Biologie des centrosomes et de l’instabilité génétique

Publications de l’équipe

Année de publication : 2012

Renata Basto (2012 Oct 10)

A centriolar lifeline.

Nature reviews. Molecular cell biology : 686 : DOI : 10.1038/nrm3460 En savoir plus
Résumé

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Véronique Marthiens, Matthieu Piel, Renata Basto (2012 Sep 8)

Never tear us apart–the importance of centrosome clustering.

Journal of cell science : 3281-92 : DOI : 10.1242/jcs.094797 En savoir plus
Résumé

The presence of more than two centrosomes (centrosome amplification) at the onset of mitosis has long been associated with multipolar spindle formation, and with the generation of genetic instability. However, in recent years, several studies have shown that a process termed ‘centrosome clustering’ actively contributes to bipolar division by promoting the gathering of extra centrosomes in two main poles. In this Commentary, we describe the main proteins that are involved in centriole duplication and discuss how centrosome amplification can be generated both in vitro and in vivo. We then summarize what is currently known about the processes that contribute to bipolar spindle formation when extra centrosomes are present, and which forces contribute to this process. Finally, we discuss how extra centrosomes might contribute to tumorigenesis, giving emphasis to the role of centrosome amplification in promoting genetic instability.

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Année de publication : 2011

Dora Sabino, Nicholas H Brown, Renata Basto (2011 Mar 16)

Drosophila Ajuba is not an Aurora-A activator but is required to maintain Aurora-A at the centrosome.

Journal of cell science : 1156-66 : DOI : 10.1242/jcs.076711 En savoir plus
Résumé

The LIM-domain protein Ajuba localizes at sites of epithelial cell-cell adhesion and has also been implicated in the activation of Aurora-A (Aur-A). Despite the expected importance of Ajuba, Ajuba-deficient mice are viable, which has been attributed to functional redundancy with the related LIM-domain protein LIMD1. To gain insights into the function of Ajuba, we investigated its role in Drosophila, where a single gene (jub) encodes a protein closely related to Ajuba and LIMD1. We identified a key function in neural stem cells, where Jub localizes to the centrosome. In these cells, mutation in jub leads to centrosome separation defects and aberrant mitotic spindles, which is a phenotype similar to that of aur-A mutants. We show that in jub mutants Aur-A activity is not perturbed, but that Aur-A recruitment and maintenance at the centrosome is affected. As a consequence the active kinase is displaced from the centrosome. On the basis of our studies in Drosophila neuroblasts, we propose that a key function of Ajuba, in these cells, is to maintain active Aur-A at the centrosome during mitosis.

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Delphine Gogendeau, Ilse Hurbain, Graca Raposo, Jean Cohen, France Koll, Renata Basto (2011 Feb 2)

Sas-4 proteins are required during basal body duplication in Paramecium.

Molecular biology of the cell : 1035-44 : DOI : 10.1091/mbc.E10-11-0901 En savoir plus
Résumé

Centrioles and basal bodies are structurally related organelles composed of nine microtubule (MT) triplets. Studies performed in Caenorhabditis elegans embryos have shown that centriole duplication takes place in sequential way, in which different proteins are recruited in a specific order to assemble a procentriole. ZYG-1 initiates centriole duplication by triggering the recruitment of a complex of SAS-5 and SAS-6, which then recruits the final player, SAS-4, to allow the incorporation of MT singlets. It is thought that a similar mechanism (that also involves additional proteins) is present in other animal cells, but it remains to be investigated whether the same players and their ascribed functions are conserved during basal body duplication in cells that exclusively contain basal bodies. To investigate this question, we have used the multiciliated protist Paramecium tetraurelia. Here we show that in the absence of PtSas4, two types of defects in basal body duplication can be identified. In the majority of cases, the germinative disk and cartwheel, the first structures assembled during duplication, are not detected. In addition, if daughter basal bodies were formed, they invariably had defects in MT recruitment. Our results suggest that PtSas4 has a broader function than its animal orthologues.

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Année de publication : 2010

Philippe Benaroch, Elisabeth Billard, Raphaël Gaudin, Michael Schindler, Mabel Jouve (2010 Apr 9)

HIV-1 assembly in macrophages.

