InteractiveFly: GeneBrief

Gamma-tubulin ring protein 84: Biological Overview | References


Gene name - Gamma-tubulin ring protein 84

Synonyms -

Cytological map position - 18D1-18D1

Function - cytoskeleton

Keywords - mitotic and meiotic microtubule organization, γ-tubulin ring complex component

Symbol - Grip84

FlyBase ID: FBgn0026430

Genetic map position - X: 19,455,373..19,459,629 [-]

Classification - Spc97/Spc98 family

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

γ-Tubulin, a protein critical for microtubule assembly, functions within multiprotein complexes. However, little is known about the respective role of γ-tubulin partners in metazoans. For the first time in a multicellular organism, the function of Dgrip84, the Drosophila orthologue of the Saccharomyces cerevisiae γ-tubulin-associated protein Spc97p, has been investigated. 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, Dgrip84 is involved 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 γ-tubulin complex necessary for proper microtubule organization in the metazoans is discussed (Colombié, 2006).

The mechanisms of microtubule nucleation remain unclear, although it has been demonstrated that γ-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. In vitro, γ-tubulin monomers enhance the assembly of alpha/beta heterodimers and block the minus ends of microtubules. However, in vivo, γ-tubulin does not seem to act as a monomer, but rather in a variety of protein complexes. The simplest one called γ-tubulin small complex (γ-TuSC) has been well characterized in Saccharomyces cerevisiae. It is formed by γ-tubulin and two associated proteins in a 2:1:1 stoichiometry. 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. Besides γ-TuSC, other larger γ-tubulin-containing complexes have been described. One termed γ-tubulin ring complex (γ-TuRC) has been characterized in multicellular organisms such as D. melanogaster, Xenopus laevis, and Homo sapiens. It is assumed that the γ-TuRC results from the association of several γ-TuSCs with at least four other proteins. Three of them show sequence homologies (grip motifs) with the two γ-tubulin-associated proteins in the γ-TuSC, whereas the fourth exhibits five WD repeats. The function of γ-tubulin has been studied extensively in several organisms and in cultured cells using antibody microinjection, mutants or gene silencing. For example, in Drosophila where two γ-tubulin isotypes are expressed, homozygous γ-tub 23C mutants die during late larval stage, exhibiting atypical mitotic spindles and abnormal centrosomal structures, whereas disruption of the γ-tub 37CD gene results in abnormal female meiotic spindles. However, knowledge about the function of the γ-tubulin-interacting proteins is limited. The role of the two grip proteins associated with γ-tubulin in the γ-TuSC has been studied both in budding and fission yeasts. Deletions of each of these genes (SPC97 and SPC98 in S. cerevisiae, Alp4 and Alp6 in Schizosaccharamyces pombe) are lethal; they induce mitotic defects and abnormal long cytoplasmic microtubules. However conditional mutants show phenotypic differences. In S. cerevisiae, Spc98p seems critical for γ-tubulin anchorage to the inner spindle plaque via direct interaction with Spc110p, whereas Spc97p seems essential for a correct SPB duplication and separation (Knop, 1997s). In fission yeast, Alp4 mutations compromise γ-tubulin localization to the SPB, but they do not affect the assembly of the large γ-tubulin complex. Discrepancies exist about the role of the two grip motif γ-TuSC subunits, in particular in the SPB duplication/separation process and in the γ-tubulin anchorage. Moreover, results obtained in yeasts are difficult to transfer to metazoans for several reasons: (1) the morphology of the microtubule-organizing centers is different; (2) the amino acid sequences of these proteins are poorly conserved because Spc97p and its Drosophila orthologue (Dgrip84) exhibit only 10% identity and 22% similarity; (3) in contrast with S. cerevisiae where γ-tubulin relocalizes to the SPB as γ-TuSC, in multicellular organisms, it is assumed that γ-tubulin is recruited to the centrosome as γ-TuRC. In metazoans, the silencing of γ-tubulin-associated proteins (Dgrip91, the Spc98p orthologue, and Dgrip75) has been performed essentially in Drosophila and does not induce similar phenotypes. Dgrip91 is an essential protein, required for correct bipolar spindle assembly during mitosis and male meiosis. In contrast, Dgrip75, a protein restricted to the large γ-tubulin complex, is essential for female fertility, but not for viability. Mutations in Dgrip75 prevent the correct localization of some morphogenetic determinants during oogenesis, suggesting a role in the organization/dynamics of some subsets of microtubules (Colombié, 2006).

A functional analysis of Dgrip84 during mitosis and male meiosis has been performed using two independent strategies (mutant strains and RNA interference [RNAi] in cultured cells). The role of this γ-TuSC protein in the organization of microtubule cytoplasmic arrays has been examined. Dgrip84, as γ-tubulin and Dgrip91, is essential for viability attesting that none of these three proteins is fully redundant (Colombié, 2006).

The function of the three constituents of the γ-TuSC has been examined using genetic approaches only in unicellular organisms. These analyses must be further investigated in animals where γ-tubulin is assumed to be recruited to the centrosome as γ-TuRC. Functional analysis of γ-tubulin-associated proteins in metazoans has been mainly restricted to Drosophila: Dgrip91, a γ-TuSC protein, and Dgrip75, a protein that specifically belongs to the large complex. The study of Dgrip84, the third component of the small complex in D. melanogaster, completes the functional characterization of the γ-TuSC, allowing for the first time a comparison of the phenotypes resulting from the disruption of each γ-TuSC component in a same metazoan (Colombié, 2006).

