Дисертації з теми "Centriole to centrosome conversion"

The centrosomes play an essential role in cell and tissue homeostasis, therefore, their structure, function, and number are highly regulated to ensure natural organisms development through the assembly of a multiplicity of protein complexes. Since the organization and integrity of the centrosome depend on its centrioles and pericentriolar material (PCM), understanding the dynamics of these organelles is crucial to decipher the centrosome behaviour. To date, we have a fairly detailed knowledge of the centriole composition and structure and also of the process of duplication and centrosomal maturation. Something is understood about the process of centriole elimination during gametogenesis, but very little is known about how centrioles are eliminated in post-mitotic differentiated cells. During the development of the Drosophila eye, the centrioles of the differentiating retinal cells do not recruit γ-Tubulin, suggesting that they are unable to organize functional microtubule-organizing centers (MTOCs). Consistent with this hypothesis, this study shows that in Drosophila third instar larvae Cnn and Spd-2, proteins that allow γ-Tubulin recruitment, and DPlp, which is involved in the organization of the pericentriolar material, are not accumulated by centrioles of eye imaginal disc cells. Despite the loss of these essential components of the pericentriolar material, the centrioles are structurally intact and can recruit Asl and ANA-1. Usually, the accumulation of Asl and ANA-1 allows the daughter centrioles to acquire the motherhood condition. Indeed, mother centrioles accumulate properly Plk-4; however, they are not able to duplicate. These findings show that, in this model, the accumulation of Plk-4 is not sufficient to allow centriole duplication. During the progression of pupal development, the centriole number progressively decreases, and structural defects can be observed. These phenotypes suggest that during Drosophila eye development centriole elimination begins with the loss of the structural integrity, rather than with the PCM reduction as occurs in other models. Furthermore, Asl, ANA-1 and Sas-4 are still detectable, indicating that these proteins by themselves are not able to ensure the maintenance of centriole integrity. Among the essential cellular functions played by centrioles, there is their ability to act as basal bodies to nucleate the axoneme, the supporting structure of cilia and flagella, which perform crucial cellular functions such as signal transduction and cell motility. Given the critical role of centrioles and cilia in cell physiology, mutations in numerous centriolar proteins cause various disorders, including microcephaly, dwarfism and ciliopathies. Therefore, it is crucial to understand better the mechanisms that regulate the dynamics of centrioles and cilia. In this study, the cilia of Drosophila melanogaster type I sensory neurons have been analysed, to understand the role played by the centriolar proteins Klp10A, Cnb, Gorab and Rcd4 in the dynamics of centrioles and cilia. In Drosophila wild type sensory neurons, Klp10A (Kinesin-like protein 10A), a member of the kinesin-13 family, is located in the distal part of the transition zone (TZ), just above the UNC–GFP signal. This study shows that mutations in klp10A result in substantial structural defects of sensory neurons such as the over elongation of both centrioles in opposite directions. It has also been observed that the extensions of both centrioles, called proximal and distal basal bodies, show doublets surrounded by electron-dense material and short lateral projections as found in the control TZ. Therefore, the elongated distal regions of the centrioles in klp10A mutants may be equivalent to a TZ. The phenotype observed in klp10A mutant is deeply different from that observed in sensory neurons of mutants for other TZ proteins that are limited to the proximal portion of the TZ. This suggests that Klp10A could be a core component of the ciliary transition zone in Drosophila, specifically associated with the distal region of the TZ where it plays an essential role in centriole elongation and the assembly and maintenance of the ciliary axoneme. Centrobin (Cnb) is a centrosome-associated protein that localizes specifically at the daughter centrioles. It has been shown that a cnb mutation makes the daughter centrioles, called PBBs in this model, able to act as distal basal bodies (DBBs) to nucleate supernumerary axonemes. This is confirmed by the present study performed on a different cnb mutant strain, suggesting that Cnb acts as a negative regulator of ciliogenesis. Recently a new centriolar protein required for centriole duplication, called Gorab, has been discovered in Drosophila melanogaster. The cnb-gorab double mutant sensory neurons analysed in this study, show a stronger centriole reduction compared to the single gorab mutant. Consequently, the number of cilia is also severely affected. These findings suggest that in the cnb-gorab mutant, the centriole duplication fails before the basal body formation. Recent works have identified the human protein called PPP1R35 (Rcd4 in Drosophila - Reduction in Cnn dots 4), that is involved in centriole-to-centrosome conversion (CCC) and centriole elongation. Here we demonstrate that rcd4 mutant sensory neurons show a severe centriole and cilia reduction, accompanied by centriolar fragmentation. This suggests that Rcd4 could be involved in the CCC similarly to its human counterpart.
