Roles for the APC/C in the centrosome cycle and the centrosomal regulation of the APC/C activity

为了保持从一代细胞到下一代细胞的基因活性的正确平衡，在细胞分裂过程中高保真地分割遗传物质是至关重要的。两个子细胞应该从它们的母细胞获得相同数量的染色体。携带所有遗传信息的染色体被称为有丝分裂纺锤体的细胞装置平等地分离成两个子细胞。中心体位于有丝分裂纺锤体的两端，集中在构成大多数动物细胞纺锤体的分子纤维或微管上。中心体的丢失或过度放大会导致有丝分裂纺锤体的缺陷，从而导致灾难性的细胞分裂，导致遗传信息的丢失，并可能促进癌症等疾病的发展。在过去的十年中，中心体通过组织干细胞持续更新所需的纺锤体的特定方向，在干细胞的维持中发挥着至关重要的作用。因此，进一步了解中心体复制和功能的分子机制将具有重要的科学和医学意义。中心体包括两个亚结构：中心粒和着丝粒周围物质(PCM)。中心粒是圆柱形的结构，其中两个在中心体中相互连接。中心粒在每个细胞分裂周期复制一次，并遗传到两个子细胞中。中心粒复制是一个进化保守的过程，因此可以在果蝇黑腹果蝇的模式生物中进行研究。通过这种方式，我们的团队发现Plk4是控制中心粒复制的关键酶。PCM含有许多组织微管所需的蛋白质。在有丝分裂中，它聚集在中心体，以形成有丝分裂纺锤体。Polo和Aurora A蛋白激酶是触发这一过程的主要因素，通过在PCM蛋白上添加磷酸基团来改变其性质。细胞分裂后，PCM变小，中心体变得不那么活跃。我们称之为“中心体回归”的这一过程仍未得到充分的研究。为了确保基因组信息的正确遗传，中心体结构和功能的变化必须与细胞分裂过程相协调。最近，我们和其他人发现，这在一定程度上涉及泛素-蛋白酶体途径，这是一种在不再需要蛋白质时破坏蛋白质的机制。一种称为后期促进复合体或环体的蛋白质复合体(APC/C)专门针对选定的蛋白质进行途径破坏，是细胞分裂的关键调节因子之一。有了这笔BBSRC赠款，我们想要探索APC/C如何参与协调中心体活动和复制与细胞分裂周期。我们将确切地确定哪些中心体蛋白与APC/C相互作用，并将研究这种相互作用在中心体周期中的作用。我们发现Plk4与APC/C结合，提示APC/C可能与功能相似的互补复合体SCF一起调节中心粒复制。虽然人们早就知道APC/C在细胞分裂结束时以Polo和Aurora A为目标降解，但这在中心体是如何调节的还没有研究。我们还发现，一种特定的PCM蛋白Spd-2与一种名为泡沫相关的蛋白强烈结合，该蛋白调节APC/C与其靶标的关联。因此，我们想要了解这些相互作用如何调节特定蛋白质的破坏的空间和时间控制，以调节中心体的复制和功能。

To maintain the correct balance of gene activity from one generation of cells to the next, it is crucial to partition the genetic material during cell division with the high fidelity. The two daughter cells should receive equal number of chromosomes from their mother cell. Chromosomes, which carry all the genetic information, are segregated equally into the two daughter cells by the cellular apparatus named the mitotic spindle. The centrosome is a body located at both ends of the mitotic spindle that focuses the molecular fibres or microtubules that constitute the spindle in most of animal cells. The loss or the over-amplification of centrosomes results in defective mitotic spindles, thereby disastrous cell division that leads to loss of genetic information and potentially facilitates development of diseases such as cancer. In the last decade it has also become clear that the centrosome plays a crucial role in the maintenance of stem cells by organising the specific orientation of the spindle required for constant stem cell renewal. Thus, increased understanding of the molecular mechanisms of centrosome duplication and function will have significant scientific and medical implications. The centrosome comprises two substructures: centrioles and the pericentriolar material (PCM). Centrioles are cylindrical structures, two of which are connected to each other in the centrosome. Centrioles duplicate once every cell division cycle and are inherited into the two daughter cells. Centriole duplication is an evolutionarily conserved process and so can be studied in a model organism, the fruit fly Drosophila melanogaster. In this way our group discovered that the PLK4 is a critical enzyme for controlling the centriole duplication. The PCM contains numerous proteins required to organise microtubules. It accumulates at centrosomes in mitosis in order to form the mitotic spindle. Polo and Aurora A protein kinases are the primal factors that trigger this process by adding phosphate groups onto PCM proteins to change their properties. After cell division the PCM diminishes in size and the centrosome becomes less active. This process that we term 'centrosome regression' remains entirely understudied. In order to ensure the proper inheritance of the genomic information, changes in centrosome structure and function have to be coordinated with the process of cell division. Recently, we and others discovered that in part this involves the ubiquitin-proteasome pathway, a mechanism for destroying proteins when they are no longer needed. A protein complex called the anaphase-promoting complex or cyclosome, (APC/C) specifically targets selected proteins for destruction by pathway and is one of the key regulators of cell division. With this BBSRC grant, we would like to explore how the APC/C participates in coordinating centrosome activity and duplication with the cell division cycle. We will determine exactly which centrosomal proteins interact with the APC/C and will investigate the roles of this interaction in the centrosome cycle. We have found that PLK4 binds to the APC/C, suggesting that the APC/C might regulate the centriole duplication together with a complementary complex of similar function called the SCF. Although it has been long known that the APC/C targets Polo and Aurora A for degradation in the end of cell division, how this is regulated at the centrosome has not been investigated. We have also discovered that a particular PCM protein, Spd-2, strongly binds to a protein called Fizzy-related that regulates the association of the APC/C with its targets. Hence, we would like to understand how these interactions mediate the spatial and temporal control of the destruction of specific proteins to regulate centrosome replication and function.