Biomedical subjects

Michel Wright

Publications and source records attributed to Michel Wright.

12 recordsLinked to original sources

PubMed2006

Drosophila melanogaster gamma-TuRC is dispensable for targeting gamma-tubulin to the centrosome and microtubule nucleation.

In metazoans, gamma-tubulin acts within two main complexes, gamma-tubulin small complexes (gamma-TuSCs) and gamma-tubulin ring complexes (gamma-TuRCs). In higher eukaryotes, it is assumed that microtubule nucleation at the centrosome depends on gamma-TuRCs, but the role of gamma-TuRC components remains undefined. For the first time, we analyzed the function of all four gamma-TuRC-specific subunits in Drosophila melanogaster: Dgrip75, Dgrip128, Dgrip163, and Dgp71WD. Grip-motif proteins, but not Dgp71WD, appear to be required for gamma-TuRC assembly. Individual depletion of gamma-TuRC components, in cultured cells and in vivo, induces mitotic delay and abnormal spindles. Surprisingly, gamma-TuSCs are recruited to the centrosomes. These defects are less severe than those resulting from the inhibition of gamma-TuSC components and do not appear critical for viability. Simultaneous cosilencing of all gamma-TuRC proteins leads to stronger phenotypes and partial recruitment of gamma-TuSC. In conclusion, gamma-TuRCs are required for assembly of fully functional spindles, but we suggest that gamma-TuSC could be targeted to the centrosomes, which is where basic microtubule assembly activities are maintained.

Animals↗

PubMed2006

NEDD1-dependent recruitment of the gamma-tubulin ring complex to the centrosome is necessary for centriole duplication and spindle assembly.

The centrosome is the major microtubule organizing structure in somatic cells. Centrosomal microtubule nucleation depends on the protein gamma-tubulin. In mammals, gamma-tubulin associates with additional proteins into a large complex, the gamma-tubulin ring complex (gammaTuRC). We characterize NEDD1, a centrosomal protein that associates with gammaTuRCs. We show that the majority of gammaTuRCs assemble even after NEDD1 depletion but require NEDD1 for centrosomal targeting. In contrast, NEDD1 can target to the centrosome in the absence of gamma-tubulin. NEDD1-depleted cells show defects in centrosomal microtubule nucleation and form aberrant mitotic spindles with poorly separated poles. Similar spindle defects are obtained by overexpression of a fusion protein of GFP tagged to the carboxy-terminal half of NEDD1, which mediates binding to gammaTuRCs. Further, we show that depletion of NEDD1 inhibits centriole duplication, as does depletion of gamma-tubulin. Our data suggest that centriole duplication requires NEDD1-dependent recruitment of gamma-tubulin to the centrosome.

Cell Line↗

PubMed2005

The Drosophila gamma-tubulin small complex subunit Dgrip84 is required for structural and functional integrity of the spindle apparatus.

Gamma-tubulin, a protein critical for microtubule assembly, functions within multiprotein complexes. However, little is known about the respective role of gamma-tubulin partners in metazoans. For the first time in a multicellular organism, we have investigated the function of Dgrip84, the Drosophila orthologue of the Saccharomyces cerevisiae gamma-tubulin-associated protein Spc97p. Mutant analysis shows that Dgrip84 is essential for viability. Its depletion promotes a moderate increase in the mitotic index, correlated with the appearance of monopolar or unpolarized spindles, impairment of centrosome maturation, and increase of polyploid nuclei. This in vivo study is strengthened by an RNA interference approach in cultured S2 cells. Electron microscopy analysis suggests that monopolar spindles might result from a failure of centrosome separation and an unusual microtubule assembly pathway via centriolar triplets. Moreover, we point to an involvement of Dgrip84 in the spindle checkpoint regulation and in the maintenance of interphase microtubule dynamics. Dgrip84 also seems essential for male meiosis, ensuring spindle bipolarity and correct completion of cytokinesis. These data sustain that Dgrip84 is required in some aspects of microtubule dynamics and organization both in interphase and mitosis. The nature of a minimal gamma-tubulin complex necessary for proper microtubule organization in the metazoans is discussed.

Animals↗

PubMed2005

DNA damage induce gamma-tubulin-RAD51 nuclear complexes in mammalian cells.

Rad51 protein plays an essential role in recombination repair of DNA double-strand breaks and DNA crosslinking adducts. It is part of complexes which can vary with the stage of the cell cycle and the nature of the DNA lesions. During a search for Rad51-associated proteins in CHO nuclear extracts of S-phase cells by mass spectrometry of proteins immunoprecipitated with Rad51 antibodies, we identified a centrosomal protein, gamma-tubulin. This association was confirmed by the reverse immunoprecipitation with gamma-tubulin antibodies. Both proteins copurified from HeLa cells nuclear extracts following a tandem affinity purification of double-tagged Rad51. Immunofluorescence analysis showed colocalization of both Rad51 and gamma-tubulin in discrete foci in mammalian cell nuclei. The number of colocalized foci and their overlapping area increased in the presence of DNA damage produced by genotoxic treatments either during S phase or in exponentially growing cells. These variations did not result from an overall stress because microtubule cytoskeleton poisons devoid of direct interactions with DNA, such as taxol or colcemid, did not lead to an increase of this association. The recruitment of Rad51 and gamma-tubulin in the same nuclear complex suggests a link between DNA recombination repair and the centrosome function during the cell cycle.