Retrovirology : 29 : DOI : 10.1186/1742-4690-7-29 En savoir plus
Résumé

The molecular mechanisms involved in the assembly of newly synthesized Human Immunodeficiency Virus (HIV) particles are poorly understood. Most of the work on HIV-1 assembly has been performed in T cells in which viral particle budding and assembly take place at the plasma membrane. In contrast, few studies have been performed on macrophages, the other major target of HIV-1. Infected macrophages represent a viral reservoir and probably play a key role in HIV-1 physiopathology. Indeed macrophages retain infectious particles for long periods of time, keeping them protected from anti-viral immune response or drug treatments. Here, we present an overview of what is known about HIV-1 assembly in macrophages as compared to T lymphocytes or cell lines.Early electron microscopy studies suggested that viral assembly takes place at the limiting membrane of an intracellular compartment in macrophages and not at the plasma membrane as in T cells. This was first considered as a late endosomal compartment in which viral budding seems to be similar to the process of vesicle release into multi-vesicular bodies. This view was notably supported by a large body of evidence involving the ESCRT (Endosomal Sorting Complex Required for Transport) machinery in HIV-1 budding, the observation of viral budding profiles in such compartments by immuno-electron microscopy, and the presence of late endosomal markers associated with macrophage-derived virions. However, this model needs to be revisited as recent data indicate that the viral compartment has a neutral pH and can be connected to the plasma membrane via very thin micro-channels. To date, the exact nature and biogenesis of the HIV assembly compartment in macrophages remains elusive. Many cellular proteins potentially involved in the late phases of HIV-1 cycle have been identified; and, recently, the list has grown rapidly with the publication of four independent genome-wide screens. However, their respective roles in infected cells and especially in macrophages remain to be characterized. In summary, the complete process of HIV-1 assembly is still poorly understood and will undoubtedly benefit from the ongoing explosion of new imaging techniques allowing better time-lapse and quantitative studies.

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Année de publication : 2009

Vincent Popoff, Gonzalo A Mardones, Siau-Kun Bai, Valérie Chambon, Danièle Tenza, Patricia V Burgos, Anbing Shi, Philippe Benaroch, Sylvie Urbé, Christophe Lamaze, Barth D Grant, Graça Raposo, Ludger Johannes (2009 Oct 31)

Analysis of articulation between clathrin and retromer in retrograde sorting on early endosomes.

Traffic (Copenhagen, Denmark) : 1868-80 : DOI : 10.1111/j.1600-0854.2009.00993.x En savoir plus
Résumé

Clathrin and retromer have key functions for retrograde trafficking between early endosomes and the trans-Golgi network (TGN). Previous studies on Shiga toxin suggested that these two coat complexes operate in a sequential manner. Here, we show that the curvature recognition subunit component sorting nexin 1 (SNX1) of retromer interacts with receptor-mediated endocytosis-8 (RME-8) protein, and that RME-8 and SNX1 colocalize on early endosomes together with a model cargo of the retrograde route, the receptor-binding B-subunit of Shiga toxin (STxB). RME-8 has previously been found to bind to the clathrin uncoating adenosine triphosphatase (ATPase) Hsc70, and we now report that depletion of RME-8 or Hsc70 affects retrograde trafficking at the early endosomes-TGN interface of STxB and the cation-independent mannose 6-phosphate receptor, an endogenous retrograde cargo protein. We also provide evidence that retromer interacts with the clathrin-binding protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) not only via SNX1, as previously published (Chin Raynor MC, Wei X, Chen HQ, Li L. Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor. J Biol Chem 2001;276:7069-7078), but also via the core complex component Vps35. Hrs codistributes at the ultrastructural level with STxB on early endosomes, and interfering with Hrs function using antibodies or mild overexpression inhibits retrograde transport. Our combined data suggest a model according to which the functions in retrograde sorting on early endosomes of SNX1/retromer and clathrin are articulated by RME-8, and possibly also by Hrs.

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Delphine Gogendeau, Renata Basto (2009 Jun 25)

Centrioles in flies: the exception to the rule?

Seminars in cell & developmental biology : 163-73 : DOI : 10.1016/j.semcdb.2009.07.001 En savoir plus
Résumé

Centrioles and basal bodies are MT based structures that present a highly conserved ninefold symmetry. Centrioles can be found at the core of the centrosome where they participate in PCM recruitment and organization, contributing to cytoplasmic MT nucleation. Basal bodies are normally located closely to the plasma membrane where they are responsible for axoneme assembly to form structures such as cilia or flagella. While it is well accepted that these organelles have important roles in cell and tissue organization, their contribution to certain phases of animal development is still not entirely established. Here we review the role of centrosomes and cilia in Drosophila melanogaster and briefly discuss the implications of these findings to other model organisms.

