Analysis of the molecular mechanisms of the Golgi-based G2/M cell cycle checkpoint

This thesis is focused on the role of Golgi fragmentation in the regulation of the G2/M transition of the cell cycle, and it is based on previous findings that Golgi fragmentation is required to enter into mitosis. The Golgi complex is composed of many cisternal stacks that are interconnected by tubules, to form a continuous 'ribbon-like' structure. During mitosis, the Golgi ribbon undergoes extensive fragmentation through a multi stage process that promotes its correct partitioning and inheritance by the daughter cells. The first part of my work is focused on the understanding of the mechanisms which block cells in G2 when Golgi fragmentation is inhibited. I show that the Golgi-dependent G2 arrest is mediated by a failure of centrosome maturation, an event that is essential to achieve activation of the CdkllCyclinB (Cdkl/CycB) complex, the master regulator of mitosis. Indeed, the failure of Golgi fragmentation inhibits the recruitment to and activation at the centrosome of the kinase Aurora-A. This kinase is essential for the activation of Cdkl/CycB at the centrosome. This part of the thesis contributes to the definition of a previously unidentified point of dialogue between the Golgi apparatus and the centrosome in the regulation of G2/M transition. The second part of the thesis describes the development of three novel experimental approaches to induce the block of Golgi fragmentation. They integrate a previously developed assay that is based on the microinjection of blockers of Golgi fragmentation, a reliable but demanding approach. The assays that I have developed are based on the ability of the GRASP65 protein to regulate Golgi fragmentation. As well as being essential for inducing the Golgi checkpoint in a wide cell population, they are also useful for the unravelling of the mechanism through which GRASP65 acts in the Golgi checkpoint.EThOS - Electronic Theses Online ServiceGBUnited Kingdo