Rôle des centrosomes dans la régulation du point de contrôle en G2/M en réponse aux dommages à l'ADN

Les centrosomes sont les centres organisateurs des microtubules et jouent un rôle crucial dans l’organisation du fuseau bipolaire pendant la mitose. Plus récemment, le rôle des centrosomes dans la régulation de l’entrée en mitose a été mis en évidence. Les centrosomes semblent également contribuer à l’activation du point de contrôle en G2/M en réponse aux lésions de l’ADN en servant de point de rencontre pour les régulateurs du cycle cellulaire et les gènes de réponse aux dommages à l’ADN. L’amplification du nombre de centrosomes est une caractéristique des cellules tumorales mais de façon intéressante, elle constitue aussi une réponse des cellules aux dommages à l’ADN. Les mécanismes qui régulent l’homéostasie et la dynamique des centrosomes sont encore mal compris. Pour mieux comprendre le rôle des centrosomes dans la régulation du point de contrôle en G2/M en réponse aux dommages à l’ADN, le recrutement et/ou l’activation au niveau des centrosomes des kinases impliquées dans les voies de signalisation de ce point de contrôle ont été étudiés par immunofluorescence indirecte sur cellules HeLaS3 ou par Western blot sur des fractions enrichies en centrosomes. Nos résultats montrent que les kinases ATM, ATR, CHK1 et CHK2 sont actives dans les centrosomes de cellules en phase G2. En réponse à l’activation du point de contrôle en G2/M, les formes actives de ces kinases diminuent au niveau des centrosomes. Pour identifier de nouveaux acteurs centrosomaux potentiellement impliqués dans la régulation de ce point de contrôle, une analyse comparative des protéomes de centrosomes purifiés a également été réalisée par spectrométrie de masse. Pour étudier plus particulièrement la fonction de CHK2 au niveau des centrosomes, nous avons développer des outils moléculaires qui serviront à déterminer le rôle de la sous population de CHK2 localisée aux centrosomes 1) dans la régulation de l’entrée en mitose au cours d’un cycle normal 2) dans l’activation et la stabilité du point de contrôle en G2/M en réponse aux lésions l’ADN et 3) dans l’homéostasie et la dynamiques des centrosomes en réponse aux dommages à l’ADN. Cette étude permettra de mieux comprendre la fonction des centrosomes dans la réponse cellulaire au stress génotoxiques anti-cancereux et de révéler de nouvelles fonctions potentielles pour la kinase CHK2.Centrosomes function primarily as microtubule-organizing centres that play a crucial rôle in the equal segregation of chromosomes by organizing the bipolar spindle during mitosis. Recent studies have revealed the involvement of centrosomes in regulating G2/M transition during normal cell cycle progression. Moreover, increasing evidence suggests that centrosomes also play roles in the DNA damage response and cell cycle checkpoint signalling by serving as “meeting points” where DNA-damage-responsive genes and cell cycle regulators communicate. Numerical centrosome aberrations, or centrosome amplification, is a common feature of most human cancers that promotes aneuploidy and is involved in tumorigenesis as well as tumor progression. Interestingly, centrosome amplification and fragmentation have also been shown to constitute a cellular response to impaired DNA integrity that triggers cell death by mitotic failure. Although their roles are critical in tumorigenesis and the DNA damage response, the mechanisms that regulate centrosome homeostasis and dynamics remain poorly understood. To gain a better understanding of the role of the centrosomes in checkpoint regulation at G2/M transition in response to DNA damage; the recruitment and/or centrosomal activation of the kinases implicated in this checkpoint pathways were studied by indirect immunofluorescence on HeLaS3 cells or western blot on purified centrosomal fractions. Our results showed that the kinases CHK1, CHK2, ATM and ATR are activated at the centrosomes in cell synchronised in G2. However, after activation of the G2/M checkpoint, these activated kinases moved from centrosomes. Finally, to identify new centrosomal actors potentially involved in the regulation of this checkpoint, a comparative analysis of the proteome of purified centrosomes was also realized by mass spectrometry. To study more specifically the function of CHK2 at the centrosomes, we developped molecular tools wich will serve to determine the role of the sub-population of CHK2 localized at the centrosomes in 1) in regulating entry into mitosis during unperturbed cell cycle progression, 2) in checkpoint regulation at G2/M transition in response to DNA damage induced by ionizing radiations and genotoxic drugs and 3) in centrosomal homeostasis and dynamics by regulating centrosomal amplification, fragmentation and clustering during normal cell cycle progression and in response to genotoxic drugs. This study will further elucidate the importance of centrosomes in regulating the cell response to genotoxic stress induced by anti-cancer treatments and reveal potential new functions for the kinase CHK2 in unperturbed cell cycle progression and in response to DNA damage

