gamma-Tubulin at 37C


REGULATION

γTubulin ring complex behavior during mitotic catastrophe

In syncytial Drosophila embryos, damaged or incompletely replicated DNA triggers centrosome disruption in mitosis, leading to defects in spindle assembly and anaphase chromosome segregation. The damaged nuclei drop from the cortex and are not incorporated into the cells that form the embryo proper. A null mutation in the Drosophila checkpoint kinase 2 tumor suppressor homolog (Chk2) blocks this mitotic response to DNA lesions and also prevents loss of defective nuclei from the cortex. In addition, DNA damage leads to increased Chk2 localization to the centrosome and spindle microtubules. Chk2 is therefore essential for a 'mitotic catastrophe' signal that disrupts centrosome function in response to genotoxic stress and ensures that mutant and aneuploid nuclei are eliminated from the embryonic precursor pool (Takada, 2003).

γTuRC localization was examined following DNA damage. Wild-type embryos treated with DNA damaging agent camptothecin show mitosis-specific loss of γ-tubulin from the centrosomes and increased labeling over the anastral central spindle microtubules. Note that this response is observed in both the syncytial embryo and the pole cells, which are germline precursors and the first true cells to form during embryogenesis. Centrosome inactivation, therefore, is not restricted to the syncytial divisions. In mnk embryos, camptothecin has no effect on γ-tubulin localization, which accumulated at centrosomes in both the syncytium and pole cells. However, in mnk mutants carrying a wild-type mnk transgene, camptothecin induces wild-type loss of γ-tubulin localization in both the syncytium and pole cells. Based on these observations, it is conclude that Drosophila Chk2 is essential to centrosome inactivation in response to replication defects and a wide range of genotoxic lesions (Takada, 2003).

Based on these observations, it is proposed that Chk2 functions at two points during the early embryonic response to genotoxic stress. At the onset of mitosis, the presence of DNA lesions leads to Chk2 activation, which targets proteins involved in maintaining γ-tubulin ring complex (γTuRC) localization and centrosomal microtubule nucleating activity. The resulting anastral spindle is functionally compromised and anaphase chromosome segregation fails. Following division failure, Chk2 is required for a second process that appears to disrupt the link between centrosomes and nuclei, or prevent reestablishment of this link. Because the centrosomes anchor nuclei at the cortex, this Chk2-dependent response to DNA damage leads to loss of nuclei from the cortical monolayer. Only cortical nuclei are incorporated into the cells that will form the blastoderm embryo. The two-step Chk2-dependent response to genotoxic stress thus blocks propagation of abnormal nuclei and prevents their transmission to the embryo proper (Takada, 2003).

In Drosophila larvae and other systems, Chk2 appears to function upstream of p53 during DNA damage induced apoptosis. Because the p53 gene has been implicated in control of centrosome duplication, it is speculated that Chk2 functions through p53 during this mitosis-specific response to DNA damage. However, p53 null mutant embryos show a wild-type centrosome inactivation response. Localization of the γTuRC is disrupted during centrosome inactivation, and localization of Chk2 to the centrosome in response to DNA damage and in checkpoint defective embryos raises the possibility that this conserved kinase directly modifies one or more components of this complex. However, nuclear loss following division failure also requires Chk2, and this response is not dependent on loss of microtubule nucleating function during mitosis. Chk2 thus appears to target distinct factors involved in maintaining or establishing the link between nuclei and centrosomes. Mutations in cytoplasmic dynein lead to centrosome detachment from the nucleus during the syncytial divisions, suggesting that Chk2 could modify dynein or a dynein binding protein on mitotic exit in response to DNA damage. Identification of the downstream targets of Chk2 on mitotic onset and exit should shed light on both the mitotic DNA damage response and normal centrosome function (Takada, 2003).

Drosophila melanogaster γTuRC is dispensable for targeting γ-tubulin to the centrosome and microtubule nucleation

In metazoans, γ-tubulin acts within two main complexes, γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs). In higher eukaryotes, it is assumed that microtubule nucleation at the centrosome depends on γ-TuRCs, but the role of γ-TuRC components remains undefined. This study analyzed the function of all four γ-TuRC–specific subunits in Drosophila: Dgrip75, Dgrip128, Dgrip163, and Dgp71WD. Grip-motif proteins, but not Dgp71WD, appear to be required for γ-TuRC assembly. Individual depletion of γ-TuRC components, in cultured cells and in vivo, induces mitotic delay and abnormal spindles. Surprisingly, γ-TuSCs are recruited to the centrosomes. These defects are less severe than those resulting from the inhibition of γ-TuSC components and do not appear critical for viability. Simultaneous cosilencing of all γ-TuRC proteins leads to stronger phenotypes and partial recruitment of γ-TuSC. In conclusion, γ-TuRCs are required for assembly of fully functional spindles, but it is suggested that γ-TuSC could be targeted to the centrosomes, which is where basic microtubule assembly activities are maintained (Verollet, 2006; full text of article).

Individual depletion of Dgrip75, Dgrip128, or Dgrip163 induced a strong decrease in cytoplasmic γ-TuRCs in cultured S2 cells. This is consistent with previous data obtained in vitro showing that immunodepletion of Xgrip210, the Xenopus laevis orthologue of Dgrip163, blocks the reassembly of purified and salt-treated γ-TuRCs. In contrast to the strong decrease of γ-TuRCs observed after the depletion of grip-motif proteins, the down-regulation of Dgp71WD did not produce a significant effect on the ratio and the sedimentation coefficients of the γ-tubulin complexes. However, the possibility that residual amounts of this protein are sufficient to maintain the overall structure of these complexes cannot be excluded. Dgp71WD is probably associated with the γ- TuRCs at a low stoichiometry, explaining why no change in the sedimentation coefficient could be detected after its depletion. The data suggest that although the grip-motif proteins are essential for the assembly and stability of γ-TuRCs, Dgp71WD appears dispensable for these processes (Verollet, 2006).

In Dgp71WD-depleted cells, no obvious effect was observed on the level of cytosolic γ-TuRCs and on the recruitment of the different γ-TuRC components to the mitotic centrosomes, suggesting that γ-TuRCs depleted of Dgp71WD had not completely lost their ability to be targeted to the poles. However, γ-tubulin localization along spindle microtubules was impaired and monopolar figures with poorly separated poles appeared with a high occurrence. These monopolar phenotypes are similar to those observed after depletion of Nedd1, the human Dgp71WD orthologue. The mitotic defects could result from a partial functionality of the γ-tubulin complexes recruited to the centrosomes or from modification in the properties of microtubule arrays that were no longer decorated by γ-tubulin. Thus, it appears that the complete γ-TuRC is required for normal mitotic progression (Verollet, 2006).