Per via del suo ruolo essenziale nell'omeostasi cellulare e tissutale, la struttura, la funzione e il numero di centrosomi sono altamente regolati per garantire il naturale sviluppo degli organismi, attraverso l'assemblaggio di una molteplicità di complessi proteici. Poiché l'organizzazione e l'integrità del centrosoma dipendono dai centrioli e dal materiale pericentriolare (PCM) che lo compongono, comprendere la dinamica di questi organelli è fondamentale per decifrare il comportamento del centrosoma. Ad oggi, abbiamo una conoscenza abbastanza dettagliata della composizione e della struttura dei centrioli e anche di ciò che riguarda il processo di duplicazione e maturazione centrosomale. Si conosce qualcosa del processo di eliminazione dei centrioli durante la gametogenesi, ma si sa molto poco su come i centrioli vengono eliminati nelle cellule differenziate post-mitotiche. Durante lo sviluppo dell'occhio di Drosophila, i centrioli delle cellule retiniche in differenziazione non reclutano la γ-Tubulina, suggerendo che non sono in grado di organizzare centri di organizzazione dei microtubuli (MTOC) funzionali. Coerentemente con questa ipotesi, questo studio mostra che Cnn e Spd-2, proteine che consentono il reclutamento di γ-tubulina, e DPlp, che è coinvolta nell'organizzazione del materiale pericentriolare, non vengono accumulati dai centrioli delle cellule del terzo stadio larvale. Nonostante la perdita di questi componenti essenziali del materiale pericentriolare, i centrioli sono strutturalmente intatti e possono reclutare Asl e ANA-1. Di solito, l'accumulo di Asl e ANA-1 consente ai centrioli figli di acquisire la condizione di maternità. Infatti, i centrioli madre accumulano correttamente Plk-4; tuttavia, non sono in grado di duplicare. Questi risultati mostrano che, in questo modello, l'accumulo di Plk-4 non è sufficiente per consentire la duplicazione di centrioli. Durante la progressione dello sviluppo della pupa, il numero di centrioli diminuisce progressivamente, e iniziano a essere osservati difetti strutturali. Questi fenotipi suggeriscono che l'eliminazione dei centrioli inizia con la perdita dell'integrità strutturale, piuttosto che con la riduzione del PCM, come mostrato in altri modelli. Inoltre, Asl, ANA-1 e Sas-4 sono ancora rilevabili, sottolineando che queste proteine da sole non sono in grado di garantire il mantenimento dell'integrità dei centrioli. Tra le funzioni cellulari essenziali svolte dai centrioli, vi è la loro capacità di agire come basal bodies per nucleare l'assonema, la struttura portante di ciglia e flagelli, che svolgono importanti funzioni cellulari come la trasduzione del segnale e la motilità cellulare. Dato il ruolo critico dei centrioli e delle ciglia nella fisiologia cellulare, le mutazioni di numerose proteine centriolari causano vari disturbi, tra cui microcefalia, nanismo e ciliopatie. Pertanto, è fondamentale comprendere meglio i meccanismi che regolano la dinamica dei centrioli e delle ciglia. In questo studio sono state analizzate le ciglia dei neuroni sensoriali di tipo I della Drosophila melanogaster, per comprendere il ruolo svolto dalle proteine centriolari Klp10A, Cnb, Gorab e Rcd4 nelle dinamiche di centrioli e ciglia. Nei neuroni sensoriali di tipo I di Drosophila, Klp10A (Kinesin-like protein 10A), un membro della famiglia delle kinesine 13, si localizza nella parte distale della zona di transizione (TZ), appena sopra il segnale UNC-GFP. Questo studio mostra che la mutazione di klp10A provoca sostanziali difetti strutturali dei neuroni sensoriali, come l'eccessivo allungamento di entrambi i centrioli in direzioni opposte. È stato anche osservato che le estensioni di entrambi i centrioli, chiamati basal bodies prossimale e distale, mostrano doppietti circondati da materiale elettrondenso e brevi sporgenze laterali come si quelle che si trovano nella TZ di controllo. Pertanto, le regioni distali allungate dei centrioli dei mutanti per klp10A, possono essere equivalenti a TZ. Il fenotipo osservato nel mutante klp10A è profondamente diverso da quello osservato nei neuroni sensoriali dei mutanti per altre proteine della TZ che sono limitate alla porzione prossimale. Ciò suggerisce che Klp10A potrebbe essere un componente chiave della zona di transizione ciliare in Drosophila, specificamente associato alla regione distale della TZ dove svolge un ruolo essenziale nell'allungamento dei centrioli e nell'assemblaggio e nell mantenimento dell'assoneema ciliare. La Centrobina (Cnb) è una proteina centrosomale che si localizza specificamente nei centrioli figli. È stato dimostrato che la mutazione della cnb rende i centrioli figli, chiamati PBB in questo modello, in grado di agire come basal body distali (DBB) per nucleare assonemi soprannumerari. Ciò è confermato da questo studio condotto in un diverso ceppo