gammaTubulin at 23C


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).

Microtubule anchoring by cortical actin bundles prevents streaming of the oocyte cytoplasm; Actin bundles at the cortex of the oocyte which are involved in the cortical localisation of γTubulin

The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. This study reports a careful analysis of the oocyte cytoskeleton. Thick actin bundles are identified at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, the data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. Actin bundle formation requires Profilin but not Capu and Spire. Thus, these results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, these data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. The release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent (Wang, 2007).

This study reports the existence of actin bundles at the cortex of the oocyte which are involved in the cortical localisation of γTubulin. γTubulin is part of the γTubulin ring complex that is stabilising the minus ends of MTs. The presence of γTubulin alone does not allow distinguishing whether the protein is part of a microtubule organising centre (MTOC) that nucleates MTs or whether it only protects existing MTs from depolymerisation. Here, γTubulin was used solely as a maker for the MT minus ends, and it was shown that these are embedded within the cortical actin bundles before stage 10b (Wang, 2007).

The cytoskeletal rearrangements at stage 10b include the disassembly of the cortical actin bundles, the redistribution of the MT minus ends from the cortex to subcortical regions and the formation of MT arrays parallel to the oocyte cortex. Concomitantly with these cytoskeletal changes, the transition from slow to fast cytoplasmic streaming is triggered. What is the causal relationship between these events? The finding that interfering with actin bundle formation by drug treatment and GFPactin5c overexpression results in MT minus ends redistribution, MT array formation and premature fast streaming indicates that actin bundling acts upstream of MT reorganisation and streaming. The analysis of Khc mutants allows to further dissect the subsequent steps reorganising the MT cytoskeleton. In the absence of streaming, caused by the loss of Khc function, the redistribution of MT minus ends occurs normally, while the formation of MT arrays is abolished. Thus, minus end redistribution is upstream of streaming, and array formation is downstream. It is therefore proposed that streaming is initiated by the disassembly of the cortical actin bundles resulting in loss of cortical MT minus end anchoring. It is further proposed that the redistribution of the minus ends to subcortical regions is important for the reorganisation of the MT cytoskeleton into arrays that run parallel to the oocyte cortex. At this step a previously suggested self amplifying loop could be initiated, in which MT array formation and Kinesin movement enhance each other. In this loop the Kinesin driven streaming helps to sweep MTs into parallel arrays, which in turn allow more robust currents in the cytoplasm (Wang, 2007 and references therein).

How do the actin binding proteins Capu, Spire and Profilin act on the oocyte cytoskeleton to prevent premature cytoplasmic streaming? chic/Profilin mutants and latrunculin A treatment both interfere with bundle formation. Latrunculin A treatment inhibits actin polymerisation by binding to and sequestering actin monomers. Profilin is involved in actin polymerisation by delivering actin monomers to the growing ends of actin filaments. Thus, latrunculin A and Profilin mutants appear to interfere with bundling by limiting the pool of monomers that can be added to growing actin filaments. In contrast, capu and spire mutants are not required for the formation of actin bundles. It is proposed that Capu and Spire anchor the MT minus ends in a scaffold provided by the cortical actin bundles. The lack of Capu and Spire activity in the mutants prevents cortical MT anchoring and allows streaming in the presence of actin bundles. This model is supported by the work of Rosales-Nieves (2006) who have shown that Capu and Spire proteins are able to crosslink F-actin and MTs in vitro, and that both proteins localise to the oocyte cortex (Wang, 2007).

The regulation of fast ooplasmic streaming could be controlled at the level of the cortical localisation of Capu and Spire. The displacement of the two proteins from the cortex at stage 10b might result in loss of MT minus end anchoring, and thereby induce fast streaming. To test this, the localisation of GFP-Capu and GFP-Spire was analysed in cross sections of oocytes. However, no difference were in the localisation of the two proteins before and after onset of fast streaming. In addition, no displacement of GFP-Capu and GFP-Spire was detected after induction of premature streaming by latrunculin A treatment. Thus, Capu and Spire activities are not regulated at the level of their localisation (Wang, 2007).

A different mode of Capu and Spire regulation is suggested by their genetic and biochemical interaction with Rho1. This interaction led to a model in which Rho1 initiates fast streaming by regulating the crosslinking activities of Capu and Spire (Rosales-Nieves, 2006). The prevention of streaming requires not only capu and spire but also the presence of actin bundles. The formation of these bundles occurs, however, independently of capu and spire. This suggests that the onset of fast streaming is not only controlled by regulating Capu and Spire activities, but also by disassembly of the actin bundles (Wang, 2007).

Genes were also tested that are involved in actin regulation in the oocyte but do not induce premature streaming. For capulet, swallow and moesin mutants the formation of ectopic actin clumps has been reported reflecting defects in the organisation of the oocyte actin cytoskeleton. The presence was confirmed of ectopic F-actin in the oocyte cytoplasm in these mutants, but nevertheless the formation cortical actin bundles was detected. Thus, actin defects in the oocyte do not necessarily affect cortical actin bundling (Wang, 2007).

Zfrp8, the Drosophila ortholog of PDCD2, functions in lymph gland development and controls cell proliferation

Zfrp8 is essential for hematopoiesis in Drosophila. Zfrp8 (Zinc finger protein RP-8) is the Drosophila ortholog of the PDCD2 (programmed cell death 2) protein of unknown function, and is highly conserved in all eukaryotes. Zfrp8 mutants present a developmental delay, lethality during larval and pupal stages and hyperplasia of the hematopoietic organ, the lymph gland. This overgrowth results from an increase in proliferation of undifferentiated hemocytes throughout development and is accompanied by abnormal differentiation of hemocytes. Furthermore, the subcellular distribution of γ-Tubulin and Cyclin B is affected. Consistent with this, the phenotype of the lymph gland of Zfpr8 heterozygous mutants is dominantly enhanced by the l(1)dd4 gene encoding Dgrip91, which is involved in anchoring γ-Tubulin to the centrosome. The overgrowth phenotype is also enhanced by a mutation in Cdc27, which encodes a component of the anaphase-promoting complex (APC) that regulates the degradation of cyclins. No evidence for an apoptotic function of Zfrp8 was found. Based on the phenotype, genetic interactions and subcellular localization of Zfrp8, it is proposed that the protein is involved in the regulation of cell proliferation from embryonic stages onward, through the function of the centrosome, and regulates the level and localization of cell-cycle components. The overproliferation of cells in the lymph gland results in abnormal hemocyte differentiation (Minakhina, 2007).

The developmental mechanisms of human and Drosophila blood systems show remarkable parallels. In humans, several blood cell types with specific functions develop from the same pluripotent stem cells. In Drosophila, only a few specialized cell types exist, with functions similar to human cells. These are thought to originate from a common set of hematopoietic precursors. The development and specification of blood cells in humans and flies are controlled by conserved signaling pathways. Because of its relative simplicity, hematopoiesis in Drosophila is frequently used as a model to investigate the genetic control of hematopoiesis in flies and humans (Minakhina, 2007).

In Drosophila, mature hemocytes arise from two distinct sources: the mature larval circulating hemocytes derive from the embryonic head mesoderm, whereas the lymph gland hemocytes are normally released into circulation at the onset of metamorphosis and perdure into the adult stage. As in vertebrate blood and vascular systems, the Drosophila lymph gland hemocytes and heart cells derive from a common progenitor, called the hemangioblast or cardiogenic mesoderm, which further splits into the lymph gland and cardiogenic progenitors (Mandal, 2004; Minakhina, 2007).

Among the earliest requirements for the specification of blood progenitors in mammals and Drosophila are the highly conserved, GATA zinc-finger transcription factors. The Drosophila GATA-factor Pannier (Pnr) is required for early specification of the hemangioblast/cardiogenic mesoderm. Another GATA-factor, Serpent (Srp), plays a central role in committing mesodermal precursors to the hemocyte fate (Minakhina, 2007).

By the end of embryogenesis, the lymph gland is fully formed and contains mostly pro-hemocytes. The third instar larval lymph gland contains a pair of primary and several secondary lobes. Each primary lobe is subdivided into (1) the medullary zone, populated by slowly proliferating pro-hemocytes; (2) the cortical zone, containing differentiated hemocytes; and (3) the posterior signaling center (PSC), first defined as a small group of cells expressing the Notch ligand Serrate (Ser). Under the control of the EBF-homolog (early B-cell factor) collier (col; knot), PSCs function as a hematopoietic niche to maintain a population of blood cell precursors. The blood cell precursors differentiate into three groups of hemocytes: plasmatocytes, crystal cells and lamellocytes. All three are released into the open circulating hemolymph during the onset of metamorphosis or as a part of an immune reaction. Differentiated plasmatocytes and crystal cells are found in both the cortical zone of the lymph gland and the larval hemolymph, but lamellocytes are rare (Minakhina, 2007).

Plasmatocytes, the predominant form of hemocytes in larvae, perform phagocytic functions and secrete extracellular matrix components and immune peptides similar to human white blood cells. Crystal cells are non-adhesive hemocytes responsible for melanization during wound healing and encapsulation of parasites. Crystal cell differentiation requires the cell-autonomous expression of the transcription factor Lozenge (Lz), homologous to the mammalian acute myeloid leukemia 1 protein (Aml1 or Runx1) (Minakhina, 2007).

Lamellocytes function in encapsulation. Their number is significantly increased at the onset of metamorphosis and in response to infection. Differentiation of lamellocytes is connected to two major pathways - the Drosophila Toll/NF-kappaB and the JAK/STAT - that regulate blood cells proliferation and activation during immune response. Constitutive activation of either pathway leads to overproliferation of circulating and lymph gland hemocytes, an increase in lamellocytes and activation of the cellular immune response (Minakhina, 2007).

A newly identified gene, Zfrp8, is essential for lymph gland growth and for the normal development of Drosophila larvae. Mutant larvae show hyperplasia of the hematopoietic organs. This phenotype is not linked to apoptosis but rather to an increase in cell proliferation. Mutant lymph glands also show a drastic increase in the number of lamellocytes (Minakhina, 2007).

These phenotypes are suppressed by mutations in the GATA factor gene pnr. Mutations in the two cell-cycle genes Cdc27 and l(1)dd4 [lethal (1) discs degenerate 4], have the opposite effect as they enhance the lymph gland overgrowth phenotype of Zfrp8/+. Cdc27 encodes a subunit of the APC complex, responsible for the turnover of cyclins, and l(1)dd4 encodes Dgrip91, a component of the centrosome involved in γ-Tubulin anchoring. In the Zfrp8 mutant lymph gland cells, both Cyclin B (CycB) and γ-Tubulin exhibit abnormal subcellular distribution, suggesting that Zfrp8 plays an important role in their regulation (Minakhina, 2007).

In the literature, the Zfrp8 vertebrate ortholog, PDCD2, is routinely referred to as an apoptotic gene solely because it was upregulated during steroid-induced programmed cell death in rat thymocytes. Subsequent studies, using different cells and assay conditions, found no connection between PDCD2 expression and programmed cell death (Minakhina, 2007 and references therein).

It is unlikely that a reduction in cell death is the cause of the lymph gland overgrowth observed in Zfrp8 mutant larvae. Very few or no apoptotic cells are detected in wild-type larval lymph glands. This study found a statistically insignificant increase in the number of apoptotic cells in Zfrp8 mutants. No other evidence of change in programmed cell death in Zfrp8 mutant animals, no increase in apoptotic gene expression, no change in caspase cleavage and no genetic interaction of Zfrp8 with known apoptotic genes were found (Minakhina, 2007).

The results are consistent with an increase in cell division in Zfrp8 mutants throughout development. This conclusion is supported by the observation that Zfrp8 lymph glands are already twice the size of their normal counterparts in late-stage embryos, and that the number of cells in mitosis is about 30% higher in the mutant glands than in wild type (Minakhina, 2007).

Detailed analysis of Zfrp8 lymph glands shows that its phenotype is different from that of Drosophila hematopoietic/immunity mutants. Unlike hematopoietic/immunity mutants, the increase in lymph gland cell numbers is much larger than the increase in circulating hemocytes. Furthermore, the blood cell overproliferation in Zfrp8-null mutants is not accompanied by constitutive activation of immunity. Zfrp8 larvae show normal induction of immune peptide genes in response to bacterial challenge and normal wound clogging and wound melanization. That the requirements are different for Zfrp8 and known hematopoiesis and immunity genes is underlined by the absence of their genetic interaction (Minakhina, 2007).

In normal lymph glands, plasmatocytes are found mostly in the cortical region and very few lamellocytes are detected. The PSC is formed at the base of each primary lobe. The presence of additional PSCs in mutant lymph glands might indicate that additional primary lobes are formed by the large number of cells (Minakhina, 2007).

PSCs are essential for maintaining the undifferentiated hemocyte population in the medullary zone and that they control lamellocyte differentiation during parasitic infection. Lack of the transcription factor collier, essential for PSC maintenance, leads to a decrease in the pro-hemocyte population and abolishes lamellocyte differentiation. Loss of Zfrp8 leads to the opposite phenotype - an increase in pro-hemocyte proliferation, beginning during embryogenesis, and an increased number of cells acquiring the lamellocyte fate. Expansion of the PSCs alone does not account for this phenotype. Ectopic expression of the homeotic gene Antennapedia results in expansion of the PSCs, and a concomitant increase of the medullar zone, but not the gland overgrowth. Therefore, it is unlikely that Zfrp8 is directly involved in the establishment of PSCs (Minakhina, 2007).

The results link the Zfrp8 overgrowth phenotype to a defect in normal cell proliferation. In mutant lymph glands, the cell-cycle markers γ-Tubulin and CycB are misregulated. Zfrp8 genetically interacts with at least two genes functioning in the cell cycle, Cdc27 encoding a subunit of the anaphase-promoting complex (APC), and l(1)dd4 encoding the Drosophila gamma-ring protein Dgrip91 (Minakhina, 2007).

Dgrip91 and γ-Tubulin are components of the γ-TuRC microtubule-nucleating complex anchored to centrosomes. Beyond the conventional role in microtubule organization, centrosomes also serve as a scaffold for anchoring a number of cell-cycle regulators. For instance, centrosome-association of Cdc27 and CycB proteins plays an important role in CycB activation, degradation and entrance into mitosis (Minakhina, 2007).

The link between the phenotypes described above and Zfrp8 function became clear when it was discovered that a proportion of Zfrp8 protein localizes adjacent to the centrosome in wild-type tissue. This subcellular localization is consistent with a function of Zfrp8 in centrosome organization and in the anchoring of proteins such as γ-Tubulin and CycB to this organelle (Minakhina, 2007).

Zfrp8 might also affect the expression of bona fide cell-cycle regulators. The protein contains a zinc-finger domain, MYND, present in a number of transcriptional regulators, that fosters protein-protein interactions and recruits co-repressors. PDCD2/Zfrp8 is known to interact with the HCF-1 transcriptional regulator, which suggests that PDCD2/Zfrp8 might be involved in regulating the cell cycle at the transcriptional level (Minakhina, 2007).

Zfrp8 might have a dual function, through its association with the centrosome and as a transcriptional regulator of the cell cycle. Several transcriptional regulators have been found to localize to the centrosome, but their centrosomal function has not been documented (Minakhina, 2007).

Zfrp8 function is essential for the control of cell proliferation already in the embryo. With this being the case, it functions upstream from most of the conserved signaling pathways involved in fly hematopoiesis and immunity. Because of the similarity of the protein in flies and vertebrates, it is possible that PDCD2 has a similar function in vertebrate hematopoiesis (Minakhina, 2007).

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).

RacGAP50C directs perinuclear gamma-tubulin localization to organize the uniform microtubule array required for Drosophila myotube extension

The microtubule (MT) cytoskeleton is reorganized during myogenesis as individual myoblasts fuse into multinucleated myotubes. Although this reorganization has long been observed in cell culture, these findings have not been validated during development, and proteins that regulate this process are largely unknown. A novel postmitotic function has been identified for the cytokinesis proteins RacGAP50C (Tumbleweed) and Pavarotti as essential regulators of MT organization during Drosophila myogenesis. The localization of the MT nucleator gamma-tubulin changes from diffuse cytoplasmic staining in mononucleated myoblasts to discrete cytoplasmic puncta at the nuclear periphery in multinucleated myoblasts, and this change in localization depends on RacGAP50C. RacGAP50C and gamma-tubulin colocalize at perinuclear sites in myotubes, and in RacGAP50C mutants gamma-tubulin remains dispersed throughout the cytoplasm. Furthermore, the mislocalization of RacGAP50C in pavarotti mutants is sufficient to redistribute gamma-tubulin to the muscle fiber ends. Finally, myotubes in RacGAP50C mutants have MTs with non-uniform polarity, resulting in multiple guidance errors. Taken together, these findings provide strong evidence that the reorganization of the MT network that has been observed in vitro plays an important role in myotube extension and muscle patterning in vivo, and also identify two molecules crucial for this process (Guerin, 2009).

The reorganization of the actin and MT cytoskeletons during myogenesis has long been observed in cell culture as individual myoblasts fuse into multinucleated myotubes. Although the actin cytoskeleton has been shown to be indispensable for mediating myoblast fusion, little is known about how the MT network is remodeled during muscle development or the developmental significance of this event. This study provides evidence that RacGAP, a known regulator of the MT cytoskeleton during cytokinesis, and Pav, a kinesin-like RacGAP-binding protein, play a novel and important role in MT organization in vivo by localizing γ-tubulin to perinuclear sites in myotubes. Furthermore, the organization of the MT network in multinucleated myotubes is important for muscle attachment site (MAS) selection. Muscles that are mutant for RacGAP or pav have defects in MT polarity and fail to properly extend towards their attachment sites, resulting in defects in somatic muscle patterning (Guerin, 2009).

The current model for MT organization in differentiated myotubes has come primarily from cell culture studies, which describe MTs that run parallel to the long axis of the cell and do not appear to be directly associated with any one organizing center. Further studies have demonstrated that proteins involved in MT organization, such as γ-tubulin, are redistributed from the centrosome of individual myoblasts to discrete cytoplasmic puncta as well as along the nuclear membrane in multinucleated myotubes and that these sites are associated with MT growth (Bugnard, 2005; Musa, 2003). The diffuse cytoplasmic distribution of γ-tubulin that is observed in Drosophila myoblasts differs from that in cultured vertebrate myoblasts, in which γ-tubulin is associated with centrosomes. Nonetheless, in both cases, the MT cytoskeleton must be reorganized from either a centrosomal or broadly distributed array in individual myoblasts, to a parallel array in multinucleated myotubes with the plus ends directed outwards. This study shows that RacGAP plays a crucial role in this reorganization. In the absence of RacGAP, MTs are not uniform in their polarity and γ-tubulin remains dispersed throughout the cytoplasm rather than accumulating at the nuclear periphery of multinucleated myotubes. Furthermore, in pav mutants, mislocalization of RacGAP is sufficient to redistribute γ-tubulin to the ends of myotubes (Guerin, 2009).

To date, the perinuclear localization of γ-tubulin in myotubes has only been weakly detected in vitro (Bugnard, 2005). This study shows that the association of γ-tubulin with the nucleus also occurs in vivo and is dependent at least in part on RacGAP. What is the function of γ-tubulin localization to the nuclear periphery in myotubes? One likely possibility is to anchor MT minus ends. Because the nuclei in multinucleated myotubes cluster in the interior of the myotube, this would allow for the polarization of the MT network, which is aligned along the axis of cell migration, with the plus ends at the leading edge. What is the purpose of MT polymerization at the ends of myotubes? Although, conventionally, the driving force for cell motility has been thought to be provided mainly by the reorganization of the actin cytoskeleton, there is increasing evidence that MTs are indispensable for cell migration. It has been hypothesized that MTs form longitudinal arrays in bipolar myotubes in order to facilitate elongation by 'active crawling' of the two ends of the myotube during MAS selection (Musa, 2003). The data point to a MT-based mechanism for myotube extension and MAS selection. In the absence of RacGAP or Pav, the MT network shows non-uniform polarity and many muscle fibers are abnormally shaped and display guidance errors. The effect of RacGAP and pav mutations on muscle morphology is consistent with previous findings in which both RacGAP and Pav have been implicated in regulating axonal outgrowth and maintaining dendritic morphology. RacGAP was identified in a genetic screen by the increased dendritic branching phenotype observed in tum mutants. RacGAP and Pav have also been shown to play a role in regulating the morphogenesis of postmitotic mushroom body neurons in the Drosophila brain. In addition, disruption of the mammalian form of Pav, KIF23 (CHO1; MKLP1), in postmitotic cultured neurons resulted in the rearrangement of MT polarity and in the disruption of dendrite morphology (Guerin, 2009).

There is increasing evidence that morphological processes require regulated coordination of the cytoskeleton by linking actin and MTs. For example, in Drosophila the Rho activator RhoGEF2 is implicated both in Myosin II localization and MT organization via the localization of the plus-end protein Eb1. Likewise, RacGAP provides a connection between the actomyosin ring and the peripheral central-spindle MTs during cytokinesis via its interaction with the actin-binding protein Anillin. In addition, proper formation of the cleavage furrow is dependent on a complex between RacGAP, the Rho activator Pebble, and the plus-end-directed MT protein Pav. The current data show that similar to its function during cytokinesis, the function of RacGAP in postmitotic myotubes depends on its association with the MT-binding protein Pav. However, the role of RacGAP in regulating γ-tubulin distribution appears to be independent of its interaction with Anillin and the actin cytoskeleton. scraps mutants do not show defects in muscle patterning. Furthermore, the organization of the actin cytoskeleton and two known actin-dependent processes, myoblast fusion and muscle attachment, are not significantly affected in RacGAP mutants. These findings demonstrate a newly described function for RacGAP that is restricted to the modulation of MTs, but not the actin cytoskeleton, in postmitotic cells (Guerin, 2009).