If one assumes that γ-tubulin is recruited to the centrosome in the form of γ-TuRCs, depletion of γ-TuRCs and γ-tubulin would be expected to lead to similar phenotypes. In fact, depletion of grip-motif components of the γ-TuRCs, such as Dgrip75, did not fully mimic the effects of depletion of γ-TuSC subunits on mitotic progress. Less severe defects were observed as indicated by the lower occurrence of monopolar spindles, the efficient distribution of centrosomes to the two poles, the maintenance of bipolar spindles with astral microtubules and focused poles. These phenotypes had been confirmed in vivo, using mutant larval neuroblasts. These moderate defects were consistent with the viability of Dgrip75 mutants and the weak percentage of aneuploidy. Surprisingly, despite a significant reduction of the cytosolic pool of γ-TuRCs, the tethering of the three γ-TuSC proteins to the centrosomes was not impaired, as judged by immunofluorescence analyses both in mutant neuroblasts and in cultured cells. Similar phenotypes were observed after individual silencing of the two other grip-motif proteins, Dgrip128 and Dgrip163. It was noteworthy that after an RNAi treatment against any one of the three grip-motif proteins specific of the γ-TuRCs, a decrease in the level of the two others was noticed, suggesting some coregulation between this set of proteins. Altogether, these results show that the complete γ-TuRC is not a prerequisite for the centrosomal accumulation of γ-TuSC proteins (Verollet, 2006).

Because γ-tubulin could be recruited to the centrosomes independently of the γ-TuRCs, characterization of the complex that mediates its targeting was of interest. Sucrose gradient and immunofluorescence analyses after depletion of individual γ-TuRC components strongly suggest that γ-tubulin is recruited to the centrosomes as γ-TuSC. Codepletion of the four γ-TuRC–specific proteins still allowed the polar accumulation of the γ-TuSC components in most of the cells. This latter experiment supports the hypothesis that the γ-TuSC is a vector competent for targeting γ-tubulin to the centrosome. However, since RNAi treatments do not completely remove all protein, it is possible that the residual γ-TuRC–specific proteins participate in the formation of a γ-TuRC–like structure that is unstable in the cytoplasm, but stabilized upon assembly within the pericentriolar matrix. The idea that γ-TuSC could be recruited to the poles is consistent with data reported in Saccharomyces cerevisiae, in which no orthologues of the γ-TuRC–specific proteins have been reported. In this organism, the main interactions of γ-tubulin with the microtubule-organizing centers are mediated by the two γ-tubulin–associated proteins in the γ-TuSC. Similarly, in mammals, the γ-TuRCs can be tethered to the centrosomes via interactions of the orthologues of Dgrip84 and Dgrip91 with the centrosomal anchoring proteins kendrin and centrosome- and Golgi-localized protein kinase N–associated protein. In Drosophila, the calmodulin-binding protein CP309 has been proposed to anchor γ-TuRCs to the centrosome by direct binding to the γ-TuSC. However, γ-tubulin recruitment to the centrosomes is probably a complex process, as additional proteins, including ninein and centrosomin, provide alternative sites for γ-tubulin anchorage. Although evidence suggests that γ-tubulin can be targeted to the centrosomes in the form of γ-TuSCs, it cannot be ruled out that in Drosophila other proteins at low abundance, previously uncharacterized proteins such as the Dgrip79 and Dgrip223 identified by in silico analyses, or different combinations of known γ-tubulin partners could be involved in this recruitment. Moreover, after concomitant depletion of all γ-TuRC–specific proteins, spindles were nonfunctional and the amount of γ-TuSC recruited to the mitotic centrosomes was reduced. Because of the observation that the small complexes are less active than γ-TuRCs in promoting nucleation, it is proposed that the decrease in the amount of γ-TuSCs under a threshold can be critical for spindle functionality (Verollet, 2006).

Interestingly, the transient association of γ-tubulin to the spindle, as well as to the midbody, was no longer detectable after individual or concomitant RNAi treatment against Dgrip75, Dgrip128, Dgrip163, and/or Dgp71WD. Hence, mitotic γ-tubulin localization to structures outside the pericentriolar material requires the fully assembled γ-TuRCs, and Dgp71WD could play an active role in this process. The absence of γ-tubulin recruitment along the spindle microtubules and at the midbody can also be explained by distinct properties of docking proteins at centrosomes and at noncentrosomal sites. Actually, novel proteins in charge of the recruitment of γ-tubulin complexes along spindle microtubules have recently been identified in fission yeast (Verollet, 2006).

In contrast with the impairment of microtubule assembly from the pericentriolar material after removal of γ-TuSC components, the pericentriolar material appears to remain active as a microtubule-nucleating center after down-regulation of specific γ-TuRC proteins, as shown by the presence of astral microtubules identified both by immunofluorescence and electron microscopy. Moreover, microtubules containing 13 protofilaments were still nucleated, challenging the 'template model'. This indicates that preassembly of cytosolic γ-TuRCs does not seem required for the formation of canonical microtubules. It is not excluded that a ringlike structure could be assembled from γ-TuSCs alone or in combination with small amounts of γ-TuRC–specific proteins at the pericentriolar level, although the purified γ-TuSC does not assemble into larger structures in vitro. In that case, such ringlike complexes would exhibit stoichiometries and protein compositions different from γ-TuRCs (Verollet, 2006).

These observations could reconcile the findings of microtubule nucleation in animal cells and in S. cerevisiae. Moreover, after depletion of γ-TuRC–specific components, recruitment of γ-TuSC appeared sufficient, in most of the cases, to control the formation of spindles competent for chromosome segregation. However, mitotic processes were partly disrupted in cells lacking γ-TuRCs, leading to a transient prometaphase accumulation and a poor density of spindle microtubules. Several possibilities could account for these defects, such as a lower efficiency of γ-TuSCs compared with γ-TuRCs in microtubule nucleation, the presence of distinct binding sites for γ-TuSCs and γ-TuRCs, or the loss of γ-TuRC recruitment along spindle microtubules, which would affect the organization or the dynamics of specific microtubule arrays (Verollet, 2006).