mutante di cnb che suggerisce che la Cnb agisce come regolatore negativo della ciliogenesi. In Drosophila melanogaster, è stata scoperta una nuova proteina centriolare essenziale per la duplicazione dei centrioli, Gorab. I neuroni sensoriali del doppio mutante cnb-gorab analizzati in questo studio, mostrano una riduzione più forte dei centrioli rispetto al singolo mutante gorab. Di conseguenza, anche il numero di ciglia è gravemente colpito. Questi risultati suggeriscono che nel mutante cnb-gorab, la duplicazione dei centrioli fallisce prima della formazione del basal body. Lavori recenti hanno identificato la proteina umana chiamata PPP1R35 (Rcd4 in Drosophila - Reduction in Cnn dots 4), che è coinvolta nella conversione centriolocentrosoma (CCC) e nell’allungamento di centriolo. Le analisi dei neuroni sensoriali mutanti di Rcd4 mostrano una forte riduzione dei centrioli e delle ciglia e anche la frammentazione centriolare. Ciò suggerisce che Rcd4 potrebbe essere coinvolto nella CCC in modo simile alla sua controparte umana.

La nucléoline est une des protéines les plus abondantes des nucléoles. Ses fonctions ne sont cependant pas restreintes à la biogénèse des ribosomes. En absence de nucléoline, un phénotype d’amplification du nombre de centrosomes en mitose, associé à des fuseaux multipolaires a été récemment rapporté. Notre étude vise à comprendre l’implication de la nucléoline dans l’apparition de ce phénotype et notamment ses conséquences sur l’organisation des microtubules.Par immunofluorescence, nous mettons en évidence que la fraction centrosomale de la nucléoline est spécifiquement associée au centriole mature en interphase, alors qu’en mitose seule une forme phosphorylée y est détectée.En interphase, les cellules déplétées en nucleoline présentent une amplification de leurs centrioles immatures, entourés par un réseau anormal de péricentrine, dénotant une perte de structuration de la matrice péricentriolaire. De plus, une désorientation du réseau microtubulaire causée par une capacité de nucléation ralentie et une perte d’ancrage des microtubules au centrosome mature est observée. Par des expériences de co-immunoprécipitation avec la tubuline γ, un lien avec le complexe d’initiation de la nucléation des microtubules est dévoilé.En conclusion, les résultats de ma thèse révèlent que structurellement la nucléoline est associée au centriole mature des cellules en interphase et que fonctionnellement elle stimule la nucléation des microtubules et participe à leur ancrage au centrosome mature pour orienter le réseau microtubulaire en interphase. La nucléoline pourrait ainsi être un des acteurs de la synchronicité entre centrosomes et nucléoles pour la régulation du cycle cellulaire
Nucleolin is an abundant non-ribosomal protein of the nucleolus. Nevertheless its functions are not restricted to ribosome biogenesis. Without nucleolin, a phenotype of abnormally high centrosome numbers was recently reported in mitosis, associated with multipolar spindle formation. The purpose of our study is to understand nucleolin’s involvement in the appearance of this phenotype and specifically consequences on microtubule network organisation. By immunofluorescence, visual evidences of a centrosomal fraction of nucleolin are provided, specifically associated with the mature centriole of interphase cells. In mitosis, only a phosphorylated form of nucleolin is detected at the spindle poles.In interphase, nucleolin depleted cells exhibit immature centriole amplification surrounded by an abnormal mesh of pericentrine, showing a loss of pericentriolar matrix structuration. Furthermore, in most nucleolin depleted cells, a complete disorganisation of microtubule network is observed, caused by a slower microtubule nucleation capacity and a loss of microtubule anchoring at the mature centriole. Using co-immunoprecipitation with γ-tubulin, a major centrosomal protein, a link with the microtubule nucleation complex was highlighted.Taken together my thesis results reveal that in interphase cells, nucleolin is structurally associated with the mature centriole, and functionally stimulates microtubule nucleation and participates in their anchoring at the mature centrosome to orient microtubule network. Thus, nucleolin could be a major actor in the synchronicity between centrosome and nucleoli for cell cycle regulation