What is the developmental significance of the actin-independent function of RacGAP in myotube extension? The answer might lie in the complex process of myogenesis itself. Building a mature muscle fiber requires the coordination of many morphological processes, including myoblast fusion, myotube extension and muscle attachment. The uncoupling of actin- and MT-based cytoskeletal processes might allow for actin-based myoblast fusion and MT-based myotube elongation to occur simultaneously. This idea is supported by previous findings showing that myoblasts continue to fuse as the myotube elongates to find its attachment sites. In addition, fusion-defective mutant FCs have been observed to extend and attempt to migrate to their targets, demonstrating that the migration machinery is not perturbed in mutants in which fusion is disrupted (Guerin, 2009).

It is not yet clear what serves as the trigger for MT reorganization upon myoblast fusion or how RacGAP is recruited for this process. It also remains to be determined whether RacGAP promotes the nucleation of new MTs at the nuclear periphery, or reorganizes existing MTs from fusing myoblasts. Changes in MT architecture could be regulated through a direct physical interaction between RacGAP and γ-tubulin, or indirectly through a complex with downstream targets of the GAP domain of RacGAP (Guerin, 2009).

γ-Tubulin ring complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning

γ-Tubulin is critical for microtubule (MT) assembly and organization. In metazoa, this protein acts in multiprotein complexes called γ-Tubulin Ring Complexes (γ-TuRCs). While the subunits that constitute γ-Tubulin Small Complexes (γ-TuSCs), the core of the MT nucleation machinery, are essential, mutation of γ-TuRC-specific proteins in Drosophila causes sterility and morphological abnormalities via hitherto unidentified mechanisms. This study demonstrates a role of γ-TuRCs in controlling spindle orientation independent of MT nucleation activity, both in cultured cells and in vivo and examines a potential function for γ-TuRCs on astral MTs. γ-TuRCs locate along the length of astral MTs, and depletion of γ-TuRC-specific proteins increases MT dynamics and causes the plus-end tracking protein EB1 to redistribute along MTs. Moreover, suppression of MT dynamics through drug treatment or EB1 down-regulation rescues spindle orientation defects induced by γ-TuRC depletion. Therefore, a role is preposed for γ-TuRCs in regulating spindle positioning by controlling the stability of astral MTs (Bouissou, 2014).

Using cultured cells and Drosophila neuroblasts, this study has demonstrated a novel role of γ-TuRCs in spindle positioning. It is proposed that spindle positioning is controlled by a balance created by antagonistic factors, exerting stabilizing and destabilizing effects on astral MTs. γ-TuRCs and EB1 were characterized as representative examples for these two types of factors. Moreover, EB1 redistribution was shown to be is concomitant with an increase of GTP-tubulin islands along MTs. This suggests that γ-TuRC-dependent changes of MT dynamics involve switches of tubulin conformation that could affect EB1 localization (Bouissou, 2014).

γ-TuRCs were shown to stabilize astral MTs. These results are consistent with previous studies on γ-TuRCs associated to the lattice of interphase MTs that regulate dynamics by preventing MT depolymerization beyond the sites of γ-TuRC attachment. In comparison to interphase, the relative increase of MT dynamics upon γ-TuRC depletion is weaker during mitosis. Cell cycle differences, such as the composition of the cortical area or the balance of MT-associated proteins, may affect MT behaviour. Consistent with a role of γ-TuRCs in regulating astral MT dynamics, regular spindle orientation can be restored in the absence of γ-TuRCs at least partially, when cells are simultaneously treated with drugs that reduce MT dynamics, or when the level of plus-end-binding proteins that promote MT dynamics is lowered (Bouissou, 2014).

To investigate the mechanisms by which γ-TuRCs regulate the stability of astral MTs, the localization of γ-TuRCs was studied in mitotic cells. Astral localization of γ-TuRCs was observed, evidenced by different immunofluorescence procedures and confirmed by live-imaging. Such a localization pattern has not been described before, likely for three reasons. First, this localization may have been obscured by a large background of cytoplasmic γ-TuRCs. Second, only a small fraction of γ-tubulin is localized on MTs, and the faint, punctuate staining may have been overlooked previously. Third, astral MTs mostly appear as individual MTs, not organized in bundles like kinetochore fibers, and consequently associated proteins appear less concentrated. This study also demonstrates that the recruitment of γ-TuRCs to astral MTs in mammalian cells is, at least partially, dependent on the augmin complexes, whereas augmin proteins, required for centrosome-independent microtubule generation within the spindle, are still present along MTs after γ-TuRC disassembly. These data suggest a recruitment of the augmin complexes on astral MTs prior to γ-tubulin complexes, or a pre-requisite of large complexes (including augmin and γ-TuRCs) forming in the cytoplasm before γ-TuRC recruitment. In addition to augmins, the protein Cdk5Rap2 may also be involved in binding γ-tubulin complexes to the MTs, since Cdk5Rap2 is known to interact with γ-TuRCs and concentrates partially at MT plus-ends, where it contributes to the regulation of MT dynamics (Bouissou, 2014).

So far, the main function that has been attributed to γ-TuRCs attached to the MT surface via augmin complexes is the nucleation of secondary MTs, to increase the density of kinetochore fibers. However, this study as well as previously published data on plant cells argue against a sole function of MT-bound γ-TuRCs in secondary nucleation and suggest that these complexes have additional functions. First of all, measurements of a homogeneous tubulin immunofluorescence intensity along astral MTs indicate that no additional MTs have been nucleated at these specific sites. Moreover in plant cells, the majority of γ-TuRC foci associated to interphase MTs are not nucleating any other MTs, and the subset of γ-tubulin complexes active in generating new MTs appear enriched in the homolog of Mozart1. Finally, a recently published study in Arabidopsis shows that the augmin subunit 8 binds to the MT plus-ends, regulates the dynamics of MT plus-ends and by this way controls MT reorientation in hypocotyls (Bouissou, 2014).

All these data lead to a proposal of certain heterogeneity or plasticity in γ-TuRCs associated to the MT surface. In addition to their nucleation activity, some γ-TuRCs, by attaching to MTs, could exert a stabilizing effect on individual MTs, independent of their dormant potential to nucleate secondary MTs. γ-TuRCs along the MT surface may function in an analogous manner as a Microtubule Associated Protein (MAP), or if localized to the plus-end, increase stability similar to a cap. It is suggested that the spindle orientation defects that were observe upon γ-TuRC-disassembly are not due to altered mechanisms of MT nuleation. First, it was shown that the activity of nucleation at the poles is comparable in control and γ-TuRC-deficient cells and mitotic MTs in cells lacking the full γ-TuRC proteins possess a regular structure, with 13 protofilaments per diameter. Moreover, suppression of MT dynamics is sufficient to restore spindle orientation. Besides, data in yeast suggest that mutations in individual components of the γ-tubulin complexes influence MT dynamics in a post-nucleation manner. In addition, depletion of Dgp71WD or of an augmin subunit does not modify the quantity and the elongation of de novo nucleated MTs associated with acentriolar centers but does affect their stability. This is consistent with previous data showing that in Drosophila cells the soluble pool of α/β tubulin is not significantly changed after depletion of individual γ-TuRC grip-motif proteins. Even if the soluble pool were slightly increased, the effects on MT dynamics would probably be minimal, since MT dynamics in cells, in contrast to the situation described in vitro, appear much more sensitive to the regulation by MT-associated proteins than to the concentration of free α/β tubulin (Bouissou, 2014).

Consistent with the hypothesis on altered MT dynamics, a change was observed in EB1 localization following depletion of γ-TuRC-specific subunits. EB1 is no longer concentrated at the MT plus-ends but rather distributed along MT side walls. Similar EB1 redistribution has been reported in other experimental setups affecting MT dynamics, for example following depletion of EB1 interactors, such as the XMAP215/Dis protein family or the p150Glued subunit of dynactin, or immunodepletion of γ-tubulin from frog egg extracts. However, a mechanistic explanation is lacking so far. One possibility would be that reduced affinity of EB1 to MT tips increases the pool of EB1 that might now bind to lower affinity sites on the MT lattice. Alternatively, and not mutually exclusive, changes in the size and intensity of EB1 staining after γ-TuRC depletion may result from alterations in GTP caps or GTP-remnants on MTs. This possibility was explored using the antibody hMB11, known to recognize a conformational state of tubulin, acquired upon binding of GTP. EB1 and hMB11 antibodies have higher affinity for GTP-MTs compared to GDP-MTs in vitro and both can colocalize as patches on the lattice of axonal MTs when EB1 is overexpressed. So, the extended MT surface that was stained with both EB1 and hMB11 after γ-TuRC depletion may be due to altered tubulin conformation or a modified guanosine nucleotide status. This interpretation makes sense with a recent study that proposes that the effects between EB1 and the MT-associated protein XMAP215 on MT dynamics don't rely on any direct interaction but rather on allosteric interaction through MT ends. Abnormal loading of EB1 to the MTs after γ-TuRC disassembly may have itself a feedback on the balanced loading of +TIP complexes and also on the MT binding to the cell cortex. Therefore it may enhance any primary effects following the loss of γ-TuRCs, especially on spindle orientation. In any of the above discussed scenarios, γ-TuRCs may not interact directly with EB1. This is based on the observations (1) that EB1 is generally not enriched at sites of γ-TuRC localization, (2) that a decrease of γ-TuRC along MTs leads to an increase of the length of EB1 staining, and (3) that EB1 appears absent from sucrose gradient fractions corresponding to γ-TuRCs (Bouissou, 2014).

Oriented cell divisions appear critical for proper development and maintenance of tissue homeostasis. Moreover, emerging evidence reveals a link between spindle mis-orientation and a number of developmental diseases as well as tumorigenesis. The study of spindle positioning is therefore fundamental to both developmental biology and human pathologies. This study provide new mechanistic information for understanding this key event. These results invoke γ-TuRCs as novel players in spindle orientation by indirectly controlling localization of EB1, a key protein involved in the physical interaction between mitotic spindle and cell cortex (Bouissou, 2014).

Protein Interactions

Centrosomal microtubule-associated proteins associated with gammaTubulin

gammaTubulin is a component of a previously isolated complex of Drosophila proteins that contains at least two centrosomal microtubule-associated proteins (CP190 and CP60). Like CP190 and CP60, the gammaTubulin in extracts of early Drosophila embryos binds to microtubules, although this binding may be indirect. Unlike CP190 and CP60, however, only 10-50% of the gamma-tubulin in the extract is able to bind to microtubules. gamma-tubulin binds to a microtubule column as part of a complex, and a substantial fraction of this gamma-tubulin is tightly associated with CP60. As neither alpha- nor beta-tubulin bind to microtubule columns, the gammaTubulin cannot be binding to such columns in the form of an alpha:gamma or beta:gamma heterodimer. These observations suggest that gammaRubulin, CP60, and CP190 are components of a centrosomal complex that can interact with microtubules (Raff, 1993).

In Drosophila the male gamete is presumed to provide the active microtubule organizing center (MTOC). Zygotic centrosome assembly in fertilized Drosophila eggs was analyzed with the aid of an antiserum Rb188, previously shown to be specific for CP190, a 190 kDa centrosome-associated protein. The CP190 protein is detected in two discrete spots, associated with the anterior and posterior ends of the elongating nucleus of Drosophila spermatids. As the spermatids mature this labelling gradually disappears and is no longer visible in sperm dissected from spermathecae and ventral receptacles. gamma-Tubulin is also found in association with the posterior end of the sperm nucleus during spermatogenesis, but is not detected in mature sperm. This suggests that CP190 and gamma-tubulin are not present in detectable quantities in fertilizing sperm. CP190 is not detected in association with the sperm nucleus of newly fertilized eggs removed from the uterus, whereas many CP190-positive particles are associated with microtubules of the sperm aster from anaphase I to anaphase II. It is thought that this CP190 is of maternal origin. These particles disappear during early telophase II; only one pair of CP190-positive spots remain visible at the microtubule focus of the sperm aster. These spots are associated with one aster through telophase, and then move away to form two smaller asters from which the first mitotic spindle is organized. Colchicine treatment suggests that at least some CP190 protein is an integral part of the centrosome rather than merely being transported along microtubules. Centrosomal localization of the CP190 antigen is prevented by incubation of the permeabilized zygote in 20 mM EDTA. At the end of mitosis the female MTOC is lost. The difference in behavior between the male and female MTOCs is probably due to the lack of important constituents in the female spindle that are necessary for the assembly of a centrosome which can reproduce and nucleate the first mitotic spindle. The most obvious difference between the male and female MTOCs is the presence of the paternal centrole at the center of the sperm aster; this centriole (and any associated pericentriolar material) could be the target for the assembly of the maternal components necessary for centrosome reproduction and nucleation of the mitotic spindle (Riparbelli, 1997).

CP190 is associated with the centrosomes during mitosis, and relocates to chromatin during interphase. Staining of salivary gland chromosomes of third instar Drosophila larva, with antibodies specific to CP190, indicates that the protein is associated with a large number of loci on these interphase polytene chromosomes. The central region of the predicted amino acid sequence of the CP190 protein contains four CysX2CysX12HisX4His zinc-finger motifs, similar to those described for several well characterised DNA binding proteins. The data suggest that the function of CP190 involves cell cycle dependent associations with both the centrosome, and with specific chromosomal loci (Whitfield, 1995).

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 and 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 1997b). 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).

In agreement with others working on Drosophila centrosomal proteins, the names for DMAP190 and DMAP60 have been changed to CP190 and CP60, respectively, to give these proteins a consistent nomenclature. In Drosophila, CP190 is a microtubule-associated protein that is localized to the centrosome. Affinity chromatography was used to identify proteins that interact with CP190, allowing the identification of CP60. Like CP190, CP60 interacts with microtubules and is localized to the centrosome. The two proteins associate as part of a multiprotein complex. The amino acid sequence of CP60 is not homologous to any protein in the database, although it contains six consensus sites for phosphorylation by cyclin-dependent kinases. As judged by in situ hybridization, the gene for CP60 maps to chromosomal region 46A. Antibodies that recognize CP60 reveal that it is localized to the centrosome in a cell cycle-dependent manner. The amount of CP60 at the centrosome is maximal during anaphase and telophase, and then drops dramatically sometime between late telophase and early interphase. This dramatic disappearance of CP60 may be due to specific proteolysis, because CP60 contains a sequence of amino acids similar to the "destruction box" that targets cyclins for proteolysis at the end of mitosis. Starting with nuclear cycle 12, CP60 and CP190 are both found in the nucleus during interphase. CP60 isolated from Drosophila embryos is highly phosphorylated, and dephosphorylated CP60 is a good substrate for cyclin B/p34cdc2 kinase complexes. A second kinase activity capable of phosphorylating CP60 is present in the CP60/CP190 multiprotein complex. CP60 binds to purified microtubules, and this binding is blocked by CP60 phosphorylation (Kellogg, 1995).

CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner, moving between the nucleus (during interphase), and the centrosome (during mitosis). A single bipartite 19-amino acid nuclear localization signal was detected that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the nucleus during interphase. The domain responsible for microtubule binding overlaps the domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Both CP190 and CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules (Oegema, 1995).

Both the nucleus and the centrosome are complex, dynamic structures whose architectures undergo cell cycle-specific rearrangements. CP190 and CP60 are two Drosophila proteins of unknown function that shuttle between centrosomes and nuclei in a cell cycle-dependent manner. These two proteins are associated in vitro, and localize to centrosomes in a microtubule independent manner. Fluorescently labeled, bacterially expressed CP190 and CP60 were injected into living Drosophila embryos and their behavior followed during the rapid syncytial blastoderm divisions (nuclear cycles 10-13). CP190 and CP60 cycle between nuclei and centrosomes asynchronously with the accumulation of CP190 leading that of CP60 both at centrosomes and in nuclei. During interphase, CP190 is found in nuclei. Immediately following nuclear envelope breakdown, CP190 localizes to centrosomes where it remains until telophase, thereafter accumulating in reforming nuclei. Unlike CP190, CP60 accumulates at centrosomes primarily during anaphase, where it remains into early interphase. During nuclear cycles 10 and 11, CP60 accumulates in nuclei simultaneous with nuclear envelope breakdown, suggesting that CP60 binds to an unknown nuclear structure, which persists into mitosis. During nuclear cycles 12 and 13, CP60 accumulates gradually in nuclei during interphase, reaching peak levels just before nuclear envelope breakdown. Once in the nucleus, both CP190 and CP60 appear to form fibrous intranuclear networks that remain coherent even after nuclear envelope breakdown. The CP190 and CP60 networks do not co-localize extensively with one another or with DNA. This work provides direct evidence, in living cells, of a coherent protein network that may represent a nuclear skeleton (Oegema, 1997).

Structure of the gamma-tubulin ring complex: a template for microtubule nucleation.

The gamma-tubulin ring complex (gammaTuRC) is a protein complex of relative molecular mass ~2.2 x 106 that nucleates microtubules at the centrosome. Electron-microscopic tomography and metal shadowing has been used to examine the structure of isolated Drosophila gammaTuRCs and the ends of microtubules nucleated by gammaTuRCs and by centrosomes. The gammaTuRC is a lockwasher-like structure made up of repeating subunits, topped asymmetrically with a cap. A similar capped ring is also visible at one end of microtubules grown from isolated gammaTuRCs and from centrosomes. Antibodies against gamma-tubulin label microtubule ends, but not walls, in centrosomes. These data are consistent with a template-mediated mechanism for microtubule nucleation by the gammaTuRC (Moritz, 2000).

The reconstructions that were obtained lead to the proposal of models for the arrangement of gammaTuRC proteins within the complex as well as for points of contact between the gammaTuRC and the microtubule. The repeating subunits in the ring wall are most probably made up of gamma-tubulin, Dgrip84 and Dgrip91. The latter two proteins (or their orthologs yeast Spc97 and Spc98, and human GCP2 and GCP3/HsSpc98p) are known to interact with each other and with gamma-tubulin. In Drosophila and S. cerevisiae, these proteins have been found to form a small complex that contains two gamma-tubulins and one molecule each of Dgrip84 (or Spc97) and Dgrip91 (or Spc98). In addition, stoichiometric studies of the Drosophila proteins (and some of their Xenopus counterparts) in the gammaTuRC show that these three proteins are the most abundant in the complex, indicating that they may comprise the ring wall, where many repeating subunits are visible. Moreover, it is likely that one of the paired, V-shaped subunits corresponds to one gamma-tubulin small complex (gammaTuSC in Drosophila). Since the height of each column in the subunit (~10 nm) is too great for it to be made up of a single molecule of one of these proteins, it is proposed that a tandem arrangement of Dgrip91 or Dgrip84 with gamma-tubulin exists. In this model, the gamma-tubulins are located most proximally to the face of the ring that does not contain the cap, and are therefore available to bind to alpha/beta-tubulin, thereby promoting microtubule growth (Moritz, 2000).

Characterization and reconstitution of Drosophila gamma-Tubulin ring complex subunits

The gamma-tubulin ring complex (gammaTuRC) is important for microtubule nucleation from the centrosome. In addition to gamma-tubulin, the Drosophila gammaTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small gamma-tubulin complex gammaTuSC) and the gammaTuRC, while the remaining subunits are found only in the gammaTuRC. To study gammaTuRC assembly and function, gammaTuSC was reconstituted using the baculovirus expression system. Using the reconstituted gammaTuSC, it has been shown that this subcomplex of the gammaTuRC has microtubule binding and capping activities. Two new gammaTuRC subunits, Dgrips128 and 163, have been characterized and they are shown to be centrosomal proteins. Sequence comparisons among all known gammaTuRC subunits reveal two novel sequence motifs, which have been named grip motifs 1 and 2. Dgrips128 and 163 can each interact with gammaTuSC. However, this interaction is insufficient for gammaTuRC assembly (Gunawardane, 2000).

With the reconstitution of the gammaTuSC, an important step has been made toward understanding the assembly and function of the gammaTuRC. By both functional and biochemical criteria, the reconstituted gammaTuSC is identical to the endogenous gammaTuSC. In addition to its nucleating activity, the gammaTuSC can also bind and cap the minus ends of MTs. This observation is consistent with the finding that the monomeric gamma-tubulin and gammaTuRC can also bind and cap the minus ends of MTs. Compared with gammaTuRC, gammaTuSC is a weak MT nucleator. Consistent with its weak nucleating activity, the capping activity of gammaTuSC is significantly less than that of the gammaTuRC. For example, ~50% of MTs nucleated in the presence of gammaTuRC were capped, while under the same assay conditions, gammaTuSC could only cap up to 20% of MTs. One explanation for this weak nucleating and capping activity of gammaTuSC is that gammaTuSC contains only two gamma-tubulin molecules, while the intact gammaTuRC contains ~12 gamma-tubulin molecules (Gunawardane, 2000).