Animals↗

PubMed2004

Elongation of centriolar microtubule triplets contributes to the formation of the mitotic spindle in gamma-tubulin-depleted cells.

The assembly of the mitotic spindle after depletion of the major gamma-tubulin isotype by RNA-mediated interference was assessed in the Drosophila S2 cell line. Depletion of gamma-tubulin had no significant effect on the cytoskeletal microtubules during interphase. However, it promoted an increase in the mitotic index, resulting mainly in monopolar and, to a lesser extent, asymmetrical bipolar prometaphases lacking astral microtubules. This mitotic accumulation coincided with the activation of the mitotic checkpoint. Immunostaining with an anti-Asp antibody revealed that the spindle poles, which were always devoid of gamma-tubulin, were unfocused and organized into sub-spindles. Despite the marked depletion of gamma-tubulin, the pericentriolar proteins CP190 and centrosomin were recruited to the spindle pole(s), where they formed three or four dots, suggesting the presence of several centrioles. Electron microscopic reconstructions demonstrated that most of the monopolar spindles exhibited three or four centrioles, indicating centriole duplication with a failure in the separation process. Most of the centrioles were shortened, suggesting a role for gamma-tubulin in centriole morphogenesis. Moreover, in contrast to metaphases observed in control cells, in which the spindle microtubules radiated from the pericentriolar material, in gamma-tubulin-depleted cells, microtubule assembly still occurred at the poles but involved the elongation of centriolar microtubule triplets. Our results demonstrate that, after depletion of gamma-tubulin, the pericentriolar material is unable to promote efficient microtubule nucleation. They point to an alternative mechanism of centrosomal microtubule assembly that contributes to the formation of abnormal, albeit partially functional, mitotic spindles.

Animals↗

PubMed2003

Basal body/centriole assembly and continuity.

The long-standing interest in centrioles and basal bodies stems from the evolutionary conservation of their structural design and from their dual mode of assembly (templated versus de novo), revealed by electron microscopic studies nearly four decades ago and unique for a subcellular organelle. Molecular dissection of the assembly pathway during the past few years has recently progressed, essentially through direct and reverse genetic approaches. These studies revealed essential roles for centrins and the gamma-, delta-, epsilon - and eta-tubulins in assembly or as specific signals for centriole duplication. Identification of further components of basal bodies and centrioles might help to unravel the two assembly pathways and their regulation.

Animals↗

PubMed2003

Gamma-tubulin and MTOCs in Paramecium.

The characterization of the two Paramecium gamma-tubulin genes, gammaPT1 and gammaPT2, allowed us to raise Paramecium-specific antibodies, directed against their most divergent carboxy-terminal peptide and to analyze the localization and dynamics of gamma-tubulin throughout the cell cycle. As in other cell types, a large proportion of the protein was found to be cytosolic, but in contrast to the general situation, gamma-tubulin was found to be permanently associated to four types of sites: basal bodies, the micronuclear compartment--within which mitotic and meiotic spindles develop without membrane breakdown, the pores of the contractile vacuoles and the cytoproct which are cortical microtubular organelles fulfilling excretory functions. In addition, a transient site of gamma-tubulin and microtubule assembly was observed at the site of nuclear exchange during conjugation. This complexity accounts for the nucleation of most of the numerous and diverse microtubule arrays present in Paramecium. The sites and mode of nucleation of the microtubule bundles formed in the macronuclear compartment during division remain unclear. These observations lead us to discuss the relationships between microtubules, gamma-tubulin and MTOCs.

Amino Acid Sequence↗

PubMed2002

The beta2-adaptin clathrin adaptor interacts with the mitotic checkpoint kinase BubR1.

The adaptor AP2 is a heterotetrameric complex that associates with clathrin and regulatory proteins to mediate rapid endocytosis from the plasma membrane. Here, we report the identification of the mitotic checkpoint kinase BubR1 as a novel binding partner of beta2-adaptin, one of the AP2 large subunits. Using two-hybrid experiments and in vitro binding assays, we show that beta2-adaptin binds to BubR1 through its amino-terminal beta2-'trunk' domain, while the beta2-binding region of BubR1 maps to the carboxy-terminal kinase domain. Subcellular immunolocalization studies suggest that the interaction between BubR1 and beta2-adaptin could take place in the cytosol at any time during the cell cycle. In addition, we found that BubR1 and the BubR1-related kinase, Bub1, also bind to beta-adaptins of other AP complexes. Together, these results support a model in which the mitotic checkpoint kinases BubR1 and BuB1, by binding to beta-adaptins, may play novel roles in the regulation of vesicular intracellular traffic.