Le centrosome est le centre organisateur des microtubules dans les cellules animales, il nucléé les microtubules interphasiques ainsi que le fuseau mitotique. Les centrosomes sont produits par duplication, mécanisme rigoureusement régulé au cours du cycle cellulaire. En effet, un centrosome comporte deux centrioles qui se dupliquent une fois par cycle cellulaire. Des erreurs de duplication conduisant à plus de deux centrosomes induisent la formation de fuseaux multipolaires et provoquent des défauts de ségrégation des chromosomes. Chez la levure Schizosaccharomyces pombe, un organisme modèle pour l’étude de la division cellulaire, les homologues des centrosomes sont les SPBs (pour Spindle Pole Body). Une structure annexe spécifique liée aux SPBs est appelée demi-pont (quand les SPBs ne sont pas dupliqués) puis pont (quand elle relie les deux SPBs dupliqués). Les deux principaux composants du pont chez la levure S. pombe sont Cdc31 et Sfi1. Sfi1 est une protéine linéaire formée de répétitions en hélice α formant des sites de liaison pour la Centrine/Cdc31. Sfi1 s’assemble en réseau de molécules parallèles interagissant avec le SPB via leur domaine N-terminal. Lors de la première partie de ma thèse, j’ai démontré que Sfi1 est requis pour la duplication et la séparation des deux SPBs. Dans la première partie de ma thèse, je me suis intéressée aux fonctions de Sfi1 chez la levure. Cette étude a permis de démontrer que Sfi1 est un composant du demi-pont et qu’il est essentiel pour la duplication des SPBs et l’assemblage d’un fuseau bipolaire. De plus, nous avons déterminé que le pont est dupliqué en fin de mitose. Enfin, nous avons aussi montré que la déstabilisation du pont menant à sa rupture en mitose, dépend de la phosphorylation de Cdc31 par la kinase mitotique Cdk1. Lors de la seconde partie de ma thèse, je me suis intéressée au complexe Sfi1/Centrine dans les cellules humaines. J’ai confirmé que Sfi1 est localisée aux centrioles. De plus, j’ai montré que la déplétion de Sfi1 dans les cellules RPE1, conduit à une perte de localisation de la Centrine, suggérant soit un défaut de recrutement, soit une déstabilisation. De plus, en absence de Sfi1, les cellules RPE1 ne sont plus capables de former de cil primaire. Ce résultat suggère que Sfi1 et la Centrine sont requis pour la ciliogénèse. Enfin, j’ai aussi démontré que la déplétion deSfi1 induit un arrêt de cycle cellulaire dans les cellules non tumorales RPE1. Dans les cellules cancéreuses, HeLa, le cycle n’est pas arrêté mais j’ai pu observer une prolongation du temps de mitose. En conclusion mes travaux montrent que bien que la fonction de Sfi1/Centrin ne soit pas conservée, le complexe reste essentiel pour l’intégrité structurale et fonctionnelle du centrosome
The centrosome is the main microtubule organizing center. It nucleates and organizes interphase microtubule and contributes to the assembly of the bipolar mitotic spindle. To do so, the centrosome, present in one copy at the beginning of the cell cycle, duplicates to produce a second copy. The duplication process is tightly controlled and regulated since centrosome over-duplication can lead to multipolar mitotic spindles and promote genome instability and tumorigenesis. The duplication of the yeast centrosome, the SPB (Spindle pole body), begins with the duplication of the half bridge. This appendage is composed of Sfi1/Cdc31 complex organized in a parallel array attached to the core SPB. SPB duplication relies on the assembly of a second array of Sfi1/Cdc31, anti-parallel to the first one, creating thereby an assembly site for the new SPB. Therefore Sfi1 is essential for SPB duplication and our work defined the timing of half-bridge duplication and some of the regulatory mechanisms that favor bridge splitting to release duplicated centrosomes and allow spindle assembly at mitotic onset. Sfi1 and Cdc31/Centrins are conserved in human cells where the centrosome is composed of two centrioles surrounded by the pericentriolar material. Centrins are concentrated in the distal end of centrioles. Sfi1 has also been localized to centrioles, but its function remained unknown. Thus, we started investigating Sfi1 function in human cells. We found that Sfi1 depletion leads to a decrease in Centrin recruitment to the centrioles. It also leads to a cell cycle arrest in G1 in RPE1 cells, an event previously observed in presence of defects in centriole biogenesis. In HeLa cells where the cell cycle is not affected, Sfi1 depletion leads to a mitotic delay. Moreover, Sfi1 depletion leads to cilium assembly. To conclude, these results altogether point towards a role of human Sfi1 in centriole biogenesis