The existence of the conserved sequence motifs in all five of the grips suggests that gammaTuRC assembly may be mediated by conserved structural surfaces defined by these motifs. A provocative idea is that grip motif 2, which is present in all five grips, is involved in interacting with a common protein in the gammaTuRC (e.g., gamma-tubulin). Consistent with these ideas, gamma-tubulin coimmunoprecipitates with each of the five Dgrips when coexpressed in pairs. Furthermore, using similar methods, it has been found that the Dgrips also interact with each other. It will be important to study the nature of these interactions and test whether they are mediated by the grip motifs (Gunawardane, 2000).

Alternatively, the grip motifs could be involved in binding the gammaTuRC to its centrosomal docking site. Using in vitro centrosome assembly assays in Xenopus egg extracts, it has been shown that the removal of Xgrip210 (Dgrip163 homolog) blocks the localization of Xgrip109 (Dgrip91 homolog) to the centrosome and vice versa. This suggests that the grip motifs present in Xgrips109 and 210 are not sufficient for the binding of individual grips to the centrosome. Therefore, it is unlikely that the individual grip motifs per se are sufficient to mediate the recruitment and binding to the centrosomes. Instead, a certain combination of the grip motifs may be required for centrosomal docking to take place, and such a structural surface may only occur in the intact gammaTuRC (Gunawardane, 2000).

Based on the structural features of the gammaTuRC, four possible models are proposed for gammaTuRC assembly. In the first model, the cap structure of the gammaTuRC acts as a scaffold onto which multiple gammaTuSCs assemble to form a ring. In this assembly pathway, the formation of the ring requires preassembly of the cap structure. In the second model, multiple gammaTuSCs oligomerize and individual cap subunits add onto this oligomer to form the gammaTuRC. In this model, prior assembly of a cap structure is not required and gammaTuSC polymerization drives the assembly process. The third model features the preassembly of both a cap structure and gammaTuSC oligomers. In this model, the gammaTuSC oligomers are stabilized by the preformed cap structure to form a gammaTuRC. Finally, the fourth model predicts that the gammaTuRC is assembled sequentially from several distinct intermediates (Gunawardane, 2000).

The majority of the reconstituted and purified gammaTuSC migrates as a 10 S complex on sucrose gradients. However, a small fraction of the gammaTuSC appears to oligomerize and migrate faster than the 10 S complex. This observation suggests that oligomerization of gammaTuSC could contribute toward gammaTuRC assembly. This success in gammaTuSC reconstitution should allow the further testing of conditions that promote gammaTuSC oligomerization and aid the study of gammaTuRC assembly (Gunawardane, 2000).

Although coexpressing Dgrips128 and 163 with gammaTuSC does not promote gammaTuSC oligomerization or gammaTuRC assembly, both these proteins can interact with gammaTuSC independent of each other. These interactions may give rise to the assembly intermediates, as suggested by the sequential pathway of gammaTuRC assembly. In addition, in vitro assays using Xenopus egg extract showed that the Dgrip163 homolog Xgrip210 is essential for gammaTuRC assembly. Based on these observations, it is suggested that Dgrips128 and 163 are essential but not sufficient for gammaTuRC formation. If all gammaTuRC subunits are needed for its assembly, the identification and expression of Dgrip75s (the remaining subunits of gammaTuRC) should permit the reconstitution of the gammaTuRC and the testing of the various models for gammaTuRC assembly (Gunawardane, 2000).

The gamma-tubulin ring complex (gammaTuRC), purified from the cytoplasm of vertebrate and invertebrate cells, is a microtubule nucleator in vitro. Structural studies have shown that gammaTuRC is a structure shaped like a lock-washer and topped with a cap. Microtubules are thought to nucleate from the uncapped side of the gammaTuRC. Consequently, the cap structure of the gammaTuRC is distal to the base of the microtubules, giving the end of the microtubule the shape of a pointed cap. The cloning and characterization of a new subunit of Xenopus gammaTuRC, Xgrip210, is reported. Xgrip210 is a conserved centrosomal protein that is essential for the formation of gammaTuRC. Using immunogold labeling, it has been found that Xgrip210 is localized to the ends of microtubules nucleated by the gammaTuRC and that its localization is more distal, toward the tip of the gammaTuRC-cap structure, than that of gamma-tubulin. Immunodepletion of Xgrip210 blocks not only the assembly of the gammaTuRC, but also the recruitment of gamma-tubulin and its interacting protein, Xgrip109, to the centrosome. These results suggest that Xgrip210 is a component of the gammaTuRC cap structure that is required for the assembly of the gammaTuRC (Zhang, 2000).

Polar organization of gamma-tubulin in acentriolar mitotic spindles of Drosophila melanogaster cells

The spindle pole localization of gamma-tubulin was compared in wild type and acentriolar cultured Drosophila cells using polyclonal antibodies specifically raised against the carboxy terminal amino acid sequence of Drosophila gamma-tubulin-1. During interphase, gamma-tubulin is present in the centrosome of wild type cells and accumulates around this organelle in a cell cycle dependent manner. In contrast, no such structure is observed in acentriolar cells. Wild type mitoses are homogeneously composed of biconical spindles, with two centrosome-associated gamma-tubulin spots at the poles. The mitotic apparatuses observed in the acentriolar cells are heterogeneous; multipolar mitoses, bipolar mitoses with a barrel-shaped spindle and bipolar mitoses with biconical spindles were observed. In acentriolar cells, gamma-tubulin accumulation at mitotic poles is dependent on spindle microtubule integrity. Most acentriolar spindles present a dispersed gamma-tubulin labeling at the poles. Only well polarized and biconical acentriolar spindles show a strong gamma-tubulin polar spot. Finally, acentriolar mitotic poles are not organized around true centrosomes. In contrast to wild type cells, in acentriolar cells the Bx63 centrosome-associated antigen is absent and the gamma-tubulin containing material dispersed readily following microtubule disassembly. These observations confirm that gamma-tubulin plays an essential role in the nucleation of microtubules even in the absence of mitotic polar organelles. In addition the data suggest that the mechanisms involved in the bipolarization of wild type and acentriolar mitoses are different, and that centrioles play a role in the spatial organization of the nucleating material containing gamma-tubulin (Debec, 1995).

Protein phosphatase 4 is an essential enzyme required for organisation of microtubules at centrosomes in Drosophila embryos

The protein serine/threonine phosphatase 4 (PP4), which localises to centrosomes/spindle pole bodies in human cells, is shown to exhibit a similar localization in Drosophila cells and embryos and possess a highly conserved (from humans to invertebrates) amino acid sequence (91% identical). A homozygous Drosophila melanogaster strain mutant in the PP4 gene at 19C1-2 has been produced using P element mutagenesis. This strain, termed centrosomes minus microtubules (cmm), has reduced amounts of PP4 mRNA (~25% of normal PP4 protein in early embryos) and exhibits a semi-lethal phenotype with only 10% viability under certain conditions. Reversion mutagenesis shows that the phenotype is due to the presence of the P element in the PP4 mRNA. In early cmm embryos, nuclear divisions become asynchronous and large regions containing centrosomes with no well defined radiating microtubules are visible. In such areas, most nuclei arrest during mitosis with condensed DNA, and mitotic spindle microtubules are either absent, or aberrant and unconnected to the centrosome. A reduction in the staining of gamma-tubulin at centrosomes in cmm embryos suggests a conformational change or relocation of this protein, which is known to be essential for initiation of microtubule growth. The total level of gamma-Tubulin is identical in cmm and wild-type embryo extracts. Therefore it appears that there must be a relocation or conformational change of gamma-Tubulin in cmm embryos. To confirm that centrosomes are still present in severely abnormal embryos, the localization of CP190 was examined and found to be similar in the wild-type and cmm embryo, even when gamma-Tubulin staining was very weak in the latter. These findings indicate that PP4 is required for nucleation, growth and/or stabilization of microtubules at centrosomes/spindle pole bodies (Helps, 1998).

Mutation of a Drosophila gamma tubulin ring complex subunit encoded by discs degenerate-4 differentially disrupts centrosomal protein localization

The Drosophila gene discs degenerate-4 (dd4) has been cloned and found to encode a component of the gamma-tubulin ring complex (gammaTuRC) homologous to Spc98 of budding yeast. This provides the first opportunity to study decreased function of a member of the gamma-tubulin ring complex, other than gamma-tubulin itself, in a metazoan cell. gamma-tubulin is no longer found at the centrosomes but is dispersed throughout dd4 cells and yet bipolar metaphase spindles do form, although these have a dramatically decreased density of microtubules. Centrosomin (CNN) remains in broad discrete bodies but only at the focused poles of such spindles, whereas Asp (abnormal spindle protein) is always present at the presumptive minus ends of microtubules, whether or not they are focused. This is consistent with the proposed role of Asp in coordinating the nucleation of mitotic microtubule organizing centers. The centrosome associated protein CP190 is partially lost from the spindle poles in dd4 cells supporting a weak interaction with gamma-tubulin, and the displaced protein accumulates in the vicinity of chromosomes. Electron microscopy indicates not only that the poles of dd4 cells have irregular amounts of pericentriolar material, but also that they can have abnormal centrioles. In six dd4 cells subjected to serial sectioning, centrioles were missing from one of the two poles. This suggests that in addition to its role in nucleating cytoplasmic and spindle microtubules, the gammaTuRC is also essential to the structure of centrioles and the separation of centrosomes (Barbosa, 2000).

The major microtubule organizing center (MTOC) in animal cells is the centrosome, which nucleates the slowly growing minus ends of microtubules allowing the plus ends to extend into the cytoplasm. In most animal cells, centrosomes are essential for definition of the interphase MT arrays, for determination of cell polarity, and for the formation and function of the spindle in mitosis. There are two main components of the centrosome: a pair of centrioles comprising cylinders of nine triplet microtubules and the pericentriolar material (PCM) that appears to provide nucleation centers for cytoplasmic and spindle microtubules. Little is known about the organization of the PCM, although both pericentrin and gamma-tubulin have been described as forming a protein complex organized into a lattice like structure. gamma-tubulin is a conserved member of the tubulin family found at MTOCs, including animal cell centrosomes, and the equivalent organelles of yeasts, the spindle pole bodies (SPBs). The gamma-tubulin of S. cerevisiae forms a 6S complex with Spc98p and Spc97p, associated with both the inner and outer plaques of the SPB. Temperature sensitive mutants of its structural gene, tub4, show defects in microtubule nucleation at the newly formed SPB as well as in the assembly of a mitotic spindle. The SPC98 gene was identified as a dosage-dependent suppressor of the tub4-1(ts) allele. Its gene product appears essential for mitotic spindle formation because cells harboring the temperature sensitive allele spc98-1 or over expressing wild-type protein, duplicate and separate their SPBs but form a defective mitotic spindle. The gene encoding the other main component of this complex, SPC97, was isolated as a suppressor of the spc9-2(ts) mutant. Its temperature sensitive alleles show phenotypes similar to tub4 and spc98 mutants as well as defects in SPB duplication. Spc98p docks the Tub4p complex to the inner plaque of the SPB through the N terminus of Spc110p, a protein that forms a bridge between the inner and central plaques. The Tub4 complex is formed in the cytoplasm. It is transported to the nucleus via the nuclear localization signal (NLS) present in Spc98p. Spc98p at the inner plaque of the SPB is phosphorylated in a cell cycle-dependent manner, whereas Spc98p in the outer plaque does not appear to undergo such modification (Barbosa, 2000).

In higher eukaryotes gamma-tubulin occurs in a 25S-32S complex that has been shown by electron microscopy to have a ring shape leading to the name gamma-tubulin ring complex (gammaTuRC). The Xenopus complex comprises seven proteins: alpha-, beta-, and gamma-tubulin, and additional proteins of 75, 109, 133, and 195 kD. The 109 kD Xenopus protein (Xgrip109) is a homolog of yeast Spc98p. It interacts directly with gamma-tubulin and is essential for microtubule nucleation. The major human and Drosophila gammaTuRCs have very similar protein profiles. The 100 and 101 kD human proteins hGCP2 and hGCP3 correspond to Spc97p and Spc98p, respectively. In Drosophila, a second smaller 240 kD gamma-tubulin complex has been described comprising only gamma-tubulin and the Spc97/98 homolog Dgrip84 and Dgrip91. It is proposed that this is assembled into the complete 3 MDa gamma-TuRC, which contains multiple copies of the heterotrimer plus ancillary proteins (Barbosa, 2000 and references therein).

A model of the gammaTuRC in Drosophila suggests that the complex is assembled in the cytoplasm from the heterotrimer subunits and recruited onto the centrosome, where it nucleates microtubules. In mitosis it functions in concert with Asp, or a protein with equivalent function in other organisms, to organize the spindle microtubules (do Carmo Avides, 1999). This implies that the centrosome might be essential for MT nucleation and therefore for the formation of spindles. However, in the female meiotic divisions of Drosophila the spindles form in the absence of centrioles and without detectable concentration of gamma-tubulin at the poles. Moreover, loss of centrosomes has been observed from the spindle poles in the syncytial embryos of several mutants, and although these generally lead to the accumulation of mitotic defects, several rounds of mitosis can take place on such spindles. These observations, together with the ability to build spindles without centrosomes in vitro that are able to undertake metaphase and anaphase, has been taken to mean that centrosomes might be dispensable in the formation of a functional spindle in some systems. Recent observations that a functional spindle can still form in mammalian cells after laser ablation of the centrosomes now reinforce this idea (Barbosa, 2000 and references therein).

Because dd4 encodes a component of the gamma-tubulin ring complex, the organization of the mitotic spindle and its poles were examined in cells from the central nervous system of dd4 mutants. The localization of gamma-tubulin was examined in relation to centrosomin (CNN), another component of the Drosophila centrosome. In mitotic cells from wild-type brains, these two proteins colocalize to the two centrosomes. In cells of all three dd4 mutant alleles, gamma-tubulin staining can still be detected; however, it is no longer found in a well-defined body but rather is distributed throughout the cell. In contrast, distinct CNN-containing bodies can be seen in every mitotic cell. However, whereas wild-type cells invariably contained two such bodies (the functional centrosomes), some mutant cells contained only one body stained with CNN, while others contained two (Barbosa, 2000).

In cells stained to reveal the spindle microtubules, the CNN-containing bodies always appear to be associated with a microtubule organizing center. In wild-type cells, these are spherical centrosomes at the spindle poles and astral microtubules. In the dd4 mutant cells, the CNN staining bodies are often less tightly defined structures and astral microtubules are not seen. In some cells the CNN containing body appears to have fragmented, and a satellite body can be seen near the main pole. In those cells having only one CNN-staining body, prominent arrays of microtubules extend between this pole and the chromosomes. Conversely, microtubules extending from the chromosomes to the pole lacking the CNN body exhibit reduced staining, and in some cells this pole appears not to have organized microtubules (Barbosa, 2000).

In addition to the gamma-tubulin ring complex, the Asp protein is also known to be required to nucleate asters of microtubules (do Carmo Avides, 1999). In wild-type mitotic cells, the Asp protein is found on the face of the centrosome that makes contact with spindle microtubules. This close juxtaposition of gamma-tubulin and Asp is no longer seen in dd4cells in which the gamma-tubulin is dispersed, but Asp maintains a punctate distribution. This punctate staining can be clustered around the spindle poles or clustered in one area and scattered throughout the remaining part of the cell, but is never diffuse as is gamma-tubulin. Immunostaining to reveal microtubules shows that Asp protein is always found at the poles of bipolar spindles, either as a well organized body, but more usually in clustered aggregates. In spindles that had only one focused pole, individual bundles of the microtubules could be seen to extend both from this focus and from small punctate caps of Asp at the unfocused pole towards the chromosomes (Barbosa, 2000).

CP190 is an abundant protein which, together with its partner CP60, associates with mitotic centrosomes in Drosophila cells. Although frequently used as a marker to follow centrosome behavior, its function remains unknown. In wild-type cells, CP190 is found associated with the centrosomes at the spindle pole. In the weakest allele, dd4S, some of the antigen is lost from the poles and appears in the central part of the spindle in the region occupied by the mitotic chromosomes. In the stronger alleles, only a small proportion of CP190 remains at the poles; the remainder is clustered around the chromosomes. Thus, intact gamma-tubulin ring complex appears to be required at the poles in order to maintain the polar association of the CP190 protein (Barbosa, 2000).

In addition to being present in the PCM, gamma-tubulin is also found within the centrioles themselves and appears to be required for centriolar function. To determine whether dd4 mutants showed any irregularity of centriolar structure, the ultrastructure of centrosomes was examined by electron microscopy of serial sections of cells from the larval central nervous system. These data consist of complete sets of serial sections through some four wild-type prometaphase cells with chromosomes undergoing congression, one wild-type cell at metaphase, and six dd41mutant cells that are in a metaphase-like state. The centrosomes have well defined centrioles showing typical arrays of triplet microtubules surrounded by the electron-dense PCM. Arrays of spindle microtubules extend from these centrosomes towards condensed chromosomes. These microtubules occur in bundles that make contact with well defined kinetochore plates on the chromosomes. All of the dd4 cells examined had two opposing poles, generally broad, but secondary poles were often present. Centrioles were present only at one of the two main poles in all six of the cells, but there were up to four of them; they varied in size and were not consistently arranged as perpendicular pairs. The amount of PCM varied from one pole to another showing no correlation with the pattern of centrioles. The density of spindle microtubules in dd4 cells is strikingly reduced compared to the wild-type spindle, consistent with the impression gained from immunostaining. The spindle microtubules make poor contact with the mitotic chromosomes and, in contrast to wild-type cells, it was difficult to see organized plate-like kinetochores on dd4 chromosomes (Barbosa, 2000).

It is of interest to compare phenotypes of mutations in dd4 with mutations in the gamma-tubulin genes. Drosophila has two genes for gamma-tubulin; the one at 23C is expressed in a variety of tissues including brains, imaginal discs and testes, whereas expression of the second at 37C is restricted to ovaries and embryos. Like the dd4 mutants, cells from gamma-tub23C brains display abnormally high levels of chromosome condensation, spindles with defective or absent poles, and polyploidy. However, whereas the mitotic index of dd4 cells is dramatically elevated, the mitotic index of gamma-tub23C cells is reduced relative to wild-type and anaphase figures are very rare. The reasons for these differences are not clear. It is as though reduction in levels of the 23C gamma-tubulin lead to a limited numbers of chromosome duplication cycles in the absence of mitosis, but this is followed by interphase cell cycle arrest. In contrast, the dramatic increase in metaphase figures in dd4 mutants resembles a more typical spindle checkpoint arrest. The frequency of anaphases, not dissimilar in total number to wild type, suggests that cells can evade this checkpoint at some frequency, as has been described in several organisms. The phenotypes of various allelic combinations suggest that the strongest allele, dd41, is not completely amorphic by genetic criteria. Western blotting indicates that there is 80%-90% reduction of the Dgrip91 protein in this allele. Although it is uncertain whether this residual protein has any function, this and the genetic observations suggest there may be some residual function of the gamma-TuRC even in the dd41 mutant (Barbosa, 2000).

It is generally thought that the late survival of larvae with extreme mitotic defects reflects perdurance of maternal contribution to the oocyte from the heterozygous mother, as has been shown for other cell-cycle genes. In the case of dd4, this assumption has been challenged by the report of normal oocytes from homozygous dd41 mitotic ovarian clones arising in heterozygous females. However, preliminary observations of embryonic development in eggs produced from allelic combinations weak enough to give viable escaper females (dd4S/dd4S and dd4S/dd43) do indicate that there is a vital maternal contribution to the oocyte: such eggs appear to have parental DNA but they fail to undergo any development. These observations suggest that if mothers carrying weak enough allelic combinations to be compatible with survival to adulthood cannot build a viable egg, then either the observations of no maternal effect are in error, or the observed clones have sufficient perdurance of the wild-type product to build eggs indistinguishable from normal heterozygotes (Barbosa, 2000).

Together, Dgrip84, Dgrip91, and gamma-tubulin form the three major components of the gamma-TuRC and are homologous to the budding yeast proteins Spc97, Spc98, and Tub4. Genetic and molecular studies show interactions between these genes in budding yeast, and their requirement for SPB structure, duplication, and separation. Interactions between members of this complex and other components of the SPB and spindle are only beginning to be understood. Spc98, for example, binds to the N-terminal region of Spc110p, a coiled-coil protein that spans the inner and central plaques of the SPB. The calmodulin binding C terminus of this protein contacts the central plaque and the N-terminal region, the inner plaque. Thus Spc98 might form an essential link between Spc110 and the spindle microtubules that emanate from the inner plaque and the defective spindle structures seen in spc98 mutants may be a direct consequence of defects in this interaction. The phenotypes of spc98 mutants thus have some parallels with dd4 mutants in abnormal spindle microtubule density and organization, and it will be of interest to determine whether Dgrip91 has similar interactions with specific components of the centrosome (Barbosa, 2000).

