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No description available.

No description available.

No description available.

No description available.

Bacterial cell division relies on the formation and contraction of the Z-ring, coordinated and regulated by a dynamic protein complex called divisome. Here, we show that ZapB, a newly identified cell division factor in Escherichia coli, is recruited to the division site in the early stages of Z-ring assembly by ZapA. Inactivation or overproduction of ZapA caused ZapB delocalization and diffusion. Bacterial two-hybrid and in vitro assays showed that ZapB interacts directly with ZapA and, through it, with FtsZ and that the three proteins together can form a highmolecular- weight complex. Furthermore, during the cell cycle ZapB closely followed FtsZ dynamic localization but interestingly, using high-resolution 3D reconstruction microscopy, we found that it formed a ring located on the inside of the Z-ring, consistently in all cells in consecutive cell division events. Only in the absence of the bacterial actin homologue MreB, ZapB was not able to constrict ahead of FtsZ and instead co-localized seemingly perfectly with the Z-ring. Morphological analysis of cells carrying a zapB deletion and the ftsZ84 allele exhibited a synthetic detrimental phenotype and cell division defects. A model in which ZapB further increases the lateral association of FtsZ filaments by cross-linking ZapA molecules bound to adjacent FtsZ filaments is supported by light scattering assays and analysis of structures formed by FtsZ-ZapA-ZapB using electron microscopy. Surprisingly, ZapB seemed to be active in cell division in the absence of ZapA raising the possibility that ZapB might have a secondary ZapA-independent function.

Entry into mitosis results in a dramatic reorganization of the cellular architecture to allow for segregation of duplicated DNA to nascent daughter cells. This complex biomechanical feat is orchestrated by members of the Cyclin-dependent, Aurora, Polo-like and NIMA-related kinase families. Four NIMA-related kinase proteins are implicated in regulation of mitotic progression, Nek2, Nek9, Nek6 and Nek7. Nek6 and Nek7 are closely related in sequence, encode little more than a catalytic domain and have been implicated in a mitotic NIMA-related kinase cascade downstream of Nek9. Functional data on Nek9 has implicated it in regulation of mitotic spindle architecture and thus Nek6 and Nek7 are also thought likely to function in spindle organization. However, functional data validating such a role is sparse and the roles of Nek6 and Nek7 remain poorly defined. In this thesis I set out to carry out a detailed functional analysis of Nek6 and Nek7, focusing on their proposed roles in mitotic progression. We show that expression of Nek6 and Nek7 mutants whose kinase activity is compromised results in mitotic arrest leading to apoptosis. However, whilst mutants with no activity cause an arrest at metaphase with fragile mitotic spindles, hypomorphic mutants, which retain intermediate levels of activity, result in an arrest in late mitosis. Nek6 and Nek7 interact with γ-tubulin, and interference with Nek6 or Nek7 disturbs the centrosomal localization of γ-tubulin. Nek6 localizes to the microtubules of the mitotic spindle and RNAi depletion of Nek6 leads to destabilization of the spindle microtubules. Thus, Nek6 and Nek7 may function during metaphase to regulate microtubule nucleation both from the spindle poles and from within the spindle itself. Furthermore, we identified the spindle components, Hsp70 and β-tubulin as Nek6 substrates, providing a possible mechanism by which Nek6 may achieve such a role. Finally, we also identified Cortactin A, a regulator of actin dynamics, as a Nek6 substrate. Cortactin A and Nek6 localize to the cleavage furrow of late mitotic cells raising the possibility that Nek6 may be involved in the regulation of membrane dynamics during cytokinesis. Together, the functional data suggests that Nek6 and Nek7 may regulate multiple events during mitosis and the identification of Nek6 substrates provides possible mechanisms by which they might achieve these functions.

The spindle assembly checkpoint (SAC) functions to prevent anaphase onset until all chromosomes are correctly bi-oriented on the mitotic spindle and aligned at the metaphase plate. Cohesion between sister chromatids is essential for this biorientation. In animal cells, most cohesin is removed from chromosome arms during prophase and prometaphase. Cohesin at centromeres is refractory to removal at this stage and persists until metaphase, whereupon its Sccl subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? 1 show that depletion of Sgol by RNA interference in HeLa cells causes precocious loss of centromeric cohesin from chromosomes in prometaphase and a permanent cell cycle arrest, presumably due to inactivation or the spindle 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.