gammaTubulin at 23C



During early embryogenesis of Drosophila, mutations in the DNA-replication checkpoint lead to chromosome-segregation failures. These segregation failures are associated with the assembly of an anastral microtubule spindle, a mitosis-specific loss of centrosome function, and dissociation of several components of the gamma-tubulin ring complex from a core centrosomal structure. The DNA-replication inhibitor aphidicolin and DNA-damaging agents trigger identical mitotic defects in wild-type embryos, indicating that centrosome inactivation is a checkpoint-independent and mitosis-specific response to damaged or incompletely replicated DNA. It is proposed that centrosome inactivation is part of a damage-control system that blocks chromosome segregation when replication/damage checkpoint control fails (Sibon, 2000).

It has been proposed that a component of the DNA-synthesis machinery becomes rate-limiting during the later syncytial divisions, and that activation of the DNA-replication checkpoint thus produces the increases in embryonic cell-cycle length that characterize these divisions. This model predicts that checkpoint-mutant embryos will initiate mitosis before completing DNA replication specifically during the later syncytial divisions, when the mitotic defects first appear. It was therefore speculated that the centrosome defects and segregation failures in checkpoint-mutant embryos resulted from incomplete DNA replication at mitosis, and that the checkpoint pathway is not specifically required to maintain centrosome function. In wild-type embryos, the DNA-replication inhibitor aphidicolin triggers cell-cycle delays, but mitosis is eventually initiated before S phase is completed. Thus, to test the hypothesis, mitosis was examined following treatment of wild-type embryos with aphidicolin. Under these conditions, gamma-tubulin, and the gamma-tubulin ring complex components Dgrip84 and Dgrip91 are displaced from a core centrosome structure. Aphidicolin triggers mitosis-specific loss of centrosomal gamma-tubulin, Dgrip84 and Dgrip91 during all of the syncytial divisions, in both wild type and checkpoint mutants. This structural response to aphidicolin is therefore replication-checkpoint-independent and is not specific to the later syncytial divisions. This structural response to aphidicoline occurs in grp and mei-41 mutant embryos, and is therefore replication-checkpoint-independent (Sibon, 2000).

To analyse the effects of aphidicolin on spindle assembly and chromosome segregation directly, embryos were injected with a mixture of aphidicolin, rhodamine-labelled tubulin and the DNA label Oligreen and were examined by time-lapse confocal microscopy. The results showed that aphidicolin leads to loss of tubulin foci at nuclear envelope breakdown (NEB), assembly of anastral spindles, and defects in chromosome congression and segregation. Furthermore, the centrosomal foci are re-established on exit from mitosis. Thus aphidicolin treatment accurately phenocopies the mitotic defects observed in checkpoint mutants, indicating that centrosome inactivation and the associated chromosome-segregation failures in these mutants are probably triggered by the presence of incompletely replicated DNA at mitosis (Sibon, 2000).

To determine whether photodamage to DNA also triggers centrosome inactivation and chromosome-segregation defects, laser illumination was used to induce DNA damage. Bipolar spindles are established shortly after NEB, at the time that microtubules originating at the poles interact with each other and with the mitotic chromosomes, and chromosomes rapidly congress to the metaphase plate. During mitosis 12, anaphase is initiated roughly 4 min after NEB, and chromosome segregation is completed and the nuclear envelope begins to reform ~3 min later. However, when the intensity of laser illumination is increased to 90% of maximum, the centrosomal tubulin foci decrease in intensity at NEB and disorganized microtubule bundles form around the condensed chromosomes. Bipolar spindles with broad poles eventually assemble, but the chromosome arms never fully compact at the metaphase plate and chromosome segregation fails. On intense laser illumination in checkpoint mutants and in wild-type embryos, small tubulin-containing dots are often found near poles of the anastral spindles. These structures do not appear to nucleate microtubules during mitosis, but gain nucleating function on exit from mitosis. These structures thus appear to be centrosomes that are inactivated at NEB and reactivated on exit from mitosis. X-ray treatments and ultraviolet light also trigger mitosis-specific loss of centrosomal gamma-tubulin, anastral spindle assembly, and defects in chromosome congression and segregation. On the basis of these observations, it is concluded that a variety of DNA-damaging agents and replication defects can trigger mitosis-specific centrosome inactivation and mitotic chromosome-segregation defects (Sibon, 2000).

Thus it has been shown that the chromosome-segregation defects in grp and mei-41 checkpoint-mutant embryos are linked to loss of centrosome function and dissociation of several components of the gamma-tubulin ring complex from a core centrosomal structure. However, this does not reflect a specific requirement for grp or mei-41 in maintaining centrosome function. The alleles analysed here are null, yet the mutant embryos proceed through 11-12 normal mitotic divisions before mitotic defects are observed. In addition, replication inhibitors or DNA-damaging agents trigger cytologically identical centrosome defects in wild-type embryos. It is therefore concluded that the loss of centrosome function and mitotic failures in grp and mei-41 mutants reflect activation of a replication-checkpoint-independent response to incomplete DNA replication or DNA damage at mitosis (Sibon, 2000).