The more drastic disruption of purified preparations of centrosomes with the salt KI in vitro removes a set of proteins, including the gamma-TuRC, CP60, CP190, CNN, and Asp, thus destroying their ability to organize microtubules. The salt treatment appears to leave behind unidentified core centrosomal components, since the structure of the PCM is changed very little when examined by electron microscopy. In contrast to salt extraction, reduction of functional Dgrip91 has a differential effect upon the loss of centrosomal antigens. CNN remains in distinct bodies at most of the well focused poles, indicating that its centrosomal association is not dependent upon the presence of the gamma-TuRC. The defects of centrosomin(cnn) mutants have been characterized for a number of alleles that show maternal effects and male sterility. These indicate that its function is required for the integrity of both centrosomal and centriolar structures. Syncytial embryos derived from centrosomin mutant mothers undertake up to 12 rounds of mitosis upon spindles whose poles have no astral microtubules and have very little or none of the centrosomal proteins CP60, CP190, or gamma-tubulin. Together, these data imply that CNN appears to be more important in holding the structure of the centrosome together than does the gamma-TuRC, and this is perhaps to be expected from the predicted coiled-coil nature of CNN (Barbosa, 2000).

It is clear that mitotic spindles can form and function in the absence of centrosomes. Repeated rounds of mitosis are known to take place in the absence of centrosomes in the unfertilized eggs of Sciara flies. Moreover, in Drosophila eggs derived from polo mothers, the four products of female meiosis are capable of undergoing many rounds of mitosis on acentriolar spindles. These spindles strongly resemble the meiotic spindles of female Drosophila in which gamma-tubulin cannot be detected by immunostaining at these spindle poles, even though it is apparently needed for spindle function. The ability to build a functional spindle in Xenopus extracts in the absence of centrosomes is also well documented and requires minus end directed motors such as dynein to focus the poles. The consequences of removing centrosomes from cells that have robust checkpoints to monitor spindle assembly can vary, and could reflect either or both the cell line studied and exactly how the experiment was performed. Microsurgical removal of centrosomes has been reported to block future cycles of cell division. However, laser directed ablation of either one or both centrosomes does not prevent assembly of spindles that could successfully undertake anaphase. The high mitotic index resulting from partial disruption of the centrosome in dd4 mutants suggests a mitotic delay likely to result from activation of the spindle integrity checkpoint known to be functional in larval brain cells (Barbosa, 2000).

The distribution of the Asp following the apparent breakdown of the gamma-TuRC gives insight into how these proteins might cooperate in microtubule nucleation. It is known that, following KI depletion of centrosomes, their ability to organize asters of microtubules can only be restored by supplying a complementary cytoplasmic extract that contains both the gamma-TuRC and functional Asp protein. In wild-type cells, Asp forms a hemispherical cup-like structure on the face of the spindle microtubules suggesting that it is contacting the minus ends of these, and not the astral microtubules. Astral microtubules are not seen in dd4 mutant cells at either the light or EM levels, and the spindle poles exhibit varying degrees of disorganization. Nevertheless, the Asp protein is invariably present at the spindle poles even in those extreme cases where individual bundles of microtubules are no longer held together at a single poorly focused pole. In such cases Asp appears at the very tips of these tubules as if it is providing some capping property to their minus ends (Barbosa, 2000).

It is difficult to compare the effects of gamma-tub23C and dd4 mutations upon the structure of the centrosome itself. Nevertheless, although the centrosome had abnormal morphology judged by the distribution of CP190 (Bx63 antigen) in the gamma-tub23C mutant, the antigen was only noted as being at pole-like structures. Unfortunately, there are currently no known mutants of the CP190 gene, and its function remains unknown. CP190 exists in a complex with CP60, and both proteins are known to be nuclear during interphase and move onto centrosomes at mitosis. The extent of interaction between these proteins and gamma-tubulin is also unclear. Two complexes containing gamma-tubulin have been purified from Drosophila embryos; the 3 MD gamma-TuRC itself, and a smaller complex of 240 kD that appears to be a subunit of the larger one. The CP190-CP60 complex does not appear to be present in either of these gamma-tubulin complexes from which it was separable by gel-filtration. However, low levels of gamma-tubulin can be detected in the eluate from immunoaffinity columns constructed from antibodies to CP190 and CP60. This has led to the speculation that although these proteins may not assemble with each other in stoichiometric ratios, they may still show interactions, either on an affinity column in vitro or during centrosome assembly. Consistent with this is the observation that following loss of the majority of the gamma-TuRC from the centrosome in dd41, some CP190 remains in the centrosome, whereas some dissociates and clusters in punctate arrays in the region of the spindle occupied by the condensed chromosomes. In this sense CP190 may be obeying elements of a nuclear localization signal that directs its interphase location, the nuclear envelope undergoing incomplete breakdown during mitosis in Drosophila to form a fenestrated envelope around the spindle (Barbosa, 2000).

Despite the differences in fixation procedures, several aspects of the ultrastructure of the mitotic apparatus in dd4 cells as seen by electron microscopy, such as the microtubule density, are concordant with observations by immunofluorescence. Chromosomes are abnormally condensed and the number and density of spindle microtubules is greatly reduced in the mutant cells. The dispersion of the gamma-tubulin, which is assumed to be the primary consequence of the dd4 mutations, is reflected by disorganization of the PCM and altered centriole morphology. Some gamma-tubulin has been shown to be localized to the core of the centriole, and inactivation of the gamma-tubulin gene in Paramecium leads to inhibition of the duplication of the related structures, the basal bodies. The finding of fewer than four centrioles in the serial EM sections of some dd4 mutant cells suggests a failure of centriole duplication. However, the failure to find centrioles at one of the poles in six dd4 mutant cells suggests that centrosome separation is also dependent upon a functional gamma-TuRC. This may be related to a function in correctly holding centrioles together, because mother and daughter centrioles are rarely perpendicular. The extent to which other centrosomal components found principally in the PCM can contribute to structure of the centriole is not clear. Nonetheless, it is interesting in this context that an isoform of CNN expressed during spermatogenesis is localized both to the centrosomes and to the basal body and has been shown by mutational analysis to be required for the organization of the flagellar axoneme that develops from the spermatid basal body (Barbosa, 2000).

Drosophila Aurora-A is required for centrosome maturation and actin-dependent asymmetric protein localization during mitosis

During asymmetric cell division in the Drosophila nervous system, Numb segregates into one of two daughter cells where it is required for the establishment of the correct cell fate. Numb is uniformly cortical in interphase, but in late prophase, the protein concentrates in the cortical area overlying one of two centrosomes in an actin/myosin-dependent manner. What triggers the asymmetric localization of Numb at the onset of mitosis is unclear. The mitotic kinase Aurora-A is required for the asymmetric localization of Numb. In Drosophila sensory organ precursor (SOP) cells mutant for the aurora-A allele aurA37, Numb is uniformly localized around the cell cortex during mitosis and segregates into both daughter cells, leading to cell fate transformations in the SOP lineage. aurA37 mutant cells also fail to recruit Centrosomin (Cnn) and gamma-Tubulin to centrosomes during mitosis, leading to spindle morphology defects. However, Numb still localizes asymmetrically in cnn mutants or after disruption of microtubules, indicating that there are two independent functions for Aurora-A in centrosome maturation and asymmetric protein localization during mitosis. Using photobleaching of a GFP-Aurora fusion protein, it has been shown that two rapidly exchanging pools of Aurora-A are present in the cytoplasm and at the centrosome and might carry out these two functions. These results suggest that activation of the Aurora-A kinase at the onset of mitosis is required for the actin-dependent asymmetric localization of Numb. Aurora-A is also involved in centrosome maturation and spindle assembly, indicating that it regulates both actin- and microtubule-dependent processes in mitotic cells (Berdnik, 2002).

A mitotic function for Drosophila Aurora-A has been described before. In strong alleles of aurora-A, centrosomes fail to separate, leading to the generation of abnormal monopolar mitotic spindles, defects in chromosome segregation, and the formation of polyploid cells. aurA37 mutant cells, in contrast, complete mitosis and divide into two daughter cells and can therefore be used to characterize other aspects of Aurora-A function. To analyze mitotic spindles in aurA37 mutants, control and mutant pupae were stained for DNA and alpha-Tubulin. Bipolar mitotic spindles are formed in aurA37 mutants, but while microtubule minus ends converge on the centrosome in wild-type, they are less focused in aurA37 mutants. This spindle morphology phenotype could reflect a defect in centrosome function, and therefore aurA37 mutants were stained for the centrosomal marker gamma-Tubulin. In control SOP cells, gamma-Tubulin staining is weak during interphase, but two strong dots appear during mitosis, indicating that gamma-Tubulin is recruited to centrosomes. In 67% of the aurA37 mutant mitotic SOP cells, no strong dots of gamma-Tubulin staining were visible, indicating a failure to recruit the protein to centrosomes. This defect is not completely penetrant, since one dot was observed in 17% of the cells, and, in another 17%, two closely spaced dots were seen. Defects in mitotic recruitment of gamma-Tubulin have been described before in flies mutant for cnn, a centrosomal core component that is dispersed in interphase but localized to centrosomes during mitosis. aurA37 mutant pupae were therefore double stained for Cnn and gamma-Tubulin. In contrast to wild-type, where Cnn is detected in two strong dots at either spindle pole, the protein is undetectable on centrosomes of most aurA37 mutant SOP cells. Thus, Aurora-A is required for recruiting both gamma-Tubulin and Cnn to centrosomes during mitosis, and these defects in centrosome maturation might be the cause of the abnormal spindle morphology. Despite the spindle defects in aurA37 mutants, however, the two daughter cells of SOPs are still preferentially arranged along the anterior-posterior axis, indicating that spindle orientation is unaffected (Berdnik, 2002).

Cnn has been shown to be required for localization of gamma-Tubulin to centrosomes during mitosis. The defects in gamma-Tubulin localization in aurA37 mutants could therefore be an indirect consequence of the failure to recruit Cnn. To test whether the defects in Numb localization are also caused by the failure to recruit Cnn, cnn null mutant Drosophila pupae were stained for Numb, gamma-Tubulin, and DNA. Even though no Cnn protein could be detected, these flies are viable. As in wild-type, Numb localizes into a cortical crescent during late prophase in all cnn mutant SOP cells even though, in 9% of the mitotic SOP cells (n = 22), the Numb crescent is mispositioned and does not correlate with the orientation of the metaphase plate. Since gamma-Tubulin is not recruited to centrosomes in these mutants, it is concluded that neither Cnn nor gamma-Tubulin recruitment to mitotic centrosomes is required for the asymmetric localization of Numb (Berdnik, 2002).

The failure to localize Numb asymmetrically in aurA37 mutants could still be caused by the spindle defects. In neuroblasts, Numb localization still occurs after complete disruption of the mitotic spindle. To test the requirement of a mitotic spindle for Numb localization in SOP cells, wild-type pupae were incubated in 20 µg/ml colcemid for 1 or 2 hr and stained for Numb and gamma-Tubulin. After 1 hr of treatment, on average, nine SOP cells per pupal notum showed the mitotic arrest phenotype typical of microtubule inhibitors: chromosomes were no longer aligned in the metaphase plate, and centrosomes were distributed at random positions. Numb was still asymmetrically localized in 78% of these colcemid-arrested SOP cells. After 2 hr of treatment, the average number of metaphase-arrested SOP cells per notum increased to 27, and Numb was asymmetrically localized in 81% of them, indicating that new Numb crescents can be formed in the absence of a functional mitotic spindle. No effect on centrosome position was observed in a control experiment in which colcemid was omitted and Numb crescents were observed in 71% of the mitotic SOP cells. Thus, neither a functional mitotic spindle nor recruitment of Centrosomin and gamma-Tubulin to centrosomes are required for asymmetric localization of Numb. It is concluded that the defects in Numb localization observed in aurA37 mutants are not indirect consequences of the spindle or centrosome defects. Rather, they indicate an independent role for Aurora-A in asymmetric protein localization during mitosis (Berdnik, 2002).

Characterization of a new gammaTuRC subunit with WD repeats

The γ-tubulin ring complex (γTuRC), consisting of multiple protein subunits, can nucleate microtubule assembly. Although many subunits of the γTuRC have been identified, a complete set remains to be defined in any organism. In addition, how the subunits interact with each other to assemble into γTuRC remains largely unknown. This study reports the characterization of a novel γTuRC subunit, Drosophila gamma ring protein with WD repeats (Dgp71WD). With the exception of γ-tubulin, Dgp71WD is the only γTuRC component identified to date that does not contain the grip motifs, which are signature sequences conserved in γTuRC components. By performing immunoprecipitations after pair-wise coexpression in Sf9 cells, it was shown that Dgp71WD directly interacts with the grip motif-containing γTuRC subunits, Dgrips84, 91, 128, and 163, suggesting that Dgp71WD may play a scaffolding role in γTuRC organization. Dgrips128 and 163, like Dgrips84 and 91, can interact directly with γ-tubulin. Coexpression of any of these grip motif-containing proteins with γ-tubulin promotes γ-tubulin binding to guanine nucleotide. In contrast, in the same assay Dgp71WD interacts with γ-tubulin but does not facilitate nucleotide binding (Gunawardane, 2003).

The Drosophila γTuRC consists of ~8 polypeptides of which γ-tubulin and Dgrips75 (76p), 84, 91, 128, and 163 have been identified and characterized. Interestingly, all of these Dgrips contain conserved grip motifs. On the basis of the protein profile of the purified Drosophila γTuRC, it is estimated that two to three additional Dgrips with apparent molecular masses of ~75 kDa remain to be cloned and characterized. Through database searches, it was found that several peptide sequences that were obtained by microsequencing of the purified γTuRC matched a Drosophila partial EST. The full-length cDNA was cloned and found to contained five WD repeats. Therefore, this novel protein is referred to as Dgp71WD, which stands for Drosophila gamma ring protein of 71 kDa with WD repeats (Dgp71WD, accession number AF461267). Interestingly, Dgp71WD did not contain the grip motifs found in all of the other Dgrips identified thus far. Database searches revealed a large number of proteins from various organisms sharing sequence homology with the WD-repeat regions of Dgp71WD. The alignment the five WD repeats in Dgp71WD align with the WD repeats of the Arabidopsis photomorphogenesis repressor COP1 and the mouse Nedd1. Interestingly, the presently uncharacterized mouse Nedd1 is similar in size to Dgp71WD and both proteins have the WD repeats at their N-termini. Pair-wise alignment showed that the overall amino acid identity between Nedd1 and Dgp71WD is 20.8% and that the homology between the two proteins extends beyond the WD repeats (Gunawardane, 2003).

To determine if Dgp71WD is a γTuRC subunit, rabbit polyclonal antibodies against the C-terminal peptide of γ-tubulin (DrosC) or against a peptide corresponding to amino acids 399-417 of Dgp71WD were used to immunoprecipitate Dgp71WD from Drosophila embryo extract. Anti-Dgp71WD antibody was found to immunoprecipitate the same set of γTuRC subunits as does the anti-γ-tubulin antibody, suggesting that Dgp71WD is a subunit of the γTuRC. Furthermore, the antibody against Dgp71WD recognizes a protein of ~71 kDa in γTuRC immunoprecipitated with γ-tubulin antibodies. In addition, when Drosophila embryo extracts are subjected to sucrose gradient sedimentation, Dgp71WD comigrate with the other γTuRC subunits. Because the previously characterized γTuRC subunits localize to the centrosome throughout the cell cycle, whether Dgp71WD shows a similar localization pattern was tested. Dgp71WD was found to colocalize with γ-tubulin at centrosomes in Drosophila embryos during interphase and mitosis. Taken together, these results showed that Dgp71WD is a subunit of the Drosophila γTuRC (Gunawardane, 2003).

Because Dgp71WD contains WD repeats and no grip motifs, it was reasoned that it might play a scaffolding role by binding to the grip motif-containing subunits. If this is true, Dgrips84, 91, 128, and 163 should directly interact with Dgp71WD. To test this possibility, Dgp71WD was coexpressed with each of the Flag-tagged Dgrips84, 91, 163, or untagged Dgrip128, in Sf9 cells. Specific antibodies that recognize Dgp71WD, Dgrips84, 91, 128, 163, or Flag antibody were used to immunoprecipitate each of the proteins. The immunoprecipitates were first separated by SDS-PAGE and the proteins were either stained by Coomassie Blue or probed by Western blotting with specific antibodies. It was found that the Dgp71WD antibody immunoprecipitates Dgrips84, 91, 128, and 163 . Conversely, antibodies that immunoprecipitate each of the Dgrips also immunoprecipitate Dgp71WD. On the basis of these results, it was concluded that Dgp71WD interacts with each of the four grip-motif-containing γTuRC subunits (Gunawardane, 2003).

Next, it was asked whether Dgp71WD also directly interacts with γ-tubulin. Dgp71WD and γ-tubulin were coexpressed in Sf9 cells and antibodies against each of the proteins were used for immunoprecipitation. It was found that although the antibody against γ-tubulin immunoprecipitates Dgp71WD, the antibody against Dgp71WD does not immunoprecipitate γ-tubulin. The lack of reciprocal immunoprecipitation could reflect a lack of interaction between γ-tubulin and Dgp71WD. Alternatively, it is possible that Dgp71WD and γ-tubulin do interact with each other, but the binding of this particular antibody to Dgp71WD disrupts this interaction. The disruption is possible if the Dgp71WD antibody bind to the region of Dgp71WD involved in its binding to γ-tubulin. In contrast, DrosC and Dγ2 antibodies do immunoprecipitate Dgp71WD. On the basis of these results, it is concluded that Dgp71WD and γ-tubulin directly interact with each other (Gunawardane, 2003).

Attempts were made to develop an assay to compare the functional consequences of the binding of Dgp71WD to γ-tubulin with that of the grip motif-containing proteins. Because γ-tubulin in γTuRC and γTuSC can bind to guanine nucleotides, it was reasoned that the nucleotide binding of γ-tubulin could be dependent on its interaction with other γTuRC subunits. Therefore, it was asked whether γ-tubulin expressed alone or together with Dgrip84 or Dgrip91 could bind to GTP. Using a UV cross-linking assay, it was found that γ-tubulin when expressed alone and purified, does not bind to GTP. However, when γ-tubulin is coexpressed with either Dgrip84 or Dgrip91 in Sf9 cells and purified, it is able to bind to GTP in the cross-linking assay. As expected, γ-tubulin in the baculovirus reconstituted γTuSC also binds to GTP. These findings suggest that the interactions between the Dgrip84 and Dgrip91 with γ-tubulin facilitate γ-tubulin binding to GTP (Gunawardane, 2003).

These finding prompted a test of whether Dgp71WD, Dgrip128, and Dgrip163 could also facilitate γ-tubulin binding to GTP. γ-Tubulin was coexpressed with Dgp71WD, Dgrip128, or Dgrip163, isolated, and assayed for GTP binding using the UV cross-linking assay as above. It was found that γ-tubulin binds to GTP when coexpressed with either Dgrip128 or Dgrip163, but not when coexpressed with Dgp71WD. Furthermore, γ-tubulin binds to GTP when coexpressed with all three subunits, suggesting that Dgp71WD does not inhibit GTP binding. These studies suggest that the interaction between γ-tubulin and the grip motif-containing subunits facilitate γ-tubulin binding to GTP, but the interaction between γ-tubulin and Dgp71WD does not. From these studies, it is concluded that the nature of the interaction of Dgp71WD with γ-tubulin differs from that of the grip motif-containing γTuRC subunits (Gunawardane, 2003).

The finding that all Dgrips interact with γ-tubulin directly is intriguing because it suggests that the Dgrips75, 79, 128, and 163 that were originally thought to be the cap subunits, directly contact γ-tubulin in the γTuRC. If this is the case, the current γTuRC model, which hypothesizes that the γTuRC ring consists exclusively of γTuSCs, would need to be revised. It is speculated that each of the Dgrips75, 79, 128, and 163 can form dimers with γ-tubulin molecules. These dimers along with several γTuSCs may be required to form the ring of the γTuRC (Gunawardane, 2003).

Structural studies revealed that WD repeats form a ß-propeller fold that mediate protein-protein interactions. This study suggests that Dgp71WD could provide a scaffold via its WD-repeats to tether all of the Dgrips together in the γTuRC. Consistent with this idea, it was found that four Dgrips (84, 91, 128, and 163) interact directly with Dgp71WD. Clearly, further structural and biochemical studies are necessary to determine whether some or all of the Dgrips75, 79, 128, and 163 participate in the formation of the γTuRC ring. Also, it is important to determine whether and how any of these Dgrips participate in the formation of the cap structure of the γTuRC (Gunawardane, 2003).

Coexpressing any one of the grip motif-containing subunits, Dgrips84, 91, 128, and 163, with γ-tubulin is sufficient to promote γ-tubulin to bind to GTP. However, coexpressing γ-tubulin with Dgp71WD, which does not contain grip motifs, does not facilitate γ-tubulin binding to GTP. This finding shows that the interactions between γ-tubulin and Dgrips have a significantly different consequence from the interaction between γ-tubulin and Dgp71WD. Furthermore, it suggests that the grip motif-containing subunits play a role in regulating the GTP binding properties of γ-tubulin. Because GTP is important for alpha- and ß-tubulin function, it is suspected that Dgrips may be important for γ-tubulin function. Consistent with this idea, it was found that γ-tubulin expressed alone does not incorporate into γTuRC in vitro; but coexpressing one of the Dgrips with γ-tubulin is sufficient for the incorporation (Gunawardane, 2003).

Previously, human and Chlamydomonas γ-tubulins expressed alone using rabbit reticulocyte lysate and baculovirus expression systems, respectively, were used to study the microtubule binding and nucleating activities of γ-tubulin. However, whether the γ-tubulins could bind to guanine nucleotides was not determined. Because it was found that γ-tubulin expressed alone does not bind GTP, caution should be used in analyzing the function of γ-tubulin in the absence of Dgrips (Gunawardane, 2003).