Their depletion has no obvious effect on the cytoskeleton organization during interphase. However, in Dgrip84- or γ-tubulin-depleted cells, regrowth experiments allow the appearance of abnormally long and less numerous cytoplasmic microtubules compared with controls. It could be indicative of a role of γ-TuSC proteins in the assembly and the maintenance of the length of interphase microtubules. Long microtubules could also result from an increase of the concentration of free tubulin in consequence of low microtubule assembly. Similar phenotypes displaying long microtubules have been observed after mutations in the γ-tubulin encoding gene both in budding yeast and A. nidulans and in γ-TuSC components in S. pombe. In S2 cells, this effect could be hidden because of the density and the complexity of microtubule arrays. Whatever the outset of interphase microtubules in Drosophila cells, functional γ-TuSC seems required in some aspects of microtubule dynamics and organization (Colombié, 2006).

Disruption of each γ-TuSC component promotes a moderate increase of the mitotic index. The arrest is weak compared with the one induced after a microtubule poison treatment. Moreover, Dgrip84-RNAi depletion previously to drug exposure reduces significantly the blockage extent, suggesting a deregulation of the spindle checkpoint or an activation of another cell cycle checkpoint. Inactivation of Drosophila γ-tubulin, Dgrip84, or Dgrip91 results in large polyploid nuclei (Sunkel, 1995; Barbosa, 2000; this study). This observation strengthens the idea that when the γ-TuSC integrity is affected, cells could escape prematurely from the mitotic checkpoint. These results are consistent with several sets of data. Human γ-tubulin mutants expressed in S. pombe allow cytokinesis to proceed in spite of spindle abnormalities. Some alleles of the A. nidulans γ-tubulin gene exhibit a slight delay in mitosis and could enter interphase without correct division, even after a microtubule-destabilizing treatment. In S. pombe, mutations in genes encoding γ-tubulin interacting proteins bypass the spindle assembly checkpoint and cause the untimely activation of the septation initiation network. Together, these results suggest a role of the γ-TuSC in the mitotic checkpoint control (Colombié, 2006).

Mitotic figures of Dgrip84-depleted cells exhibit monopolar morphology or microtubule arrays that fail to define oriented polar structures and correct chromosome congression. This is also a characteristic feature of cells lacking Dgrip91 or γ-tubulin (Sunkel, 1995; Barbosa, 2000; Raynaud-Messina, 2004). In monopolar spindles, Cnn- and Asp-labeling patterns are consistent with the presence of supernumerary centrioles at the poles. Electron microscopy analysis supports this view, suggesting that centrosomes fail to segregate. This abnormality has also been observed both in Dgrip91 mutant neuroblasts and after 23Cγ-tubulin depletion in S2 cells (Barbosa, 2000; Raynaud-Messina, 2004). Moreover, Dgrip84 mutant spermatocytes exhibit monopolar structures. Similar figures, described in γ-tub23C and Dgrip91 mutant spermatocytes, have been shown to be the consequence of the collapse of the two poles. Drosophila γ-TuSC assembly seems necessary for the proper separation of the centrosomes. Similar observations were obtained after γ-tubulin depletion in Caenorhabditis elegans, where separated asters are reapproaching in late prophase. The simplest interpretation is that the microtubule network involved in the maintenance of centrosome separation is defective either in its dynamics or its density (Colombié, 2006).

These studies emphasize that, after Dgrip84 depletion, some assembly and organization of spindles still occur and point to mechanisms of nucleation independent of γ-tubulin complexes. Microtubule organization still takes place at the poles. However, instead of emerging from pericentriolar material, some microtubules occur in continuity with centriolar triplets. Although this mechanism is unlikely to be dominant in the wild-type spindle assembly, it could account for the nucleation of some microtubules in Dgrip84-depleted cells that are characterized by anastral spindles. Some microtubules are able to establish contact with kinetochores showing that they retain some of their functional properties. Strong BubR1 signal in RNAi-treated cells is consistent with kinetochore-microtubule occupancy but alterations in microtubule tension (Colombié, 2006).

Depletion of Dgrip84 prevents both γ-tubulin and Dgrip91 localization at the poles. Moreover, γ-tubulin depletion impairs Dgrip84 and Dgrip91 mitotic recruitment (Raynaud-Messina, 2004). Therefore, it is likely that γ-TuSC components must be assembled in complexes before relocalization to the poles or that γ-TuSC proteins are required for γ-tubulin polar attachment. This view, supported by studies in S. cerevisiae, has been recently extended to mammalian cells: the γ-TuRCs could be anchored to the centrosome via interactions of GCP2 (Dgrip84 orthologue) and GCP3 (Dgrip91 orthologue) with the centrosomal proteins CG-NAP and pericentrin (Takahashi, 2002; Zimmerman, 2004). In Drosophila, the calmodulin-binding protein CP309 has been proposed to tether the γ-TuRC to the centrosome through direct binding with γ-TuSC components (Kawaguchi, 2004). So, the γ-TuSC grip-motif proteins seem essential, due to their critical role in the attachment of γ-tubulin complexes to the pericentriolar matrix. In contrast, the proteins specific for the γ-tubulin large complexes characterized so far, like Drosophila Dgrip75 and S. pombe Alp16p (Dgrip163 orthologue) or Gfh1p (Dgrip75 orthologue), are not essential for cell viability . It could be hypothesized that these γ-tubulin partners play a role in the organization or in the dynamics of a subset of microtubules (Colombié, 2006).

Analysis of meiosis in mutant spermatocytes reveals that Dgrip84, like Dgrip91 and γ-tubulin (Sampaio, 2001; Barbosa, 2003), is required for completion of the two meiotic divisions. In the absence of one of the γ-TuSC components, abnormal mitotic spindles can evolve toward a central spindle-like structure as viewed by the recruitment of different markers (Polo Kinase, Klp3A, Asp, Pav-KLP). Nevertheless, these proteins exhibit abnormal localization and cytokinesis is strongly asymmetrical and/or clearly abortive (Colombié, 2006).

