The role of Cenp-F and Nudel in chromosome segregation

大图：人类卵子与精子的受精产生了一个单细胞，经过连续的细胞分裂，产生了一个由数十亿细胞组成的人。在每次分裂之前，细胞必须复制和分离其基因组，以便两个子细胞都能获得进一步生长和发育所需的所有遗传信息。由于人类基因组分为46条染色体，分离过程对细胞提出了挑战：关键是每个子细胞接受每条染色体的一个副本。如果分离过程不准确，细胞可能获得或失去染色体。不幸的是，这类事件确实会发生，而且往往与与年龄有关的疾病有关。例如，当一个孩子继承了21号染色体的额外副本时，唐氏综合症就会出现。此外，人类癌细胞通常会出错，因此染色体数量非常不规则。我们有兴趣了解细胞用来准确分离染色体的分子机制，以及这些机制在与衰老相关的疾病中是如何出错的。着丝粒确保准确的染色体分离：当一个细胞准备分裂时，它组装一个双极微管纺锤体，染色体附着在上面。当所有的染色体连接在一起时，它们纵向分裂成两个姐妹染色单体，这两个染色单体被拉到相反的两极。然后细胞沿着纺锤体赤道分裂，这样每个子细胞接收到每个染色单体的一个拷贝。如果要准确地分离染色体，必须满足两个条件。首先，染色体必须是双向的，即姐妹必须连接到相反的极点。其次，因为染色体是同步分裂的，分裂必须在每条染色体都是双向的时候才会发生。着丝粒，特殊的染色体亚结构域在这一过程中起着两个关键作用。首先，它们组装着丝点，这是一种捕获微管并在纺锤体上移动染色体的蛋白质结构。着丝点背靠背地位于每个染色单体上，从而促进了双向定位。重要的是，着丝点还调节着一种监视机制，即纺锤体检查点，它可以防止分裂，直到所有染色体都是双取向的。其次，着丝粒是将姐妹染色单体连接在一起的位置，这一过程被称为内聚。重要的是，必须保持内聚，直到所有的染色体都是双向的：如果过早溶解，姐妹将无法连接到相反的纺锤杆。为了了解着丝粒如何发挥这些功能，我们的策略是关注关键蛋白质，并在分子水平上剖析它们的特性。我们的计划：Cenp-F是一种定位于着丝点的大型多功能蛋白。最近的研究表明，Cenp-F是着丝点-微管相互作用和染色单体内聚所必需的。我们发现Cenp-F结合了一种名为Nudel的蛋白质，这表明Cenp-F的功能可能是通过Nudel介导的。因此，这项建议的一个主要部分是剖析Nudel的作用并确定其与Cenp-F的关系。然而，由于迄今为止用于研究Cenp-F函数的方法的局限性，其确切作用仍然存在争议。此外，Cenp-F可能在染色体分离之外发挥作用。特别是，Nudel在有丝分裂后神经元中明显起作用。此外，Cenp-F是APC-Cdh1的底物，APC-Cdh1是轴突生长和模式以及突触发育和功能所需的酶复合物。此外，cenp - f相关蛋白是肌肉形成所必需的。因此，为了明确地定义Cenp-F的作用，并在整个生物体的更广泛背景下研究它，我们将产生一种携带Cenp-F基因突变的小鼠。这种方法不仅允许我们在体内研究Cenp-F的功能，而且还将提供强大的新的体外系统来梳理这种知之甚少的蛋白质的各种功能

THE BIG PICTURE: The fertilisation of a human egg by a sperm generates a single cell, which, following successive rounds of cell division, creates a person comprised of billions of cells. Before each division, the cell must replicate and segregate its genome such that both daughter cells receive all the genetic information required for further growth and development. Because the human genome is divided into 46 chromosomes, the segregation process presents a challenge to the cell: it is critical that each daughter cell receives one copy of each chromosome. If the segregation process is not accurate, a cell may either gain or lose chromosomes. Unfortunately, such events do occur and they are often associated with age-related diseases. For example, Downs syndrome arises when a child inherits an extra copy of chromosome 21. In addition, human cancer cells routinely make errors and consequently have highly irregular numbers of chromosomes. We are interested in understanding the molecular mechanisms cells use to accurately segregate their chromosomes, and how these mechanisms go wrong in diseases associated with aging. CENTROMERES ENSURE ACCURATE CHROMOSOME SEGREGATION: When a cell is ready to divide, it assembles a bipolar microtubule spindle to which the chromosomes attach. When all the chromosomes are attached, they spilt longitudinally into two sister chromatids which are pulled to opposite poles. The cell then divides down the spindle equator such that each daughter cell receives one copy of each chromatid. If the chromosomes are to be segregated accurately, two criteria must be satisfied. First, the chromosomes must biorient, i.e. sisters must attach to opposite poles. Second, because chromosomes split synchronously, splitting must not occur until every chromosome is bioriented. Centromeres, specialised chromosomal-subdomains play two key functions in this process. Firstly, they assemble kinetochores, protein-structures which capture microtubules and move chromosomes on the spindle. Kinetochores sit back-to-back on each chromatid thus facilitating biorientation. Importantly, kinetochores also regulate a surveillance mechanism, the spindle checkpoint, which prevents splitting until all the chromosomes are bioriented. Second, centromeres are the sites which hold the sister chromatids together, a process know as cohesion. Importantly, cohesion must be maintained until all the chromosomes are bioriented: if prematurely dissolved, sisters will not be able to attach to opposite spindle poles. To understand how centromeres perform these functions, our strategy is to focus on key proteins and dissect their properties at the molecular level. OUR PLAN: Cenp-F is a large multi-functional protein which localises to kinetochores. Recent studies indicate that Cenp-F is required for kinetochore-microtubule interactions and chromatid cohesion. We have discovered that Cenp-F binds a protein called Nudel, suggesting that Cenp-F's function might be mediated via Nudel. A major part of this proposal therefore is to dissect the role of Nudel and define its relationship with Cenp-F. However, because of the limitation of the methodology used thus far to study Cenp-F function, its exact role remains controversial. In addition, Cenp-F may play roles outside of chromosome segregation. In particular, Nudel clearly plays roles in post-mitotic neurons. In addition, Cenp-F is a substrate of APC-Cdh1, an enzyme complex required for axonal growth and patterning, as well as synaptic development and function. Furthermore, Cenp-F-related proteins are required for myogenesis. Therefore, to unambiguously define Cenp-F's role and to study it in the wider context of the whole organism, we will generate a strain of mice harbouring a mutation in the CENP-F gene. Not only will this approach allow us to investigate Cenp-F's function in vivo, but it will also provide powerful new in vitro systems to tease apart the various functions of this poorly understood protei