The interaction observed between Dgp71WD and γ-tubulin should also be interpreted cautiously. Because γ-tubulin found in γTuRC and γTuSC can be cross-linked to GTP, the inability of γ-tubulin to bind to GTP when coexpressed with Dgp71WD may indicate that γ-tubulin does not assume the same structural conformation as in the γTuRC. One possibility is that the γ-tubulin coexpressed with Dgp71WD is not folded properly. If so, the interaction between Dgp71WD and γ-tubulin that observed may not reflect a true interaction in the γTuRC. Alternatively, it is possible that the γ-tubulin expressed with Dgp71WD is folded correctly, but assumes a slightly different conformation to disfavor GTP binding in the in vitro assays. If so, the interaction between Dgp71WD and γ-tubulin is likely to reflect a true interaction in the γTuRC. Structural studies of γTuRC will be important to resolve whether γ-tubulin directly contact Dgp71WD in the complex (Gunawardane, 2003).

Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. This study shows that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, Centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of γ-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with γ-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring γ-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN (Terada, 2003).

In animal cells, microtubules are organized from the centrosome/microtubule-organizing center (MTOC), composed of a pair of centrioles and the surrounding pericentriolar material. Individual microtubules are nucleated from an ~25-nm γ-tubulin–containing ring complex (γ-TuRC). At the onset of M phase, the centrosome becomes 'mature' and organizes more microtubules, which is accompanied with an increased level of γ-tubulin accumulation at each spindle pole. One of the molecules that has been implicated in the mechanism of centrosome maturation is Aurora-A, a mitosis-specific Ser/Thr kinase located at mitotic poles and spindle microtubules. The kinase, originally identified as a gene product important in spindle assembly and function in Drosophila, has recently been shown to be in the Ran-signaling pathway and to play an important role in efficient transmission of Ran-GTP gradient established by the condensed chromosomes for the control of spindle assembly and dynamics. Aurora-A binds to spindle components, such as TACC/XMAP215 and TPX2. Although possible functions of those molecules and their interaction with Aurora-A in bipolar spindle formation have been elucidated, mechanisms of how Aurora-A stimulates the recruitment of γ-tubulin to the centrosome at spindle poles have not yet been evaluated. To address this question, centrosomal proteins were sought that interact with Aurora-A and regulate the process of microtubule nucleation onto the centrosome (Terada, 2003).

By screening of a Drosophila two-hybrid library, two clones were isolated encoding a molecule capable of interaction with Aurora-A. The sequence corresponds to the COOH-terminal domain of centrosomin (CNN), a core component of the centrosome important for assembly of mitotic centrosomes in Drosophila. Although the truncated polypeptide covered by clone CNN-C1 appears to be sufficient for interaction with Aurora-A, the binding intensity was weaker than CNN-C. Endogenous Aurora-A, but not Aurora-B, immunoprecipitates with HA-tagged CNN expressed in S2 cells. Specificity of the COOH-terminal domain of CNN for interaction with Aurora-A was further confirmed by in vitro binding assays (Terada, 2003).

To investigate the role of protein interaction in the centrosome, S2 cells were prepared from which Aurora-A or CNN was depleted by RNA interference (RNAi). In cells lacking Aurora-A, not only CNN, but also γ-tubulin, were absent at each spindle pole. When CNN was depleted, neither γ-tubulin nor Aurora-A was seen at the spindle pole. In cells with partially depleted Aurora-A or CNN, comparable amounts of γ-tubulin and CNN or Aurora-A were detected at each pole. Besides γ-tubulin, other centrosome proteins, CP190 (a–c) and CP60 (d–f), become dislocated from the spindle poles in RNAi cells. Therefore, it is concluded that CNN and Aurora-A are mutually dependent for localization at spindle poles, which is required for proper targeting of other centrosomal proteins to the centrosome. This is consistent with previous observations (Barbosa, 2000) that the centrosomal association of CNN is not dependent on the presence of γ-tubulin/γ-TuRC (Terada, 2003).

To confirm the role of CNN in recruiting γ-tubulin, protein interaction was analyzed in vitro. Nickel beads conjugated with His-tagged CNN were mixed with cell extracts prepared from colcemid-treated S2 cells. γ-Tubulin was specifically sedimented by the full and NH2-terminal sequence, but not the COOH-terminal sequence of CNN. Because neither in vitro binding assays nor two-hybrid screens demonstrated direct binding between two molecules, CNN may interact with a γ-tubulin complex, rather than γ-tubulin directly. Further, HA-tagged CNN was expressed in S2 cells. Exogenous proteins caused formation of γ-tubulin–containing cytoplasmic aggregates capable of microtubule formation and association with microtubule asters. These results clearly indicate that the NH2-terminal domain of CNN interacts with γ-tubulin/γ-TuRC and plays an important role in assembly of MTOCs (Terada, 2003).

γ-Tubulin/γ-TuRC–mediated microtubule assembly is believed to be common among species. Thus, it is highly likely that an Aurora-A–binding molecule(s) equivalent to CNN is functioning in a variety of organisms. Although Drosophila CNN was unable to associate with mammalian Aurora-A in transfected mammalian cells as well as by two-hybrid screens, the NH2-terminal domain of CNN still interacts with γ-tubulin/γ-TuRC in mammalian cells as in S2 cells. To analyze a possible role of CNN–γ-tubulin interaction in initiation of microtubule assembly, Drosophila CNN was overexpressed in mammalian cells. HA-tagged CNN induces cytoplasmic foci in various sizes and numbers. Significantly, the pattern of microtubule distribution is profoundly affected as a result of microtubule association with virtually every dot containing CNN. These sites can initiate microtubule formation as evidently shown in cells where short microtubules are assembled during brief recovery from nocodazole treatment. All cells overexpressing CNN induced microtubule-organizing sites, which were associated with centrosome proteins, such as pericentrin and Cep135. Particularly prominent was γ-tubulin, which was probably recruited from a large cytoplasmic pool. In support of this view, GFP-tagged exogenous γ-tubulin became colocalized with HA-CNN to participate in the formation of microtubule-nucleating sites. This was in striking contrast with cells expressing γ-tubulin alone, where cytoplasmic aggregates induced by γ-tubulin expression could not contribute to microtubule formation. These results suggest that microtubules are directly nucleated from the CNN aggregates through the mechanism mediated by γ-tubulin/γ-TuRC (Terada, 2003).

To confirm the microtubule-nucleating activity of the CNN aggregates, microtubules were polymerized in vitro by incubating isolated GFP-tagged CNN dots with X-rhodamine–conjugated brain tubulin. There was always a dot positive in GFP fluorescence at the center of the microtubule asters. Although variable numbers of microtubules emanated from the center, more microtubules tended to polymerize onto the GFP dots in larger sizes. The process of aster formation was monitored by time-lapse microscopy. A fluorescence image taken 10 min after mounting the sample on a microscopic stage revealed several microtubules growing from a GFP-positive site. As time progressed, more microtubules appeared to emanate from the center, indicating that microtubules were formed by direct polymerization onto the CNN-containing foci, rather than that preformed microtubules were gathered around the center (Terada, 2003).

Microtubules are nucleated from the pericentriolar material that surrounds the centrioles of the centrosome. To compare ultrastructure of microtubule-initiating sites induced by CNN with that of the pericentriolar material/centrosome, CHO cells expressing GFP-tagged CNN were examined by EM. Two microtubule asters were seen formed in cells that were briefly extracted before fixation. Located at each focal point of microtubule asters was an electron-dense particle in various sizes and shapes. Unlike the pericentriolar material, which has been described as an ill-defined amorphous cloud, the entire structure induced by CNN was well delineated by electron-dense materials to which microtubules were attached. In favorable sections, microtubules could be seen penetrating to the interior region of the aggregates. Neither centrioles nor centrosomal substructures, such as satellites, appendages, and CHO cell–specific virus particles, were generally seen at the site induced by CNN expression. Because CNN is a coiled-coil structural protein (Heuer, 1995), the dense particles likely represent the aggregated form of overexpressed CNN proteins (Terada, 2003).

Multiple centrosomes/MTOCs have been detected in cells in which the mechanism of centrosome duplication coupled with the cell cycle control becomes deregulated. In the case of CNN-containing MTOCs, their number and size formed during relatively short periods (8–12 h) varied greatly according to the level of protein expression. Moreover, no centrioles were found at ectopic MTOCs by EM and immunostaining with centriole-specific centrin-2 antibodies. Therefore, it is plausible that CNN expression causes the formation of protein aggregates that acquire the microtubule-nucleating capacity by recruiting γ-tubulin/γ-TuRC. This unique property of CNN to generate microtubule-nucleating sites by interacting with γ-tubulin/γ-TuRC suggested CNN may function as an adaptor for connecting γ-tubulin to the centrosome (Terada, 2003).

By expressing truncated polypeptides, it was concluded that CNN's ability to interact with γ-tubulin/γ-TuRC and induce ectopic microtubule-nucleating sites resides in the NH2-terminal sequence of CNN from which the Aurora-A–binding domain is omitted. In contrast, cytoplasmic aggregates formed in cells expressing the COOH-terminal domain failed to initiate microtubule formation in both S2 and mammalian cells. These results lead to the conclusion that CNN consists of two functionally distinct subdomains: the Aurora-A–binding site is at the COOH terminus capable of formation of the protein complex to be recruited to the spindle pole, and the NH2-terminal sequence is involved in assembling centrosomes/MTOCs by recruiting γ-tubulin/γ-TuRC. Although no CNN homologues have yet been identified outside Drosophila, Aurora-A would likely be involved in the control of microtubule nucleation through its association with the COOH terminus of a CNN-related molecule(s) in mammalian cells (Terada, 2003). Control of mitotic spindle assembly onto the centrosome could be achieved by several mechanisms, including nucleation of individual microtubules onto γ-tubulin–containing protein complexes, stimulation of microtubule nucleation and stabilization of polymerized microtubules by MAPs, and recruitment of minus ends of preexisting microtubules by the action of motor activity to the centrosome. Aurora-A binds not only CNN but also the D-TACC/MSPS/XMAP215 complex. These components appear to be required for microtubule assembly on mitotic centrosomes/poles controlled through the distinct mechanisms from that of γ-tubulin recruitment. Therefore, it is reasonable that Aurora-A plays a role in regulating the overall process of centrosome maturation by orchestrating multiple pathways of microtubule assembly during mitosis. It is worth mentioning that individual mechanisms of microtubule assembly may show a distinct requirement for protein phosphorylation and the Aurora-A kinase activity; although both Aurora-A and CNN are still able to locate at the centrosome, D-TACC/MSPS complex failed to be recruited to spindle poles in the absence of enzymatic activity of Aurora-A kinase (Terada, 2003).

Aurora kinases are highly expressed in cells derived from many human tumor cell types, which frequently contain multiple centrosomes. Because defects in the number, structures, and function of centrosomes are closely associated with the genetic instability in transformed cells, Aurora-A might be involved in tumorigenesis by inducing abnormal numbers of MTOCs as a result of inappropriate distribution of CNN-like molecule(s) (Terada, 2003).

Characterization of a Drosophila centrosome protein CP309 that shares homology with Kendrin and CG-NAP; CP309 is required for microtubule nucleation mediated by centrosomes and it interacts with the gamma-tubulin small complex

The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle. A large coiled-coil centrosome protein identified in Drosophila can bind to calmodulin. Biochemical studies reveal that this novel centrosome protein, centrosome protein of 309 kDa (Cp309 or Pericentrin-like protein), cofractionates with the gamma-tubulin ring complex and the centrosome-complementing activity. CP309 is required for microtubule nucleation mediated by centrosomes and it interacts with the gamma-tubulin small complex. These findings suggest that the microtubule-nucleating activity of the centrosome requires the function of CP309 (Kawaguchi, 2004).

It was reasoned that CaM-binding proteins such as Kendrin and CG-NAP, that are important for centrosome function, should exist in Drosophila. Database searches by using the full-length Kendrin or CG-NAP did not reveal any Drosophila proteins sharing overall sequence homology with Kendrin or CG-NAP. However, several predicted Drosophila proteins were found to contain the highly conserved CaM-binding motif that is found in Spc110, Kendrin, and CG-NAP. Interestingly, only one of these proteins, encoded by CG6735, is predicted to contain largely coiled-coil sequences 5' to the C-terminal CaM-binding domain such as Spc110, Kendrin, and CG-NAP. Because CG6735 encodes a potential homolog of Kendrin and CG-NAP, the gene product of CG6735 was analyzed further (Kawaguchi, 2004).

Sequence analyses reveal that CG6735 encodes a complete open reading frame of 1109 amino acids. This predicted protein is significantly shorter than Kendrin (3246 amino acids) and CG-NAP (3899 amino acids). Therefore, it was asked whether there is a longer splicing variant(s) of CG6735 in Drosophila by using RACE analyses starting from the 5' end of CG6735. This led to the identification of a complete open reading frame that encodes a predicted protein of 2726 amino acids and 309 kDa. This protein will be referred to as CP309 (Kawaguchi, 2004).

CP309 is similar to Kendrin and CG-NAP in the following three aspects. (1) All three proteins contain the conserved pericentrin AKAP450 centrosome targeting (PACT) domain of 200 amino acids at their C termini; (2) they have similar coiled-coil organizations N-terminal to the PACT domain; (3) they share a conserved region of 40 amino acids located in the N-terminal half of these proteins. Based on these analyses, it is suggested that CP309 is the Drosophila equivalent of Kendrin or CG-NAP. Although CP309 can bind to CaM in the absence of Ca2+, Ca2+ significantly enhances the binding (Kawaguchi, 2004).

Previous studies have suggested that Kendrin and CG-NAP interact with a mammalian-gammaTuSC component (Takahashi, 2002). Consistent with this, biochemical studies have shown that CP309 cofractionates with gammaTuSC and gammaTuRC in Drosophila embryo extracts. However, it was found that gammaTuRC/gammaTuSC and CP309 do not coimmunoprecipitate with each other in the embryo extracts. This suggests that the interaction between CP309 and gammaTuRC/gammaTuSC is weak. Because both CP309 and gammaTuSC could be co-expressed in Sf9 cells to levels higher than those in the embryo extracts, it was examined whether CP309 and gammaTuSC interact with each other in Sf9 cells. FLAG-tagged CP309, gamma-tubulin, and untagged Dgrips84 and 91 were expressed in Sf9 cells, and reciprocal immunoprecipitations were carried out using antibodies against CP309, Dgrip84, and gamma-tubulin. Either preimmune serum for CP309 antibody or random nonimmunized rabbit IgGs were used as control. It was found that the antibody against CP309 immunoprecipitates all three gammaTuSC subunits. Because the antibodies against gamma-tubulin and Dgrip84 also immunoprecipitate CP309, it is concluded that CP309 interacts with gammaTuSC. The above-mentioned experiments were carried out in the presence or absence of Ca2+ and CaM; they did not affect the interaction between CP309 and gammaTuSC. Therefore, Ca2+ or CaM does not seem to regulate the interaction between CP309 and gammaTuSC in vitro (Kawaguchi, 2004).

Drosophila Wee1 interacts with members of the γTURC and is required for proper mitotic-spindle morphogenesis and positioning

Wee1 kinases delay entry into mitosis by phosphorylating and inactivating cyclin-dependent kinase 1 (Cdk1). Loss of this activity in many systems, including Drosophila, leads to premature mitotic entry. Drosophila Wee1 (dwee1) mutant embryos show mitotic-spindle defects that include ectopic foci of microtubule organization, formation of multipolar spindles from adjacent centrosome pairs, and promiscuous interactions between neighboring spindles. Furthermore, centrosomes are displaced from the embryo cortex in mutants. These defects are not observed to the same extent in embryos in which nuclei also enter mitosis prematurely as a result of a lack of checkpoint control or in embryos with elevated Cdk1 activity. dWee1 physically interacts with members of the γ-tubulin ring complex (γTuRC), and γ-tubulin is phosphorylated in a dwee1-dependent manner in embryo extracts. Some of the abnormalities in dwee1 mutant embryos cannot be explained by premature entry into mitosis or bulk elevation of Cdk1 activity. Instead, dWee1 is also required for phosphorylation of gamma-tubulin, centrosome positioning, and mitotic-spindle integrity. A model is proposed to account for these requirements (Stempff, 2005).

dwee1 mutant embryos enter mitosis prematurely and form abnormal mitotic spindles. To determine whether premature mitotic entry and consequent centrosome inactivation account for the spindle abnormalities seen, dwee1 mutants, grp mutants, and irradiated wild-type embryos were compared. In all three cases, evidence of centrosome inactivation was seen: diminished astral microtubules and dispersal of both γ-tubulin and Dgrip84 from the centrosome in fixed embryos. Similar results were obtained from analyses of live dwee1 mutant embryos carrying the 17238-GFP transgene, in which GFP is inserted into a gene of unknown function and localizes to microtubules and centrosomes. dwee1 and grp mutant embryos and irradiated wild-type embryos show the loss of GFP signal on astral microtubules and at spindle poles during the syncytial blastoderm division cycles M12 and M13. In addition, all three groups display monopolar spindles, the inability to form a central spindle in M12 and M13, and the failure to fully separate centrosomes during interphase (Stempff, 2005).

Live analysis revealed three additional, phenotypes in cycles 12 and 13 of dwee1 mutants: (1) spindles with one to two ectopic microtubule-organizing centers (MTOC) within the single microtubule network (the ectopic sites change location dramatically within the confines of the spindle); (2) promiscuous interactions between adjacent spindles (in these situations, microtubules from one spindle appear to pull on a neighboring spindle), and (3) multipolar spindles that result from centrosome pairs of neighboring nuclei that interact. The last two phenotypes are also seen in fixed embryos. The first, however, was not seen -- ectopic MTOC may be too dynamic to be preserved during fixation. Importantly, these three phenotypes are absent in live irradiated wild-type embryos and present only at low frequencies in live grp mutants. These phenotypes are referred to as 'dwee1-specific' spindle defects (Stempff, 2005).

Centrosome inactivation in irradiated or grp mutant embryos is dependent on Chk2 function. Therefore, dwee1, chk2 double mutants were analyzed to further assess whether dwee1 spindle defects are caused by induction of the checkpoint. chk2 mutations are known to rescue cellularization in dwee1 mutants. As expected, mutation of chk2 also rescues the phenotypes, such as anastral spindles and the dispersal of γTURC in dwee1 mutants, that are characteristic of centrosome inactivation. However, dwee1, chk2 double mutants still display spindle interactions and form multipolar spindles (11 of 12 embryos in M12 or M13): the two dwee1-specific spindle defects are discernable in fixed embryos. On the basis of these data and the finding that grp mutants or irradiated embryos do not display dwee1-specific spindle defects to the same extent, it is concluded that spindle interactions and multipolar spindles in dwee1 mutants are not due to premature mitotic entry or to induction of chk2-dependent centrosome inactivation (Stempff, 2005).

Cdk1 activity is elevated during cortical syncytial cycles in dwee1 mutant embryos, and elevation of Cdk1 activity has been shown previously to affect spindle morphogenesis in precortical syncytial cycles. Therefore, the possibility that elevated Cdk1 activity is the cause of dwee1-specific spindle defects was addressed. To this end, fixed embryos from a fly stock with six copies of cyclin B, six cycB, were analyzed. Increasing cyclin B levels in embryos is known to increase Cdk1 activity. Consistently, it was found that (six cycB) embryos harbor higher CycB-Cdk1 activity than do wild-type embryos. More Cdk1 coprecipitates with cyclin B from six cycB embryos than from wild-type or dwee1 mutant embryos, suggesting that this increase in activity is due to the presence of more-active complexes, an idea that is consistent with previous observations that Cdk1 levels are not limiting in embryos. six cycB embryos display defects such as asynchronous divisions, but dwee1-specific spindle defects were not detected. It is concluded that a bulk elevation of Cdk1 activity cannot account for dwee1-specific spindle defects (Stempff, 2005).

CycB levels are reproducibly lower in dwee1 embryos than in wild-type embryos. The reason for this finding is not known, but nonetheless the possibility that reduction of CycB levels is the cause of dwee1-specific phenotypes was ruled out. This was done by analyzing embryos from mothers that are hemizygous for cycB and therefore have lower CycB levels. No evidence was found of spindle interactions or centrosome-positioning changes that resemble those of dwee1 mutant embryos (Stempff, 2005).

The interactions between neighboring centrosomes and spindles in dwee1 mutants are limited to cortical syncytial divisions. These are a subset of syncytial divisions that follow the migration of nuclei to the embryo cortex. During cortical divisions, nuclei and centrosomes closely abut the cortex, and their position is maintained by microtubule-filament- and actin-filament-dependent mechanisms. Specifically, astral microtubules nucleated by the centrosomes are proposed to interact with cortical actin to mediate the attachment of centrosomes, along with their associated nuclei, to the cortex. Physical separation of mitotic spindles in a syncytium is thought to occur by reorganization of F-actin caps into pseudocleavage furrows that surround each dividing nucleus. Pseudocleavage furrows form during prophase and metaphase, retract in anaphase, and are mostly absent in late anaphase and telophase (Stempff, 2005).