In conclusion, the characterization of the third and last component of a metazoan γ-TuSC has been presented in this study. Together with previous studies, these data strongly suggest that the integrity of this complex must be maintained to ensure γ-tubulin recruitment, proper microtubule organization and an efficient mitotic checkpoint. In contrast, the depletion of γ-TuRC-specific proteins does not impair viability and γ-tubulin accumulation. It is suggested that the γ-TuSC could be a universal and minimal subunit required for γ-tubulin recruitment to the cytoskeleton structures and proper microtubule nucleation in eukaryotic cells (Colombié, 2006).

Distinct Dgrip84 isoforms correlate with distinct γ-tubulins in Drosophila

γ-Tubulin is an indispensable component of the animal centrosome and is required for proper microtubule organization. Within the cell, γ-tubulin exists in a multiprotein complex containing between two (some yeasts) and six or more (metazoa) additional highly conserved proteins named gamma ring proteins (Grips) or gamma complex proteins (GCPs). γ-Tubulin containing complexes isolated from Xenopus eggs or Drosophila embryos appear ring-shaped and have therefore been named the γ-tubulin ring complex (γTuRC). Curiously, many organisms (including humans) have two distinct γ-tubulin genes. In Drosophila, where the two γ-tubulin isotypes have been studied most extensively, the γ-tubulin genes are developmentally regulated: the 'maternal' γ-tubulin isotype (named γTub37CD according to its location on the genetic map) is expressed in the ovary and is deposited in the egg, where it is thought to orchestrate the meiotic and early embryonic cleavages. The second γ-tubulin isotype (γTub23C) is ubiquitously expressed and persists in most of the cells of the adult fly. In those rare cases where both γ-tubulins coexist in the same cell, they show distinct subcellular distributions and cell-cycle-dependent changes: γTub37CD mainly localizes to the centrosome, where its levels vary only slightly with the cell cycle. In contrast, the level of γTub23C at the centrosome increases at the beginning of mitosis, and γTub23C also associates with spindle pole microtubules. This study shows that γTub23C forms discrete complexes that closely resemble the complexes formed by γTub37CD. Surprisingly, however, γTub23C associates with a distinct, longer splice variant of Dgrip84. This may reflect a role for Dgrip84 in regulating the activity and/or the location of the γ-tubulin complexes formed with γTub37CD and γTub23C (Wiese, 2008).

The temporal and spatial control of microtubule nucleation and organization are essential for many fundamental cellular processes, including cell shape changes, intracellular transport, cell motility, partitioning of polarity signals during embryonic development, and, perhaps most importantly, the faithful segregation of chromosomes during cell division. In most animal cells, microtubules are nucleated and organized by the centrosome, which is composed of a pair of centrioles surrounded by the amorphous pericentriolar material (PCM) that appears to be the site of microtubule nucleation and anchoring. Although efforts to generate an inventory of centrosomal components have made great progress in recent years, the molecular organization of the centrosome itself is not well understood, and mechanistic descriptions of how centrosomal proteins contribute to microtubule formation and organization are yet to be obtained (Wiese, 2008).

γ-Tubulin is an indispensable component of the animal centrosome required for microtubule nucleation and organization in all eukaryotes. Human γ-tubulin can restore viability to Schizosaccharomyces pombe lacking endogenous γ-tubulin, suggesting that γ-tubulins are functionally conserved in phylogenetically distant organisms. Within the cell, γ-tubulin exists in a multiprotein complex containing between two (in the case of Saccharomyces cerevisiae) and five or more (S. pombe, Xenopus laevis, Drosophila melanogaster, and humans) additional proteins named gamma ring proteins (Grips) or gamma complex proteins (GCPs). γ-Tubulin containing complexes isolated from Xenopus eggs or Drosophila embryos appear ring-shaped and have therefore been named γ-tubulin ring complex. One of the major subcomplexes of the γTuRC, which has been named the γ-tubulin small complex (γTuSC; Oegema, 1999), appears to be a tetramer composed of two molecules of γ-tubulin and one molecule each of two other Grips known as Spc97p and Spc98p in S. cerevisiae, Dgrip84 and Dgrip91 in Drosophila melanogaster, or GCP2 and GCP3 in mammals (reviewed in Wiese, 2006). The tetramer was first described in S. cerevisiae (Knop, 1997b), where it may be the only soluble γ-tubulin complex. The metazoan γTuRC is thought to be composed of six or seven tetramers held together and capped by several additional proteins that copurify with γ-tubulin from Xenopus egg extracts or Drosophila embryos (Zheng, 1995; Oegema, 1999; Gunawardane, 2003), but subcomplexes between γ-tubulin and non-γTuSC Grips may also contribute to γTuRC structure (Gunawardane, 2000; Wiese, 2008 and references therein).

Consistent with a role for the γTuRC in microtubule nucleation, γ-tubulin and its binding partners are localized to the PCM at the minus ends of the microtubules (reviewed in Wiese, 2006). However, the molecular mechanism(s) for the function of the γTuRC in microtubule nucleation are still unclear. In addition to facilitating nucleation of microtubules, γ-tubulin also seems to play a role in interphase microtubule organization, and spindle assembly checkpoint regulation. Little is known to date about how γ-tubulin or the Grips participate in each of these functions (Wiese, 2008).