Études fonctionnelles de deux nouvelles protéines centrosomales, NPHP5 et Cep76, et leurs implications dans les maladies humaines

Les centrosomes sont de petits organites qui régulent divers processus cellulaires comme la polarité ou la mitose dans les cellules de mammifères. Ils sont composés de deux centrioles entourés par une matrice péricentriolaire. Ces centrosomes sont les principaux centres organisateurs de microtubules. De plus, ils favorisent la formation de cils, des protubérances sur la surface des cellules quiescentes qui sont critiques pour la transduction du signal. Une grande variété de maladies humaines telles que les cancers ou les ciliopathies sont liées à un mauvais fonctionnement des centrosomes et des cils. C’est pourquoi le but de mes projets de recherche est de comprendre les mécanismes nécessaires à la biogénèse et au fonctionnement des centrosomes et des cils. Tout d'abord, j’ai caractérisé une nouvelle protéine centrosomale nommée nephrocystine - 5 (NPHP5). Cette protéine est localisée dans les cellules en interphase au niveau de la région distale des centrioles. Sa déplétion inhibe la migration des centrosomes à la surface cellulaire lors de l’étape précoce de la formation des cils. NPHP5 interagit avec la protéine CEP290 via sa région C-terminale qui est essentielle pour la ciliogenèse. Elle interagit également avec la calmoduline ce qui empêche son auto-agrégation. J’ai démontré que les domaines de liaison de NHPH5 à CEP290 et à la calmoduline, ainsi que son domaine de localisation centrosomale sont séparables. De plus, j’ai démontré que les protéines NPHP5 présentant des mutations pathogènes ne peuvent plus interagir avec CEP290 et ne sont plus localisées aux centrosomes, rendant ainsi ces protéines non fonctionnelles. Enfin, en utilisant une approche pharmacologique pour moduler les événements en aval dans la voie ciliogénique, j’ai montré que la formation des cils peut être restaurée même en absence de NPHP5. D’autre part, j’ai étudié le rôle de NPHP5 dans l'assemblage et le trafic du complexe BBSome dans le cil. Le BBSome est composé de huit sous-unités différentes qui s’assemblent en un complexe fonctionnel dont on sait peu de chose sur la régulation spatiotemporelle de son processus d'assemblage. J’ai précédemment montré que NPHP5 favorisait la formation des cils et que son dysfonctionnement contribuait au développement de néphronophtise (NPHP). Bien que la NPHP et le syndrome de Bardet-Biedl (BBS) soient des ciliopathies qui partagent des caractéristiques cliniques communes, la base moléculaire de ces ressemblances phénotypiques n’est pas comprise. J’ai constaté que NPHP5, localisé à la base du cil, contient deux sites de liaison distincts pour le BBSome. De plus, j’ai démontré que NPHP5 et son partenaire CEP290 interagissent de façon dynamique avec le BBSome pendant la transition de la prolifération à la quiescence. La déplétion de NPHP5 ou CEP290 conduit à la dissociation d’au moins deux sous-unités du BBSome formant alors un sous-complexe dont la capacité de migration dans le cil n’est pas compromise. J’ai montré que le transport des cargos vers le compartiment ciliaire par ce sous-complexe n’est que partiellement altéré. Enfin, j’ai également concentré mes recherches sur une autre protéine centrosomale peu caractérisée. La protéine centrosomale de 76 kDa (Cep76) a été précédemment impliquée dans le maintien d’une duplication unique des centrioles par cycle cellulaire, et dans une interaction avec la kinase cycline-dépendante 2 (CDK2). Cep76 est préférentiellement phosphorylée par le complexe cycline A/CDK2 sur le site unique S83. Cet événement est essentiel pour supprimer l'amplification des centrioles en phase S. J’ai démontré que Cep76 inhibe cette amplification en bloquant la phosphorylation de Plk1 au niveau des centrosomes. D’autre part, Cep76 peut être acétylée au site K279 en phase G2, ce qui régule