Collectively, the results strongly suggest that the assembly of γ-TuRCs is not essential for γ-tubulin–dependent microtubule nucleation at the centrosome, but instead is required for other, noncentrosomal localization of γ-tubulin. In Drosophila, γ-TuRCs may target a fully organized 'nucleation machinery' to the sites of nucleation; γ-TuSC would act as a functional unit, whereas γ-TuRC–specific proteins would rather play a more refined role in regulating or optimizing of microtubule arrays during mitosis. γ-Tubulin can be targeted to the poles by the direct docking of γ-TuSCs that exert basic nucleation activity. This γ-TuSC recruitment could be a 'salvage pathway,' involved only when the dominant microtubule assembly mechanism mediated by γ-TuRCs is impaired or as a physiological pathway acting in parallel to γ-TuRC nucleation activity. Altogether, the data should prompt the reexamination of the current nucleation models (Verollet, 2006).

The gammaTuRC components Grip75 and Grip128 have an essential microtubule-anchoring function in the Drosophila germline

The γ-tubulin ring complex (γTuRC) forms an essential template for microtubule nucleation in animal cells. The molecular composition of the γTuRC has been described; however, the functions of the subunits proposed to form the cap structure remain to be characterized in vivo. In Drosophila, the core components of the γTuRC are essential for mitosis, whereas the cap component Grip75 is not required for viability but functions in bicoid RNA localization during oogenesis. The other cap components have not been analyzed in vivo. This study reports the functional characterization of the cap components Grip128 and Grip75. Animals with mutations in Dgrip128 or Dgrip75 are viable, but both males and females are sterile. Both proteins are required for the formation of distinct sets of microtubules, which facilitate bicoid RNA localization during oogenesis, the formation of the central microtubule aster connecting the meiosis II spindles in oocytes and cytokinesis in male meiosis. Grip75 and Grip128 anchor the axoneme at the nucleus during sperm elongation. It is proposed that Grip75 and Grip128 are required to tether microtubules at specific microtubule-organizing centers, instead of being required for general microtubule nucleation. The γTuRC cap structure may be essential only for non-centrosome-based microtubule functions (Vogt, 2006).

γTuRC function in microtubule nucleation is crucial for viability; mutations in Grip91/l(1)dd4, Grip84 and γTub23C are lethal. 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 γTuRC and that the γTuRC formed in these mutants is sufficient for microtubule function in somatic cell types of the fly. However, depletion of cap components such as Grip75, Grip128 or Grip163 by RNAi leads to a higher mitotic index in S2 cells, but the cap components are not absolutely essential for mitotic progression. This is not surprising as even mutants with centrosomal defects can survive. Furthermore, γ-tubulin is recruited to centrosomes in Grip75 or Grip128 mutant spermatocytes, Grip75 mutant neuroblasts and in S2 cells depleted for cap components, showing that γ-tubulin targeting to the centrosome does not depend on cap components. It has been proposed that γ-tubulin can be recruited to centrosomes as part of the γTuSC, as the amount of large γ-tubulin-containing complexes is severely reduced in cells depleted for cap components. Using buffers with lower salt concentrations, we observe large γ-tubulin containing complexes in Grip75 and Grip128 mutants, albeit in reduced amounts compared with wild type. It is likely that these complexes are indeed γTuRCs that lack parts of the cap structure, as they are similar in size to the γTuRC; in addition, Grip128 is present in Grip75 mutant complexes. The mutant γTuRCs might still be capable of nucleating microtubules (Vogt, 2006).

Whether γ-tubulin forms γTuSCs or incomplete γTuRCs, the cap subunits are dispensable for microtubule nucleation and γ-tubulin recruitment to centrosomes. Moreover, a γTuRC has not been described in Saccharomyces cerevisiae and homologs of the cap components have not been identified in yeast, further supporting the notion that microtubule nucleation can occur in the absence of the cap structure (Vogt, 2006).

In Drosophila, it is not known whether individual γTuRC complexes vary in their subunit composition and whether the γTuRC-specific subunits have similar functions. The human γTuRC has been shown to contain all of the described subunits. The cap components Grip75, Grip128 and Grip163 depend on each other for their stability. Furthermore, individual depletion of Grip75, Grip128 or Grip163 results in a similar increase of the mitotic index in treated cells. Moreover, Grip128;Grip75 double mutants show the same phenotypes as the single mutants in the Drosophila germline. Taken together, the data support the view that Grip163, Grip128 and Grip75 function in the same processes and are part of the same complexes (Vogt, 2006).

By contrast, Grip71 appears to have a distinct function. On the one hand, depletion by RNAi does not impair protein levels of the other γTuRC-specific proteins or their recruitment to centrosomes; on the other hand, the mitotic phenotypes are much stronger in Grip71 mutants when compared with Grip75 mutants (Vogt, 2006).

Genetic and cell biological data suggest that an intact cap structure is not necessary for microtubule nucleation; thus, the function of the cap is still in question. It could be required for efficient assembly of the γTuRC, for a higher microtubule nucleation rate or for tethering the complex to MTOCs. The former two possibilities predict that all microtubules would be affected to a similar degree, and therefore the most sensitive microtubule-dependent processes would be disrupted in Grip75 and Grip128 mutants. The latter possibility predicts that phenotypes would arise when redundant anchoring mechanisms were not available (Vogt, 2006).

Mutants with global defects in microtubule function such as hypomorphic αtub84B mutants show a wide range of phenotypes such as polyphasic lethality, cuticle defects, short life span and sterility. Similarly, hypomorphic Grip91/l(1)dd4 mutants are lethal and display both mitotic and meiotic defects in spermatogenesis. As Grip75 and Grip128 mutants show very specific phenotypes, a function for the γTuRC cap structure in microtubule anchoring at MTOCs is more conceivable. This is supported by the observed detachment of axonemes from their respective nuclei without any aberrations in axoneme architecture and the undisturbed orb RNA localization in Grip128 mutants. Microtubule recruitment to or anchoring at centrosomes has been shown to depend on a number of factors, such as pericentrin or motor proteins. Redundant mechanisms might act to focus microtubules at conventional MTOCs in somatic cells, but this might not be the case at nonconventional MTOCs in the Drosophila germline (Vogt, 2006).

The Drosophila pericentrin-like protein D-PLP recruits or anchors γ-tubulin to centrosomes, possibly by direct interaction with γTuSC components. Interestingly, D-PLP is only required for efficient anchoring of γ-tubulin to the centrosome in early phases of mitosis, suggesting that a D-PLP independent pathway can recruit and anchor centrosomal components. Maybe D-PLP and the γTuRC cap structure act redundantly in anchoring γ-tubulin at the pericentriolar material during mitosis (Vogt, 2006).