Curiously, the genomes of several organisms, including humans and flies, encode two distinct γ-tubulin genes. In Drosophila, where the two γ-tubulin isotypes have been studied most extensively, the γ-tubulin genes are developmentally regulated: the 'maternal' γ-tubulin isotype (named γTub37CD according to its location on the genetic map) is highly expressed in the ovary and is deposited in the egg, where it is thought to orchestrate the early embryonic cleavages. Consistent with this idea, mutation of γTub37CD results in female sterility. In contrast, γTub23C is essentially ubiquitous and is required for viability, microtubule organization during somatic mitotic divisions, and male meiosis. Although both isotypes are present in early embryos, only γTub37CD is detected at centrosomes in syncytial embryos, whereas punctate structures containing γTub23C distribute throughout the cytoplasm. Later during development, both γTub23C and γTub37CD localize to centrosomes in cellularized embryos. The extent to which each γ-tubulin isotype contributes to the assembly of female meiotic spindles remains to be resolved (Wiese, 2008 and references therein).

Differences in the timing of their association with the centrosome have also been reported for the two γ-tubulin isotypes in Drosophila tissue culture cells (Raynaud-Messina, 2001). In Kc23 cells, the centrosomal levels of γTub37CD have been reported to vary only slightly throughout the cell cycle, whereas centrosomal γTub23C levels increased sharply during late G2. Furthermore, γTub23C was also recruited to the microtubules of the mitotic spindle, whereas γTub37CD was never found on spindle microtubules. This suggests the existence of mechanisms that allow the cell to distinguish between the two γ-tubulin isotypes. This study set out to gain a better understanding of the biochemistry of γTub23C. Drosophila γTub23C isolated from tissue culture cells forms bona fide ring complexes that include most of the same Grips as the γTuRCs formed by γTub37CD. However, it was surprising to find that γTub23C and γTub37CD associate with distinct splice variants of Dgrip84/GCP2 that differ by 74 amino acids in their amino termini. It is speculated that Dgrip84 may be involved in regulating the subcellular localization of γ-tubulin (Wiese, 2008).

This study analyzed γ-tubulin complexes in two different systems: in Drosophila embryos, where γTub37CD is the major γ-tubulin isotype, and in Schneider S2 tissue culture cells, where γTub23C is the major γ-tubulin isotype and γTub37C plays only a minor role, if any (Raynaud-Messina, 2004). Based on the observation that γ-tubulin and its interacting proteins are highly conserved among distantly related species, it was reasonable to suspect that γTub23C forms complexes in Drosophila cells that closely resemble the γTuRC37CD. This study has provides the first experimental evidence that this is indeed the case. Previously identified γ-tubulin-interacting proteins (Dgrips) copurify with γTub23C isolated from tissue culture cells, and together these proteins form a ring-shaped complex that closely resembles the γTuRC37CD when viewed by negative staining electron microscopy. These studies revealed two unexpected results: 1) γTub23C forms a large complex in early embryos that is distinct from the γTuRC, and 2) expression of different Dgrip84 isoforms is developmentally regulated and correlates with expression of different γ-tubulin isoforms. These findings support the notion that the two Drosophila γ-tubulins interact with different binding partners and perhaps interact differently with similar binding partners (Wiese, 2008).

In embryos, γTub23C distributes in punctate patterns not associated with centrosomes, whereas γTub37CD localizes to centrosomes and spindle poles during early embryonic divisions (Wilson, 1997). It has been postulated that only γTub37CD is utilized at centrosomes during embryonic cleavages (Wilson, 1997). Consistent with this, female meiosis and nuclear divisions during early embryogenesis specifically require γTub37CD, and γTub37CD mutants are female sterile despite the presence of γTub23C in the embryos. This suggests that the γ-tubulin isotypes are not functionally redundant during embryogenesis. The current results provide a potential biochemical explanation for these observations. It is proposed that the embryonic γTub23C complex may represent a type of 'storage form' of γTub23C. This is suggested by the observation that γTub23C could not be immunoprecipitated from early embryos, indicating that the epitope recognized by the antibody was not accessible. It is speculated that the storage particle might include molecular chaperones of the TriC protein family, which have been shown to interact with and aid in the folding of γ-tubulin. It was found that the large γTub23C complexes persisted for at least the first 8 h of development, suggesting that the availability of γTub23C is very tightly regulated during early fly development. Full analysis of the embryonic γTub23C complex awaits methods to purify the complex that do not rely on antibody affinity chromatography. How the association of γTub23C with the putative storage particle is regulated and how γTub37CD escapes recruitment to the particle remains a mystery (Wiese, 2008).

To date, Drosophila Dgrip84 is the only member of the GCP2 protein family that has been reported to exist as more than one splice variant. Database analysis reveals that at least two Drosophila species possess the 74-amino acid N-terminal extension, D. melanogaster and D. pseudoobscura. It is possible that unique structural features of one or the other Dgrip84 isoform facilitate the particularly rapid centrosome assembly of the early Drosophila embryo, as has previously been postulated for γTub37CD (Wilson, 1997). Consistent with this hypothesis, Dgrip84 has recently been implicated in centrosome assembly and separation (Colombié, 2006). GCP2 (and GCP3) family members have also been implicated in γTuRC recruitment to the spindle pole body in S. cerevisiae and to the centrosome in metazoa. The precise role of GCP2 family members in γTuRC function and recruitment remains to be elucidated (Wiese, 2008).

