管附着和染色体分离准确性或时间的Astrin突变体，以了解它们促进细胞衰老的程度，无论是立即（几小时内）或长期（几天内），通过跟踪压力标记，DNA损伤和修复。这将是第一个纳米级的动态变化研究，在外部-动粒，其保护正确附着并防止染色体错误分离。同时跟踪附着缺陷、分离不准确和过早老化的单细胞使该项目在分离导致老化的有丝分裂错误方面具有独特和宝贵的价值。这些知识可以帮助建立生物标志物来预测和跟踪动物和人类的过早衰老。这里关于微管介导的染色体拉动或推动的基本发现将广泛用于我们体内的其他微管介导的过程。例如，神经元生长、纺锤体旋转、免疫信号传导和细胞迁移都依赖于调节开关来感知和稳定细胞不同部位的微管。因此，这项研究将广泛用于了解细胞内的力产生机制。

When a mother cell divides into two, its chromosomes are pulled apart into two equal sets by rope-like microtubules. Errors in chromosome-microtubule attachment can result in the loss or gain of chromosomes, leading to irregular chromosome numbers in cells - a hallmark of animal and human infertility and several premature ageing syndromes. To precisely pinpoint why chromosome numbers are incorrect in some diseases, a clear molecular understanding of how microtubules capture and pull chromosomes apart is essential.Chromosome-microtubule attachment is mediated by a macromolecular structure - the kinetochore - made of nearly 100 proteins. The Draviam group reported a protein complex Astrin-SKAP that is recruited to kinetochores soon after the formation of correct chromosome-microtubule attachments, and the complex is required for maintaining correct attachments. How Astrin senses attachment status and how it stabilises them are not known. These will be addressed to explain how Astrin ensures the accurate segregation of chromosomes.By combining methods in structural biology (Pickersgill lab) and evolutionary biology (Martin-Duran lab), the Draviam group showed that Astrin interacts with an outer-kinetochore protein, HEC1, and Astrin delivers an enzyme PP1-phosphatase. Astrin-mediated delivery of PP1 is carefully scheduled to selectively stabilise correct attachments. In other words, Astrin works like a 'messenger' arriving selectively at correctly attached kinetochores to deliver a 'tool' that is needed to stabilise the attachments. This means that determining how Astrin-HEC1 and Astrin-PP1 interactions are controlled will unravel how cells ensure proper chromosome-microtubule attachments and prevent chromosome missegregation.This project is timely as it takes advantage of a Super-resolution microscope (funded by BBSRC) to track dynamic changes at the outer kinetochore, at the highest spatial resolution possible. First, Astrin's arrival at kinetochores will be correlated with nanoscale structural changes at the outer-kinetochore to learn about changes specific to correct attachments. Second, the regions of Astrin essential for HEC1 or PP1 interaction will be determined, and mutants of Astrin that cannot bind to HEC1 or PP1 will be expressed in cells to study how Astrin senses attachments and how cells schedule Astrin-PP1 interaction to ensure the accurate segregation of chromosomes.To further strengthen the research program, two collaborations have been planned: (i) Pull-downs using Owenia embryo lysates to take clues from evolutionarily conserved Astrin interactions.(ii) Computational modelling of protein structure to take clues from HEC1 and PP1 crystal structures for designing Astrin interaction mutants.Astrin mutants that disrupt chromosome-microtubule attachment and chromosome segregation accuracy or timing will be studied for the extent to which they promote cellular ageing, either immediately (within hours) or in the long-term (in days), by tracking markers for stress, DNA damage and repair.This will be the first nano-scale study of dynamic changes at the outer-kinetochore which protect correct attachments and prevent chromosome missegregation. Simultaneous single-cell tracking of attachment defects, segregation inaccuracy and premature ageing make this project unique and invaluable for isolating mitotic errors that cause ageing. This knowledge can help build biomarkers to predict and track premature ageing in animals and humans.Fundamental discoveries made here about microtubule-mediated pulling or pushing of chromosomes will be widely useful for other microtubule-mediated processes in our body. For instance, neuronal growth, spindle rotation, immune signalling and cell migration are all reliant on regulatory switches to sense and stabilise microtubules in different parts of the cell. Thus the study will be broadly useful to understand force generation mechanisms within cells.