Dissection of kinetochore structure and function in Drosophila

将复制的染色体正确分离到子细胞是生物学的基本组成部分，因为它确保了遗传物质的正确传递。当这个过程在人类卵子的发育过程中没有正确发生时，就会导致后代因为染色体数量错误而不能正确发育。在我们身体的分裂细胞中，染色体分离的错误会使这些细胞容易变成肿瘤细胞。这是因为失去一条携带野生型肿瘤抑制基因副本的染色体，只会留下携带功能错误基因的对应染色体。因此，了解调节染色体分离的机制对于我们理解这些医学科学的关键方面是很重要的。染色体有一个特殊的组成部分，着丝点，它与有丝分裂纺锤体的微管相互作用，有丝分裂纺锤体是染色体进入两个子细胞所必需的分子机器。着丝点为调节染色体附着到微管的分子提供了一个平台，这些分子与监测这一过程是否正确发生的分子一起传递。在这里，我们提出研究果蝇的着丝点是如何构建的，果蝇是一种模式生物，已经被用来研究染色体遗传近100年。了解着丝点是如何构成的将有助于我们理解它的功能。酵母的着丝点包含70多种蛋白质，动物细胞的着丝点可能包含更多。人们认为所有物种的着丝点都包括四个主要的亚复合物。我们的初步研究已经确认了3种蛋白质的身份，这些蛋白质是其中两个亚复合物的成员。这为使用遗传和生化方法的结合来研究这个模型系统中着丝点的组织和功能提供了一条“途径”。我们将研究破坏这两个着丝点亚复合体的影响，通过一个过程，使我们能够从培养细胞中消耗这些蛋白质，并通过研究它们在发育中的苍蝇中的突变体。对于这两种类型的研究，我们将使用延时显微镜来跟踪纺锤体微管，这些微管带有一种蛋白质，使它们发出绿色荧光，而染色体携带一种蛋白质，带有另一种蛋白质，发出红色荧光。这将使我们能够跟踪这个复杂分子机器行为中的缺陷。我们还将研究破坏其中一个亚复合体对其余着丝点组装的影响。在此过程中，我们不仅要研究着丝点的核心成分在最终结构中的招募，还要研究调节染色体附着的分子和机器内单个马达的分子。我们将调整培养的果蝇细胞，使它们产生“标记”的着丝点成分，这样它们就可以从细胞中存在的复杂蛋白质混合物中捞出来。这也会把它们结合的蛋白质捞出来。我们可以用一种仪器来鉴定这种蛋白质，这种仪器可以分析蛋白质分解产物的精确质量与电荷比。这提供了一个特征签名，可以在果蝇的基因蓝图中识别出来。在确定了着丝点的组成部分之后我们将能够研究它们相互作用的方式以及这是如何影响功能的。由于将带负电荷的磷酸基团置于蛋白质上的酶可以影响蛋白质之间的相互作用，因此我们将研究这些酶对构成着丝点的蛋白质复合物内蛋白质相互作用的影响。

The correct segregation of duplicated chromosomes to daughter cells is a fundamental part of biology as it ensures the correct transmission of the genetic material. When this process does not occur correctly in the development of the human egg, it leads to offspring that do not develop correctly because they have the wrong number of chromosomes. Mistakes in chromosome segregation in the dividing cells of our bodies can predispose these cells to turn into tumour cells. This is because loss of one chromosome carrying a wild-type copy of a tumour suppressor gene can leave only its counterpart carrying a mis-functioning gene. Thus an understanding of the mechanisms that regulate chromosome segregation are important for our understanding of these critical aspects of medical science. Chromosomes have a specialised component, the kinetochore, that interacts with microtubules of the mitotic spindle, the molecular machine required for chromosomes to be directed into the two daughter cells. The kinetochore provides a platform for the molecules that mediate chromosome attachment to microtubules and their transmission together with molecules that monitor whether this process occurs correctly. Here we propose to study how a kinetochore is built in the fruit fly, a model organism that has been used to study chromosome inheritance for almost 100 years. An understanding of how a kinetochore is constructed will help us to understand its function. The kinetochores of yeasts comprise over 70 proteins and those of animal cells could contain more. It is thought that kinetochores from all species comprise four main sub-complexes. Our pilot studies have confirmed the identity of 3 proteins that are members of two of these sub-complexes. These gives a 'way in' to the study of the organisation and function of the kinetochore in this model system using a combination of genetic and biochemical approaches. We will study the effects of disrupting these two kinetochore sub-complexes by a process that allows us to deplete these proteins from cultured cells and by studying their mutants in the developing fly. For both types of study we will use time-lapse microscopy to follow spindle microtubules tagged with a protein that makes them fluoresce green and chromosomes that carry a protein tagged with another protein that fluoresces red. This will allow us to follow the defects in the behaviour of this complex molecular machine. We will also examine the consequences of disrupting one of the sub-complexes for the assembly of the rest of the kinetochore. In so doing we will examine not only the recruitment of the core components of the kinetochore into the final structure but also the molecules that regulate chromosomes attachment and those that are individual motors within the machine. We will adapt cultured fruitfly cells so that they make kinetochore components that have been 'tagged' in such a way that they can be fished out from the complex mix of proteins present in the cell. This also fishes out the proteins that they bind to. We can identify such proteins in an instrument that analyses the precise mass to charge ratio of breakdown products from the proteins. This gives a characteristic signature that can be recognised in the genetic blueprint of the fruit fly. Having identified the components of the kinetochore we will be able to study the way in which they interact and how this affects function. As protein-protein interactions can be influenced by enzymes that place negatively charged phosphate groups on proteins, we will study the effects of these enzymes upon the protein interactions within the complexes of proteins that make up the kinetochore.