Incomplete DNA replication or DNA damage could lead to kinetochore defects, which trigger the spindle-assembly checkpoint; centrosome inactivation could be a response to activation of this checkpoint. However, the localization of gamma-tubulin to the centrosome is not significantly affected by treatment with colchicine, which triggers metaphase arrest through the spindle-assembly checkpoint pathway. In addition, centrosome inactivation does not block centromere alignment, indicating that kinetochore function is not severely compromised when the centrosomes are inactivated. Therefore, DNA-replication- and DNA-damage-associated centrosome inactivation are both independent of the replication and the spindle checkpoints, and appear to reflect the action of a new pathway that links centrosome function to the physical state of the genome (Sibon, 2000).

Mitotic spindle assembly is normally dominated by astral microtubules, which are nucleated at centrosomes. The chromosome-segregation defects in checkpoint mutants and in wild-type embryos treated with aphidicolin or DNA-damaging agents are tightly linked to loss of centrosome-organized microtubules. These observations indicate that asters may be required for spindle function. However, anastral spindles are common during female meiosis and are found in some unusual mitotic systems and cell-free extracts. In addition, spindles in Drosophila embryos mutant for cnn have severely reduced asters, and these anastral spindles may mediate chromosome segregation. Therefore, DNA replication or damage could induce centrosome inactivation and modifications to other functions that are essential for anastral spindle function (Sibon, 2000).

Whacked and Rab35 polarize dynein-motor-complex-dependent seamless tube growth

Seamless tubes form intracellularly without cell-cell or autocellular junctions (see Labarsky, 2003). Such tubes have been described across phyla, but remain mysterious despite their simple architecture. In Drosophila, seamless tubes are found within tracheal terminal cells, which have dozens of branched protrusions extending hundreds of micrometres. This study has found that mutations in multiple components of the dynein motor complex block seamless tube growth, raising the possibility that the lumenal membrane forms through minus-end-directed transport of apical membrane components along microtubules. Growth of seamless tubes is polarized along the proximodistal axis by Rab35 and its apical membrane-localized GAP, Whacked. Strikingly, loss of whacked (or constitutive activation of Rab35) leads to tube overgrowth at terminal cell branch tips, whereas overexpression of Whacked (or dominant-negative Rab35) causes formation of ectopic tubes surrounding the terminal cell nucleus. Thus, vesicle trafficking has key roles in making and shaping seamless tubes (Schottenfeld-Roames, 2013).

Three tube types -- multicellular, autocellular and seamless -- are found in the Drosophila trachea. Most tracheal cells contribute to multicellular tubes or make themselves into unicellular tubes by wrapping around a lumenal space and forming autocellular adherens junctions, but two specialized tracheal cell types, fusion cells and terminal cells, make 'seamless' tubes. How seamless tubes are made and how they are shaped are largely unknown. One hypothesis holds that seamless tubes are built by 'cell hollowing', in which vesicles traffic to the centre of the cell and fuse to form an internal tube of apical membrane, whereas an alternative model proposes that apical membrane is extended internally from the site of intercellular adhesion. In both models, transport of apical membrane would probably play a key role. As terminal cells make seamless tubes continuously during larval life, they serve as an especially sensitive model system in which to dissect the genetic program (Schottenfeld-Roames, 2013).

Tracheal cells are initially organized into epithelial sacs with their apical surface facing the sac lumen. During tubulogenesis, γ-tubulin becomes localized to the lumenal membrane of each tracheal cell, generating microtubule networks oriented with minus ends towards the apical membrane. Terminal and fusion cells are first selected as tip cells that undergo a partial epithelial-to-mesenchymal transition and initiate branching morphogenesis: they lose all but one or two cell-cell contacts and become migratory. Branchless-FGF signalling induces a subpopulation of tip cells to differentiate as terminal cells. During larval life, terminal cells ramify on tissues spread across several hundred micrometres, with branching patterns that reflect local hypoxia. A single seamless tube forms within each branched extension of the terminal cell (Schottenfeld-Roames, 2013).

How trafficking contributes to seamless tube morphogenesis is unknown. Despite clues that vesicle transport plays a role in the genesis of seamless tubes, the tube morphogenesis genes remain elusive. This study characterized the cytoskeletal polarity of larval terminal cells, shows that a minus-end-directed microtubule motor complex is required for seamless tube growth, and characterizes mutations in whacked (wkd) that uncouple seamless tube growth from the normal spatial cues. Sequence analysis indicates that wkd encodes a RabGAP, and it was shown that Rab35 is the essential target of Wkd, and that together, Wkd and Rab35 can polarize the growth of seamless tubes (Schottenfeld-Roames, 2013).