Additionally, microtubule motors focus microtubules at the mitotic centrosome. Inhibition of the dynein-dynactin complex results in disorganized spindles that lack well-focused poles, while analysis of Dhc64C mutations in Drosophila suggests that dynein is required for the attachment of spindle poles at centrosomes. The kinesin-related Ncd is a minus-end directed microtubule motor that also functions in spindle assembly during mitosis. Depletion of Ncd by RNAi in S2 cells results in frequent release of microtubules from the spindle pole (Vogt, 2006).

Although the roles of D-PLP and the above mentioned microtubule motors are fairly well established in centrosomes, their contributions to other MTOCs, such as the Grip75- and Grip128-dependent ones, are not as well studied. D-PLP has been shown to maintain the structural integrity of centrioles in male meiosis I; however, a possible function in γ-tubulin anchoring in male meiosis II is difficult to address because of the centriolar defects. Ncd organizes the female meiosis I spindle and also localizes to the meiosis II spindle. ncd mutants do not form a structured central aster in meiosis II, but this could be a consequence of defects in meiosis I. It is proposed that redundant mechanisms focus or anchor microtubules at conventional centrosomes during mitosis. However, some MTOCs in the germline crucially depend on the anchoring function of the γTuRC cap subunits Grip128 and Grip75. Hence, these proteins allow the organization of distinct microtubule populations at particular positions in complex cells, independently of centrosomes (Vogt, 2006).

Interestingly, mutations in Grip75 or Grip128 fully disrupt the function of only certain MTOCs. As Grip128 and Grip75 mutants are viable, most microtubule-dependent processes in somatic cells function at least to an extent that allows survival of the organism, even though mitosis is delayed. These processes are directed by microtubules associated with classical centrosomes, suggesting that somatic centrosomes are less sensitive to the lack of Grip128 and Grip75 function than the specialized MTOCs in the male and female germline (Vogt, 2006).

Grip128, Grip75 and γTub37C participate in the formation of a new MTOC at stage 10b, which directs the relocalization of bcd RNA during stage 10b. They are specifically involved in bcd RNA localization; other microtubule-dependent processes in the oocyte such as oocyte specification, nuclear migration, cytoplasmic streaming, and orb, grk and osk RNA transport are normal in the respective mutants. It has been proposed that different subsets of microtubules could perform this variety of functions. Alternatively, loss of Grip128 or Grip75 function could lead to a reduction in microtubule number or function, thus impairing only the most sensitive microtubule-dependent processes. Three lines of evidence support a selective function of Grip128 in the organization of the anteriorly originating microtubules during stage 10b and 11. The subcortical microtubule network appears to be normal in mutant oocytes, whereas the anterior set of microtubules is not present. Cytoplasmic streaming is undisturbed in Grip128 mutants. orb RNA localization has been demonstrated to be more sensitive to microtubule-depolymerizing drugs than bcd RNA localization; however, orb RNA is correctly localized in Grip128 mutant oocytes. These data argue against a general microtubule impairment in Grip128 mutants (Vogt, 2006).

Female meiosis requires the activities of Grip128 and Grip75 during the second meiotic division. Spindle formation in female meiosis is atypical, with the anastral and acentrosomal first meiotic spindle forming in a chromatin-driven fashion. The second meiotic division is characterized by two tandemly arranged spindles, which are connected by a central microtubule aster. This central aster has been proposed to be necessary for correct spacing and alignment of the meiosis II spindles. It contains γ-tubulin, whereas the distal poles are devoid of γ-tubulin. The absence of the central microtubule aster in Grip75 and Grip128 mutants could be due either to reduced microtubule nucleation from the MTOC or to a failure in MTOC assembly. The latter hypothesis is favored, since the inner half spindles are formed in the mutants, and the absence of a robust central microtubule aster is also observed in cnn and polo mutants (Vogt, 2006).

As in females, meiosis in males displays special features, such as the reductional segregation of centrioles in the second meiotic division. Thus, the second meiotic spindle is built from centrosomes, which contain a single centriole each, thereby giving rise to unicentriolar cells. Centrioles in spermatocytes are large and associated with very little pericentriolar material when compared with mitotic centrioles. These meiotic centrosomes might depend on Grip75 and Grip128 for correct microtubule organization. Alternatively, the central spindle, which is essential for cytokinesis, has been postulated to use transient microtubule organizing centers present between the two daughter nuclei. Grip75 and Grip128 could function in these transient MTOCs to organize the central spindle (Vogt, 2006).

Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle

Since the discovery of γ-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of γ-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for γ-tubulin might reside elsewhere. An RNA interference (RNAi) screen, carried out in Drosophila cultured cells, has identified five genes (Dgt2-6) required for localizing γ-tubulin to spindle microtubules. The Dgt proteins interact, forming a stable complex. Spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex, named 'augmin' functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. A functional human homologue of Dgt6 has also been identified. These results suggest that an important mitotic function for γ-tubulin may lie within the spindle, where augmin and γ-tubulin function cooperatively to amplify the number of microtubules (Goshima, 2008).

Thus, Dgt proteins associate to form a stable complex. Based upon its role in increasing MT numbers in the spindle, it is proposed that this complex be called 'augmin' from the Latin verb augmentare, which means to increase. The role of augmin in increasing spindle MT density appears to be very important for building K-fibers and enabling chromatin-mediated MT nucleation to proceed toward the formation of a bipolar-shaped spindle, particularly when centrosome function is attenuated (Goshima, 2008).

The results suggest a model for how centrosome-, chromosome-, and spindle-based MT nucleation processes may cooperate to build mitotic spindles. After NEBD in somatic cells, the most obvious generation of new MTs occurs at centrosomes, producing astral MT arrays. MTs are also nucleated in the vicinity of chromosomes immediately after NEBD. These processes are critical for generating the first set of mitotic MTs, enabling the process of spindle assembly to begin. However, after the first sets of MTs are formed, it is proposed that augmin-γ-TuRC nucleates MT growth from existing MTs, thus providing a powerful mechanism for rapidly amplifying the number of MTs within the spindle to facilitate chromosome capture and K-fiber formation (Goshima, 2008).

It is postulated that kinetochore MTs, initially generated via the centrosome or chromatin pathway, are used as templates for binding augmin, which in turn recruits and activates γ-TuRC for MT nucleation. The outer γ-TuRC subunits appear to be critical for this specific spindle function of γ-tubulin because knockdown of these subunits produces a very similar phenotype to augmin knockdown, although evidence has not yet been obtained for a direct interaction between augmin and γ-TuRC. An intriguing possibility is that augmin may dock onto an MT in a manner that positions γ-TuRC to preferentially nucleate new MT growth with the same polarity as the parent MT (Goshima, 2008).