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Sandrine Moutel, Ahmed El Marjou, Ole Vielemeyer, Clément Nizak, Philippe Benaroch, Stefan Dübel, Franck Perez (2009 Feb 28)

A multi-Fc-species system for recombinant antibody production.

BMC biotechnology : 14 : DOI : 10.1186/1472-6750-9-14 En savoir plus
Résumé

Genomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural antibodies.

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Sandrine Moutel, Ole Vielemeyer, Hulin Jin, Séverine Divoux, Philippe Benaroch, Franck Perez (2009 Jan 22)

Fully in vitro selection of recombinant antibodies.

Biotechnology journal : 38-43 : DOI : 10.1002/biot.200800246 En savoir plus
Résumé

Antibodies are essential for the identification and characterization of proteins. In the current postgenomic era the need for highly specific antibodies has further increased not only for research applications but also because they represent one of the most promising therapeutic options, especially in the field of cancer treatment. One appealing approach for rapid and inexpensive antibody generation is the use of phage display. This technique allows for a fast and animal-free selection of highly functional alternatives to classical antibodies. However, one strong limitation of this recombinant approach has been the difficulty in producing and purifying antigens. These steps have to be adjusted for each new target, are time consuming and sometimes present an insurmountable obstacle. Here we report the development of new antibody selection approach where antigens are produced through in vitro translation and are used directly and without the need for purification. With this approach we were able to rapidly select recombinant antibodies directed against GFP and the mammalian protein tsg101, respectively. We believe that our method greatly facilitates antigen preparation and thus may broaden the use of the recombinant approach for antibody generation, especially since the technique could in the future be adapted to a high-throughput technology, thus further accelerating antibody selection.

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Année de publication : 2008

Charlotte Sadaka, Marie-Annick Marloie-Provost, Vassili Soumelis, Philippe Benaroch (2008 Nov 19)

Developmental regulation of MHC II expression and transport in human plasmacytoid-derived dendritic cells.

Blood : 2127-35 : DOI : 10.1182/blood-2008-10-178152 En savoir plus
Résumé

Plasmacytoid predendritic cells (pDCs) play a key role in antiviral immunity through their capacity to produce large amounts of type I interferons in response to Toll-like receptor triggering, and to differentiate into dendritic cells (DCs). However, their antigen processing and presentation pathways remain poorly characterized. In this study, we analyzed major histocompatibility complex class II (MHC II) synthesis and transport in primary human pDCs. We show that stimulation of pDCs with influenza virus leads to a sustained neosynthesis of MHC II molecules, which rapidly accumulate in antigen loading compartments organized around the microtubule organization center. MHC II endocytosis as well as antigen internalization remain active during the entire process of pDC differentiation into DCs, suggesting a capacity to constantly renew surface peptide-MHC II complexes. Formation of the intracellular pool of MHC II in activated pDCs is nuclear factor-kappaB-dependent and associated with acquisition of a dendritic phenotype, but independent of the IRF7-type I interferon-dependent pathway, suggesting that innate and adaptive functions of pDCs are differentially regulated. Our data demonstrate that the regulation of MHC II expression and transport is drastically different in pDCs compared with conventional DCs, indicating distinct and potentially complementary immunoregulatory functions.

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Fanni Gergely, Renata Basto (2008 Sep 4)

Multiple centrosomes: together they stand, divided they fall.

Genes & development : 2291-6 : DOI : 10.1101/gad.1715208 En savoir plus
Résumé

Cells with extra centrosomes rely entirely on centrosome clustering mechanisms to assemble a bipolar spindle and to divide in a bipolar fashion. To identify the pathways involved in suppression of multipolarity, Kwon, Godinho, and colleagues (pp. 2189-2203) have set up a genome-wide screen in Drosophila S2 cells. Surprisingly, they found that efficient clustering requires a large number of proteins associated with a variety of cellular functions.