Apical-basal polarity and cytoskeletal organization was examined in mature larval terminal cells. The lumenal membrane was decorated by puncta of Crumbs, a definitive apical membrane marker. Actin filaments were found enriched in three distinct subcellular domains: surrounding seamless tubes, decorating filopodia and outlining short stretches of basolateral membrane. The microtubule cytoskeleton also seemed polarized, with γ-tubulin lining the seamless tubes and enriched at tube tips. These data are consistent with tracheal studies in the embryo. EB1::GFP analyses of growing (plus-end) microtubules demonstrated that some are oriented towards the soma and others towards branch tips. Stable acetylated microtubules ran parallel to the tubes and extended beyond the lumen at branch tips where they may template tube growth. Consistent with such a role, microtubule-tract-associated fragments of apical membrane were observed distal to the blind ends of the seamless tubes. Filopodia extended past the stable microtubules as expected. These data indicate that mature terminal cells maintain the polarity and organization described for embryonic terminal cells. On the basis of γ-tubulin localization, it is inferred that a subset of microtubules is nucleated at the apical membrane, and that apically targeted transport along such microtubules would require minus-end motor proteins. Indeed, homozygous mutant Lissencephaly-1. As γ-tubulin lines the entire apical membrane, growth through minus-end-directed transport might be expected to occur all along the length of seamless tubes, and indeed, a pulse of CD8::GFP (transmembrane protein tagged with GFP) synthesis uniformly labelled the apical membrane as it first became detectable (Schottenfeld-Roames, 2013).

The cytoplasmic dynein motor complex drives minus-end-directed transport of intracellular vesicles in many cell types; to test for its requirement in seamless tube formation, terminal cells were examined mutant for any of four dynein motor complex genes: Dynein heavy chain 64C (Dhc64C), Dynein light intermediate chain (dlic), Dynactin p150 (Glued) and Lis-1. Mutant terminal cells showed a cell autonomous requirement for these genes. Mutant terminal cells had thin cytoplasmic branches that lacked air-filling, and antibody staining revealed that seamless tubes did not extend into these branches although acetylated microtubules often did. It was also noted that formation of filopodia at branch tips is disrupted in dynein motor complex mutants, which may account for the decreased number of branches in mutant terminal cells. Ectopic seamless tubes that were not air-filled were detected near the nucleus, as described below. Interestingly, discontinuous apical membrane fragments (similar to those in Lis-1 embryos) were found in terminal branches lacking seamless tubes, and were associated with microtubule tracts. Whereas γ-tubulin was enriched on truncated tubes and on these presumptive seamless tube intermediates, diffuse γ-tubulin staining was detected throughout the mutant cells, indicating that assembly of apical membrane is required to establish or maintain γ-tubulin localization. Likewise, Crumbs seemed reduced and aberrantly localized. Reduced levels of acetylated microtubule staining in these cells may reflect loss of apical γ-tubulin. Importantly, these data show that stable microtubules extend through cellular projections that lack seamless tubes. Thus, without minus-end-directed transport, stable microtubules are insufficient to promote seamless tube formation, but stable cellular projections are formed and maintained in the absence of seamless tubes (Schottenfeld-Roames, 2013).

In contrast to these defects in seamless tube generation, mutations in wkd confer overly exuberant tube growth. Examination of wkd terminal cell tips revealed a 'U-turn'; phenotype in which seamless tubes executed a series of 180 degree turns - the possibility is entertained that branch retraction, similar to that observed in talin mutants, could contribute to the U-turn defect (Schottenfeld-Roames, 2013).

Homozygous wkd animals survived until pharate adult stages, and, other than the seamless tube defects, had normal tracheal tubes at the third larval instar. Mosaic analysis revealed a terminal cell autonomous requirement for wkd. Mutant clones in multicellular tubes, and in unicellular tubes that lumenize by making autocellular adherens junctions, were of normal morphology. Strikingly, fusion cells, which also form seamless tubes, were unaffected by loss of wkd (Schottenfeld-Roames, 2013).

To determine the molecular nature of wkd, a positional cloning approach was taken. Mapping techniques defined a candidate gene interval of ~ 75 kilobases (kb). Focused was placed on CG5344 as it encodes a protein containing a TBC (Tre2/Bub2/Cdc16) domain characteristic of Rab GTPase-activating proteins, and hence was likely to participate in vesicular trafficking, a process that could lie at the heart of seamless tube formation. A single nucleotide change was identified that resulted in mis-sense (PC24) and non-sense (220) mutations in the CG5344 coding sequence. Pan-tracheal knockdown of wkd by RNA-mediated interference (RNAi) caused terminal cell-specific U-turn defects (other defects characteristic of the ethyl methanesulfonate (EMS)-induced alleles of wkd were detected at a low frequency). A genomic rescue construct for CG5344 rescued wkd mutants, confirming gene identity. On the basis of these results, it is concluded that wkd is CG5344 and that it probably regulates vesicular trafficking during seamless tube morphogenesis (Schottenfeld-Roames, 2013).

