The centrosome is the major microtubule organising centre in vertebrate cells. CDK5RAP2 is a human protein that localises to the centrosome. At the start of this thesis work, the function of CDK5RAP2 was uncharacterised. Significantly, cdk5rap2 is one of several centrosomal genes that are mutated in the developmental disorder Primary Microcephaly, where affected individuals have smaller brains than expected for the age- and sex-adjusted mean. Orthologues of CDK5RAP2 in the fruit fly (Centrosomin/Cnn) and in fission yeast (Mod20p) have been well characterised and are known to have important roles in maintaining centrosome structure and in regulating microtubule nucleation. CDK5RAP2 shares two evolutionarily conserved domains with Cnn, known as CNN motif 1 and 2. Using the chicken B-cell line, DT40, I have used gene-targeting methods to disrupt both of these domains in CDK5RAP2. This revealed a function for CDK5RAP2 in attaching centrosomes to mitotic spindle poles. Centrosome attachment to spindle poles is mediated by a binding partner of CDK5RAP2, AKAP450. AKAP450 also localises to centrosomes and provides anchorage sites for spindle poles in the centrosome. Disruption of the CNN1 and CNN2 domains of CDK5RAP2 causes mislocalisation of AKAP450 from the centrosome and detachment of centrosomes from spindle poles. My studies in DT40 and in human cell lines revealed that CDK5RAP2 and AKAP450 also cooperate during interphase to maintain the two centrioles in the centrosome as a pair. In addition to a structural role in the centrosome, I also find that CNN motif 1 of CDK5RAP2 plays a role in the cellular response to DNA damage. In the absence of CNN motif 1, cells no longer efficiently arrest the cell cycle in response to damage. Centrosome-mediated mitotic spindle alignment and the DNA damage response have both been implicated in microcephaly. Therefore, defects in these functions of CDK5RAP2 may explain how mutations in cdk5rap2 may lead to microcephaly.

Le juste positionnement du noyau durant la formation musculaire semble important pour la fonction musculaire et des défauts ont été associés à plusieurs maladies musculaires. Le positionnement nucléaire dépend des microtubules (MTs), qui sont réorganisés depuis le centrosome, dans les myoblastes proliférants, vers l’enveloppe nucléaire (EN), dans les myotubes différenciés. Cette réorganisation s'accompagne de la redistribution des protéines centrosomales vers l’EN qui adopte le rôle de centre organisateur des microtubules (MTOC) lors de la différenciation myogénique. Néanmoins, les mécanismes sous-jacents restent inconnus. Ici, nous avons identifié les protéines Nesprin-1 et Sun1/2, localisées respectivement à la membrane nucléaire externe et interne, comme impliquées dans le recrutement de la fonction MTOC à l’EN. Les cellules déficientes en Nesprin-1 ou Sun1/2 ont montré une localisation altérée des protéines centrosomales dans le cytoplasme et l’absence des MTs depuis l’EN. De plus, Nesprin-1alpha, une myo-isoforme de Nesprin-1, s’associait aux protéines centrosomales Akap450, Pericentrin et Pcm1 dans les myotubes C2C12 et était suffisante pour corriger les défauts observés dans des cellules déplétées en Nesprin-1. Parmi les protéines centrosomales recrutées par Nesprin-1alpha, seule Akap450 semble nécessaire à la nucléation des MTs à l’EN. Ce processus, médié par Akap450 et Nesprin-1alpha, s’est avéré important pour le positionnement nucléaire lors du développement des myotubes. Ces résultats renforcent notre compréhension sur le lien causal entre des défauts lors de la formation du MTOC à l’EN et des défauts de positionnement nucléaire dans les dystrophies musculaires. / The accurate position of the nucleus during skeletal muscle formation seems to be important for muscle function, and defects have been associated with numerous muscle diseases. Nuclear positioning requires microtubules (MTs) which are reorganized from the centrosome in proliferating myoblasts to the nuclear envelope (NE) in differentiated myotubes. This dramatic MT reorganization is accompanied by a redistribution of proteins from the centrosome to the NE which thus takes over the function as a microtubule-organizing center (MTOC) during myogenic differentiation. However, the underlying mechanisms are still unknown. Here, we identified Nesprin-1 and Sun1/2, outer and inner nuclear membrane proteins, respectively, to be involved in the recruitment of MTOC function to the NE. Nesprin-1 or Sun1/2 deficient cells displayed mislocalization of centrosomal proteins to the cytoplasm and failed to regrow MTs from the NE. Moreover, the muscle-specific isoform of Nesprin-1, namely Nesprin-1alpha, was shown to be highly associated with the centrosomal proteins Akap450, Pericentrin and Pcm1 in C2C12 myotubes and to be sufficient to rescue the observed defects in Nesprin-1 depleted cells. Among the centrosomal proteins localizing at the NE during myogenic differentiation, solely Akap450 seemed to be required for MT nucleation. Akap450-Nesprin-1alpha-mediated MT nucleation from the NE was demonstrated to play an important role in nuclear positioning during myotube formation. These findings strengthen our understanding on how defects in MTOC formation at the NE can link to nuclear positioning defects in muscular dystrophies.