Centrioles and basal bodies with their characteristic 9+2 structure are found in all major eukaryotic lineages. The correlation between the occurrence of centrioles and the presence of cilia/flagella, but not centrosome-like structures, suggests that the ciliogenesis function of centrioles is ancestral. Here, it is demonstrated that the centriole domain of centrosomes emerged within the Metazoa from an ancestral state of possessing a centriole with basal body function but no functional association with a centrosome. Centrosome structures involving a centriole are metazoan innovations. When an axoneme is still present but no longer fully functional, such as the sensory cilia of Caenorhabditis elegans or, as depicted here, the flagellum of the intracellular amastigote stage of the Leishmania mexicana parasite, the basal body structure is less constrained and can depart from the canonical structure. A general view has emerged that classifies axonemes into canonical motile 9+2 and noncanonical, sensory 9+0 structures. This study reveals this view to be overly simplistic, and additional axonemal architectures associated with potential sensory structures should be incorporated into prevailing models. Here, a striking similarity between the axoneme structure of Leishmania amastigotes and vertebrate primary cilia is revealed. This striking conservation of ciliary structure, despite the evolutionary distance between Leishmania and mammalian cells, suggests a sensory function for the amastigote flagellum. Adding weight to a sensory hypothesis, close examination of Leishmania positioning inside the parasitophorous vacuole revealed frequent contact between the flagellum tip and the vacuole membrane. A sensory function could also explain the retention of a flagellum in Trypanosoma cruzi amastigotes, an intracellular stage that, as shown in this study, emerged independently to the Leishmania amastigote. Basal body appendages, such as pro-basal bodies and microtubule rootlets, also vary widely in their structure. Choanoflagellates, a sister group to the Metazoa, posses an extensive microtubule rootlet system that provides support for their characteristic collar tentacles. This atypical structure is reflected in the underlying molecular components of the choanoflagellate basal body. The importance of choanoflagellates as the closest known relative of metazoans was first revealed by their similarity to choanocytes, the feeding cells of sponges. Although phylogenetic analyses leave little doubt that choanoflagellates are a sister group of animals, comparisons of molecular and structural components of appendages associated with the collar tentacles highlight significant differences and questions the extent to which the collar structures of choanoflagellates and choanocytes can be assumed to be homologous. Finally, the confinement of a centriole-based centrosome to the Metazoa provides little support for the flagellar synthesis constraint as an explanation for the origin of multicellularity. There is, indeed, an apparent constraint; no flagellated or ciliated metazoan cell ever divides. This constraint, however, did not arise until after the incorporation of centrioles into the centrosome in the metazoan lineage and the co-option of centrioles as a structural and functional component of the centrosome. The flagellar synthesis constraint is therefore not an explanation for the origin of multicellularity but a consequence of it.

Centrosomes are cellular organelles present in most animal cells, and are formed of two main components: the centrioles and the pericentriolar material (PCM). Centrosomes perform a variety of functions: they are the main microtubule organising centre in the cell, and are important localisation hubs for kinases involved in regulating the cell cycle. Hundreds of proteins are thought to localise to centrosomes, but work in the last decade has narrowed down this list to a handful of proteins that are thought to be essential for centrosome structure and function in Drosophila. Asl, Ana2, DSas-4, DSas-6 and Sak have been identified as essential components for centriole duplication, while Cnn and DSpd-2 are thought to be important in establishing the PCM. However, the relative position of these 7 components in the pathway of centrosome assembly in Drosophila embryos remains elusive, as a genetics analysis of this process is hampered by the absence of centrioles in most mutant embryos for these proteins. In this thesis I elucidate the pathway of centrosome assembly in Drosophila by using SAPs (DSas-6/Ana2 particles that form in Drosophila unfertilised eggs upon moderate expression of DSas-6 and Ana2) as proxy models of centrosomes. I show SAPs are very similar to centrosomes in composition and dynamics but unlike centrosomes are able to form even in the absence of some of the essential centriolar components. SAP analysis in the absence of each of the main centrosome components reveals that: Sak is not required for the recruitment of downstream components; DSas-4 is necessary for Ana2 and DSas-6 to interact; Asl is the most upstream component of the PCM recruitment pathway, followed by DSpd-2; it is likely that there is an additional PCM recruitment pathway. I then take advantage of some of these results to examine how centrosome formation is potentiated after egg activation. My work allows me to propose an improved description of the pathway of centrosome formation in Drosophila.