To determine the Rab target(s) of Wkd regulation, whether tracheal expression of constitutively active 'GTP-locked'; Rab isoforms (henceforth, RabCA) might phenocopy wkd was investigated. RabCA for 31 of the 33 Drosophila Rabs were tested individually in the tracheal system. Rab35CA alone conferred terminal-cell-specific U-turns defects (Schottenfeld-Roames, 2013).

To evaluate Wkd overexpression, UAS-wkd was expressed in wild-type animals in a pan-tracheal pattern. Excess Wkd caused formation of ectopic seamless tubes surrounding the terminal cell nucleus. At higher levels of expression, small spheres of apical membrane were found adjacent to the nucleus and less abundantly at more distal sites. Consistent with Wkd regulation of vesicle trafficking by modulation of Rab35, expression of a dominant-negative Rab35 (henceforth, Rab35DN) caused formation of ectopic proximal tubules (Schottenfeld-Roames, 2013).

Attempts were made to determine whether Rab35 was the essential target of Wkd GAP activity. Wkd primary structure is equally conserved in three human RabGAPs. All three act as Rab35GAPs, although each has been proposed to have additional targets. To further determine if Wkd acts as a Rab35GAP, whether Rab35DN could suppress wkd mutants was examined; tracheal-specific expression of Rab35DN strongly suppressed the 'U-turn'; defects of wkd-null animals and, surprisingly, also rescued the lethality of wkd. Since mutant Rab35 isoforms phenocopy wkd gain and loss of function, Rab35DN bypasses the requirement for wkd and human Wkd orthologues are Rab35GAPs, it is concluded that the critical function of Wkd is as a GAP for Drosophila Rab35 (Schottenfeld-Roames, 2013).

In other systems Rab35 is implicated in polarized membrane addition to plasma membrane compartments - for example, immune synapse, cytokinetic furrow and so on - or, in actin regulation. A role for actin in fusion cell seamless tube formation has been proposed, so whether Wkd and Rab35 act by modulation of the terminal cell actin cytoskeleton was examined. As the actin-bundling protein Fascin (Drosophila singed) was recently identified biochemically as a Rab35 effector, a role of singed in terminal cell tubes was examined, but found no evidence was found for one. Furthermore, overexpression of Wkd, or of Rab35DN, did not significantly alter the terminal cell actin cytoskeleton, leading to the conclusion that actin regulation is not a primary function of Wkd/Rab35 during seamless tube morphogenesis (Schottenfeld-Roames, 2013).

The alternative model -- that Rab35 acts in polarized membrane addition -- was found to be attractive, because extra Rab35-GTP activity promoted seamless tube growth at branch tips whereas depletion of Rab35-GTP promoted tube growth at the cell soma. To test this model, advantage was taken of the information that expression of an activated Breathless-FGFR (lambdaBtl in terminal cells induces robust growth of ectopic seamless tubes surrounding the nucleus; whether growth of the ectopic tubes could be redirected from the soma to the branch tips was investigated by eliminating wkd. The activated FGFR phenotype was not altered in wkd heterozygotes, but in wkd mutant animals (or wkd-RNAi animals) the site of ectopic seamless tube growth was strikingly different. In some cells, extra tubes were found throughout the cell - in the soma and at branch tip - whereas in others extra tubes were present only at the branch tip. Thus, the position of seamless tube growth is dependent on Wkd activity, although Wkd itself is not essential for tube formation. These data provide evidence against branch retraction (as occurs in talin mutants) as the mechanism for generating a U-turn phenotype, because branch retraction would not redirect ectopic tube growth (Schottenfeld-Roames, 2013).

To better understand how Wkd and Rab35 determined the site of seamless tube growth, their subcellular distribution was examined. Pan-tracheal expression of mKate2-tagged Wkd (Wkd::mKate2) rescued wkd-null animals. The steady-state subcellular localization of Wkd::mKate2 was restricted to the lumenal membrane with higher accumulation at the growing tips of seamless tubes. At lower levels, cytoplasmic puncta of Wkd::mKate2 were noted that could reflect vesicular localization, as well as labeling of filopodia. It was found that YFP::Rab35 was distributed in a diffuse pattern throughout the terminal cell cytoplasm with some apical enrichment, and notable localization to filopodia. Substantial co-localization of Wkd::mKate2with YFP::Rab35 was found at the apical membrane, in cytoplasmic puncta, and in filopodia. Among endosomal Rabs, Rab35 seemed uniquely abundant within filopodia, and showed the greatest overlap with Wkd at the apical membrane. Substantial overlap was noted between Wkd/Rab35 and acetylated microtubules, including at positions distal to the blind end of seamless tubes. The enrichment of Wkd along seamless tubes indicates that Rab35 functions in an apical membrane trafficking event, leading to the speculation that recycling endosomes at filopodia might be targeted to the growing seamless tube by minus-end motor transport (Schottenfeld-Roames, 2013).

