Differential properties of the two Drosophila γ-tubulin isotypes

Raynaud-Messina, Brigitte; Debec, Alain; Tollon, Yvette; Garès, Michèle; Wright, Michel

doi:10.1078/0171-9335-00195

Cited by 21 publications

(24 citation statements)

References 39 publications

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“…Rabbit anti-␥-tubulin: R46 [␥Tub37C (Raynaud-Messina et al, 2001)], 1:500 dilution, Rb1011 [␥Tub37C (Tavosanis and Gonzalez, 2003)] 1:5, R77 [␥Tub23C (Debec et al, 1995)] 1:500. Mouse anti-␤-galactosidase (Promega), 1:200.…”

Section: Immunohistochemistry and In-situ Hybridizationmentioning

confidence: 99%

The centrosome-nucleus complex and microtubule organization in theDrosophilaoocyte

et al. 2006

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Molecular motors transport the axis-determining mRNAs oskar, bicoid and gurken along microtubules (MTs) in the Drosophila oocyte. However, it remains unclear how the underlying MT network is organized and how this transport takes place. We have identified a centriole-containing centrosome close to the oocyte nucleus. Remarkably, the centrosomal components, ␥-tubulin and Drosophila pericentrin-like protein also strongly accumulate at the periphery of this nucleus. MT polymerization after cold-induced disassembly in wild type and in gurken mutants suggests that in the oocyte the centrosome-nucleus complex is an active center of MT polymerization. We further report that the MT network comprises two perpendicular MT subsets that undergo dynamic rearrangements during oogenesis. This MT reorganization parallels the successive steps in localization of gurken and oskar mRNAs. We propose that in addition to a highly polarized microtubule scaffold specified by the cortex oocyte, the repositioning of the nucleus and its tightly associated centrosome could control MT reorganization and, hence, oocyte polarization.

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Section: Immunohistochemistry and In-situ Hybridizationmentioning

confidence: 99%

The centrosome-nucleus complex and microtubule organization in theDrosophilaoocyte

et al. 2006

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“…Protein extracts from cultured S2 cells (Raynaud-Messina et al, 2001) and from total brains were subjected to Western blot analyses as described previously. Total brain extracts were prepared from about 10 brains of each genotype, dissected in saline (0.7% NaCl), boiled for 3 min in 2ϫ Laemmli sample buffer and diluted twice.…”

Section: Western Blottingmentioning

confidence: 99%

TheDrosophilaγ-Tubulin Small Complex Subunit Dgrip84 Is Required for Structural and Functional Integrity of the Spindle Apparatus

Colombié

Vérollet

Sampaio

et al. 2006

MBoC

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␥-Tubulin, a protein critical for microtubule assembly, functions within multiprotein complexes. However, little is known about the respective role of ␥-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 ␥-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 ␥-tubulin complex necessary for proper microtubule organization in the metazoans is discussed. INTRODUCTIONThe mechanisms of microtubule nucleation remain unclear, although it has been demonstrated that ␥-tubulin, a universal component of the microtubule-organizing centers, plays an essential role in microtubule nucleation. The molecular details of this process are still poorly understood (Oakley and Oakley, 1989;Oakley et al., 1990;Erickson and Stoffler, 1996;Gunawardane et al., 2003). In vitro, ␥-tubulin monomers enhance the assembly of ␣/␤ heterodimers and block the minus ends of microtubules (Li and Joshi, 1995;Leguy et al., 2000). However, in vivo, ␥-tubulin does not seem to act as a monomer, but rather in a variety of protein complexes (Murphy et al., 1998;Oegema et al., 1999;Fujita et al., 2002). The simplest one called ␥-tubulin small complex (␥-TuSC) has been well characterized in Saccharomyces cerevisiae , Drosophila melanogaster (Oegema et al., 1999), and vertebrates (Murphy et al., 1998). It is formed by ␥-tubulin and two associated proteins in a 2:1:1 stoichiometry Oegema et al., 1999;Murphy et al., 2001). This small complex is recruited at the inner and outer spindle plaques of S. cerevisiae spindle pole bodies (SPB) where it is responsible for microtubule nucleation Schiebel, 1997, 1998;Pereira et al., 1998). Besides ␥-TuSC, other larger ␥-tubulin-containing complexes have been described (Zheng et al., 1995;Fujita et al., 2002). One termed ␥-tubulin ring complex (␥-TuRC) has been characterized in multicellular organisms such as D. melanogaster, Xenopus laevis, and Homo sapiens (Zheng et al., 1995;Oegema et al., 1999;Murphy et al., 2001). It is assumed that the ␥-TuRC results from the associa...

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“…The rabbit antibodies R62 and R46 were used for the detection of the 13 C-terminal amino acids of 23C γ-tubulin and the pre-C-terminal amino acid sequence of 37CD γ-tubulin, respectively (Raynaud-Messina et al, 2001). Antibody Rb188 was used to detect CP190 (Whitfield et al, 1988).…”

Section: Immunofluorescence Stainingmentioning

confidence: 99%

“…Cell protein extracts were prepared and subjected to western-blot analysis as previously described (Raynaud-Messina et al, 2001). The mouse monoclonal antibody GTU-88 was raised against the amino acid region encompassing residues 38-53 of human γ-tubulin (SigmaAldrich, Saint Quentin Fallavier, France).…”

Section: Western-blot Analysismentioning

confidence: 99%

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

Raynaud-Messina

Mazzolini

Moisand

et al. 2004

Journal of Cell Science

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The assembly of the mitotic spindle after depletion of the major γ-tubulin isotype by RNA-mediated interference was assessed in the Drosophila S2 cell line. Depletion of γ-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 γ-tubulin, were unfocused and organized into sub-spindles. Despite the marked depletion of γ-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 γ-tubulin in centriole morphogenesis. Moreover, in contrast to metaphases observed in control cells, in which the spindle microtubules radiated from the pericentriolar material, in γ-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 γ-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.

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Differential properties of the two Drosophila γ-tubulin isotypes

Cited by 21 publications

References 39 publications

The centrosome-nucleus complex and microtubule organization in theDrosophilaoocyte

The centrosome-nucleus complex and microtubule organization in theDrosophilaoocyte

TheDrosophilaγ-Tubulin Small Complex Subunit Dgrip84 Is Required for Structural and Functional Integrity of the Spindle Apparatus

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

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