As an alternative to this model, augmin might increase the number of spindle MTs by activating an MT-severing enzyme, although this is unlikely because RNAi of katanin and other AAA ATPases (e.g., spastin) does not give rise to the same mitotic phenotype as Dgt or γ-TuRC RNAi. Another class of models is that augmin increases spindle MT density by either stabilizing, elongating, or transporting MTs that are nucleated at the kinetochore/chromatin and that augmin-γ-tubulin are not involved in MT nucleation within the spindle. Although this possibility cannot be ruled out, FRAP results showing the concomitant recovery of fluorescence at the center of the spindle and the spindle poles suggest that MTs are being formed throughout the spindle and not just near the chromatin. The notion of an MT amplification process is also supported by in vitro studies of the assembly of MT asters in X. laevis extract and accompanying computational simulations). The exact mechanism by which augmin increases MT density awaits future work, most decisively by in vitro reconstitution of the process with purified proteins (Goshima, 2008).

In addition to the mitotic spindle, there are other cellular MT networks in animals that are unlikely to be exclusively built by centrosome-based nucleation and assembly processes, such as axons or the meiotic spindle in oocytes. An MT-templated MT nucleation reaction could constitute an excellent means for generating new MTs while preserving the polarity of an existing MT array. Augmin is a candidate to play a role in this and numerous other cases where polarized noncentrosomal MTs are generated (Goshima, 2008).

Protein Interactions

Additonal information about gammaTubulin protein interactions can be found at the gammaTubulin at 23C site.

The colocalization of gammaTub37C and Grip75GFP at the anterior pole at stage 10b and their reported molecular interaction in cell culture (Fava, 1999) led to an analysis of the interaction of gammaTub37C and Grip75 in ovary extracts. Grip75 could be precipitated from wild-type and swa mutant, but not from gammaTub37C and Grip75 mutant ovaries, using the gammaTub37C-specific antibody. Similarly, the Grip75 antibody precipitates gamma-tubulin from wild-type and swa, but not from gammaTub37C and Grip75 mutant ovaries. This shows that gammaTub37C and Grip75 form a stable complex, presumably the gamma Tubulin ring complex (gammaTuRC), during oogenesis. This complex is not disrupted in swa mutants. In addition to Grip75, Swa protein is also coimmunoprecipitated with the gammaTub37C antibody. The binding of Swa to gammaTub37C is less stable than the gammaTub37C interaction with Grip75, suggesting that Swa is not a core component of the gammaTuRC. The Swa-gammaTub37C interaction is lost in Grip75 and in all swa mutants analyzed. This shows that Grip75 is necessary for the efficient binding of Swa to gammaTuRC. The mutant Swa proteins from swaTG31 or swa384 ovaries, which are not localized at the anterior pole of the oocyte (Schnorrer, 2000), do not bind to the gammaTuRC (Schnorrer, 2002).

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, 1997), 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).


DEVELOPMENTAL BIOLOGY

Embryonic

Microtubule nucleation in vivo requires gamma-tubulin, a highly conserved component of microtubule-organizing centers. In Drosophila melanogaster there are two gamma-tubulin genes: gammaTUB23C and gammaTUB37C. The 37C protein can only be detected in ovaries and embryos. Antibodies against this isoform predominantly label the centrosomes in embryos from early cleavage divisions until cycle 15, but fail to reveal any particular localization of gamma-tubulin in the developing egg chambers. The loss of function of this gene results in female sterility and has no effect on viability or male fertility. Early stages of oogenesis are unaffected by mutations in this gene, as judged both by morphological criteria and by localization of reporter genes, but the female meiotic spindle is extremely disrupted. Nuclear proliferation within the eggs laid by mutant females is also impaired. It is concluded that the expression of the 37C gamma-Tubulin isoform of D. melanogaster is under strict developmental regulation and that the organization of the female meiotic spindle requires gamma-tubulin (Tavosanis, 1997).

Effects of Mutation or Deletion

Components of the cytoskeletal machinery, which localizes bcd RNA to the anterior pole of the oocyte, might have multiple functions during development. Therefore, the following approach was chosen in order to identify novel factors essential for bcd RNA localization. As part of a large F1 genetic screen for EMS-induced maternal effect mutations, mutant females were isolated whose germ line clone-derived eggs arrest early in development, as judged by the absence of cuticular structures. The ovaries of those females were inspected by in situ hybridization using a bcd RNA antisense probe. Three mutations, 139-14, 175-14, and 224-26, were isolated displaying an abnormal bcd RNA localization with 100% penetrance. 175-14 and 224-26 are two alleles of the same gene, while 139-14 represents a single allele (Schnorrer, 2002).

Using a set of overlapping chromosomal deficiencies, 139-14 was mapped to cytological region 37C. Database searches revealed gamma-Tubulin37C (gammaTub37C) as a candidate gene for the 139-14 mutation. 139-14 indeed failed to complement gammaTub37C alleles gammaTub37C1 and gammaTub37C3 and showed no detectable gammaTub37C protein on a Western blot with an antibody specific for the C terminus of gammaTub37C. Therefore, it was concluded that 139-14 bears a mutation in the gammaTub37C locus. gammaTub37C mutants are viable, but females are sterile. As reported by Llamazares (1999) and Tavosanis (1997), the eggs produced by mutant mothers show an arrest of nuclear divisions during early embryogenesis because both microtubule polymerization and (as a consequence) spindle formation are blocked (Schnorrer, 2002).

Mutations in the second gene, 175-14 and 224-26, are also viable. The mutations map to region 31D-F, where Grip75 (gamma-tubulin ring complex protein of 75 kDa) is annotated on the basis of the sequence homology to human Grip76. The human homolog is part of the gamma-tubulin ring complex and is located at the centrosomes of eukaryotic cells, but no mutants are described (Fava, 1999). The Grip75 gene from 175-14 and 224-26 flies was sequenced and molecular lesions were identified in both mutants. The Grip75 gene of 175-14 has a nonsense mutation at base pair 911 of its mRNA, resulting in a truncation of the predicted protein after amino acid 290. 224-26 contains a missense mutation at the splice acceptor site of the 6th intron, resulting in an mRNA with a 1 bp insertion after base pair 1580, corresponding to amino acid 513. Two internal fragments of Grip75 were used to generate a Grip75 polyclonal antibody. The antiserum recognizes a band of about 75 kDa that is missing in both Grip75 alleles, indicating that both Grip75 alleles are protein null alleles. Grip75 fused to GFP was expressed using a maternal gamma-tubulin GAL4 line in a 175-14 background. This Grip75GFP fusion protein rescues the female sterility of 175-14 and its bcd RNA mislocalization defect. In conclusion, 175-14 and 224-26 are mutations in the Grip75 locus and will be referred to as Grip75175 and Grip75224, respectively (Schnorrer, 2002).