In a similar vein, it is speculated that vesicles might be transported from the soma towards branch tips in a process regulated by Wkd and Rab35. Disruption of such transport might explain why overexpression of Wkd leads to ectopic seamless tube growth in the soma. Whether Wkd::mKate2 localization was compromised in dynein motor complex mutants was examined. As these cells have branches that lack apical membrane/seamless tubes, disruption in the localization pattern of Wkd was anticipated, but it was wondered whether co-localization with acetylated tubulin would be intact, indicative of a microtubule association independent of dynein motor transport. It was found that Wkd::mKate2 is broadly distributed throughout the cytoplasm of dynein motor complex mutants, and does not show enrichment on acetylated microtubule tracts; indeed, substantial basal enrichment was detected of Wkd::mKate2. If Wkd/Rab35-dependent trafficking of apical vesicles was dynein motor complex dependent, ectopic seamless tubes would be expected in the soma of dynein motor complex mutants, similar to those seen with Wkd overexpression or expression of Rab35DN. In fact, such ectopic tubes were consistently found in the dynein motor complex mutants, consistent with dynein-dependent trafficking of Rab35 vesicles. It cannot be ruled out that these defects are due to dynein-dependent processes unrelated to Wkd and Rab35; however, whether the ectopic tubes could be redirected distally by expression of Rab35CA, or elimination of Wkd, was examined. The motor complex ectopic tube phenotype could not be altered, indicating that the phenotype does not arise as an indirect consequence of altered Wkd localization or Rab35 activity (Schottenfeld-Roames, 2013).

The roles of RabGAP proteins have started to become clear only in recent years. Historically, it has been difficult to determine which Rab proteins are substrates of specific RabGAPs. Tests of in vitro GAP activity produced conflicting results, and in some cases did not seem indicative of in vivo function. Indeed, the specificity of Carabin (also known as Wkd orthologue TBC1D10C) has been controversial: it was first shown to act as a RasGAP, whereas later studies indicate a Rab35-specific GAP activity. The in vivo genetic data for wkd, together with recent studies characterizing the function of all three human Wkd-like TBC protein, make a compelling case that this family of proteins acts as GAPs for Rab35. Furthermore, this study establishes a role for classical vesicle trafficking proteins in seamless tube growth. As seamless tubes, but not multicellular or autocellular tracheal tubes, are affected by mutations in wkd and Rab35, this study also establishes an in vivo cell-type-specific requirement for trafficking genes in tube morphogenesis (Schottenfeld-Roames, 2013).

It is concluded that Wkd and Rab35 regulate polarized growth of seamless tubes, and it is speculated that Wkd and Rab35 direct transport of apical membrane vesicles to the distal tip of terminal cell branches (when equilibrium is shifted towards active Rab35-GTP), or to a central location adjacent to the terminal cell nucleus (when equilibrium is shifted towards inactive Rab35-GDP). Analogous to its previously described roles in targeting vesicles to the immune synapse in T cells, the cytokinetic furrow in Drosophila S2 cells and the neuromuscular junction in motor neurons, Rab35 would promote transport of vesicles from a recycling endosome compartment to the apical membrane. It is further speculated that Breathless-FGFR activation at branch tips may couple terminal cell branching with seamless tube growth within that new branch (Schottenfeld-Roames, 2013).

Golgi outposts shape dendrite morphology by functioning as sites of acentrosomal microtubule nucleation in neurons

Microtubule nucleation is essential for proper establishment and maintenance of axons and dendrites. Centrosomes, the primary site of nucleation in most cells, lose their function as microtubule organizing centers during neuronal development. How neurons generate acentrosomal microtubules remains unclear. Drosophila dendritic arborization (da) neurons lack centrosomes and therefore provide a model system to study acentrosomal microtubule nucleation. This study investigated the origin of microtubules within the elaborate dendritic arbor of class IV da neurons. Using a combination of in vivo and in vitro techniques, it was found that Golgi outposts can directly nucleate microtubules throughout the arbor. This acentrosomal nucleation requires gamma-tubulin and CP309, the Drosophila homolog of AKAP450, and contributes to the complex microtubule organization within the arbor and dendrite branch growth and stability. Together, these results identify a direct mechanism for acentrosomal microtubule nucleation within neurons and reveal a function for Golgi outposts in this process (Ori-McKenney, 2012).

Microtubules are organized into dynamic arrays that serve as tracks for directed vesicular transport and are essential for the proper establishment and maintenance of neuronal architecture. The organization and nucleation of microtubules must be highly regulated in order to generate and maintain such complex arrays. Nucleating complexes, in particular, are necessary because spontaneous nucleation of new tubulin polymers is kinetically limiting both in vivo and in vitro. Gamma(Γ)-tubulin is a core component of microtubule organization centers and has a well-established role in nucleating spindle and cytoplasmic microtubules. Previous studies have proposed that in mammalian neurons, microtubules are nucleated by γ-tubulin at the centrosome, released by microtubule severing proteins, and then transported into developing neurites by motor protein. Indeed, injection of antibodies against γ-tubulin or severing proteins inhibited axon outgrowth in neurons cultured for one day in vitro (DIV1) (Ori-McKenney, 2012).