Pseudocleavage furrows in dwee1 mutants form with normal timing and reach similar depths as in wild-type embryos. Nuclei in dwee1 mutants, however, are positioned beyond the deepest part of the furrows in metaphase. Quantification of centrosome-cortex distance in wild-type embryos and dwee1 mutants in cycle 12 illustrates this phenotype. This cycle was chosen because it occurs after completion of cortical nuclear migration but before the onset of the centrosome-inactivation checkpoint, as evidenced by anastral spindles. Displacement of centrosomes from the cortex is observed in both interphase and mitosis of cycle 12 in dwee1 mutants and is only partially rescued by the chk2 mutation. It is suggested that centrosome and nuclear displacement in dwee1 mutants has two underlying components: one is a consequence of the Chk2-mediated checkpoint, and the other is a more direct result of loss of dwee1. That is, dwee1 is required to promote centrosome-cortex interaction, which is thought to be dependent on centrosomal microtubules and cortical actin. The displacement of centrosomes from the cortex in dwee1 mutants could distance mitotic spindles from the protection of pseudocleavage furrows in prophase and metaphase, thereby allowing interactions between adjacent spindles (Stempff, 2005).

six cycB embryos show normal localization of nuclei and centrosomes, suggesting that bulk elevation of Cdk1 activity in dwee1 mutants cannot account for the cortical detachment of centrosomes. Additionally, spindle interactions in dwee1 mutants also initiate when pseudocleavage furrows are normally absent (anaphase and telophase). Therefore, a furrow-independent mechanism may operate to keep spindles apart in anaphase and telophase in a dwee1-dependent fashion (Stempff, 2005).

To further address the requirement for dWee1 in centrosome and spindle function, dWee1-containing protein complexes were purified and dWee1-interacting proteins were identified by mass spectrometry. The heat-inducible HA-dWee1 transgene used as a source of dWee1 has been shown to partially rescue dwee1 mutant embryos when expressed in the mothers, indicating that the product is functional. HA-dWee1 was induced in embryos, purified on an anti-HA antibody column, and eluted with a HA-dipeptide. Analysis of eluates by SDS-PAGE and mass spectrometry identified peptides that matched dWee1 and Drosophila γ-tubulin ring proteins (Dgrips) 163, 128, 91, 84, and 71. The identities of Dgrip84, Dgrip91, and HA-dWee1 were confirmed by Western blotting. Note that γ-tubulin was not detected in the HA-dWee1 eluates because a strong background band in the 50 kDa range (the MW of γ-tubulin), likely the IgG heavy chain, prevented analysis of that region of the gel (Stempff, 2005).

Because the above experiments were performed with overexpressed, tagged dWee1, it was necessary to ensure that endogenous dWee1 interacts with γTuRC. Dgrip91 and γ-tubulin was readily detected in immunoprecipitates by using an antibody against dWee1. It was not possible, however, to detect dWee1 in immunoprecipitates by using an antibody against γ-tubulin or Dgrip91. This may be because it is possible, at best, to precipitate approximately 5% of total protein present with each antibody. The presence of γ-tubulin, a structural component of the cytoskeleton, at a higher concentration than dWee1, a regulatory kinase, is a likely scenario, and it could explain why no dWee1 is seen in immunoprecipitates of γ-tubulin (Stempff, 2005).

To determine whether the kinase dWee1 influences the phosphorylation status of proteins it binds to, γ-tubulin (which is known to be phosphorylated in budding yeast) was examined. Two-dimensional (2D) gel electrophoresis followed by Western blotting revealed that Drosophila γ-tubulin separates as a series of five spots in the first dimension. Two of the more acidic isoforms are phosphatase sensitive, suggesting that Drosophila γ-tubulin is a phosphoprotein. The phosphatase-sensitive acidic forms are absent or severely diminished in extracts from dwee1 mutant embryos (Stempff, 2005).

Given that interphase is truncated in dwee1 mutants, the possibility was addressed that loss of γ-tubulin phosphorylation is a consequence of changes in cell cycle profile. However, extracts from grp mutant embryos that exhibit truncated interphases retain the phosphatase-sensitive γ-tubulin isoforms. It is concluded that interphase shortening does not lead to loss of γ-tubulin phosphorylation and that dwee1 is required for γ-tubulin phosphorylation in vivo. Despite testing a range of conditions, it has not been possible to phosphorylate γ-tubulin with recombinant dWee1 in vitro, although GST-dWee1 readily autophosphorylates and phosphorylates Cdk1 in these assays. Either dWee1 regulates γ-tubulin phosphorylation indirectly or γ-tubulin phosphorylation by dWee1 requires a cofactor (Stempff, 2005).

The known role of Wee1 homologs in cell cycle regulation is accomplished through a single substrate, Cdk1. Elevation of bulk Cdk1 activity in an otherwise wild-type background, however, does not produce dwee1-specific phenotypes. Therefore, if dWee1 influences spindle organization or positioning via Cdk1, it would have to regulate Cdk1 locally, at the embryo cortex for example. The attachment of centrosomes to the cortex is mediated by microtubules. In the absence of dWee1, Cdk1 activity would be higher locally, i.e., between the centrosome and the cortex, and could inhibit microtubule growth in this region, leading to the displacement of centrosomes. This idea is consistent with observations that increased Cdk1 activity destabilizes microtubules during nuclear migration in Drosophila embryos. In six cycB embryos, dWee1 could still inhibit Cdk1 locally to allow normal nuclear and spindle positioning. This possibility can be addressed with a Cdk1 mutant that cannot be phosphorylated by dwee1 and should mimic the loss of dwee1. Attempts were made to introduce into syncytial embryos (which are prezygotic transcription) such a mutant in which Y14 and T15 have been altered, Cdk1AF, by expressing it in females. Unfortunately, females fail to lay eggs after induction of Cdk1AF, suggesting disruption of oogenesis and precluding further analysis (Stempff, 2005).

An alternate approach to introduction of Cdk1AF is to induce dWee1-antagonizing phosphatases, Cdc25string and Cdc25twine, in embryos. Indeed, such experiments have been described before. Increasing the maternal Cdc25 gene dose by up to 4-fold in various combinations of the two Drosophila Cdc25 homologs produces increased mRNA and protein in embryos and leads to an extra syncytial nuclear division before cellularization. This division and preceding syncytial divisions, however, are normal in fixed and live embryos. No mitotic abnormalities, which are readily apparent throughout dwee1 mutant embryos, were seen in embryos with elevated Cdc25. This is consistent with the observation that bulk elevation of Cdk1 activity does not produce dwee1-specific phenotypes. Instead, localized regulation of Cdk1 by dWee1, which would still be present in embryos harboring extra Cdc25, could explain the apparently normal divisions in these embryos (Stempff, 2005).

Another explanation for spindle phenotypes in dwee1 mutants is suggested by the finding that dWee1 shows physical interaction with components of the γTuRC and that dwee1 influences the phosphorylation status of γ-tubulin in vivo. In this model, dWee1 promotes the phosphorylation of γ-tubulin, either directly or indirectly. In dwee1 mutants, loss of γ-tubulin phosphorylation could compromise microtubule-dependent attachments between centrosomes and the cortex. A test of this model will require identification and mutation of dwee1-dependent phosphoacceptor residues in γ-tubulin. Interestingly, the budding-yeast γ-tubulin homolog, Tub4p, is phosphorylated on a tyrosine residue during G1, but the responsible kinase has yet to be identified. A phosphomimetic mutation of Tub4p affects the number and organization of microtubules and causes transient nuclear-positioning abnormalities. Thus, it is possible that in both yeast and fly, the phosphorylation status of γ-tubulin plays a role in centrosome and nuclear positioning via interactions with the cortex (Stempff, 2005).

At present, it is not possible to distinguish between the two above explanations for dwee1 phenotypes; the explanations are not mutually exclusive. However, neither is predicted by previous models that describe how Wee1 homologs act to regulate entry into mitosis. Human Wee1, for example, resides in the nucleus during interphase and is proposed to prevent nuclear accumulation of Cdk1 activity and nuclear-envelope breakdown, an initiating event in mitosis. Such models can explain Wee1's role in regulating when mitosis occurs, but the current results indicate that Wee1 can also regulate where (relative to the cortex) mitosis occurs (Stempff, 2005).

The first 11 nuclear divisions proceed normally in dwee1 mutants. Therefore, dwee1-dependent regulation of spindle organization or positioning is not essential for mitosis per se. In cycle 12, nuclei are at the cortex and at twice the density (i.e., closer together) compared to those in cycle 11. It is reasoned that manifestation of dwee1-specific spindle interactions in later cortical cycles is a consequence of increasing nuclear density with each cycle that brings neighboring spindles closer together. In such a situation, protection offered by actin furrows may be essential to keep spindles apart. Detachment of centrosomes from the cortex would distance the spindles from furrows, allowing neighbors to interact (Stempff, 2005).

It is not known whether dwee1 also plays a role in spindle morphogenesis and centrosome positioning in cell cycles beyond cortical syncytial cycles. It is known, however, that dwee1 is needed to ensure fidelity of cell division in larvae; larval neuroblasts in dwee1 mutants show elevated mitotic index and ploidy. It would be interesting to determine the basis for this requirement and whether dwee1 has a role in the positioning of the spindle in cell divisions where a specific cortical attachment of the spindle is required, such as in the asymmetric cell divisions of neuroblast lineages (Stempff, 2005).

This study presents several lines of data that collectively suggest a requirement for dWee1 in centrosome function and spindle morphogenesis. Importantly, these roles translate into a requirement for dWee1 in not only temporal but also spatial regulation of mitosis. Two mechanistic models, which are not mutually exclusive, have been offered to account for these results: localized regulation of Cdk1 by dWee1 and phosphoregulation of γTuRC. Further analysis will be needed to test these models, but it is clear that the requirement for dWee1 cannot be explained by simple regulation of bulk Cdk1 activity. In this regard, Wee1 homologs may be likened to other kinases, such as Plk and Aurora B, that have multiple roles in mitosis through multiple substrates. Localized activity of master regulatory kinases such as these is likely to coordinate many distinct cell-division events (such as spindle movements, chromosome segregation, and cytokinesis) to allow faithful segregation of genetic information into daughter cells (Stempff, 2005).

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

gamma-Tubulin, a protein critical for microtubule assembly, functions within multiprotein complexes. However, little is known about the respective role of gamma-tubulin partners in metazoans. For the first time in a multicellular organism, the function of Dgrip84, the Drosophila orthologue of the Saccharomyces cerevisiae gamma-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 gamma-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 gamma-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, gamma-tubulin monomers enhance the assembly of alpha/beta heterodimers and block the minus ends of microtubules. However, in vivo, gamma-tubulin does not seem to act as a monomer, but rather in a variety of protein complexes. The simplest one called gamma-tubulin small complex (gamma-TuSC) has been well characterized in Saccharomyces cerevisiae. It is formed by gamma-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 gamma-TuSC, other larger gamma-tubulin-containing complexes have been described. One termed gamma-tubulin ring complex (gamma-TuRC) has been characterized in multicellular organisms such as D. melanogaster, Xenopus laevis, and Homo sapiens. It is assumed that the gamma-TuRC results from the association of several gamma-TuSCs with at least four other proteins. Three of them show sequence homologies (grip motifs) with the two gamma-tubulin-associated proteins in the gamma-TuSC, whereas the fourth exhibits five WD repeats. The function of gamma-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 gamma-tubulin isotypes are expressed, homozygous gamma-tub 23C mutants die during late larval stage, exhibiting atypical mitotic spindles and abnormal centrosomal structures, whereas disruption of the gamma-tub 37CD gene results in abnormal female meiotic spindles. However, knowledge about the function of the gamma-tubulin-interacting proteins is limited. The role of the two grip proteins associated with gamma-tubulin in the gamma-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 gamma-tubulin anchorage to the inner spindle plaque via direct interaction with Spc110p, whereas Spc97p seems essential for a correct SPB duplication and separation (Knop, 1997b). In fission yeast, Alp4 mutations compromise gamma-tubulin localization to the SPB, but they do not affect the assembly of the large gamma-tubulin complex. Discrepancies exist about the role of the two grip motif gamma-TuSC subunits, in particular in the SPB duplication/separation process and in the gamma-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 gamma-tubulin relocalizes to the SPB as gamma-TuSC, in multicellular organisms, it is assumed that gamma-tubulin is recruited to the centrosome as gamma-TuRC. In metazoans, the silencing of gamma-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 gamma-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 gamma-TuSC protein in the organization of microtubule cytoplasmic arrays has been examined. Dgrip84, as gamma-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 gamma-TuSC has been examined using genetic approaches only in unicellular organisms. These analyses must be further investigated in animals where gamma-tubulin is assumed to be recruited to the centrosome as gamma-TuRC. Functional analysis of gamma-tubulin-associated proteins in metazoans has been mainly restricted to Drosophila: Dgrip91, a gamma-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 gamma-TuSC, allowing for the first time a comparison of the phenotypes resulting from the disruption of each gamma-TuSC component in a same metazoan (Colombié, 2006).

Their depletion has no obvious effect on the cytoskeleton organization during interphase. However, in Dgrip84- or gamma-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 gamma-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 gamma-tubulin encoding gene both in budding yeast and A. nidulans and in gamma-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 gamma-TuSC seems required in some aspects of microtubule dynamics and organization (Colombié, 2006).

Disruption of each gamma-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 gamma-tubulin, Dgrip84, or Dgrip91 results in large polyploid nuclei (Sunkel, 1995; Barbosa, 2000; this study). This observation strengthens the idea that when the gamma-TuSC integrity is affected, cells could escape prematurely from the mitotic checkpoint. These results are consistent with several sets of data. Human gamma-tubulin mutants expressed in S. pombe allow cytokinesis to proceed in spite of spindle abnormalities. Some alleles of the A. nidulans gamma-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 gamma-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 gamma-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 gamma-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 23Cgamma-tubulin depletion in S2 cells (Barbosa, 2000; Raynaud-Messina, 2004). Moreover, Dgrip84 mutant spermatocytes exhibit monopolar structures. Similar figures, described in gamma-tub23C and Dgrip91 mutant spermatocytes, have been shown to be the consequence of the collapse of the two poles. Drosophila gamma-TuSC assembly seems necessary for the proper separation of the centrosomes. Similar observations were obtained after gamma-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 gamma-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 gamma-tubulin and Dgrip91 localization at the poles. Moreover, gamma-tubulin depletion impairs Dgrip84 and Dgrip91 mitotic recruitment (Raynaud-Messina, 2004). Therefore, it is likely that gamma-TuSC components must be assembled in complexes before relocalization to the poles or that gamma-TuSC proteins are required for gamma-tubulin polar attachment. This view, supported by studies in S. cerevisiae, has been recently extended to mammalian cells: the gamma-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 gamma-TuRC to the centrosome through direct binding with gamma-TuSC components (Kawaguchi, 2004). So, the gamma-TuSC grip-motif proteins seem essential, due to their critical role in the attachment of gamma-tubulin complexes to the pericentriolar matrix. In contrast, the proteins specific for the gamma-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 gamma-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 gamma-tubulin (Sampaio, 2001; Barbosa, 2003), is required for completion of the two meiotic divisions. In the absence of one of the gamma-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 gamma-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 gamma-tubulin recruitment, proper microtubule organization and an efficient mitotic checkpoint. In contrast, the depletion of gamma-TuRC-specific proteins does not impair viability and gamma-tubulin accumulation. It is suggested that the gamma-TuSC could be a universal and minimal subunit required for gamma-tubulin recruitment to the cytoskeleton structures and proper microtubule nucleation in eukaryotic cells (Colombié, 2006).

Making microtubules and mitotic spindles in cells without functional centrosomes

Centrosomes are considered to be the major sites of microtubule nucleation in mitotic cells, yet mitotic spindles can still form after laser ablation or disruption of centrosome function. Although kinetochores have been shown to nucleate microtubules, mechanisms for acentrosomal spindle formation remain unclear. In this study live-cell microscopy of GFP-tubulin to examine spindle formation was performed in Drosophila S2 cells after RNAi depletion of either γ-tubulin, a microtubule nucleating protein, or centrosomin, a protein that recruits γ-tubulin to the centrosome. In these RNAi-treated cells, it was show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving γ-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, it was also demonstrated that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. It is proposed that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes (Mahoney, 2006).

Drosophila S2 cells depleted of centrosomin (Cnn) formed anastral spindles and do not recruit γ-tubulin to spindle poles. γ-Tubulin, however, was present on spindle microtubules after Cnn depletion, as was also observed for wild-type cells (Mahoney, 2006).

To probe the dynamics of spindle formation after Cnn depletion, time-lapse microscopy was performed on GFP-tubulin-expressing cells. In wild-type cells, the majority of the early prophase microtubules originate from the centrosomes immediately after nuclear-envelope breakdown (NEB). However, microtubules also clearly form around the chromosomes. In Cnn-RNAi-treated cells, time-lapse microscopy revealed a very different pathway of spindle formation. Centrosomal microtubule asters did not form at prophase, but robust microtubule nucleation still occurred at chromosomes. The interphase microtubule array, which only gradually destabilized after NEB, also incorporated into the spindle, and in some cases attachment and capture of pre-existing microtubules by chromosomes was observed. After initially collecting around chromatin, microtubules then elongated and became focused to create a bipolar spindle with broad, dynamic poles, as described for meiotic spindle formation. Time-lapse microscopy also revealed that Cnn RNAi cells proceeded into anaphase without any significant delay, a result that is consistent with their normal mitotic index. Thus, even when the dominant pathway of microtubule-based search-and-capture of chromosomes by centrosomal microtubules is completely disrupted in Cnn RNAi cells, live-cell imaging reveals that chromosome-mediated nucleation and incorporation of existing microtubules generate a functional bipolar spindle in a time period comparable to that in wild-type cells (Mahoney, 2006).

Next, S2 cells were depleted of ~90% γ-tubulin by RNAi, although residual γ-tubulin may create a hypomorphic situation rather than a true null. (Note that γ-tubulin refers to the ubiquitously expressed 23C isotype; RNAi of an ovary-specific (37C) isotype did not produce a phenotype in S2 cells. Mitotic S2 cells depleted of γ-tubulin by RNAi still contained microtubules, although the mitotic spindles were virtually all abnormal. The most common morphologies were monopolar spindles (~40%) and anastral bipolar spindles with poorly focused poles (~60%). The mitotic index was elevated 3.3-fold, but anaphase cells were observed in the population. These results are consistent with prior studies showing that interfering with γ-tubulin function results in severe spindle defects, although microtubules can still form occur and chromosome alignment can occur (Mahoney, 2006).

To better understand the mechanism of spindle formation after γ-tubulin depletion, live-cell imaging was performed of GFP-tubulin-expressing cells. In cases where NEB was observed, microtubule formation from the chromosomal region was dramatically reduced or significantly delayed in comparison with wild-type and Cnn-RNAi-treated cells. This result, combined with observation of spindle localization of γ-tubulin in Cnn-depleted cells, suggests that γ-tubulin functions as a microtubule nucleator in the chromosome-mediated microtubule assembly pathway. This finding indicates that γ-tubulin need not be anchored at the centrosome to stimulate microtubule nucleation. While this paper was in review, a similar conclusion was reached in mammalian cells (Luders, 2006) on the basis of RNAi of a γ-tubulin-associated subunit (Mahoney, 2006).

The mechanism of assembly of spindle microtubules in γ-tubulin RNAi cells was difficult to decipher from time-lapse movies, and it is possible that the microtubules originate from several sources. One such source appears to be pre-existing interphase microtubules, which coalesce into bundles after NEB and can engage chromosomes as described above for Cnn-depleted cells. Fragments of former 'interphase' microtubules might also act as nucleating seeds for new microtubule growth. In addition, 'focal points' of microtubule growth were also observed, that could represent nucleation from centrioles, or from sites on the fragmenting nuclear envelope. After γ-tubulin RNAi, time-lapse imaging showed that cells usually formed a monopolar spindle initially, as seen in fixed cell images, that often converted to a bipolar spindle through the formation of a second pole. Such bipolar spindles, however, are very unstable and exhibit much more splaying and disorganization than Cnn RNAi cells. Bipolar metaphase spindles in γ-tubulin RNAi cells stall for at least twice as long as in wild-type cells, explaining the increased mitotic index, but eventually can complete anaphase and cytokinesis (Mahoney, 2006).

In conclusion, live-cell imaging reveals several redundant mechanisms for creating mitotic spindles via (1) centrosome-based nucleation, (2) chromosome-based assembly, and (3) recruitment of microtubules created at other sites. Centrosome nucleation of microtubules constitutes the dominant pathway of spindle formation in wild-type cells, but the other processes can generate spindles in the absence of centrosome function (i.e., after Cnn or γ-tubulin depletion) (Mahoney, 2006).

Time-lapse microscopy of GFP-tubulin was effective for examining the initial events in bipolar spindle formation. However, because of the high density of microtubules in the spindle, it was difficult to visualize sites of microtubule nucleation and growth during metaphase. To gain information on these issues, live-cell imaging was performed of a stable S2 cell line expressing low levels of EB1-GFP, a microtubule-plus-end-tracking protein that localizes to the terminal ~0.5 μm tip of growing microtubules and that has been used to investigate cell-cycle-dependent microtubule nucleation (Mahoney, 2006).