To identify the time point at which the bcd RNA localization pattern is disrupted in gammaTub37C and Grip75 mutants, bcd RNA distribution was analyzed at different stages of oogenesis in comparison with wild-type and swa and exu mutants. The ring-shaped localization of bcd RNA at the anterior cortex of stage 8–10a oocytes forms normally in gammaTub37C, Grip75, and swa mutants, while it is strongly reduced in exu egg chambers. In stage 10b wild-type oocytes, the bcd RNA localization changes from the ring-shaped into a disc-shaped pattern, resulting in highest bcd RNA concentration at the middle of the anterior pole. This transition is never observed in swa mutants. Instead, bcd RNA diffuses away from the anterior pole into the oocyte and is often seen in aggregates associated with the oocyte cortex. In gammaTub37C and Grip75 mutants, this swa-dependent transition occurs at least partially, since bcd RNA is found at the center of the anterior pole at stage 10b, although less in gammaTub37C compared with wild-type. Slightly later, bcd RNA spreads into the oocyte, forming aggregates similar to those seen in swa mutants, leading to a uniform distribution of bcd RNA at late stages of oogenesis. From these data it is deduced that gammaTub37C, Grip75, and swa act in sequential steps of the bcd RNA localization process. swa is essential for the 'ring to disc' transition of the bcd RNA pattern, which coincides with the anterior concentration of Swa protein at stage 10b, whereas gammaTub37C and Grip75 are required to maintain bcd RNA at the anterior pole. After stage 10b the bcd RNA mislocalization pattern is indistinguishable in all three mutants (Schnorrer, 2002).

gammaTub37C and Grip75 are both components of the microtubule cytoskeleton. gammaTub37C is known to localize at centrosomes of early Drosophila embryos and is essential for organization and control of microtubule growth (Gunawardane, 2000; Tavosanis, 1997). Before analyzing the molecular function of gammaTub37C and Grip75 during oogenesis, embryos laid from Grip75 mutant mothers were examined; as in gammaTub37C embryos, no spindles or dividing nuclei were detected. Therefore, it is concluded that Grip75 is an essential component of the gamma-tubulin ring complex, since Grip75 mutants display a complete loss of spindle function indistinguishable from gammaTub37C mutants (Schnorrer, 2002).

A maternally expressed Grip75GFP fusion rescues the spindle defect of Grip75 mutants, and nuclear divisions occur normally. Grip75GFP is concentrated at centrosomes of early embryos and colocalizes with gammaTub37C and Centrosomin (Cnn) throughout the cell cycle. These rescue data indicate that the Grip75GFP fusion protein is functional and, hence, that it is likely to recapitulate the endogenous Grip75 distribution (Schnorrer, 2002).

Interestingly, swa null mutants also show a phenotype during embryonic nuclear cleavage stages. Nuclear divisions are often abnormal from the first divisions onward: large regions in the embryo are depleted of nuclei, whereas sometimes centrosomes divide without nuclei. Probably as a consequence of the irregular distribution of centrosomes during the early divisions, centrosomes are not stably anchored when they arrive at the cell cortex but move backward and forward. The centrosomes that form a spindle in swa embryos contain normal amounts of gammaTub37C and Cnn. Endogenous Swa protein is targeted for degradation shortly after egg deposition and is undetectable after one hour of development (Schnorrer, 2000). However, a maternally expressed SwaGFP fusion perdures longer and allows the analysis of its distribution during nuclear cleavages. Strikingly, it is found concentrated together with gammaTub37C and Cnn at the centrosomes of the early embryo, suggesting that Swa may function together with gammaTub37C and Grip75 to organize and coordinate the nuclear spindles of the first divisions. Like Grip75GFP and gammaTub37C, SwaGFP is not exclusively found at the centrosomes but is also distributed on the microtubules of the mitotic figure. Time-lapse analysis of the SwaGFP distribution shows that the SwaGFP concentration at centrosomes declines after mitosis and is reestablished within the next interphase (Schnorrer, 2002).


Since gammaTub37C, Grip75, and swa mutants display spindle defects during embryogenesis, it was asked whether the microtubule network is also altered during oogenesis. To investigate function and polarity of the microtubules in gammaTub37C and Grip75 ovaries, transport to the posterior pole of the oocyte was examined. Kin:ß-gal and osk RNA distributions were examined; posterior concentration of these depends on endogenous kinesin heavy chain function. Kin:ß-gal and osk RNA localization are normal in gammaTub37C and Grip75 oocytes, and osk RNA remains concentrated at the posterior pole of early gammaTub37C and Grip75 embryos. However, the posterior association of osk RNA with the cortex is not as tight as in wild-type or swa embryos. This suggests that transport to the posterior pole is functional in gammaTub37C and Grip75 mutants, but that a late anchoring step of osk RNA is impaired (Schnorrer, 2002).

Next, transport to the anterior pole was examined. Swa localization at the anterior pole of stage 10 oocytes requires intact microtubules (Schnorrer, 2000). Hence, Swa was used to investigate the function of microtubule-dependent transport; it is localized normally at the anterior pole of gammaTub37C and Grip75 oocytes from stage 10b onward, indicating that gammaTub37C and Grip75 are not required for the anterior localization of Swa. For a further characterization of the microtubule network, the microtubule minus end marker Nod:ß-gal was used. Interestingly, a change was observed of the Nod:ß-gal localization from a ring-shaped pattern at stage 9 into a disc-shaped pattern at stage 10b, which is reminiscent of the change in the bcd RNA localization pattern. Nod:ß-gal colocalizes with Swa and bcd RNA at stage 10b of wild-type and swa mutant oocytes. This suggests that swa is not required for Nod:ß-gal transport to the anterior pole, indicating that the microtubule cytoskeleton is not generally disrupted in swa mutants. However, in gammaTub37C mutant oocytes, the anterior localization of Nod:ß-gal is reduced at stage 9, and it is not detectable anymore at the anterior pole at stage 10b, indicating that transport to the microtubule minus ends is not functioning properly in these oocytes. Furthermore, these data imply a continuous requirement of minus end-directed transport in order to maintain the anterior localization of Nod:ß-gal and of bcd RNA (Schnorrer, 2002).