This analysis of Dgrip84 isoforms showed that the isoform expressed in embryos corresponds to the shortest splice variant, whereas the isoform expressed in older embryos and S2 cells corresponds to a previously unreported variant of Dgrip84 that has an N-terminal extension but lacks the reported C-terminal insertion. To date, no experimental evidence has been found for the existence of Dgrip84 isoforms that possess the C-terminal insertion (isoforms A and B). It is possible that these isoforms are expressed only at very low levels or that they are expressed only in certain tissues. The roles of the extension or the insertion in Dgrip84 function are not yet clear, but it is noted that the developmental switch to the longer isoform of Dgrip84 correlated with the appearance of γTuRCs composed of γTub23C. Although thus far this is merely a correlation, it is tempting to speculate that the N-terminal Dgrip84 extension aids in folding or assembly of the somatic γTuRC, or is required for incorporation of γTub23C into the γTuRC. Experiments to address these questions are currently underway (Wiese, 2008).

The metazoan γTuRC contains 12-15 γ-tubulin molecules (reviewed in Wiese, 2006), most of which are thought to be arranged in tetrameric subcomplexes (γTuSCs) that contain two copies of γ-tubulin. Having two γ-tubulin isotypes in the same cell prompts a number of important questions: can both γ-tubulin isotypes coexist in the same γTuSC? Can both isotypes coexist in the same γTuRC? How does the cell distinguish between the γ-tubulin isotypes? Do the γ-tubulin isotypes perform different functions? How does the cell transition from using primarily one isotype to using the other, and how does the activity of a chimeric γTuRC (for which evidence was found by both sucrose gradient analysis and analysis of purified complexes) or γTuSC differ from each of the homogenous versions? The results presented here are beginning to provide answers to some of these questions, but gaining a true understanding of how γ-tubulin complexes that differ only slightly in their composition are perhaps used to fine-tune microtubule organization will be the subject of future studies (Wiese, 2008).

Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules, gammaTubulin small complex and gammaTubulin Ring complex

The microtubule (MT) cytoskeleton is essential for cell division and organization of the interphase cytoplasm. These functions are orchestrated by diverse and highly dynamic MT arrays generated by a variety of mechanisms, including regulation of the polymerization dynamics of MTs, of proteins that interact with and organize MTs, and of MT nucleation. The latter mechanism is possible because the spontaneous nucleation of new tubulin polymers is kinetically limiting, both in vitro when the polymerization of pure tubulin is initiated, and in vivo. Evidence for a kinetic barrier to MT nucleation in vivo comes from analysis of repolymerization of MTs after cold treatment or treatment with anti-MT agents. In many animal cells, regrowth initiates from the pericentriolar material (PCM) that surrounds the centrioles, demonstrating that the PCM promotes MT nucleation. A major breakthrough in defining the molecular basis of the MT-nucleating activity of the PCM was the discovery of gamma-tubulin: gamma-tubulin is a member of the tubulin superfamily that localizes to MT organizing centers and is found in all eukaryotes. Genetic studies have demonstrated that gamma-tubulin is required for normal cytoplasmic and spindle MT formation. In higher eukaryotes, soluble gamma-tubulin exists primarily in a large complex (between 25 and 32 S). This complex was purified from Xenopus egg extracts and shown to nucleate MTs in vitro. This complex, called the gammaTuRC (gamma-tubulin ring complex), consists of about eight proteins in addition to gamma-tubulin and has the appearance of an open ring with approximately the same diameter as a MT (Zheng, 1995). Rings of this diameter have also been observed in the PCM of centrosomes isolated from Drosophila embryos (Moritz, 1995a). In Drosophila, immunoelectron microscopy has confirmed the presence of clusters of gamma-tubulin in the ring structures and at the base of MTs nucleated by the PCM (Moritz, 1995b). Cumulatively, these results suggest that the gammaTuRC is a highly conserved structure responsible for the MT-nucleating activity of the PCM (Oegema, 1999 and references).

The gamma-tubulin in S. cerevisiae is the most divergent of all gamma-tubulins. It is only ~35%-40% identical to the other known gamma-tubulins, all of which are at least 65% identical to one another. In S. cerevisiae, the only known soluble gamma-tubulin-containing complex is ~6 S and contains three proteins: gamma-tubulin, and two related proteins, Spc97p and Spc98p (Geissler, 1996 and Knop, 1997b). Immunoprecipitation experiments with tagged proteins suggest that the S. cerevisiae complex contains one molecule of Spc97p, one molecule of Spc98p, and two or more molecules of gamma-tubulin (Knop, 1997a; Knop, 1997b). The yeast gamma-tubulin 6 S complex is thought to be anchored to the cytoplasmic side of the spindle pole body through the interaction of Spc97p and Spc98p with Spc72p (Knop, 1998), and to the nuclear side of the spindle pole body through interaction with the NH2 terminus of Spc110p (Knop, 1997b). To date, in vitro MT-nucleating activity for the yeast complex has not been demonstrated. Therefore, it remains unclear whether the yeast gamma-tubulin complex nucleates MTs directly, or whether it assembles into a larger, perhaps gammaTuRC-like structure at the spindle pole body. Interestingly, homologs of Spc97p and Spc98p in humans (hGCP2 and hGCP3/HsSpc98; Murphy, 1998, Tassin, 1998) and in Xenopus (Xgrip109; Martin, 1998) colocalize with gamma-tubulin at the centrosome and cosediment with gamma-tubulin on sucrose gradients, indicating that they are components of the large gamma-tubulin-containing complexes present in these organisms (Oegema, 1999 and references).

Understanding the role of gamma-tubulin in MT nucleation is a challenging endeavor. Low cellular concentrations make purification from native sources difficult, and the complexity of the protein complexes that contain gamma-tubulin limits expression-based studies. Analysis of MT nucleation is further complicated by the following: the complex structure of the MT lattice; the large number of tubulin molecules potentially involved in the formation of a nucleus, and the potential role of beta-tubulin GTP hydrolysis in suppressing nucleation. This difficulty is reflected by the fact that the mechanism of spontaneous nucleation of purified tubulin remains poorly understood (Oegema, 1999 and references).

