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

当母细胞分裂成两个时，它的染色体被绳状微管拉成两组相等的染色体。染色体与微管连接的错误可导致染色体的丢失或增加，导致细胞中染色体数目不规则——这是动物和人类不孕症和几种早衰综合征的标志。为了精确地查明为什么染色体数目在某些疾病中是不正确的，对微管如何捕获和拉开染色体的清晰的分子理解是必不可少的。染色体与微管的连接是由一种大分子结构介导的，即由近100种蛋白质组成的着丝点。Draviam小组报告了一种蛋白质复合物Astrin-SKAP，在正确的染色体微管附着体形成后不久就被招募到着丝点上，并且该复合物是维持正确附着体所必需的。Astrin如何感知依恋状态以及如何稳定依恋状态尚不清楚。我们将解释Astrin如何确保染色体的精确分离。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小组发现Astrin与外部着丝点蛋白HEC1相互作用，Astrin传递一种酶pp1 -磷酸酶。astrin介导的PP1递送是精心安排的，以选择性地稳定正确的附着物。换句话说，Astrin就像一个“信使”，有选择地到达正确连接的着丝点，传递稳定附件所需的“工具”。这意味着确定Astrin-HEC1和Astrin-PP1相互作用如何被控制将揭示细胞如何确保适当的染色体微管附着并防止染色体错分离。这个项目是及时的，因为它利用超分辨率显微镜（由BBSRC资助）以最高的空间分辨率跟踪外部着丝点的动态变化。首先，Astrin到达着丝点将与外部着丝点的纳米级结构变化相关联，以了解正确附着物的特定变化。其次，确定Astrin对HEC1或PP1相互作用所必需的区域，并在细胞中表达不能与HEC1或PP1结合的Astrin突变体，研究Astrin如何感知附着物以及细胞如何安排Astrin-PP1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，我们计划开展两项合作：(i)利用欧温虫胚胎裂解物进行下拉，从进化上保守的Astrin相互作用中寻找线索。（ii）蛋白质结构的计算建模，从HEC1和PP1晶体结构中获取线索，设计Astrin相互作用突变体。通过追踪压力、DNA损伤和修复的标记，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突变体在多大程度上促进细胞衰老，要么是立即（在几小时内），要么是长期（在几天内）。这将是第一次在纳米尺度上研究外着丝点的动态变化，以保护正确的附着和防止染色体错误分离。同时对单细胞的附着缺陷、分离不准确和过早老化进行跟踪，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或推的基本发现将对我们身体中其他微管介导的过程广泛有用。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，这项研究将广泛地用于理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.