In order to address the question of whether gammaTub37C and Grip75 mutants specifically affect a subset of microtubule function at the time when bcd RNA mislocalization starts, ooplasmic streaming was examined. This vigorous flow of oocyte cytoplasm from stage 10b to 12 is microtubule dependent and is inhibited by microtubule-depolymerizing drugs. However, normal ooplasmic streaming is seen in swa, gammaTub37C, and Grip75 mutants, strongly suggesting that the microtubules required for this process are not affected. Therefore, this indicates that gammaTub37C and Grip75 mutants affect only a subset of microtubules, which are required for bcd RNA localization, but not for ooplasmic streaming (Schnorrer, 2002).

One important question is whether there is a distinct MTOC responsible for localization of components at the anterior pole and, if so, what the molecular nature of such an MTOC might be. Flies expressing the functional Grip75GFP fusion during oogenesis were examined and a substantial concentration of Grip75GFP was found at the anterior pole of wild-type oocytes. This concentration is first seen at stage 10b and becomes more pronounced during stage 11. Grip75GFP colocalizes with gamma-tubulin, which was visualized with a pan anti-gamma-tubulin antibody. This anterior enrichment of gamma-tubulin and Grip75GFP is not severely affected in swa mutant oocytes. In contrast, in gammaTub37C mutants, the gamma-tubulin protein expression is severely reduced, and, as a consequence, Grip75GFP is less concentrated at the anterior pole of a gammaTub37C mutant oocyte when compared with wild-type. The anterior focus of gamma-tubulin is neither due to overexpression of Grip75GFP nor established simply by dumping of material from nurse cells to the oocyte, since a similar anterior enrichment of gamma-tubulin is detected in wild-type and quail mutants, which have a dumpless phenotype. Furthermore, gamma-tubulin was analyzed in gurken (grk) mutants and a distinct focus of gamma-tubulin was found not only at the anterior pole, but also at the posterior pole, where it colocalizes with Swa. This suggests that gamma-tubulin forms a distinct MTOC at both poles in grk mutants and only at the anterior pole in wild-type oocytes (Schnorrer, 2002).

The analysis of gamma-tubulin in the oocyte at stage 10b and later is complicated by the fact that the microtubule-rich follicle cells surround the whole oocyte. Therefore, gamma-tubulin fused to GFP was expressed specifically in the germline, using maternal gamma-tubulin GAL4; microtubules were found enriched at the anterior pole at stage 10b and extending along the oocyte cortex. The anterior gamma-tubulin focus overlaps with gamma-tubulin, consistent with the fact that gammaTub37C organizes microtubules from the anterior pole. In gammaTub37C and Grip75 mutants, the anterior enrichment of gamma-tubulin appears reduced compared with wild-type; however, microtubules are still present in the mutants (Schnorrer, 2002).

Functional analysis of gamma Tub37CD, a maternally synthesized gamma-tubulin that is highly expressed during oogenesis and utilized at centrosomes in precellular embryos. Two gamma Tub37CD mutants contained missense mutations that altered residues conserved in all gamma-tubulins and alpha- and/or beta-tubulins. A third gamma Tub37CD missense mutant identified a conserved motif unique to gamma-tubulins. A fourth gamma Tub37CD mutant contained a nonsense mutation and the corresponding premature stop codon generated a protein null allele. Immunofluorescence analysis of laid eggs and activated oocytes derived from the mutants revealed microtubules and meiotic spindles that were close to normal even in the absence of gamma Tub37CD. Eggs lacking the maternal gamma-tubulin are arrested in meiosis, indicative of a deficiency in activation. Analysis of meiosis with in vitro activation techniques showed that the cortical microtubule cytoskeleton of mature wild-type eggs is reorganized upon activation and expressed as transient assembly of cortical asters, and this cortical reorganization is altered in gamma Tub37CD mutants. In precellular embryos of partial loss of function mutants, spindles are frequently abnormal and cell cycle progression is inhibited. Thus, gamma Tub37CD functions differentially in female meiosis and in the early embryo -- while involved in oocyte activation, gamma Tub37CD is apparently not required or it plays a subtle role in formation of the female meiotic spindle, which is acentriolar, but it is essential for assembly of a discrete bipolar mitotic spindle, which is directed by centrosomes organized about centrioles (Wilson, 1998).

The mutant phenotypes brought about during early embryogenesis by mutation in the gammaTub37C gene (one of the two isoforms of gamma-tubulin that have been identified in Drosophila) have been examined. Focus was placed on fs(2)TW11 and fs(2)TW1RU34, respectively a null and a hypomorph allele of this gene, whose sequences are reported in this work. The abnormal meiotic figures observed in mutant stage 14 oocytes are not observed in laid oocytes or fertilized embryos, suggesting that these abnormal meiotic figures are not terminally arrested. Both null and hypomorph alleles led to a total arrest of nuclear proliferation during early embryogenesis. This is in contrast to their effect on female meiosis-I where hypomorph alleles display a much weaker phenotype. Finally, null and hypomorph alleles led to some distinct phenotypes. Unfertilized laid oocytes and fertilized embryos deficient for gammaTub37C do not contain polar bodies and have a few bipolar microtubule arrays. In contrast, oocytes and embryos from weaker alleles do not have these microtubule arrays, but do contain polar bodies, or polar-body-like structures. These results indicate that gammaTub37C is essential for nuclear proliferation in the early Drosophila embryo (Llamazares, 1999).