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Hélène Toussaint, François-Xavier Gobert, Michael Schindler, Carina Banning, Patrycja Kozik, Mabel Jouve, Frank Kirchhoff, Philippe Benaroch (2008 Jul 4)

Human immunodeficiency virus type 1 nef expression prevents AP-2-mediated internalization of the major histocompatibility complex class II-associated invariant chain.

Journal of virology : 8373-82 : DOI : 10.1128/JVI.00670-08 En savoir plus
Résumé

The lentiviral Nef protein has been studied extensively for its ability to induce the downregulation of several immunoreceptors on the surfaces of infected cells. However, Nef expression is unique in inducing highly effective upregulation of the major histocompatibility complex class II-associated chaperone invariant (Ii) chain complexes in different cell types. Under normal conditions, endocytosis of the Ii chain and other molecules, like the transferrin receptor and CD4, is rapid and AP-2 dependent. Human immunodeficiency virus type 1 (HIV-1) Nef expression strongly reduces the internalization of the Ii chain, enhances that of CD4, and does not modify transferrin uptake. The mutation of AP-2 binding motifs LL164 and DD174 in Nef leads to the inhibition of Ii chain upregulation. In AP-2-depleted cells, surface levels of the Ii chain are high and remain unmodified by Nef expression, further indicating that Nef regulates Ii chain internalization via the AP-2 pathway. Immunoprecipitation experiments revealed that the Ii chain can interact with Nef in a dileucine-dependent manner. Importantly, we have shown that Nef-induced CD4 downregulation and Ii chain upregulation are genetically distinguishable. We have identified natural nef alleles that have lost one of the two functions but not the other one. Moreover, we have characterized Nef mutant forms possessing a similar phenotype in the context of HIV-1 infection. Therefore, the Nef-induced accumulation of Ii chain complexes at the cell surface probably results from a complex mechanism leading to the impairment of AP-2-mediated endocytosis rather than from direct competition between Nef and the Ii chain for binding AP-2.

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Nina Peel, Renata Basto (2008 Mar 29)

The centrosome in the eukaryotic cell.

SEB experimental biology series : 113-43 En savoir plus
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Renata Basto, Kathrin Brunk, Tatiana Vinadogrova, Nina Peel, Anna Franz, Alexey Khodjakov, Jordan W Raff (2008 Feb 20)

Centrosome amplification can initiate tumorigenesis in flies.

Cell : 1032-42 : DOI : 10.1016/j.cell.2008.05.039 En savoir plus
Résumé

Centrosome amplification is a common feature of many cancer cells, and it has been previously proposed that centrosome amplification can drive genetic instability and so tumorigenesis. To test this hypothesis, we generated Drosophila lines that have extra centrosomes in approximately 60% of their somatic cells. Many cells with extra centrosomes initially form multipolar spindles, but these spindles ultimately become bipolar. This requires a delay in mitosis that is mediated by the spindle assembly checkpoint (SAC). As a result of this delay, there is no dramatic increase in genetic instability in flies with extra centrosomes, and these flies maintain a stable diploid genome over many generations. The asymmetric division of the larval neural stem cells, however, is compromised in the presence of extra centrosomes, and larval brain cells with extra centrosomes can generate metastatic tumors when transplanted into the abdomens of wild-type hosts. Thus, centrosome amplification can initiate tumorigenesis in flies.

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Année de publication : 2007

Mabel Jouve, Nathalie Sol-Foulon, Sarah Watson, Olivier Schwartz, Philippe Benaroch (2007 Nov 17)

HIV-1 buds and accumulates in « nonacidic » endosomes of macrophages.

Cell host & microbe : 85-95 En savoir plus
Résumé

Macrophages represent viral reservoirs in HIV-1-infected patients and accumulate viral particles within an endosomal compartment where they remain infectious for long periods of time. To determine how HIV-1 survives in endocytic compartments that become highly acidic and proteolytic and to study the nature of these virus-containing compartments, we carried out an ultrastructural study on HIV-1-infected primary macrophages. The endosomal compartments contain newly formed virions rather than internalized ones. In contrast to endocytic compartments free of viral proteins within the same infected cells, the virus containing compartments do not acidify. The lack of acidification is associated with an inability to recruit the proton pump vacuolar ATPase into the viral assembly compartment. This may prevent its fusion with lysosomes, since acidification is required for the maturation of endosomes. Thus, HIV-1 has developed a strategy for survival within infected macrophages involving prevention of acidification within a devoted endocytic virus assembly compartment.

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