However, proper neuron development and maintenance may not rely entirely on centrosomal sites of microtubule nucleation. Although the centrosome is the primary site of microtubule nucleation at DIV2, it loses its function as a microtubule-organizing center during neuronal development. In mature cultured mammalian neurons (DIV 11-12), γ-tubulin is depleted from the centrosome, and the majority of microtubules emanate from acentrosomal sites. In Drosophila dsas-4 mutants that lack centrioles, organization of eye-disc neurons and axon outgrowth are normal in third-instar larvae. Within the Drosophila peripheral nervous system (PNS), although dendritic arborization neurons contain centrioles, they do not form functional centrosomes, and laser ablation of the centrioles does not perturb microtubule growth or orientation (Nguyen, 2011). These results indicate that acentrosomal generation of microtubules contributes to axon development and neuronal polarity. How and where acentrosomal microtubule nucleation may contribute to the formation and maintenance of the more complex dendrites, and what factors are involved in this nucleation is unknown. Dendritic arborization (da) neurons provide an excellent system for investigating these questions. They are a subtype of multipolar neurons in the PNS of Drosophila melanogaster which produce complex dendritic arrays and do not contain centrosomes. Based on their patterns of dendrite projections, the da neurons have been grouped into four classes (I-IV) with branch complexity and arbor size increasing with class number. Class IV da neurons are ideal for studying acentrosomal microtubule nucleation because they have the most elaborate and dynamic dendritic arbor, raising intriguing questions about the modes of nucleation for its growth and maintenance (Ori-McKenney, 2012).

One potential site of acentrosomal microtubule nucleation within these neurons is the Golgi complex. A number of studies have shown that the Golgi complex can nucleate microtubules in fibroblasts. Although, in these cell types, the Golgi is tightly coupled to the centrosome, it does not require the centrosome for nucleation. It does, however, require γ-tubulin, the centrosomal protein AKAP450, and the microtubule binding proteins CLASPs. When the Golgi is fragmented upon treatment with nocodazole, the dispersed Golgi ministacks can still promote microtubule nucleation, indicating that these individual ministacks contain the necessary machinery for nucleation (Ori-McKenney, 2012 and references therein).

In both mammalian and Drosophila neurons, the Golgi complex exists as Golgi stacks located within the soma and Golgi outposts located within the dendrites. In cultured mammalian hippocampal neurons, these Golgi outposts are predominantly localized in a subset of the primary branches; however, in Drosophila class IV da neurons, the Golgi outposts appear throughout the dendritic arbor, including within the terminal branches (Ye, 2007). The Golgi outposts may provide membrane for a growing dendrite branch, as the dynamics of smaller Golgi outposts are highly correlated with dendrite branching and extension. However, the majority of larger Golgi outposts remains stationary at dendrite branchpoints and could have additional roles beyond membrane supply. It is unknown whether Drosophila Golgi outposts contain nucleation machinery similar to mammalian Golgi stacks. Such machinery could conceivably support microtubule nucleation within the complex and dynamic dendritic arbor. This study identifies a direct mechanism for acentrosomal microtubule nucleation within the dendritic arbor and reveal a role for Golgi outposts in this process. Golgi outposts contain both γ-tubulin and CP309, the Drosophila homolog of AKAP450, both of which are necessary for Golgi outpost-mediated microtubule nucleation. This type of acentrosomal nucleation contributes not only to the generation of microtubules at remote terminal branches, but also to the complex organization of microtubules within all branches of the dendritic arbor. Golgi outposts are therefore important centers of acentrosomal microtubule nucleation, which is necessary to establish and maintain the complexity of the class IV da neuronal arbor (Ori-McKenney, 2012).

This study has addressed how microtubules are organized and nucleated within the complex arbor of class IV da neurons and how essential these processes are for dendrite growth and stability. Microtubule organization within different subsets of branches in da neurons must require many levels of regulation. This study has identified the first direct mechanism for acentrosomal microtubule nucleation within these complex neurons and has uncovered a role for Golgi outposts in this process. The data are consistent with the observation that pericentriolar material is redistributed to the dendrites in mammalian neurons (Ferreira, 1993) and that γ-tubulin is depleted from the centrosome in mature mammalian neurons (Stiess, 2010). This suggests that the Golgi outposts may be one structure involved in the transport of centriole proteins such as γ-tubulin and CP309. This study found that microtubule nucleation from these Golgi outposts correlates with the extension and stability of terminal branches, which is consistent with the observation that EB3 comet entry into dendritic spines accompanies spine enlargement in mammalian neurons (Jaworski, 2009). It is striking that microtubule organization in shorter branches, but not primary branches, mimics the organization in mammalian dendrites, with a mixed microtubule polarity in the secondary branches and a uniform plus end distal polarity in the terminal branches. Kinesin-2 and certain +TIPS are necessary for uniform minus end distal microtubule polarity in the primary dendrites of da neurons. Golgi outpost mediated microtubule nucleation could also contribute to establishing or maintaining this polarity both in the terminal branches and in the primary branches. It will be of interest to identify other factors that may be involved in organizing microtubules in different subsets of branches in the future (Ori-McKenney, 2012).