Adaptor Protein Complex beta Subunits↗

PubMed2002

Differential binding to the alpha/beta-tubulin dimer of vinorelbine and vinflunine revealed by nuclear magnetic resonance analyses.

The binding of two antitumour alkaloids, vinorelbine and vinflunine, to the alpha/beta-tubulin dimer has been investigated at equilibrium by nuclear magnetic resonance (NMR) spectroscopy. Tubulin stability and assembly induced by these drugs has been checked under NMR experimental conditions, and tubulin spirals were found in majority. Then, using increasing ligand concentrations, the alkaloids were titrated against tubulin. A non-specific binding of both compounds to tubulin (K(d)>10(-5)M) was characterised by broad NMR ligand signal at 4 and 30 degrees. The tubulin dimer exhibited also 2.7 (sigma: 0.3) and 2.6 (sigma: 0.6) binding sites with a K(d)<10(-5)M for vinorelbine at 4 and 30 degrees, respectively. In contrast, if the tubulin dimer exhibited 2.7 (sigma: 0.2) binding sites for vinflunine at 4 degrees, these sites were not detected at 30 degrees. This NMR study revealed for the first time the presence of specific binding sites and a clear differential affinity of vinorelbine and vinflunine to the tubulin dimer at physiological temperatures which could possibly account for their differential cytotoxicity.

Animals↗

PubMed2002

Deregulated DNA polymerase beta induces chromosome instability and tumorigenesis.

To reach the biological alterations that characterize cancer, the genome of tumor cells must acquire increased mutability resulting from a malfunction of a network of genome stability systems, e.g., cell cycle arrest, DNA repair, and high accuracy of DNA synthesis during DNA replication. Numeric chromosomal imbalance, referred to as aneuploidy, is the most prevalent genetic changes recorded among many types of solid tumors. We report here that ectopic expression in cells of DNA polymerase beta, an error-prone enzyme frequently over-regulated in human tumors, induces aneuploidy, an abnormal localization of the centrosome-associated gamma-tubulin protein during mitosis, a deficient mitotic checkpoint, and promotes tumorigenesis in nude immunodeficient mice. Thus, we find that alteration of polymerase beta expression appears to induce major genetic changes associated with a malignant phenotype.

Aneuploidy↗

PubMed2002

Antitumor activity of Z-ajoene, a natural compound purified from garlic: antimitotic and microtubule-interaction properties.

Ajoene, a garlic stable oil-soluble sulfur rich compound was generally isolated as a mixture of two isomers [(E, Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide]. It has been described essentially as a potent inhibitor of platelet aggregation in vitro and in vivo. The antiproliferative effects of ajoene and experiments using a single isomer had received little attention. The present study aims at defining the antitumor activities of cis-Z-ajoene in vitro and in vivo. Antiproliferative activity of Z-ajoene was demonstrated against a panel of human tumor cell lines with IC(50) values varying from 5.2 mM to 26.1 mM and at a lower extent in normal marsupial kidney cells (PtK2). Meanwhile, Z-ajoene arrested HL60 cells in G(2)/M phase of cell cycle in a dose and time-dependent way. In PtK2 cells, exposure to 20 microM Z-ajoene for 6 h induced a complete disassembly of the microtubule network, that was associated with an increased number of cells blocked in early mitotic stages. An IC(50) for microtubule disassembly of 1 microM was determined by a fully automated microplate-based multi-detection reader. In vitro, a reversible inhibition of the microtubule protein assembly was observed with an IC(50) of 25 microM Z-ajoene. In vivo, Z-ajoene inhibited tumor growth by 38% and 42% in mice grafted with sarcoma 180 and hepatocarcinoma 22, respectively. For the first time, Z-ajoene was shown to be a potent inhibitor of tumor cell growth both in vitro and in vivo. The microtubule cytoskeleton appeared to be one of the Z-ajoene targets, but the mechanisms by which Z-ajoene interacted with microtubule appeared different from those of other microtubule poisons such as those of the Vinca alkaloids family. The ability of Z-ajoene to preferentially suppress the growth of neoplastic cells could provide a new approach in tumor therapy.

Animals↗

PubMed2002

Z-ajoene induces apoptosis of HL-60 cells: involvement of Bcl-2 cleavage.

Garlic organosulfur components exhibit antitumor activity, but the molecular mechanisms underlying these effects have not been well characterized. We showed that Z-ajoene, a sulfur-rich compound purified from garlic, induced time- and dose-dependent apoptosis in HL-60 cells. This process implied the activation of caspase-3 and the cleavage of the antiapoptotic protein Bcl-2. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-[OMe]-fluoromethylketone inhibited Bcl-2 cleavage and apoptosis induced by Z-ajoene. This effect was partially prevented by treatment of HL-60 cells with the antioxidant N-acetylcysteine. Hence, the transmission of apoptotic signal induced by Z-ajoene involved a reactive oxygen species-dependent pathway leading to caspase-dependent Bcl-2 cleavage.

Antineoplastic Agents↗