There are three major conclusions that can be drawn from this work regarding the function of the gammaTub37C gene. (1) Abnormal meiotic figures of the kind found in mutant stage 14 oocytes (Tavosanis, 1997) are not observed in laid oocytes or fertilized embryos. This observation suggests that these abnormal meiotic figures are not terminally arrested. The same observation has been reported by Wilson (1998). It is not known how these figures develop, but it is likely that the natural course of activation triggered by passage through the oviduct exerts some effect in the mutant oocytes; this results in the progression from the abnormal meiotic figures of the ovarian oocytes into the mutant phenotypes observed in laid eggs. (2) Hypomorph and lack-of-function alleles led to a total arrest of nuclear proliferation, during early embryogenesis. This is in contrast to their effect on female meiosis-I where hypomorph alleles display a much weaker phenotype. Thus, there seems to be a clear difference in the requirements for gamma-tubulin function between meiosis-I and mitosis so that while low levels of gamma-tubulin function may be sufficient to carry out normal female meiosis, they are not sufficient for nuclear proliferation during early embryogenesis. If the effect of the different mutant alleles on meiosis-II is similar to their effects on meiosis-I, then this observation suggests that the absence of nuclear proliferation is not just a pleiotropic effect brought about by the abnormal meiosis displayed by mutant oocytes. (3) Despite the lack of nuclear proliferation which is common to null and hypomorph alleles, each of these kinds of alleles led to some distinct phenotypes. Unfertilized laid oocytes and fertilized embryos deficient for gammaTub37C do not contain polar bodies and have one or two large bipolar microtubule arrays together with a few minor ones. In contrast, oocytes and embryos from weak alleles do not have these microtubule arrays, but do contain polar bodies, or polar-body-like structures (Llamazares, 1999).

The presence of bipolar microtubule arrays organized around chromatin in embryos derived from gammaTub37C deficient mothers is remarkable for two reasons: (1) this occurs in the same mutant background in which the female meiotic spindle often fails to be formed; (2) this takes place in the absence of the major isoform of gamma-tubulin present at that stage (Llamazares, 1999).

The presence of bipolar arrays of microtubules in laid oocytes and embryos derived from females that were unable to organize a proper meiosis-I spindle argues that these two structures, the wild-type meiotic spindle and the bipolar microtubule arrays are organized by different mechanisms. This is not surprising since a wild-type functional structure that has evolved to ensure segregation of homolog chromosomes is being compared with an aberrant microtubule array. The only suggestion of similitude between these two structures comes from their bipolar shape and the fact that they are anastral. However, this argument is not very solid. Spindle-like microtubule arrays that are bipolar and anastral can also form around microinjected naked DNA in Xenopus eggs and around beads coated with DNA in Xenopus egg extracts. Furthermore, functional spindles that are bipolar and anastral have been reported during the syncytial mitotic divisions that occur in parthenogenic Sciara embryos. Finally, even at the morphological level, there are some differences between the spindle structures found in gammaTub37C mutant derived laid oocytes and embryos and the wild-type female meiosis-I spindle. The former are often larger (sometimes more than twice as long as the wild type), considerably thicker and often contain much more chromatin. Since these spindle structures have not been followed in real time, it cannot be positively ruled out that they may drive chromosome segregation, but this is unlikely since they have never been observed in an anaphase or anaphase-like stage. In fact, the stability of these structures, which are present in relatively old embryos, indicates that they are fairly static and that chromosome segregation never occurs. Mutant derived laid oocytes and embryos always have a variable number of smaller spindle-like arrays, which also contain chromatin. The presence of these groups of large and small spindles associated with large and small masses of chromatin is interpreted as a consequence of the abnormal meiosis observed in mutant oocytes. Interestingly, similar figures are produced in a fraction of eggs from grauzone and cortex mothers, which arrest in meiosis-I (Llamazares, 1999).

Whichever their origin and nature, the microtubule arrays found in gammaTub37C deficient embryos seem to argue that microtubule polymerization is possible in the absence of this isoform. Nevertheless, this observation must be interpreted with caution. Although extremely unlikely, the possibility cannot be ruled out that non-detectable ammounts of the truncated product of the mutant allele may promote microtubule nucleation. In a gammaTub37C null embryo, microtubule polymerization could be supported by the small amounts of gammaTUB23C that are present at this stage. Alternatively, these microtubule arrays may not depend at all on gamma-tubulin for their polymerization and organization. In any case, the presence of a few microtubule arrays in gammaTub37C deficient embryos should not be interpreted as evidence that microtubule polymerization is normal since no information whatsoever is available on the dynamics of microtubule polymerization in these mutant embryos (Llamazares, 1999).

Contrary to the phenotype displayed by laid oocytes and embryos derived from females deficient for the gammaTub37C isoform, the laid oocytes and embryos from females carrying hypomorph alleles of this gene contain polar bodies. This observation is in agreement with the weak meiotic phenotype reported for this allele in which only 15% of the meiotic figures can be classified as abnormal (Tavosanis, 1997). The presence of polar bodies in this mutant condition indicates that meiotic chromosome segregation takes place, although their abnormal chromosome content and microtubule structure suggests that meiosis did not proceed normally. Both the spindle-like and the polar-body-like structures found in the two mutant conditions studied in this work seem to be able to grow although normal nuclear proliferation does not take place. In both cases, chromatin content can reach very high levels, much greater than 4N, suggesting that DNA replication is taking place within these structures. The associated microtubule mass seems to increase accordingly (Llamazares, 1999).

From the phenotypic analysis of embryos derived from gammaTub37C mutant females it is believed that the main defect caused by mutation in this gene during embryogenesis is that normal functional centrosomes cannot be assembled. This conclusion is based on the observation that organized centrosomes are always absent mutants. These few centrosomes are never observed organizing dense asters and show an erratic association with neighboring microtubule arrays. Although these observations could be partially due to a downstream effect of the abnormal meiotic divisions displayed by these oocytes, it is believed that, most likely, they reflect a direct requirement for gammaTub37C to organize functional mitotic centrosomes. This can be due to either structural or functional reasons. It is possible that the absence or reduction of gamma-tubulin levels affects centrosome organization so that the organelle cannot be assembled. Alternatively, the first centrosomes may be assembled correctly, but be unable to organize microtubules. Either case would result in the absence of functional centrosomes that could lead to a total arrest of the wild-type program of nuclear proliferation within mutant embryos (Llamazares, 1999).

It is still not known whether the mutant phenotypes produced by mutation in the gammaTub37C gene are due to a specific requirement for this isoform or simply to the fact that it is the most abundant one during these stages. Since the gammaTUB23C isoform is present at low levels during early embryogenesis (Tavosanis, 1997) and it does not seem to be associated with centrosomes (Wilson, 1997), it has been suggested that some functional incompatibility between the two isoforms may exist at this stage (Wilson, 1997). A similar argument could be made for the abnormal meiosis displayed by gammaTub37C deficient females despite the presence of small amounts of the gTUB23C isoform (Tavosanis, 1997). To unequivocally answer this question the ability of the gammaTub37C isoform to rescue these phenotypes when expressed under the control of the gammaTub37C regulatory regions is being examined (Llamazares, 1999).


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gamma-Tubulin at 37C: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 September 2008

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