In vivo and in vitro data support a role for Golgi outposts in nucleating microtubules at specific sites within terminal and primary branches. However, it is noted that not all EB1 comets originate from Golgi outposts, indicating other possible mechanisms of generating microtubules. One potentially important source of microtubules is the severing of existing microtubules by such enzymes as katanin and spastin, both of which are necessary for proper neuronal development. It is likely that both microtubule nucleation and microtubule severing contribute to the formation of new microtubules within the dendritic arbor; however, the current studies suggest that Golgi-mediated nucleation is especially important for the growth and maintenance of the terminal arbor. In γ-tubulin and CP309 mutant neurons, the primary branches contain a similar number of EB1 comets, but only a small fraction of the terminal branches still contain EB1 comets. This result indicates that severing activity or other sources of nucleation may suffice for microtubule generation within the primary branches, but γ-tubulin mediated nucleation is crucial in the terminal branches. As a result, the terminal branch arbor is dramatically reduced by mutations compromising the γ-tubulin nucleation activity at Golgi outposts (Ori-McKenney, 2012).

It is important to note that Golgi outposts are present in the dendrites, but not in the axons of da neurons; thus, this mode of nucleation is dendrite specific and likely contributes to the difference in microtubule arrays in axons and dendrites. While the axon is one long primary branch with uniform microtubule polarity, the dendrite arbor is an intricate array of branches where microtubule polarity depends on branch length. Therefore, this more elaborate branched structure may have evolved a variety of nucleation mechanisms, including Golgi outpost nucleation and microtubule severing. Intriguingly, in da neurons lacking cytoplasmic dynein function, the Golgi outposts are mislocalized to the axon, which appears branched and contains microtubules of mixed polarity (Zheng, 2008). It is speculated that in these mutants, Golgi-mediated microtubule nucleation within the axon is contributing to the mixed microtubule orientation and formation of ectopic dendrite-like branches (Ori-McKenney, 2012).

Only a subpopulation of Golgi outposts could support microtubule nucleation both in vivo and in vitro. The results show that Golgi outpost mediated microtubule nucleation is restricted to stationary outposts and dependent upon γ-tubulin and CP309, but why some outposts contain these proteins while others do not is unknown. γ-tubulin and CP309 could be recruited to the Golgi outposts in the cell body and transported on the structure into the dendrites, or they could be recruited locally from soluble pools throughout the dendritic arbor. Golgi outposts are small enough to be trafficked into terminal branches that are 150-300 nm in diameter, and therefore may provide an excellent vehicle for transporting nucleation machinery to these remote areas of the arbor. It will be interesting to determine how these nucleation factors are recruited to the Golgi outposts (Ori-McKenney, 2012).

It has been previously shown that GM130 can recruit AKAP450 to the Golgi complex, but whether the first coiled-coil domain of the Drosophila AKAP450 homolog, CP309, can also bind GM130 is unknown. Interestingly, this study has observed that predominantly stationary Golgi outposts correlated with EB1 comet formation, indicating that this specific subpopulation may contain γ-tubulin and CP309. What other factors may be necessary to properly position the Golgi outposts at sites such as branchpoints, and how this is achieved will be a fascinating direction for future studies (Ori-McKenney, 2012).

Whether the acentrosomal microtubule nucleation uncovered in this study also occurs in the dendrites of mammalian neurons is a question of great interest. Golgi outpost distribution in cultured hippocampal neurons is significantly different than that in da neurons, and hippocampal neurons do not form as elaborate arbors as da neurons. However, other types of mammalian neurons form much more complex dendritic arbors and may conceivably require acentrosomal nucleation for the growth and perpetuation of the dendrite branches (Ori-McKenney, 2012).

This study provides the first evidence that Golgi outposts can nucleate microtubules at acentrosomal sites in neurons, shedding new light on the longstanding question about the origin of the microtubule polymer in elongated neuronal processes. This source of nucleation contributes to the complex organization of microtubules within all branches of the neuron, but is specifically necessary for terminal branch development. It is thus conclude that acentrosomal microtubule nucleation is essential for dendritic branch growth and overall arbor maintenance of class IV da neurons, and that Golgi outposts are important nucleation centers within the dendritic arbor (Ori-McKenney, 2012).