In wild-type metaphase cells, EB1-GFP punctae emerged in a radial pattern from the centrosome. In addition, individual EB1-GFP punctae were visible in the microtubule-dense regions of the spindle. A semiautomated program was employed to identify and track EB1-GFP over time and create vectors plots for the growth of individual microtubules. The vector maps of EB1-GFP revealed an overall image that corresponded to the shape of the wild-type mitotic spindle, and computer-generated vectors were in good agreement with manual tracking and visual inspection of the movies. However, limitations exist in establishing the precise origin of all EB1-GFP puncta, given that some may have originated out of the plane of focus (most punctae, however, appeared suddenly as expected for nucleation in the focal plane) and that the automated program often terminated tracking when punctae crossed and overlapped in dense regions of the spindle (Mahoney, 2006).

For Cnn-RNAi-treated cells in metaphase, the radial distribution seen of vectors at the poles for wild-type cells was not observed. Surprisingly, however, EB1-GFP tracking revealed vectors originating throughout the spindle, including many vectors arising from the acentrosomal poles and traveling toward the chromosomes. This result was not anticipated, given that imaging of GFP-tubulin showed initial microtubule nucleation around chromosomes after NEB and not from a peripheral nucleating site. Thus, whereas microtubule formation initially relies upon the chromosomes, the spindle itself acquires a mechanism for forming new EB1 punctae distal from the chromosomes. However, microtubules also probably continue to form around the metaphase chromosomes in Cnn-RNAi-treated cells, because there are many EB1-GFP vectors in the chromosomal region that do not extrapolate back to the spindle poles. In γ-tubulin RNAi cells that formed anastral bipolar-like spindles, the EB1-GFP vector diagrams looked similar to those described for Cnn-RNAi-treated cells, revealing microtubule growth originating throughout the spindle and from the broad polar regions. In contrast to the selective growth of microtubules from acentrosomal poles to chromosomes in the Cnn RNAi cells, however, the orientation of the vectors in the γ-tubulin RNAi spindles tended to be more random (Mahoney, 2006).

It was of interest to determine whether EB1-GFP punctae form within wild-type spindles or whether this phenomenon is a consequence of γ-tubulin depletion/mislocalization. In some wild-type cells, the centrosome and its astral array became transiently disconnected and displaced from the kinetochore fibers, producing a clear spatial separation of the two microtubule networks in the spindle. In such cells, EB1 vectors were still observed originating from the focused minus-end region of kinetochore fibers; these acentrosomal vectors again were preferentially directed toward the chromosomes. RNAi of abnormal spindle protein (Asp) was also performed, and this treatment resulted in centrosome detachment from the main body of the spindle and splaying of kinetochore fibers. In this situation, movies of EB1-GFP also revealed fluorescent punctae originating from acentrosomal regions of splayed kinetochore fibers and moving toward the chromosomes. Thus, a process of spindle-based microtubule formation and growth occurs from acentrosomal foci of kinetochore fibers and within spindles, even in cells that possess functional centrosomes (Mahoney, 2006).

In summary, this analysis of both γ-tubulin and Cnn RNAi cells, as well as untreated cells, shows EB1-GFP punctae forming within the spindle and from acentrosomal poles and traveling toward the chromosomes. Four mechanisms are presented by which EB1 punctae might be generated in the spindle: (1) Catastrophe of stable microtubules followed by rescue can generate new growth and plus-end labeling by EB1 within the spindle, although observations of astral-microtubule dynamics show that rescue is rare and thus perhaps unlikely to be the sole source by which EB1-GFP punctae are generated within the spindle; (2) Microtubule severing of existing microtubules generates new microtubule plus ends that grow and recruit EB1; (3) γ-tubulin-mediated growth of new microtubules, potentially nucleating from the sides of pre-existing microtubules; (4) An unidentified protein nucleates new microtubules (Mahoney, 2006).

Although discussed separately, it is emphasized that these models are not mutually exclusive, and indeed, it is plausible that multiple mechanisms might be contributing to this phenomenon. One possibility for generating new EB1 punctae is through the rescue of a kinetochore microtubule that undergoes catastrophe. Observation of individual microtubule catastrophe and rescue events in the spindle is not technically possible because of the high microtubule density. Microtubule depolymerization has been induced by severing Xenopus spindles with a microneedle and it has been concluded that rescue and regrowth is rare, especially near the poles. It was not possible to image single astral microtubules in GFP-tubulin-expressing S2 cells, and observations also reveal a very low rescue frequency of depolymerizing astral microtubules (only 5% of the shrinking microtubules underwent a clear rescue event. Nevertheless, the possibility of a novel mechanism that selectively stimulates rescue in spindle versus the astral microtubule population in S2 cells cannot be excluded (Mahoney, 2006).

EB1 punctae in the spindle could also reflect the generation of additional microtubules, by either microtubule fragmentation or nucleation. An important clue in considering such models is that the EB1-GFP vectors tend to be constrained within the cone angle of the spindle and grow toward the chromosomes, in contrast to the radial nucleation/growth that occurs from centrosomes. Microtubule severing followed by regrowth of the newly created plus end could produce such results. Alternatively, a de novo templating reaction from existing spindle microtubules could occur. For example, a nucleator could bind to the side of existing microtubules and template new microtubules at a shallow angle to the mother filament, followed by crosslinking/bundling to pre-existing kinetochore microtubules. Such a mechanism is analogous to the binding and nucleation of new actin filaments by Arp2/3 bound to a pre-existing actin filament. Precedence for this idea comes from recent reports showing that γ-tubulin can nucleate microtubules from pre-existing interphase microtubules in S. pombe and in plants. γ-tubulin is present throughout the spindle in S2 cells, which might favor such a possibility in mitosis as well. The fact that γ-tubulin RNAi cells still form EB1-GFP punctae at the broad polar regions cannot necessarily be taken as evidence against γ-tubulin involvement in such a de novo microtubule nucleation mechanism, because residual γ-tubulin remains after RNAi. Moreover, EB1-GFP vectors in γ-tubulin RNAi spindles differ from those in wild-type and Cnn RNAi cells in being less dense and more random in orientation (less selective growth toward the midzone compared with γ-tubulin-containing spindles). Alternatively, a novel microtubule nucleator may be involved or could contribute in addition to γ-tubulin (Mahoney, 2006).

Clearly, further studies will be required to identify the molecule(s) responsible for generating microtubule growth at acentrosomal poles and within spindles. However, the present study illustrates that, in addition to the well-described pathways of centrosomal and chromosomal microtubule nucleation, the metaphase spindle possesses a mechanism (or mechanisms) for propagating its own architecture by promoting microtubule assembly (Mahoney, 2006).

Proper recruitment of γ-Tubulin and D-TACC/Msps to embryonic Drosophila centrosomes requires centrosomin motif 1

Centrosomes are microtubule-organizing centers and play a dominant role in assembly of the microtubule spindle apparatus at mitosis. Although the individual binding steps in centrosome maturation are largely unknown, Centrosomin (Cnn) is an essential mitotic centrosome component required for assembly of all other known pericentriolar matrix (PCM) proteins to achieve microtubule-organizing activity at mitosis in Drosophila. A conserved motif (Motif 1) has been identified near the amino terminus of Cnn that is essential for its function in vivo. Motif 1 has a higher degree of sequence conservation (40% identity/49% similarity) between Cnn and human CDK5RAP2 and is present in all homologues from S. pombe to human. Cnn Motif 1 is necessary for proper recruitment of γ-tubulin, D-TACC (the homolog of vertebrate transforming acidic coiled-coil proteins [TACC]), and Minispindles (Msps) to embryonic centrosomes but is not required for assembly of other centrosome components including Aurora A kinase and CP60. Centrosome separation and centrosomal satellite formation are severely disrupted in Cnn Motif 1 mutant embryos. However, actin organization into pseudocleavage furrows, though aberrant, remains partially intact. These data show that Motif 1 is necessary for some but not all of the activities conferred on centrosome function by intact Cnn (Zhang, 2007).

Previous studies showed that Cnn is required for centrosome assembly/maturation, for microtubule assembly from the centrosome at mitosis, and to organize actin into pseudocleavage furrows in the early embryo. It is shown here that Motif 1 of Cnn is required for specific and essential aspects of centrosome function. Centrosomes assembled in cnnβ1 embryos recruit some PCM components and are partially proficient to organize actin into pseudocleavage furrows, but do not properly recruit or maintain proteins with an established role in microtubule assembly: γ-tubulin, D-TACC, and Msps. Thus, although astral microtubules are produced at cnnβ1 mutant centrosomes, centrosome separation, a microtubule-dependent process, is severely affected. In addition, the less-understood process of satellite formation is inhibited at cnnβ1 centrosomes (Zhang, 2007).

Microtubule assembly at centrosomes is regulated by nucleation, where γ-Tub plays a key role, and by microtubule growth, which depends on a host of factors including Aurora A, D-TACC, and Msps, that promote stability. How these proteins are assembled and regulated is still largely unknown. This study shows that Cnn Motif 1 controls assembly of PCM proteins that are required for MTOC activity at centrosomes (Zhang, 2007).

γ-Tub is an essential component of MTOCs in eukaryotes for microtubule assembly. In cnn null mutant neuroblasts, imaginal disk cells, and cells depleted of Cnn by RNAi, neither γ-Tub nor astral microtubules are detected at centrosomes. However, in contrast to the above cell types, a Cnn-independent pool of γ-Tub is at the centrosome remnant in cnn null mutant early embryonic spindle poles. The small, sharp signal for γ-Tub at cnn null spindle poles implicates a centriolar pool of γ-Tub that is unique to the rapid divisions of early embryos. The level of γ-Tub at cnnβ1 mutant centrosomes is similar to the cnn null mutant, indicating that Motif 1 is required for recruitment of the Cnn-dependent pool of γ-Tub to the PCM in embryos. Drosophila Cnn and the S. pombe homolog Mto1p have been reported to coIP with γ-Tub, but a direct interaction with γ-Tub or any of the γ-TuRC proteins has not been demonstrated (Zhang, 2007).

D-TACC and Msps, and their counterparts in Xenopus (TACC3/maskin and XMAP215) and C. elegans (TAC-1 and ZYG-9) are direct binding partners required for centrosome-dependent growth of long microtubules. Mutation or depletion of D-TACC or its homologues does not affect γ-Tub localization to centrosomes, but rather appears to function with Msps in the stability of microtubules that are nucleated by γ-Tub. D-TACC and Msps are partially recruited to centrosomes in cnn null and cnnβ1 mutants, accumulating at the centrosome periphery in cnnβ1 embryos. This incomplete assembly suggests that recruitment of D-TACC and Msps to centrosomes normally involves at least two steps and that Motif 1 of Cnn is required for a secondary step in the process subsequent to docking of D-TACC at the periphery of the centrosome. Thus, Cnn Motif 1 may be required for a later phase of recruitment to the centrosome or have a role in maintaining D-TACC and Msps once they are recruited (Zhang, 2007).

Aurora A kinase is required to localize D-TACC to centrosomes and directly phosphorylates D-TACC at Ser863 to activate its microtubule-stabilizing activity. The reduced recruitment of Aurora A to cnn null centrosomes further highlights the requirement for Cnn in PCM assembly. However, Aurora A localization did not appear affected in cnnβ1 embryos, indicating that, although Aurora A is necessary to recruit D-TACC/Msps, its localization at centrosomes is not sufficient to accomplish this. Aurora A binds directly to the C-terminal half of Cnn, which remains intact in the cnnβ1 mutant. Moreover, D-TACC is phosphorylated by Aurora A in cnnβ1 embryos; however, this activated pool of D-TACC is exiled to the centrosome periphery with the bulk pool of centrosomal D-TACC. This indicates that Motif 1 of Cnn is required for anchoring or maintaining D-TACC at centrosomes subsequent to its regulatory phosphorylation by Aurora A. Alternatively, because the immunofluorescence signal for P-D-TACC was weak and P-D-TACC levels were not quantified, an effect of cnnβ1 on Aurora A activity toward D-TACC cannot be excluded (Zhang, 2007).

In cnnβ1 and cnn null embryos microtubule asters are present, particularly at early cortical cycles (cycles 10 and 11). At later cycles asters are not detected at spindle poles in cnn null embryos, coinciding with centriole loss, which is evident from the absence of Nek2 kinase (a centriolar protein) signal. Centriole displacement from the spindle poles in cnn null embryos leads to centriole loss, resulting in anastral spindle poles (Lucas and Raff, personal communication to Zhang, 2007). By comparison to cnn null embryos, PCM integrity is restored to cnnβ1 mutant centrosomes, enough to retain centrosomes at spindle poles into later cleavage cycles and with retained ability to assemble astral microtubules. Nevertheless, centrosome separation failure indicates that microtubule-dependent processes are impaired at cnnβ1 centrosomes (Zhang, 2007).

Centrosome separation is a microtubule-dependent process that is coordinated by pushing forces from interpolar microtubules and forces supplied by molecular motors that include kinesin-5, kinesin-14 (Ncd), and dynein/Lis1/dynactin. The relative contributions of motor proteins and the pushing forces generated from the assembly of interpolar centrosomal microtubules have not been determined (Zhang, 2007).

A necessary role for microtubules in centrosome separation has been demonstrated using microtubule-depolymerizing drugs in cell culture and in early Drosophila embryos. Interpolar centrosomal microtubules may represent a specialized class of microtubules, an idea supported by the recent discovery of an α-tubulin variant, α4-tubulin, which is associated with faster-growing microtubules and is enriched in interpolar microtubules. α4-tubulin is required for centrosome separation in early embryos. Cnn localized more strongly to interpolar fibers compared with spindle microtubules, suggesting that Cnn Motif 1 may regulate the organization of interpolar centrosomal microtubules to promote centrosome separation. In instances when cnnβ1 centrosomes separated, interpolar fibers formed, suggesting that interpolar fibers are obligatory to centrosome separation. Although the proposal that Motif 1 regulates microtubule assembly to achieve centrosome separation is favored, a role for Motif 1 in regulating molecular motors that are involved in this process cannot be ruled out. However, localization of the kinesin-5/Eg5 family member Klp61F to spindle poles and spindle microtubules was no different in cnnWT and cnnβ1 embryos (Zhang, 2007).

Consistent with a role for γ-Tub and D-TACC recruitment to centrosomes by Cnn Motif 1 in centrosome separation, depletion or mutation of γ-Tub, γ-TuSC proteins, and D-TACC also perturbed centrosome separation. Thus, γ-Tub at reduced levels and also astral microtubules cannot be detected, embryonic cnnβ1 centrosomes have insufficient or inappropriate microtubule assembly activity to achieve centrosome separation (Zhang, 2007).

It has been shown by live imaging of GFP-Cnn embryos that centrosomal satellites are highly dynamic structures that traffic in a microtubule-dependent and an actin-independent manner. Satellites, or 'flares,' emerge from the PCM and move bidirectionally at speeds of 4-20 µm min-1 and are produced at highest numbers at telophase/interphase, coincident with the relative intensity of astral microtubules during the cleavage cycle. cnnβ1 mutant embryos produce significantly fewer satellites. Even incipient satellites, which are apparent on cnnWT centrosomes and are present at colchicine-treated centrosomes, were nearly absent at cnnβ1 centrosomes. Satellite assembly may be an intrinsic function for Motif 1. Alternatively, fewer satellites may arise as a secondary consequence of altered MTOC activity at cnnβ1 centrosomes. Currently, it is not possible to distinguish between these two possibilities (Zhang, 2007).

The organization of actin into pseudocleavage furrows, an activity conveyed by centrosomes, is highly aberrant yet partially restored in cnnβ1 mutant embryos. This is in sharp contrast to cnn null embryos, where no apparent organization of cortical actin occurs. Although some studies have indicated that microtubules are required for cortical actin organization in the early Drosophila embryo, other evidence suggests that centrosomes organize actin and cortical polarity independent of microtubules. Because microtubule-dependent processes are disrupted in cnnβ1 embryos, the data support the model that centrosomes can organize actin independent of microtubules, but the possibility that cnnβ1 centrosomes produce sufficient astral microtubules to coordinate with actin in the assembly of furrows cannot be excluded (Zhang, 2007).

In summary, Motif 1, conserved among Cnn family members, is required for centrosome function in early embryos through the recruitment and anchoring of γ-Tub, D-TACC, and Msps, key factors in MTOC function in all eukaryotes where they have been examined. PCM architecture is partially restored in the cnnβ1 mutant compared with the cnn null, as shown by the normal distribution of CP60 and Aurora A. In addition, conspicuous yet aberrant pseudocleavage furrows assemble in cnnβ1 embryos but not in the cnn null, evidence that organization of actin by centrosomes is partially restored to cnnβ1 mutant centrosomes. This suggests that the activity to direct actin organization into cleavage furrows resides in another domain of Cnn. Identification of the direct binding partner for Cnn Motif 1 will be an important step toward understanding the relationship between Motif 1 and the MTOC functions that it governs (Zhang, 2007).

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).

Regulation of microtubule assembly and organization in mitosis by the AAA+ ATPase Pontin

To identify novel proteins important for microtubule assembly in mitosis, a centrosome-based complementation assay was used to enrich for proteins with mitotic functions. An RNA interference (RNAi)-based screen of these proteins uncover 13 novel mitotic regulators. In-depth analyses was carried out of one of these proteins, Pontin, which is known to have several functions in interphase, including chromatin remodeling, DNA repair, and transcription. Reduction of Pontin by RNAi resulted in defects in spindle assembly in Drosophila S2 cells and in several mammalian tissue culture cell lines. Further characterization of Pontin in Xenopus egg extracts demonstrates that Pontin interacts with the γ tubulin ring complex (γ-TuRC). Because depletion of Pontin leads to defects in the assembly and organization of microtubule arrays in egg extracts, these studies suggest that Pontin has a mitosis-specific function in regulating microtubule assembly (Ducat, 2008).

This study identified both Pontin and Reptin, two proteins with well-established functions in chromatin remodeling, as being present in the centrosome-complementing fraction. Using Xenopus egg extract, both Pontin and Reptin were shown to interact with γTuRC, and they are required for the egg extract to nucleate and organize robust microtubule arrays. This suggests that both proteins have a mitosis-specific function in regulating the microtubule cytoskeleton. Consistent with this, reduction of Pontin in tissue culture cells resulted in increased spindle defects. However, it is thought likely that not all of the mitotic defects observed in Pontin RNAi-treated cells were solely due to deregulation of microtubules in mitosis. For example, the mitotic cell death phenotype observed cannot be simply explained by spindle defects alone, because defective spindle assembly often triggers the spindle checkpoint, typically leading to a more prolonged mitotic arrest in the cell lines used. Mitotic death (also referred to as mitotic catastrophe) can result from imbalanced transcription of apoptotic regulators or from irresolvable DNA damage. It has been shown that reduction of RanBP1, a regulator of the Ran system, also causes mitotic cell death, likely due to combined defects in mitotic spindle assembly and spindle checkpoint signaling. Because Pontin is a component of many interphase complexes, including those involved in chromatin function, it is suggested that the mitotic cell death phenotype could be caused by a combination of cellular defects, including defects in spindle formation and chromatin organization due to lack of Pontin. In this context, it will be interesting to analyze whether Pontin reduction results in DNA damage or disorganization of centromeric DNA (which could affect kinetochore functions), and whether this contributes to the observed mitotic cell death (Ducat, 2008).

Interestingly, whereas RNAi-mediated depletion of Pontin in Drosophila and in different human cell lines caused defective spindle assembly and mitotic cell death, similar reduction of Reptin did not affect mitosis. However, Reptin depletion was able to enhance the mitotic defects observed after Pontin knockdown. One possible explanation is that Reptin functions together with Pontin to regulate microtubule assembly, but the different functions that Pontin and Reptin perform in interphase could result in different mitotic phenotypes. Consistent with this, although Pontin and Reptin are related ATPases and they are found together in several chromatin remodeling complexes, they do not always function in the same manner in interphase. In fact, expression levels of these proteins are not always similar, and certain protein complexes contain only one of these proteins. Similarly, it has been shown that overexpression of Pontin in tissue culture cells can displace Reptin from the transcription factor c-Myc. Furthermore, Pontin and Reptin oppose one another to control transcription in the β-catenin-TCF pathway in both Drosophila and zebrafish. Therefore, knockdown of Reptin alone might affect Pontin-independent pathways in interphase that obscure any mitotic phenotype or preclude entry into mitosis. Alternatively, whereas both Pontin and Reptin are needed for the full rescue of microtubule assembly in Xenopus egg extracts, it is possible that the mitotic function of Reptin in assisting Pontin assembly is masked by other proteins in tissue culture cells. Currently, these two possibilities cannot be distinguished (Ducat, 2008).

How might Pontin and Reptin regulate microtubule assembly? Both Pontin and Reptin are members of the AAA+ family of ATPases, which are typically involved in regulating protein-protein and protein-DNA interactions in many cellular contexts. Consistent with a general role in assembly of diverse cellular complexes, Pontin and Reptin have been shown to transiently associate with U3 box C/D pre-small nucleolar ribonucleoproteins and the Ino80 chromatin remodeling complex in a manner that is essential for the full assembly of each complex. Interestingly, Pontin and Reptin are also rapidly up-regulated in response to flagellar reassembly in Chlamydomonas, a process that strongly increases expression of many microtubule-related factors, heat-shock proteins, and tubulin chaperones. In this context, it is tempting to speculate that Pontin, and possibly Reptin, may function as chaperones to facilitate microtubule assembly by transiently interacting with γTuRC. Such a chaperone function could facilitate the localization of γTuRC to both spindle poles and along spindle microtubules (Ducat, 2008).