As an organelle coupling nuclear and cytoplasmic divisions, the centrosome is essential to mitotic fidelity and its inheritance could be critical to understanding cell transformation. Investigating the behavior of the centrosome in living mitotic cells, we have documented a transient and remarkable post-anaphase repositioning of this organelle which apparently controls the release of central microtubules from the midbody and the completion of cell division. We further observed that the absence of the centrosome leads to cytokinesis defects. Like the yeast SPB, the mother-centriole could possess a specifically associated activity. That activity would trigger the narrowing of the bridge, for example by disrupting the matrix anchoring MTs in the midbody. A second event would trigger abscission when the mother-centriole moves away from the bridge. The main implication of our work is that disassembly of the central spindle and of the cleavage furrow, both necessary for abscission, are distinct events and would be, like the metaphase spindle disassembly, under checkpoint control. Together with recent results in yeasts, our data point to a conserved centrosome-dependent pathway integrating spatial controls into the decision of completing cell division, which would rely on the repositioning of the centrosome organelle.

Le centrosome favorise la nucleation et l'ancrage des microtubules. Il est compose de deux centrioles et d'un materiel pericentriolaire. Les centrioles se dupliquent une fois par cycle de faÇon conservative, tous deux n'ont donc pas le meme age. Le plus vieux est appele centriole pere et possede des appendices. Les deux centrioles n'ont pas le meme comportement en interphase, le pere est immotile alors que le fils bouge. Je me suis interessee au role du centriole pere dans la ciliogenese et l'organisation des microtubules en interphase. Le centriole pere s'ancre a la membrane plasmique et sert de base pour la formation d'un axoneme. Peu de choses sont connues sur les controles de la taille du centriole, de son ancrage a la membrane et de la formation de l'axoneme. La delta-tubuline est un bon candidat pour la regulation de ces mecanismes. Elle se localise dans le materiel pericentriolaire et aux jonctions cellulaires dans les cellules mammiferes, et a la jonction centriole/axoneme chez c. Reinhardtii. Au moment ou la ciliogenese est enclenchee, l'une des formes de cette tubuline disparait. Sa surexpression entraine une hypersensibilite des microtubules au froid et en son absence, les corps basaux pourraient etre plus longs, suggerant qu'elle regule la dynamique de certains microtubules. Les microtubules en interphase sont ancres au niveau des appendices subdistaux du centriole pere. Nous avons montre que la presence du gamma-turc au centrosome ne suffit pas a ancrer les microtubules. L'ancrage necessite la presence d'une proteine du centriole pere, la nineine. Cette proteine possede la capacite d'arrimer a la fois les complexes de nucleation et d'ancrage des microtubules
The centrosome favours nucleation and anchoring of microtubules. It is composed of a centriole pair and a pericentriolar material. Centrioles duplicate once per cell cycle in a conservative manner, thus they have not the same age. The older one is named mother centriole and carried appendages. In interphase, the younger centriole moves whereas the mother is immotile. I worked on the role of the mother centriole in ciliogenesis and in microtubule organisation in interphase. The mother centriole can be anchored to the plasma membrane and promoted the formation of an axoneme. Few data are available about control of centriole size, anchoring to the membrane and of formation of the axoneme. Delta-tubulin could be a good candidate to participate in these processes. This protein is localized at the pericentrosomal material and at the cell junctions in mammal cells, and at the transition basal bodies/axonemes in c. Reinhardtii. At the onset of ciliogenesis, one of these tubulin forms disappeared. Its overexpression leads to cold hypersensitivity of microtubules, and without delta-tubulin centrioles might be longer, suggesting that this protein should regulate dynamics of some microtubules. In interphase cells, microtubules are anchored at subdistal appendages. My work has shown that the presence of the gamma-turc at the centrosome is not sufficient to anchor microtubules, suggesting that microtubule nucleation and anchoring at the centrosome are two independent processes. The microtubule anchoring seems to be dependent on the presence of the mother centriole protein ninein. This protein is able to dock the nucleation complex and the anchoring complex on these appendages