Central to an understanding of the mechanism of MT nucleation by gamma-tubulin-containing complexes will be an understanding of the relationship between gamma-tubulin and other members of the tubulin superfamily. One important aspect of this relationship is the nature of the contacts gamma-tubulin makes with itself and with alpha- or beta-tubulin. A second important aspect is how gamma-tubulin compares to other members of the tubulin family in its ability to bind and hydrolyze GTP. If gamma-tubulin binds a guanine nucleotide, it will be important to determine whether nucleotide exchange and hydrolysis contribute to its ability to assemble, disassemble, nucleate, or release MTs, or whether the bound nucleotide has a structural role, as is the case for alpha-tubulin. The functional organization of the gammaTuRC in Drosophila has been addressed by purifying and analyzing gamma-tubulin-containing complexes from Drosophila embryo extracts. In Drosophila, there are two related gamma-tubulin-containing complexes. These have been named gamma-tubulin small complex (gammaTuSC; ~280,000 D) and Drosophila gammaTuRC (~2,200,000 D). In addition to gamma-tubulin, the gammaTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The gammaTuSC is a structural subunit of the gammaTuRC, a larger complex containing about six additional polypeptides. Like the gammaTuRC isolated from Xenopus egg extracts, the Drosophila gammaTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with ~13 radially arranged structural repeats. The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity. The larger complex can be collapsed into the smaller complex by treatment with high salt. This condensation suggests that the small complex is a structural subunit of the large complex (Moritz, 1998). Both complexes have now been purified and it has been shown that the large Drosophila complex nucleates MTs much more potently than the small complex. Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily (Oegema, 1999).

To determine the protein compositions of the gammaTuSC and gammaTuRC, complexes were fractionated on a 5% to 40% sucrose gradient. The protein profile of the Drosophila gammaTuRC is reminiscent of the Xenopus gammaTuRC. Therefore, by analogy to the Xgrips (Martin, 1998), Drosophila gammaTuRC proteins have been named Dgrips and have been designated by their apparent molecular weights. Like the Xenopus gammaTuRC, the Drosophila gammaTuRC is composed of two high molecular mass proteins (Dgrip163 and Dgrip128), two prominent proteins near 100 kD (Dgrip91 and Dgrip84), and a group of three or four proteins with molecular masses near 75 kD (Dgrip75s). It is not clear whether actin is a specific component of gammaTuRC, or if it fortuitously copurifies. Depending on the purification protocol, varying amounts of alpha- and beta-tubulin copurify with Xenopus gammaTuRC (Zheng, 1995). In contrast, no alpha- or beta-tubulin copurifying with Drosophila gammaTuRC could be detected. Consistent with the idea that gammaTuSC is a structural subunit of gammaTuRC, gammaTuSC is composed of the three most prominent proteins in gammaTuRC: gamma-tubulin, Dgrip84, and Dgrip91 (Oegema, 1999).

To characterize the molecular nature of gammaTuSC, its non-gamma-tubulin components, Dgrip84 and Dgrip91 were cloned and sequenced. Dgrip84 and Dgrip91 are homologous to each other and to the Spc97/98p family of proteins identified in S. cerevisiae. This family also includes two proteins identified in humans, hGCP2 and hGCP3 (Murphy, 1998). The homology between the Drosophila proteins and the other members of this family extends over the entire length of the proteins. In comparisons of Dgrip84 and Dgrip91 with the corresponding human proteins, a one-to-one correspondence emerges. Dgrip84 is 32% identical (46% similar) to hGCP2 and only 21% identical (32% similar) to hGCP3; in contrast, Dgrip91 is 31% identical (45% similar) to hGCP3 and only 24% identical (37% similar) to hGCP2. These results suggest that Dgrip84 and hGCP2, and Dgrip91 and hGCP3 may be functionally homologous pairs. Since an estimate of the molecular mass of purified gammaTuSC from sucrose gradient sedimentation and gel filtration is 280,000 D, it is suspected that gammaTuSC contains 1 molecule of Dgrip91, 1 ;molecule of Dgrip84, and 2 molecules of gamma-tubulin. Interestingly, this corresponds to estimates of the stoichiometry of proteins in the S. cerevisiae 6 S gamma-tubulin complex (Knop, 1997a; Knop, b). If it is assumed that gammaTuRC contains only one molecule of each non-gammaTuSC component, then gammaTuRC would contain approximately six gammaTuSCs (Oegema, 1999).

If gamma-tubulin in Drosophila embryos primarily exists associated with Dgrip84 and Dgrip91 in either gammaTuSC or gammaTuRC, these three proteins would be expected to cofractionate on sucrose gradients of embryo extract and to colocalize in embryos. To test this hypothesis, rabbit polyclonal antibodies were produced that recognize Dgrip84 and Dgrip91. As expected, both Dgrip84 and Dgrip91 comigrate with gamma-tubulin in gammaTuSC and gammaTuRC when embryo extract is fractionated on sucrose gradients. In addition, the localizations of Dgrip84 and Dgrip91 in Drosophila embryos are indistinguishable from those of gamma-tubulin. Each antibody recognizes the centrosome throughout the cell cycle and shows some spindle staining during mitosis with enrichment at the spindle poles, regardless of its cognate antigen. It is proposed that Drosophila gamma-tubulin is stably associated with Dgrip91/84. Interestingly, no evidence is found for a non-gamma-tubulin associated pool of either Dgrip84 or Dgrip91 (Oegema, 1999).