Maelstrom coordinates microtubule organization during Drosophila oogenesis through interaction with components of the MTOC

The establishment of body axes in multicellular organisms requires accurate control of microtubule polarization. Mutations in Drosophila PIWI-interacting RNA (piRNA) pathway genes often disrupt the axes of the oocyte. This results from the activation of the DNA damage checkpoint factor Checkpoint kinase 2 (Chk2) due to transposon derepression. A piRNA pathway gene, maelstrom (mael), is critical for the establishment of oocyte polarity in the developing egg chamber during Drosophila oogenesis. Mael forms complexes with microtubule-organizing center (MTOC) components, including Centrosomin, Mini spindles, and γTubulin. Mael colocalizes with αTubulin and γTubulin to centrosomes in dividing cyst cells and follicle cells. MTOC components mislocalize in mael mutant germarium and egg chambers, leading to centrosome migration defects. During oogenesis, the loss of mael affects oocyte determination and induces egg chamber fusion. Finally, this study shows that the axis specification defects in mael mutants are not suppressed by a mutation in mnk, which encodes a Chk2 homolog. These findings suggest a model in which Mael serves as a platform that nucleates other MTOC components to form a functional MTOC in early oocyte development, which is independent of Chk2 activation and DNA damage signaling (Sato, 2011).

In this study, it was shown that Mael is an MTOC component and that dynamic organization of MTs does not occur in developing mael oocytes, which correlates with mislocalization of other MTOC components. It was also observed that loss of mael affects the number and position of the oocytes in egg chambers and induces fusion of egg chambers. These results indicate that Mael specifically regulates MTOC formation, and thereby plays a key role in coordinating dynamic MT organization during Drosophila oogenesis (Sato, 2011).

Initial polarization of the oocyte during the oocyte specification phase in the germarium requires replacement of the fusome by a polarized MT network, which correlates with the formation of the MTOC. Mael is concentrated in the centrosomal region and is colocalized with αTub and γTub during cyst cell divisions. γTub does not migrate to a developing oocyte in mael germariums, suggesting that Mael is required for the migration of centrioles from the cytoplasm of cysts to pro-oocytes in the germarium. Currently, the detailed mechanism by which Mael functions in MT organization is not clear. The simplest hypothesis is that Mael might serve as a platform that nucleates other MTOC components to form a functional MTOC. A previous report has shown that weaker mutant alleles of γTub affect the number of nurse cells and oocytes within the egg chamber. These γTub mutant defects are very similar to those found in mael mutants in this study. γTub is involved in the nucleation of MTs and is present in the centrosomes and MTOCs in many different systems. It was hypothesized that reduced activity of γTub could activate the oocyte determination program in one of the nurse cells by ectopically presenting MTOC material. The findings that γTub does not accumulate at centrosomes in the mael germarium and is ectopically expressed in the mael egg chamber suggest that Mael regulates localization of γTub at centrosomes through its complex formation and is thereby involved in properly organizing or positioning the MTOC (Sato, 2011).

PIWI proteins function in transposon silencing via association with piRNAs and maintain genome integrity during germline development. Recent studies have suggested that PIWI proteins in sea urchin (Seawi) and Xenopus (Xiwi) can interact with the MTs of the meiotic spindle, while fly ovarioles with mutations in any of several piRNA pathway genes, including spn-E, aub, and armi, have disorganized MTs. This raises the possibility for either a functional role of PIWI proteins in the machinery that impacts on MT organization (in addition to transposon silencing), a role of the MT cytoskeleton in piRNA generation, or both. The current findings further corroborate a link between components of the piRNA pathway and proper MT organization. Although it was found that Mael forms a complex with MTOC components, components of the piRNA pathway in this complex could not be identified. This is in contrast to observations of the mouse Mael homolog, which functions in the piRNA pathway (similar to fly Mael) and interacts with mouse PIWI proteins in the testes (Costa, 2006). Mouse Mael in the testes is almost exclusively cytoplasmic with accumulation at nuage (Soper, 2008). In contrast, fly Mael is located in both the nucleus and the cytoplasm in the ovary and is known to shuttle between them. Thus, one possibility is that in fly ovaries, there may exist nuclear Mael complexes involved in both piRNA generation and transposon silencing, which are distinct from the cytoplasmic complex containing MTOC components that were identified in this study (Sato, 2011).

Female flies with mutations in several genes in the piRNA pathway often lay eggs with axis patterning defects because of MT cytoskeletal changes that result in the mislocalization of bic, grk, and osk mRNAs within the egg chamber. These defects have been linked to the Chk2 DNA damage checkpoint that may be activated by increased retrotransposon transcript levels in mutants defective in piRNA biogenesis. However, because a mutation in mnk does not suppress the mislocalization of Osk and Grk in the mael oocyte, the axis specification defect of mael oocytes does not appear to be triggered by the activation of germline-specific DNA breaks and damage signaling through Chk2. In addition, a mutation in the mei-W68 locus, which encodes the Drosophila Spo11 homolog and induces meiotic double-strand breaks in chromosomes, cannot suppress the axis specification defect of mael oocytes. Therefore, these results suggest that the axis specification defects of mael oocytes are not a secondary consequence of DNA damage signaling. However, it has been shown that in mael mutant ovaries, Vas is post-translationally modified. These results together imply that, acting not only through Chk2, the functions of Mael in MT organization are in parallel with its function in piRNA generation and transposon silencing. There are mutants—including zuc and spn-E that are piRNA pathway genes; their axis defects cannot be rescued by mnk mutations. Vas also appears modified in these mutants, although the relationship with activated checkpoint-modified Vas is unclear (Sato, 2011).

Given that Mael is a new component of the MTOC in the Drosophila ovary, identification of a domain within Mael that is responsible for binding to other MTOC components could aid in understanding how Mael nucleates and regulates MTOC formation. Because Mael contains an evolutionarily highly conserved domain of unknown function, termed the Mael domain (Zhang, 2008), determination of its crystal structure should prove valuable in elucidating mechanisms of both MTOC formation and piRNA generation processes (Sato, 2011).

Synergy between multiple microtubule-generating pathways confers robustness to centrosome-driven mitotic spindle formation

The mitotic spindle is defined by its organized, bipolar mass of microtubules, which drive chromosome alignment and segregation. Although different cells have been shown to use different molecular pathways to generate the microtubules required for spindle formation, how these pathways are coordinated within a single cell is poorly understood. This study has tested the limits within which the Drosophila embryonic spindle forms, disrupting the inherent temporal control that overlays mitotic microtubule generation, interfering with the molecular mechanism that generates new microtubules from preexisting ones, and disrupting the spatial relationship between microtubule nucleation and the usually dominant centrosome. This work uncovers the possible routes to spindle formation in embryos and establishes the central role of Augmin, an eight-subunit complex that increases the number of spindle MTs, apparently by binding to preexisting MTs and recruiting γ-TuRC, in all microtubule-generating pathways. It also demonstrates that the contributions of each pathway to spindle formation are integrated, highlighting the remarkable flexibility with which cells can respond to perturbations that limit their capacity to generate microtubules. Therefore clear evidence has been provided that all three pathways to spindle formation (centrosomal, chromatin, and acentrosomal MT organizing center driven are dependent on a fourth: Augmin. Together with recent work demonstrating that new MTs can be produced in an Augmin-dependent manner using preexisting ones generated in vitro in Xenopus egg extracts, this evidence suggests that this conserved protein complex, once active, works on all existing mitotic MTs (Hayward, 2014).

The mitotic spindle is defined by its organized, bipolar mass of microtubules, which drive chromosome alignment and segregation. Although different cells have been shown to use different molecular pathways to generate the microtubules required for spindle formation, how these pathways are coordinated within a single cell is poorly understood. This study has tested the limits within which the Drosophila embryonic spindle forms, disrupting the inherent temporal control that overlays mitotic microtubule generation, interfering with the molecular mechanism that generates new microtubules from preexisting ones, and disrupting the spatial relationship between microtubule nucleation and the usually dominant centrosome. This work uncovers the possible routes to spindle formation in embryos and establishes the central role of Augmin, an eight-subunit complex that increases the number of spindle MTs, apparently by binding to preexisting MTs and recruiting γ-TuRC, in all microtubule-generating pathways. It also demonstrates that the contributions of each pathway to spindle formation are integrated, highlighting the remarkable flexibility with which cells can respond to perturbations that limit their capacity to generate microtubules. Therefore clear evidence has been provided that all three pathways to spindle formation (centrosomal, chromatin, and acentrosomal MT organizing center driven are dependent on a fourth: Augmin. Together with recent work demonstrating that new MTs can be produced in an Augmin-dependent manner using preexisting ones generated in vitro in Xenopus egg extracts, this evidence suggests that this conserved protein complex, once active, works on all existing mitotic MTs (Hayward, 2014).

During mitosis, microtubules (MTs), dynamic polymers of α and β tubulin, are nucleated in sufficient number so that they form a bipolar spindle apparatus, generating the force required for accurate alignment and segregation of duplicated chromosomes. Work in different model organisms has shown that the route to spindle formation can vary; for example, the MTs that constitute spindles in Xenopus egg extracts are initially nucleated by condensed chromatin, the assembly of the Drosophila early embryonic spindle is regarded as centrosome directed, while mammalian oocytes generate MTs in the cytoplasm that gradually coalesce to bipolarity In addition, MT-dependent MT generation, catalyzed by the Augmin complex, provides an additional pathway that contributes to overall spindle MT density (Goshima, 2007; Goshima, 2008; Uehara, 2009; Wainman, 2009). Given that most animal mitotic cells possess centrosomes and chromatin within a substantial cytoplasm and that Augmin is functionally conserved, one important question is whether all individual MT-generating pathways coexist in a single cell. If they do, and if their functions are integrated, it could explain why mature spindles are so robust when challenged with physical, genetic, and chemical perturbations (Hayward, 2014).

Although previous research has addressed the relationship between centrosomal and chromatin-generated MTs these studies were undertaken prior to discovery of Augmin (Goshima, 2007), and in tissue culture cells. The relationship between all the major defined MT generating pathways and their significance within a developmental context therefore remains unclear. This study used the Drosophila syncytial blastoderm embryo in order to comprehensively address how a mitotic spindle forms. This tissue, in which many hundreds of mitotic spindles form simultaneously in a common cytoplasm, allows manipulation of MT-generating pathways not only through genetics but also through immediate inactivation of proteins facilitated by interfering antibody injections. These advantages were combined with a live cold-treatment assay that allows mature embryonic mitotic spindles to be deconstructed and rebuilt and with the development and implementation of image analysis software that allows quantitative data to be extracted simultaneously from multiple spindles (Hayward, 2014).

The results demonstrate that MTs can be generated in this system by mitotic chromatin, in addition to centrosomes, using a molecular pathway dependent on the Drosophila homolog of the spindle assembly factor, HURP (FlyBase name: Mars). By disrupting the accumulation of two pericentriolar material (PCM) proteins, Spindle defective 2 (DSpd-2) and Centrosomin (Cnn), to the centrosome, it was also found that Drosophila embryos can form bipolar spindles from multiple cytosolic acentrosomal MT organizing centers (aMTOCs). This study shows that all these routes to spindle formation are supplemented by Augmin-generated MTs; inactivation of Augmin abrogates chromatin-generated and aMTOC-dependent MTs and substantially delays and reduces astral MT input. It was also demonstrated that integration does, indeed, exist between pathways. A reduction in centrosome-generated MTs leads to an increased rate of MT nucleation around chromatin, while a loss of chromatin- or Augmin-dependent MT nucleation increases the growth rate of remaining astral MTs. It was also shown that this effect is synergistic. Thus, mitotic MT generation in a cell within a developing organism comprises coordinated inputs from multiple MT-nucleating pathways, providing inherent robustness and flexibility to the mature mitotic spindle (Hayward, 2014).

These experiments demonstrate that the mature mitotic spindle in Drosophila embryos, far from being formed via a single MT-generating pathway dependent on centrosomally derived MTs, is composed of MTs whose origins are, or can be, diverse. By disrupting the molecular basis of the individual pathways, this study has assessed their relative contributions to spindle formation. In doing so, the underlying flexibility inherent within the system, in which removal of one MT-generating pathway causes the cell to respond by increasing its use of another, is demonstrated (Hayward, 2014).

The evidence supports a model in which normal, cycling embryonic centrosomes are preprimed with PCM and γ-Tubulin and exposed to α/β Tubulin dimer prior to the onset of mitosis so that they, together with amplification via Augmin, nucleate enough astral MTs to capture kinetochores quickly and efficiently within 30 s of NEB. In contrast, the chromatin, which is not exposed to α/β Tubulin dimer or Augmin until after NEB, cannot participate in MT generation to any significant extent. However, by reversing MT nucleation after mature spindle formation through cold treatment, 'rebooting' the system in midmitosis when both centrosomes and mitotic chromatin are equally exposed to Tubulin and Augmin, and quantitatively analyzing MT regrowth, it was shown that a chromatin-dependent pathway exists and, indeed, dominates over the centrosomes that are still present. This is not due to redistribution of γ-Tubulin from centrosomes, as γ-Tubulin-GFP intensity is not reduced at the centrosome in cold-treated embryos. Instead, it appears to be a consequence of sequestering and activating the SAF, D-HURP, around mitotic chromatin. Interestingly, in contrast to some other biological systems, including Drosophila S2 cells, the predominant site of new MT growth following cold treatment is not restricted to kinetochores but occurs throughout the region of the mitotic chromatin. Human HURP generates and stabilizes MTs in a Ran-dependent manner. In the Drosophila embryonic scenario, it is envisaged that cold treatment of mitotic embryos after NEB leads to cell cycle arrest in which mitotic kinases and the Ran gradient are fully active, allowing the association of D-HURP with condensed chromatin where it nucleates short MT seeds. Subsequent removal of the temperature restriction will provide the necessary conditions for MT growth. That the chromatin-dependent pathway is also part of the normal complement of spindle-forming pathways in cycling embryos, but that its input is limited until later in mitosis, is supported by the observations that removal of D-HURP in cycling embryos results in shorter mature spindles that have a higher likelihood of failing in chromosome segregation (Hayward, 2014).

In addition to astral and chromatin-dependent MT generation, this study has revealed an alternative pathway to spindle formation. A failure to stably incorporate either DSpd-2 or Cnn to the centrosome results in cytosolic MT asters that coalesce into mature bipolar spindles. These aMTOCs are quite distinct from chromatin- dependent MTs, appearing within 10 s following NEB in regions of the cytoplasm devoid of chromosomes, and are qualitatively similar to those reported for acentriolar Drosophila cell lines (Moutinho-Pereira, 2009) and mouse oocytes. They may, therefore, reflect a general mechanism of animal cell spindle formation in the absence of functioning centrosomes, where the nucleation and organization of MTs are achieved through concentration of nucleating activity at multiple cytosolic sites and bipolarity follows through their interaction and self-organization (Hayward, 2014).

This study has also provided clear evidence that all three pathways to spindle formation (centrosomal, chromatin, and aMTOC-driven) are dependent on a fourth: Augmin. Together with recent work demonstrating that new MTs can be produced in an Augmin-dependent manner using preexisting ones generated in vitro in Xenopus egg extracts (Petry, 2013), the current evidence suggests that this conserved protein complex, once active, works on all existing mitotic MTs. However, whereas in Xenopus extracts, TPX2 is required for Augmin-generated MTs (Petry, 2013), the current in vivo analysis of spindle formation in the absence of either D-TPX2 (using mei-38 null mutants) or D-HURP supports a model in which D-HURP is the dominant chromatin-directed MT nucleator in Drosophila embryos, generating MT seeds that can then be amplified by Augmin. This likely reflects either a difference in function between the Drosophila and Xenopus proteins-for example, D-TPX2 shares homology with TPX2 only in its C-terminal domain and does not possess elements such as Aurora A targeting (Goshima, 2011), or a difference in the usage of TPX2- and HURP-dependent pathways by different biological systems (Hayward, 2014).

The current work also demonstrates that astral MT nucleation is dramatically reduced in cycling embryos lacking Augmin, in a D-TPX2- and D-HURP-independent manner. Under these conditions, the remaining astral MTs are eventually able to search and capture kinetochores, producing kinetochore-kMT interactions that allow Rod poleward streaming. However, the spindle assembly checkpoint remains unsatisfied, and Rough deal Rod-GFP movement is perturbed, suggesting that some aspect of the interaction is incorrect. One possibility is that Augmin binds to and amplifies the initial kMTs, resulting in stable kMT bundles that can stream Rod poleward. Alternatively, the effect on the checkpoint may reflect the requirement of Augmin for generating many short non-kMT spindle MTs. It has recently been demonstrated that the viscoelastic properties of the Xenopus spindle, and its ability to transmit force as a unit, can be altered by reducing the density of such short, non-kMTs (Hayward, 2014).

It is therefore possible that Augmin-dependent non-kMTs transmit force exerted on individual kinetochores by kMTs throughout the spindle as part of a spindle-scale sensing mechanism, intrinsically linked to the checkpoint. Whatever the molecular mechanism at work, this study supports a model in which Augmin binds indiscriminately to preexisting MTs to generate the bulk of the embryonic mitotic spindle, placing Augmin at the heart of MT generation during spindle formation. Interestingly, this scenario was predicted by mathematical models of Drosophila embryonic spindle organization, generated approximately 10 years ago. In order for their model to recapitulate the dynamics of the anaphase spindle, the authors required the presence of multiple short MTs with origins that were distinct from centrosomes. Augmin fulfills such a role and, as such, incorporating its precise mode of action into future models of Drosophila spindle dynamics may well reveal additional features of spindle formation (Hayward, 2014).

Although clearly essential for robust spindle formation in mitotic systems, Augmin and, indeed, γ-Tubulin have been shown to be dispensable for the bulk of MTs that form the initial Drosophila female meiosis I spindle (Colombie, 2013; Hughes, 2011). Instead, the Drosophila oocyte appears to rely on the Chromosomal Passenger Complex (CPC), the MT-stabilizing protein Minispindles, and the crosslinking motor Subito to organize stable cytoplasmic MTs, generated prior to meiosis onset, into an initial spindle structure. This likely reflects the peculiarity of the pathways regulating formation of the meiotic spindle in this system. Nonetheless, it does suggest yet additional mechanisms by which a bipolar spindle can form, further highlighting the robustness of this structure (Hayward, 2014).

Finally, importantly, this study has shown that a reduction of astral input to spindle formation leads to an increase in chromatin-dependent MT generation, while removal of chromatin- or Augmin-dependent MT generation results in an increased accumulation of EB1-GFP at MT plus ends and an increase in the growth rate of the remaining astral MTs. The effect on EB1-GFP accumulation is accentuated if the number of remaining astral MTs is further reduced. These results suggest a coordinated and synergistic cellular response to perturbing mitotic MT-generating pathways. It is not known whether the increase in astral MT dynamics is a passive response to availability of resources, such as Tubulin.GTP dimer, or an active self-regulation, driven by monitoring of MT generation by the cell. In the simplest (passive) scenario, removing the MTs generated by one pathway could result in an increase in the available local concentration of Tubulin.GTP, shifting the dynamic equilibrium of remaining MTs further toward growth. Although in vitro studies have shown that increasing the concentration of Tubulin in solution increases MT growth rates and that this correlates with increased accumulation of EB1 at the growing tips, the high diffusion rate of Tubulin-GFP in the early embryo essentially rules out local depletion of resources close to individual MT tips as a source of variability. Therefore, if resource depletion is responsible for limiting MT growth, it must be a global (spindle-scale) depletion. However, the increase in EB1 comet length and MT dynamics that occurs upon the loss of MT-generating pathways was measured in the early stages of spindle regrowth. At similar time points in embryos possessing all MT-generating pathways, the EB1 fluorescence (i.e., MT growth) in the region of the chromatin continues to increase dramatically over the following 2 min. Therefore, at these early time points, Tubulin.GTP, EB1, or any other cytoplasmic molecule that stimulates MT growth, cannot be depleted and therefore cannot be limiting growth. This leaves open the intriguing possibility that the cell somehow actively monitors the overall level of MT generation during spindle formation and alters flow through available pathways accordingly. Given this possibility, an important future goal will be to identify MAPs whose association with MTs changes upon inhibition of particular MT-generating pathways (Hayward, 2014).

In summary, by revealing the presence of all the major mitotic MT-generating pathways described in animal cells within a single system, the Drosophila syncytial embryo, and by demonstrating a coordinated regulation between them, this work highlights the remarkable flexibility inherent in mitotic spindle formation. It implies that the key to building a successful spindle lies in activating a set of MT generators that together provide sufficient MTs to allow crosslinking and movement in relation to one another, regardless of how and where the MTs were initially generated. By subsequently limiting the nucleation and growth of these MTs to balance depolymerization, a steady-state spindle of defined length and physical properties is ultimately formed. Understanding the way in which a cell determines such a 'Goldilocks zone' of MT generation will undoubtedly help lead to an understanding of the overall self-regulation of this fundamental cellular structure (Hayward, 2014).

gammaTubulin at 23C: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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