négativement son activité et sa phosphorylation sur le site S83. Ces études permettent d'améliorer notre compréhension de la biologie des centrosomes et des cils et pourraient conduire au développement de nouvelles applications diagnostiques et thérapeutiques.Centrosomes are small organelles that regulate diverse cellular processes such as polarity or mitosis in mammalian cells. They are composed of two centrioles surrounded by a pericentriolar matrix. These centrosomes are the major microtubule organizing centers. Moreover, they promote the formation of cilia, protrusions on the surface of quiescent cells that are critical for signal transduction. A wide variety of human diseases such as cancers or ciliopathies are linked to a malfunction of centrosomes and cilia. Therefore the aim of my research is to understand the mechanisms necessary for the biogenesis and function of centrosomes and cilia. First, I have characterized a novel centrosomal protein called nephrocystin - 5 (NPHP5). This protein is localized, in interphase cells, in the distal region of centrioles. Its depletion inhibits the migration of centrosomes to the cell surface during the early stage of cilia formation. NPHP5 interacts with CEP290 via its C-terminal region that is essential for ciliogenesis. It also interacts with calmodulin, which prevents its self-aggregation. I have demonstrated that the Cep290- and CaM-binding domains as well as the centrosomal localization domain of NPHP5 are separable. Moreover, I have shown that NPHP5 proteins with pathogenic mutations can no longer interact with CEP290 and are not localized to centrosomes, rendering these proteins non-functional. Finally, using a pharmacological approach to modulate the downstream events in the ciliogenic pathway, I showed that cilia formation can be restored even without NPHP5. On the other hand, I studied the role of NPHP5 in the assembly and trafficking of the BBSome into the cilium. The BBSome consists of eight different subunits that assemble into a functional complex of which little is known about the spatiotemporal regulation of its assembly process. I have previously shown that NPHP5 favored the formation of cilia and its dysfunction contributes to the development of nephronophthisis (NPHP). Although the NPHP and BBS syndrome (BBS) are ciliopathies that share common clinical features, molecular basis of these phenotypic similarities is not understood. I found that NPHP5, located at the base of the cilium, contains two separate binding sites for BBSome. Furthermore, I demonstrated that NPHP5 and his partner CEP290 interact dynamically with the BBSome during the transition from quiescence to proliferation. Depletion NPHP5 or CEP290 leads to the dissociation of at least two subunits of BBSome forming a sub-complex that can still traffic into the cilium. I have shown that the transport of cargo to the ciliary compartment through this sub-complex is only partially altered. Finally, I have also focused my research on another centrosomal protein poorly characterized. The centrosomal protein of 76 kDa (Cep76) was previously involved in the maintenance of a single duplication of centrioles per cell cycle, and interacts with the cyclindependent kinase 2 (CDK2). Cep76 is preferentially phosphorylated by cyclin A/CDK2 on the single site S83. This event is essential to suppress centrioles amplification in S phase. I have demonstrated that Cep76 inhibits amplification by blocking the phosphorylation of Plk1 at the centrosome. Moreover, Cep76 can be acetylated at the K279 site in G2 phase, which negatively regulates its activity and phosphorylation on the site S83. These studies will improve our understanding of the biology of centrosomes and cilia and could lead to development of new diagnostic and therapeutic applications

Mutations specific to the Rac-GEF domain of <i>TRIO</i> cause intellectual disability and microcephaly