The homology between gamma-, alpha-, and beta-tubulins extends into domains that are involved in GTP binding by alpha- and beta-tubulin. Thus, it is tempting to speculate that gamma-tubulin can bind, and possibly hydrolyze, GTP. To determine if gamma-tubulin binds guanine nucleotide, gamma-tubulin-containing complexes were immunoisolated in the absence of GTP. The isolated complexes, either before or after sucrose gradient sedimentation, were incubated with [alpha-32P]GTP and UV cross-linked. In the peptide-eluted complexes, gamma-tubulin is the only protein that cross-links to GTP. Furthermore, gamma-tubulin in both the gammaTuRC and gammaTuSC cross-links to GTP. Competition experiments show that the cross-link can be competed by addition of excess cold GTP, GDP, and GTPgammaS but not GMP-PNP, ATP, or CTP (Oegema, 1999).

To characterize the nucleotide binding properties of gamma-tubulin, the nucleotide content of gamma-tubulin in gammaTuSC was compared to that of similarly treated alphabeta-tubulin dimer. When gammaTuSC is analyzed, the nucleotide recovered from gammaTuSC incubated in GDP buffers is exclusively GDP. Approximately 0.7 mol GDP is recovered per mole of gamma-tubulin. The exclusive presence of GDP could be explained at least three ways: (1) the guanine nucleotide binding site on gamma-tubulin subunits of gammaTuSC is freely exchangeable; (2) GDP is locked nonexchangeably into gamma-tubulin subunits of gammaTuSC, analogous to GTP bound at the N-site in alpha-tubulin; or (3) GDP is locked nonexchangeably into gammaTuSC as the product of earlier GTP hydrolysis, much like beta-tubulin bound GDP within the body of a polymerizing MT (Oegema, 1999).

To distinguish between these possibilities, gammaTuSC was isolated from GTP-containing buffer. Surprisingly, a greatly reduced amount of nucleotide was recovered. Only 0.2 mol guanine nucleotide was recovered per mole of gamma-tubulin, indicating that ~80% of the gamma-tubulin was empty at its nucleotide binding site. The low recovery of guanine nucleotide bound to gammaTuSC isolated from GTP buffer indicates that GDP is bound exchangeably to gamma-tubulin in gammaTuSC. This result also argues against the theory that the GDP bound to gamma-tubulin in gammaTuSC, isolated from GDP buffer, is being generated by earlier GTP hydrolysis. The recovery of nearly 1 mol GDP per mole of gamma-tubulin from GDP buffer and the nearly equivalent amounts of GTP and GDP in the 0.2 mol nucleotide recovered per mole of gamma-tubulin from GTP buffer, despite a GTP/GDP ratio of greater than 30 before desalting, strongly suggest that gamma-tubulin in gammaTuSC has an exchangeable guanine nucleotide binding site that has a much higher affinity for GDP than GTP (Oegema, 1999).

An important issue with respect to the in vivo roles of gammaTuSC and gammaTuRC is their relative MT nucleating activity. The fact that S. cerevisiae does not appear to contain a gammaTuRC-like complex raises the question of whether Sc gammaTuSC has nucleating activity or whether it must assemble into a larger structure at the spindle pole body to become active. Conversely, in metazoa it is possible that gammaTuRC is a storage form for gamma-tubulin and it could be gammaTuSC that nucleates MTs at centrosomes (Knop, 1997b). For the Drosophila complexes, per mole of gamma-tubulin, gammaTuRC is ~25 times more active than gammaTuSC in promoting nucleation. Combining these data with stoichiometry measurements, it is estimated that per mole of complex gammaTuRC is ~150 times more active than gammaTuSC, suggesting that organization of gammaTuSC into gammaTuRC facilitates MT nucleation activity (Oegema, 1999).

The origin of centrosomes in parthenogenetic hymenopteran insects

A longstanding enigma has been the origin of maternal centrosomes that facilitate parthenogenetic development in Hymenopteran insects. In young embryos, hundreds of microtubule-organizing centers (MTOCs) are assembled completely from maternal components. Two of these MTOCs join the female pronucleus to set up the first mitotic spindle in unfertilized embryos and drive their development. These MTOCs appear to be canonical centrosomes because they contain gamma-tubulin, CP190, and centrioles and they undergo duplication. Evidence is presented that these centrosomes originate from accessory nuclei (AN), organelles derived from the oocyte nuclear envelope. In the parasitic wasps Nasonia vitripennis and Muscidifurax uniraptor, the position and number of AN in mature oocytes correspond to the position and number of maternal centrosomes in early embryos. These AN also contain high concentrations of gamma-tubulin. In the honeybee, Apis mellifera, distinct gamma-tubulin foci are present in each AN. Additionally, the Hymenopteran homolog of the Drosophila centrosomal protein Dgrip84 localizes on the outer surfaces of AN. These organelles disintegrate in the late oocyte, leaving behind small gamma-tubulin foci, which likely seed the formation of maternal centrosomes. Accessory nuclei, therefore, may have played a significant role in the evolution of haplodiploidy in Hymenopteran insects (Ferree, 2006).


