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

Abstract

␥-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...

Stability of the small γ-tubulin complex requires HCA66, a protein of the centrosome and the nucleolus

“…Likewise, monopolar spindle formation in HCA66-depleted cells can be explained by the loss of functional γ-tubulin or γ-TuSCs, as seen in knock-down experiments or mutants in various species (Sunkel et al, 1995;Barbosa et al, 2000;Strome et al, 2001;Hannak et al, 2002;Barbosa et al, 2003;Raynaud-Messina et al, 2004;Yuba-Kubo et al, 2005;Colombié et al, 2006;Haren et al, 2006). It is likely that the spindle defects in HCA66-depleted cells lead to chromosome segregation defects and micronuclei, explaining the increased cell death observed in HCA66-depleted cultures, but we cannot exclude the possibility that HCA66 plays additional roles in interphase that are important for the survival of the cells.…”

The γTuRC components Grip75 and Grip128 have an essential microtubule-anchoring function in theDrosophilagermline

“…Grip128 and Grip75 are not essential for viability ␥TuRC function in microtubule nucleation is crucial for viability, as mutations in Grip91/l(1)dd4, Grip84 and ␥Tub23C are lethal (Barbosa et al, 2000;Colombie et al, 2006;Sunkel et al, 1995). By contrast, Grip75, Grip128 and the double mutants are viable, showing that both gene products are not essential for the microtubule-nucleating properties of the ␥TuRC and that the ␥TuRC formed in these mutants is sufficient for microtubule function in somatic cell types of the fly.…”

“…The components of the ␥TuSC appear to be required for microtubule organization. In Drosophila, mutations in Grip91 (l(1)dd4 -FlyBase) or Grip84 are lethal and display defects in spindle assembly (Barbosa et al, 2000;Colombie et al, 2006). Drosophila has two ␥-tubulin genes, ␥Tub23C and ␥Tub37C.…”

Zfrp8, theDrosophilaortholog ofPDCD2,functions in lymph gland development and controls cell proliferation

“…6 and see Fig. S3 in the supplementary material) (Colombie et al, 2006;Oegema et al, 1999). We also stained the centrosome protein CP309, which is required for microtubule nucleation (Kawaguchi and Zheng, 2004).…”