Добірка наукової літератури з теми "Centriole to centrosome conversion"

Centriole-to-centrosome conversion (CCC) safeguards centriole homeostasis by coupling centriole duplication with segregation, and is essential for stabilization of mature vertebrate centrioles naturally devoid of the geometric scaffold or the cartwheel. Here we identified PPP1R35, a putative regulator of the protein phosphatase PP1, as a novel centriolar protein required for CCC. We found that PPP1R35 is enriched at newborn daughter centrioles in S or G2 phase. In the absence of PPP1R35, centriole assembly initiates normally in S phase, but none of the nascent centrioles can form active centrosomes or recruit CEP295, an essential factor for CCC. Instead, all PPP1R35-null centrioles, although stable during their birth in interphase, become disintegrated after mitosis upon cartwheel removal. Surprisingly, we found that neither the centriolar localization nor the function of PPP1R35 in CCC requires the putative PP1-interacting motif. PPP1R35 is thus acting upstream of CEP295 to induce CCC for proper centriole maintenance.

Centrioles are self-reproducing organelles that form the core structure of centrosomes or microtubule-organizing centers (MTOCs). However, whether duplication and MTOC organization reflect innate activities of centrioles or activities acquired conditionally is unclear. In this paper, we show that newly formed full-length centrioles had no inherent capacity to duplicate or to organize pericentriolar material (PCM) but acquired both after mitosis through a Plk1-dependent modification that occurred in early mitosis. Modified centrioles initiated PCM recruitment in G1 and segregated equally in mitosis through association with spindle poles. Conversely, unmodified centrioles segregated randomly unless passively tethered to modified centrioles. Strikingly, duplication occurred only in centrioles that were both modified and disengaged, whereas unmodified centrioles, engaged or not, were “infertile,” indicating that engagement specifically blocks modified centrioles from reduplication. These two requirements, centriole modification and disengagement, fully exclude unlimited duplication in one cell cycle. We thus uncovered a Plk1-dependent mechanism whereby duplication and segregation are coupled to maintain centriole homeostasis.

The role of centrioles changes as a function of the cell cycle. Centrioles promote formation of spindle poles in mitosis and act as basal bodies to assemble primary cilia in interphase. Stringent regulations govern conversion between these two states. Although the molecular mechanisms have not been fully elucidated, recent findings have begun to shed light on pathways that regulate the conversion of centrioles to basal bodies and vice versa. Emerging studies also provide insights into how defects in the balance between centrosome and cilia function could promote ciliopathies and cancer.

The centrosome, a unique membraneless multiprotein organelle, plays a pivotal role in various cellular processes that are critical for promoting cell proliferation. Faulty assembly or organization of the centrosome results in abnormal cell division, which leads to various human disorders including cancer, microcephaly and ciliopathy. Recent studies have provided new insights into the stepwise self-assembly of two pericentriolar scaffold proteins, Cep63 and Cep152, into a near-micrometre-scale higher-order structure whose architectural properties could be crucial for proper execution of its biological function. The construction of the scaffold architecture appears to be centrally required for tight control of a Ser/Thr kinase called Plk4, a key regulator of centriole duplication, which occurs precisely once per cell cycle. In this review, we will discuss a new paradigm for understanding how pericentrosomal scaffolds are self-organized into a new functional entity and how, on the resulting structural platform, Plk4 undergoes physico-chemical conversion to trigger centriole biogenesis.

Both gain and loss of function studies have identified the Polo-like kinase Plk4/Sak as a crucial regulator of centriole biogenesis, but the mechanisms governing centrosome duplication are incompletely understood. In this study, we show that the pericentriolar material protein, Cep152, interacts with the distinctive cryptic Polo-box of Plk4 via its N-terminal domain and is required for Plk4-induced centriole overduplication. Reduction of endogenous Cep152 levels results in a failure in centriole duplication, loss of centrioles, and formation of monopolar mitotic spindles. Interfering with Cep152 function prevents recruitment of Plk4 to the centrosome and promotes loss of CPAP, a protein required for the control of centriole length in Plk4-regulated centriole biogenesis. Our results suggest that Cep152 recruits Plk4 and CPAP to the centrosome to ensure a faithful centrosome duplication process.

The two centrioles of the centrosome differ in age and function. Although the mother centriole mediates most centrosome-dependent processes, the role of the daughter remains poorly understood. A recent study has implicated the daughter centriole in centriole amplification in multiciliated cells, but its contribution to primary ciliogenesis is unclear. We found that manipulations that prevent daughter centriole formation or induce its separation from the mother abolish ciliogenesis. This defect was caused by stabilization of the negative ciliogenesis regulator CP110 and was corrected by CP110 depletion. CP110 dysregulation may be caused by effects on Neurl-4, a daughter centriole–associated ubiquitin ligase cofactor, which was required for ciliogenesis. Centrosome-targeted Neurl-4 was sufficient to restore ciliogenesis in cells with manipulated daughter centrioles. Interestingly, early during ciliogenesis, Neurl-4 transiently associated with the mother centriole in a process that required mother–daughter centriole proximity. Our data support a model in which the daughter centriole promotes ciliogenesis through Neurl-4–dependent regulation of CP110 levels at the mother centriole.

Centrosome duplication is tightly controlled in coordination with DNA replication. The molecular mechanism of centrosome duplication remains unclear. Previous studies found that a fraction of human proline-directed phosphatase Cdc14B associates with centrosomes. However, Cdc14B's involvement in centrosome cycle control has never been explored. Here, we show that depletion of Cdc14B by RNA interference leads to centriole amplification in both HeLa and normal human fibroblast BJ and MRC-5 cells. Induction of Cdc14B expression through a regulatable promoter significantly attenuates centriole amplification in prolonged S phase–arrested cells and proteasome inhibitor Z-L3VS–treated cells. This inhibitory function requires centriole-associated Cdc14B catalytic activity. Together, these results suggest a potential function for Cdc14B phosphatase in maintaining the fidelity of centrosome duplication cycle.

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.