Centrosomes are the main microtubule organizing centres in animal cells and are formed by a pair of centrioles together with surrounding pericentriolar material (PCM). Cycling cells duplicate their centrosomes strictly once per cell cycle. This process is driven by the semi-conservative duplication of the centrioles that are found at the centrosome core. During the exit from mitosis the two centrioles within the single inherited centrosome separate, and upon the start of S-phase each of these inherited mother centrioles assembles an adjacent daughter at its side. This process results in two complete centrosomes that can form the poles of the mitotic spindle, and thus segregate evenly to the next cell generation. The formation of a daughter centriole suppresses the initiation of new duplication events from the same templating mother centriole until this daughter separates - disengages - at the end of the cell cycle. This regulation - that acts to repress centriole amplification - is summarized in the 'licensing model of centriole duplication' (Tsou and Stearns, 2006). This model states that centriole disengagement provides the license for the re-duplication of mother centrioles. Importantly, experiments show that while abolishing centriole engagement is sufficient to allow mother centrioles to re-duplicate within the same cycle, it is insufficient to allow daughter centrioles the assembly of a granddaughter before they mature into mothers towards the end of their first cell cycle. The molecular nature of this daughter-to-mother transition remains mysterious. In this thesis I show that in Drosophila embryos the essential centriole duplication protein Asl is not incorporated into daughter centrioles as they assemble during S-phase, but is only incorporated once mother and daughter separate at the end of mitosis. The initial incorporation of Asterless (Asl) is irreversible, and is dependent on centriolar DSas-4. Crucially, Asl incorporation is essential for daughter centrioles to mature into mothers that can support centriole duplication. I propose that Asl acts as a permanent primary license that allows new centrioles to duplicate for the first time. Once acquired, this primary license is not lost but rather further regulation is taken over by the reduplication licensing mechanism, disengagement. This work extends the previously proposed licensing model to also explain how new centrioles are licensed for their first duplication event.

Pericentriolar satellites (PS) are electron dense granules surrounding the centrosome, the major microtubule-organizing centre in eukaryotic cells. In cycling cells the centrosome promotes spindle assembly and the faithful execution of mitosis. In non-cycling cells it is involved in forming the cilium, a plasma membrane-resident organelle, which mediates crucial signalling pathways in development and tissue homeostasis. PS are thought to contribute to centrosome formation, through the microtubule-dependent transport of centrosome components, and they are involved in ciliogenesis and stress response. Moreover, several proteins that localize to PS are mutated in human ciliopathies and neurodevelopmental disorders. The precise roles of PS in the various molecular pathways and diseases are however poorly understood, in part due to the limited knowledge of their composition. In the first part of my study I performed a comprehensive analysis of the pericentriolar satellite proteome. This was achieved by sucrose sedimentation of PS, combined with affinity purification of a key PS component, PCM1. To eliminate contamination by centrosomes, the PS proteome was determined from wild-type cells as well as from two cell lines genetically engineered to lack centrosomes. Mass spectrometry identified 170 PS components including most of the previously described PS proteins, confirming the validity of the approach. Having determined the proteomic composition of PS from DT40 cells, I then performed validation studies both in chicken and human cell lines. In the second part of my study, I aimed to use the list of PS proteins to uncover new biological roles for pericentriolar satellites. I devised two distinct approaches to gain functional insights. First, I generated a cell line lacking PCM1 as a tool to study the role(s) of PS and PS components. Second, I performed loss-of-function studies on a set of new PS proteins to determine their function(s) in maintaining the canonical PS distribution and in forming primary cilia.