Background: Neurodevelopmental disorders have challenged clinical genetics for decades, with over 700 genes implicated and many whose function remains unknown. The application of whole-exome sequencing is proving pivotal in closing the genotype/phenotype gap through the discovery of new genes and variants that help to unravel the pathogenic mechanisms driving neuropathogenesis. One such discovery includes TRIO, a gene recently implicated in neurodevelopmental delay. Trio is a Dbl family guanine nucleotide exchange factor (GEF) and a major regulator of neuronal development, controlling actin cytoskeleton dynamics by activating the GTPase Rac1.Methods: Whole-exome sequencing was undertaken on a family presenting with global developmental delay, microcephaly and mild dysmorphism. Father/daughter exome analysis was performed, followed by confirmatory Sanger sequencing and segregation analysis on four individuals. Three further patients were recruited through the deciphering developmental disorders (DDD) study. Functional studies were undertaken using patient-specific Trio protein mutations.Results: We identified a frameshift deletion in TRIO that segregated autosomal dominantly. By scrutinising data from DDD, we further identified three unrelated children with a similar phenotype who harboured de novo missense mutations in TRIO. Biochemical studies demonstrated that in three out of four families, the Trio mutations led to a markedly reduced Rac1 activation.Conclusions: We describe an inherited global developmental delay phenotype associated with a frameshift deletion in TRIO. Additionally, we identify pathogenic de novo missense mutations in TRIO associated with the same consistent phenotype, intellectual disability, microcephaly and dysmorphism with striking digital features. We further functionally validate the importance of the GEF domain in Trio protein function. Our study demonstrates how genomic technologies are yet again proving prolific in diagnosing and advancing the understanding of neurodevelopmental disorders.<br/

Mapping Neurogenesis Onset in the Optic Tectum of Xenopus Laevis

Neural progenitor cells have a central role in the development and evolution of the vertebrate brain. During early brain development, neural progenitors first expand their numbers through repeated proliferative divisions and then begin to exhibit neurogenic divisions. The transparent and experimentally accessible optic tectum of Xenopus laevis is an excellent model system for the study of the cell biology of neurogenesis, but the precise spatial and temporal relationship between proliferative and neurogenic progenitors has not been explored in this system. Here we construct a spatial map of proliferative and neurogenic divisions through lineage tracing of individual progenitors and their progeny. We find a clear spatial separation of proliferative and neurogenic progenitors along the anterior-posterior axis of the optic tectum, with proliferative progenitors located more posteriorly and neurogenic progenitors located more anteriorly. Since individual progenitors are repositioned toward more anterior locations as they mature, this spatial separation likely reflects an increased restriction in the proliferative potential of individual progenitors. We then examined whether the transition from proliferative to neurogenic behavior correlates with cellular properties that have previously been implicated in regulating neurogenesis onset. Our data reveal that the transition from proliferation to neurogenesis is associated with a small change in cleavage plane orientation and a more pronounced change in cell cycle kinetics in a manner reminiscent of observations from mammalian systems. Our findings highlight the potential to use the optic tectum of Xenopus laevis as an accessible system for the study of the cell biology of neurogenesis. This article is protected by copyright. All rights reserved

Molecular and Cellular Basis of Autosomal Recessive Primary Microcephaly

Autosomal recessive primary microcephaly (MCPH) is a rare hereditary neurodevelopmental disorder characterized by a marked reduction in brain size and intellectual disability. MCPH is genetically heterogeneous and can exhibit additional clinical features that overlap with related disorders including Seckel syndrome, Meier-Gorlin syndrome, and microcephalic osteodysplastic dwarfism. In this review, we discuss the key proteins mutated in MCPH. To date, MCPH-causing mutations have been identified in twelve different genes, many of which encode proteins that are involved in cell cycle regulation or are present at the centrosome, an organelle crucial for mitotic spindle assembly and cell division. We highlight recent findings on MCPH proteins with regard to their role in cell cycle progression, centrosome function, and early brain development