CENTROSOME CONTROL · Study of centrosome maturation as a pivotal process between G2 checkpoints and mitotic entry in mammalian cells

Final Activity Report Summary - CENTROSOME CONTROL (Study of centrosome maturation as a pivotal process between G2 checkpoints and mitotic entry in mammalian cells)

Cell division is an ordered sequence of events, whose correct execution is ensured by checkpoint control mechanisms. Central to cell division is the mitotic process, during which duplicated chromosomes segregate into daughter cells. The crucial structure operating in chromosome segregation is the mitotic spindle, which is organised by the centrosomes in somatic mammalian cells. At the G2/M transition centrosomes undergo a process termed maturation, which is required for nucleation of mitotic microtubules and hence proper spindle formation. In the proposed program for this grant, I planned to focus on the molecular mechanisms of maturation and the identification of regulators of this process. I also wished to establish whether this process is sensitive to induction of DNA damage. The major results that I have obtained in my studies are: (i) a characterisation of cell cycle processes that implicate Cep170, a novel regulator of maturation that I had identified in my research activity at the Max-Planck Institute (Munich) as a Marie Curie fellow; (ii) a characterisation of the roles, and mutual functional interplay, of two major centrosomal kinases, AuroraA and Plk1, involved in control of spindle assembly and mitotic progression. Activation of kinases generally requires not only upstream enzymes, but also adaptor molecules; among these, the microtubule-binding protein TPX2 is reported to interact with and activate AuroraA. In my postdoc at the Max-Planck Institute, I had been primarily concentrated in the identification of novel targets (or effectors) of the Plk1 kinase. Recently it has been shown that Aurora-A and Plk1 are inactivated following DNA damage in G2, suggesting that a link might exists between cell cycle checkpoints and centrosome activity. Therefore, at completion of postdoc I decided to return to Italy and initiate a new study of these mitotic kinases, from the point of view of their role in centrosome maturation, as this process might possibly represent an important integration point of cellular signals controlling the cell division cycle. To set up this work, I firstly established the RNA interference technique to analyse the function of Aurora-A, Plk1 and TPX2 in centrosome maturation and spindle formation in mammalian cells. I carried out a systematic and comparative analysis, which adds novel information on roles of these three regulators in the mitotic apparatus. To thoroughly analyse the phenotypes caused by inactivation of each of the three regulators, I have set up a small group with a student and part-time collaborations. The data we have obtained indicate that these regulators influence one another in a regulatory cascade that controls early mitotic events at centrosomes and spindle poles. In particular, we report that: (i) mitotic progression is impaired in the absence of each of the factors; (ii) Aurora-A and TPX2 are required for centriole cohesion and spindle bipolarity; (iii) TPX2 is also involved in centrosome maturation; (iv) finally, Plk1 controls both Aurora-A centrosomal localization and TPX2 recruitment to spindle microtubules. Therefore, we have identified a hierarchic relation between Plk1 and the AuroraA / TPX2 pathway, indicating for the first time a direct link between these two crucial mitotic kinases. This is a novel finding that has relevance to both our understanding of regulatory networks in cell division and to cancer research, as reported in our manuscript recently submitted. Both AuroraA and Plk1 are abnormally expressed in cancer and have recently been proposed as possible targets in cancer therapy. Thus, the understanding of their mutual interdependency is of interest for its potential applied outcomes and may help to design specific therapeutic strategies. Therefore, I plan to develop these studies further in the near future.

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