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

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.

The APC/C is a ubiquitin ligase complex, which controls critical cell cycle events through proteolysis. The centrosome is the primal microtubule organising centre in animal cells, facilitating the mitotic spindle formation. To ensure genome stability, centrosome duplication and maturation have to be tightly coupled to cell cycle progression. Although it has been known that the APC/C controls levels of some mitotic regulators such as Polo and Aurora A, its broader roles at the centrosome remain to be elucidated. Here we propose to investigate the roles of the APC/C in the cell regulation of centrosome function and duplication in Drosophila melanogaster. We will conduct biochemical and proteomic screens of centrosomal proteins for APC/C substrates and interactors. In pilot studies we have found that Plk4 and Spd-2 co-purify with the APC/C. Centriole duplication is driven by Plk4 whose level is suppressed by a second ubiquitin-protein ligase, SCF, to prevent centriole over-amplification. Additional regulation by the APC/C could account for the instability of Plk4 in anaphase and G1. We will examine whether Plk4 is subject to dual control by both APC/C and SCF and determine the precise role of its destruction in centriole duplication. Spd-2 strongly interacts with the APC/C activator Fzr and thus is a strong candidate as a platform for the Fzr-APC/C localisation and activation at the centrosome. We will investigate the role of the Spd-2-Fzr interaction in APC/C recruitment and centrosome maturation. Our recent data suggest that Spd-2 functions directly downstream of Polo to activate centrosomes in M-phase. Thus, we will examine how the APC/C-dependent regulation of Spd-2, Polo and Aurora A contributes to centrosome inactivation upon mitotic exit. Finally, we will investigate the APC/C-dependent regulation of the asymmetric centrosomes of neuroblasts in the cell divisions that both renew stem cell identity and produce differentiated daughters.

Health service provision and training There is potential impact upon our understanding of the processes of abnormal chromosome segregation as occurs during oncogenesis. Supernumerary centrosomes have been known to be a signature of cancer cells for over a century. Here we will gain understanding of how controlled protein degradation regulates centrosome number and function. The study will give increased understanding basic biology per se and provide valuable practical post-doctoral training. Pharmaceutical Industry The proteolytic degradation machinery that regulates centrosome function is only recently being recognized as a potential target for the treatment of proliferative disease. Thus increased understanding of the basic biology of its mechanism of action will add to the knowledge base that informs decisions about the development of such potential targets. Although this application is not directly focused upon cancer, it does aim to increase our understanding of the behaviour of centrosomes, a topic of relevance to cancer studies because of chromosome non-disjunction in cancer cells. An understanding of the regulation of centriole function also has relevance to a large number of ciliary diseases. Technical approaches The applicants are then aware of the possibilities of practical applications that may emerge, particular in the cancer field, from the research and will be attentive to such possibilities. Promoting the Impact of the Research To date, beneficiaries in the Health Services and Pharmaceutical Industry have been engaged through collaborative work and through presentations made to these bodies. Engagement and communication will be continued with Health Service Providers and the Pharmaceutical Industry through meetings with groups known to us. Other groups can be contacted through 'break-out' sessions in larger scientific meetings as opportunities arise. Our lab websites form an interface with the wider world for both beneficiary and lay audiences. The wider public There are excellent interactions between the University of Cambridge and its surrounding public through activities such as Science Week that encourages investigators to engage in making presentations to the general public. Health Services DMG is a member of the University Cancer Centre and has excellent contact with clinical colleagues in the University of Cambridge and at other UK and international sites. Pharmaceutical Industry: DMG works closely with Cyclacel and in the course of this work has had interactions with R & D teams of most of the major Pharmaceutical companies. This provides invaluable contacts for collaborative work and in exploiting any findings that might be made. DMG engages in presenting the work to the general public and to less specialised colleagues in Health Care and in the Pharmaceutical Industry.