Les cellules multiciliées jouent un rôle essentiel dans la propulsion des fluides physiologiques. Leur dysfonctionnement provoque des maladies chroniques. Contrairement à la plupart des cellules de mammifères qui possèdent un centrosome composé de deux centrioles, les cellules multiciliées possèdent une centaine de centrioles qui servent de base à la nucléation des cils motiles. Les mécanismes d'amplification de centrioles ou de régulation du nombre de centrioles dans ce type cellulaire étaient jusque-là inconnus. Les centrioles nouvellement formés étaient considérés comme apparaissant " de novo ". Une approche de vidéomicroscopie et de microscopie de super-résolution corrélative nous a d'abord permis de déterminer que tous les procentrioles sont générés à partir du centrosome préexistant. Nous démontrons que le centriole fils du centrosome est le site principal de nucléation de 95% de centrioles nouvellement formés dans les cellules multiciliées. Ces résultats réfutent par conséquent l'origine " de novo " des centrioles dans ce type cellulaire. Puis, nous montrons que la machinerie mitotique orchestre la progression spatio-temporelle de la dynamique centriolaire dans ces cellules post-mitotiques et en phase terminale de différentiation. L'amortissement de l'activité de Cdk1 empêche la rentrée en mitose tout en permettant la coordination du nombre de centrioles, leur croissance, et leur désengagement par des transitions phasiques nécessaires à la nucléation de cils motiles. Cette thèse aide à mieux comprendre la différentiation des cellules multiciliées, les ciliopathies, ainsi que l'amplification centriolaire pathologique associée avec le cancer et la microcéphalie
Multiciliated mammalian cells play a crucial role in the propulsion of physiological fluids. Their dysfunction causes severe chronic diseases. In contrast to the strict centriole number control in cycling cells, multiciliated cell differentiation is marked by the production of up to several hundred centrioles, each nucleating a motile cilium. The mechanisms of centriole amplification or centriole number control in these cells were unknown and new centrioles were thought to appear de novo in the cytoplasm. First, videomicroscopy combined with correlative super-resolution and electron microscopy has enabled us to determine that all procentrioles are generated via runs of nucleation from the pre-existing progenitor cell centrosome. We show that the daughter centriole of the centrosome is the primary nucleation site for 95% of the new centrioles in multiciliated cells and thus refute the de novo hypothesis. Then, we provide evidence of an activation of the mitosis regulatory network during the centriole dynamic. With single cell live imaging and pharmacological modulation of mitosis regulators, we show that the mitosis machinery orchestrates the spatiotemporal progression of centriole amplification in terminally differentiating multiciliated cell progenitors. The fine-tuning of Cdk1 activity prevents mitosis while allowing the timely coordination of centriole number, growth, and disengagement through checkpoint-like phase transitions necessary for subsequent functional motile ciliation. This PhD provides a new paradigm for studying multiciliated cell differentiation, cilia-related diseases and pathological centriole amplification associated with cancer and microcephaly

Epithelial cells are necessary building blocks of the organs they line. Their apicalbasolateral polarity, characterized by an asymmetric distribution of cell components along their apical-basal axis, is a requirement for normal organ function. Although the centrosome, also known as the microtubule organizing center, is important in establishing cell polarity the mechanisms through which it achieves this remain unclear. It has been suggested that the centrosome influences cell polarity through microtubule cytoskeleton organization and endosome trafficking. In the first chapter of this thesis, I summarize the current understanding of the mechanisms regulating cell polarity and review evidence for the role of centrosomes in this process. In the second chapter, I examine the roles of the mother centriole appendages in cell polarity during cell migration and cell division. Interestingly, the subdistal appendages, but not the distal appendages, are essential in both processes, a role they achieve through organizing centrosomal microtubules. Depletion of subdistal appendages disrupts microtubule organization at the centrosome and hence, affects microtubule stability. These microtubule defects affect centrosome reorientation and spindle orientation during cell migration and division, respectively. In addition, depletion of subdistal appendages affects the localization and dynamics of apical polarity proteins in relation to microtubule stability and endosome recycling. Taken together, our results suggest the mother centriole subdistal appendages play an essential role in regulating cell polarity. A discussion of the significance of these results is included in chapter three.

Epithelial cells are necessary building blocks of the organs they line. Their apicalbasolateral polarity, characterized by an asymmetric distribution of cell components along their apical-basal axis, is a requirement for normal organ function. Although the centrosome, also known as the microtubule organizing center, is important in establishing cell polarity the mechanisms through which it achieves this remain unclear. It has been suggested that the centrosome influences cell polarity through microtubule cytoskeleton organization and endosome trafficking. In the first chapter of this thesis, I summarize the current understanding of the mechanisms regulating cell polarity and review evidence for the role of centrosomes in this process. In the second chapter, I examine the roles of the mother centriole appendages in cell polarity during cell migration and cell division. Interestingly, the subdistal appendages, but not the distal appendages, are essential in both processes, a role they achieve through organizing centrosomal microtubules. Depletion of subdistal appendages disrupts microtubule organization at the centrosome and hence, affects microtubule stability. These microtubule defects affect centrosome reorientation and spindle orientation during cell migration and division, respectively. In addition, depletion of subdistal appendages affects the localization and dynamics of apical polarity proteins in relation to microtubule stability and endosome recycling. Taken together, our results suggest the mother centriole subdistal appendages play an essential role in regulating cell polarity. A discussion of the significance of these results is included in chapter three.