REFERENCES

Search PubMed for articles about Drosophila Grip84

Barbosa, V., Yamamoto, R. R., Henderson, D. S. and Glover, D. (2000). Mutation of a Drosophila γ tubulin ring complex subunit encoded by discs degenerate-4 differentially disrupts centrosomal protein localization. Genes Dev. 14: 3126-3139. PubMed ID: 11124805

Barbosa, V., et al. (2003). Drosophila dd4 mutants reveal that γTuRC is required to maintain juxtaposed half spindles in spermatocytes. J. Cell Sci. 116: 929-941. PubMed ID: 12571290

Colombié, N., et al. (2006). The Drosophila γ-Tubulin small complex subunit Dgrip84 is required for structural and functional integrity of the spindle apparatus. Molec. Biol. Cell 17: 272-282. PubMed ID: 16236791

Ferree, P. M., et al. (2006). The origin of centrosomes in parthenogenetic hymenopteran insects. Curr. Biol. 16: 801-807. PubMed ID: 16631588

Geissler, S., Pereira, G., Spang, A., Knop, M., Soues, S., Kilmartin, J. and Schiebel, E. (1996). The spindle pole body component Spc98p interacts with the gamma-tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment. EMBO J. 15: 3899-3911. PubMed ID: 8670895

Gunawardane, R. N., Martin, O. C., Cao, K., Zhang, L., Dej, K., Iwamatu, A. and Zheng, Y. (2000). Characterization and reconstitution of Drosophila {gamma}-tubulin ring complex subunits. J. Cell Biol 151: 1513-1524. PubMed ID: 11134079

Gunawardane, R. N., Martin, O. C., and Zheng, Y. (2003). Characterization of a new {gamma}TuRC subunit with WD repeats. Mol. Biol. Cell 14: 1017-1026. PubMed ID: 12631720

Kawaguchi, S. and Zheng, Y. (2004). Characterization of a Drosophila centrosome protein CP309 that shares homology with Kendrin and CG-NAP. Mol. Biol. Cell 15: 37-45. PubMed ID: 14565985

Knop, M., Pereira, G., Geissler, S., Grein, K. and Schiebel, E. (1997a). The spindle pole body component Spc97p interacts with the gamma-tubulin of Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication. EMBO J. 16: 1550-1564. PubMed ID: 9130700

Knop, M. and Schiebel, E. (1997b). Spc98p and Spc97p of the yeast gamma-tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p. EMBO J. 16(23): 6985-6995. PubMed ID: 9384578

Knop, M. and Schiebel, E. (1998). Receptors determine the cellular localization of a gamma-tubulin complex and thereby the site of microtubule formation. EMBO J. 17(14): 3952-3967. PubMed ID: 9670012

Martin, O. C., et al. (1998). Xgrip109: A gamma Tubulin-associated protein with an essential role in gamma tubulin ring complex (gammaTuRC) assembly and centrosome function. J. Cell Biol. 141(3): 675-687. PubMed ID: 9566968

Moritz, M., Braunfeld, M. B., Fung, J. C., Sedat, J. W., Alberts, B. M. and Agard, D. A. (1995a). Three-dimensional structural characterization of centrosomes from early Drosophila embryos. J. Cell Biol. 130: 1149-1159. PubMed ID: 7657699

Moritz, M., et al. (1995b). Microtubule nucleation by gamma-tubulin-containing rings in the centrosome. Nature 378: 638-640. PubMed ID: 8524401

Murphy, S. M., Urbani, L. and Stearns, T. (1998). The mammalian gamma-Tubulin complex contains homologues of the yeast spindle pole body components Spc97p and Spc98p. J. Cell Biol. 141(3): 663-674. PubMed ID: 9566967

Oegema, K., Wiese, C., Martin, O. C., Milligan, R. A., Iwamatsu, E., Mitchison, T. J., and Zheng, Y. (1999). Characterization of two related Drosophila γ-tubulin complexes that differ in their ability to nucleate microtubules. J. Cell Biol 144: 721-733. PubMed ID: 10037793

Raynaud-Messina, B., Mazzolini, L., Moisand, A., Cirinesi, A. M. and Wright, M. (2004). Elongation of centriolar microtubule triplets contributes to the formation of the mitotic spindle in γ-tubulin-depleted cells. J. Cell Sci. 117: 5497-5507. PubMed ID: 15479719

Sampaio, P., et al. (2001). Organized microtubule arrays in γ-tubulin-depleted Drosophila spermatocytes. Curr. Bio. 11: 1788-1793. PubMed ID: 11719222

Sunkel, C. E., et al. (1995). Gamma-tubulin is required for the structure and function of the microtubule organizing centre in Drosophila neuroblasts. EMBO J. 14(1): 28-36. PubMed ID: 7828594

Takahashi, M., Yamagiwa, A., Nishimura, T., Mukai, H. and Ono, Y. (2002). Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring γ-tubulin ring complex. Mol. Biol. Cell 13: 3235-3245. PubMed ID: 12221128

Tassin, A. M., et al. (1998). Characterization of the human homologue of the yeast Spc98p and its association with gamma-Tubulin. J. Cell Biol. 141(3): 689-701. PubMed ID: 9566969

Wiese, C., and Zheng, Y. (2006). Microtubule nucleation: γ-tubulin and beyond. J. Cell Sci 119: 4143-4153. PubMed ID: 17038541

Wiese, C. (2008). Distinct Dgrip84 isoforms correlate with distinct gamma-tubulins in Drosophila. Mol. Biol. Cell 19(1): 368-77. PubMed ID: 18003974

Wilson, P. G., Zheng, Y., Oakley, C. E., Oakley, B. R., Borisy, G. G., and Fuller, M. T. (1997). Differential expression of two {gamma}-tubulin isoforms during gametogenesis and development in Drosophila. Dev. Biol. 184: 207-221. PubMed ID: 9133431

Zheng, Y., Wong, M. L., Alberts, B., and Mitchison, T. (1995). Nucleation of microtubule assembly by a γ-tubulin-containing ring complex. Nature 378: 578-583. PubMed ID: 8524390

Zimmerman, W. C., Sillibourne, J., Rosa, J. and Doxsey, S. J. (2004). Mitosis-specific anchoring of γ-tubulin complexes by pericentrin controls spindle organization and mitotic entry. Mol. Biol. Cell 15: 3642-3657. PubMed ID: 15146056


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date revised: 20 September 2008

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