Besides gammaTubulin at 23C, there is a second gamma-tubulin gene in Drosophila, gammaTubulin at 37CD. The two gammaTubulins are differentially expressed during gametogenesis and development. Both gammaTubulins are present throughout embryogenesis and larval development but the two are differentially organized during oogenesis. During oogenesis, gammaTub23C is detected at centrosomes and in the cytoplasm of mitogenic germ cells, but is not detected in germ cells following completion of mitosis. Conversely, gammaTub37CD is not detected in proliferating germ cells, but appears to accumulate later in germ cells during egg chamber development. gammaTub37CD is usually detected in the germ cell cytoplasm of stage 4-5 and older egg chambers. Enriched accumulation of gammaTub37CD is never detected at the anterior or posterior poles of the oocyte. Neither isoform is detected at the anterior or posterior poles of developing oocytes. This is surprising since gammaTubulin has been localized to microtubule organizing centers in virtually all species examined. gammaTub37CD distribution appears to be diffuse in the germ line and not localized to MTOCs. During spermatogenesis, only gammaTub23C is detected at centrosomes, where it shows cell-cycle- and differentiation-dependent organization. During the transition into the first meiotic division, gammaTub23C becomes organized as a corpuscular focus at centrioles until completion of meiosis II. During postmeiotic spermatid differention, gammaTub23C is detected first as a rod and then as a collar-like structure near the juncture of the nucleus and the elongating flagellum, but is not detected in bundles of mature sperm. The germline-specific CDC25, encoded by twine is required for the organization of gammaTub23C into corpuscular focus in spermatocytes, but not for separation of centriole pairs in M-phase or postmeiotic organization of gammaTub23C at centrioles. Following reconstitution of a canonical centrosome at fertilization, only gammaTub37CD is detected at centrosomes in syncytial embryos, but both gamma Tubulins are detected at centrosomes in cellularized embryos. The specialized pattern of gammaTub37CD expression during cellularization raises the question of whether a unique structural feature of gammaTub37CD facilitates mitosis during early cleavage division of the embryo when centrosomes must duplicat every 9-20 minutes. Colocalization of these two isoforms suggests that both contain structural features of gamma-tubulins essential for localization to centrosomes (Wilson, 1997).

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

In the Drosophila oocyte, meiosis is arrested in the first division of metaphase, when a tapered spindle aligned parallel to the egg surface forms. The chromosomes are therefore located in the cortical region near the anterior pole, whereas fusion of parental complements occurs in the inner ooplasm. How does the female pronucleus reach the interior of the egg? The second meiotic spindles are arranged in tandem, end to end, and disposed perpendicular to the longitudinal axis of the egg with the innermost spindle carrying the female pronucleus. This pattern of spindle organization is probably involved in the migration of the female pronucleus deeper into the egg near the cytoplasmic domain of the male pronucleus. The precise time at which the mitotic spindle of Drosophila changes orientation is unknown. However, spindle rotation from a position parallel to the egg surface to a radial orientation presumably occurs during or shortly after the oocyte passes through the oviduct. How spindle orientation is achieved and maintained during meiosis is an intriguing question. Microtubules linking spindle poles to the oocyte surface have been implicted in the rotation and anchoring of the meiotic apparatus in Xenopus oocytes and in other organisms, but this does not seem to be the case in the Drosophila oocyte, since the meiotic spindles lack astral microtubels. However, the observation that a transient array of microtubules links the meiotic apparatus to discrete subcortical foci suggests that in Drosophila the orientation of the spindle also requires a functional interaction between the spindle and the oocyte cortex (Riparbelli, 1996 and references).

The microtubule array of mitosis II observed between the twin spindles at metaphase, anaphase and telophase might be an intermediate between the anastral poles of the meiotic I spindles and the astral poles of the mitotic spindles in early embryos. A complex pathway of spindle assembly takes place during resumption of meiosis at fertilization, consisting of a transient array of microtubules radiating from the equatorial region of the spindle toward discrete foci in the egg cortex. A monastral array of microtubules is observed between twin metaphase II spindles in fertilized eggs. These microtubules originating from disc-shaped material stain with Rb188 antibody specific for an antigen asssociated with the centrosome of Drosophila embryos (DMAP190 or CP190). Therefore, the Drosophila egg contains a maternal pool of centrosomal components undetectable in mature inactivated oocytes. These components nucleate microtubues in a monastral array after activation, but are unable to organize bipolar spindles (Riparbelli, 1996).

The meiosis II spindle of Drosophila oocytes is distinctive in structure, consisting of two tandem spindles with anastral distal poles and an aster-associated spindle pole body between the central poles. Assembly of the anastral:astral meiosis II spindle occurs by reorganization of the meiosis I spindle, without breakdown of the meiosis I spindle. The unusual disc- or ring-shaped central spindle pole body forms de novo in the center of the elongated meiosis I spindle, followed by formation of the central spindle poles. gamma-Tubulin transiently localizes to the central spindle pole body, implying that the body acts as a microtubule nucleating center for assembly of the central poles. The first step in formation of the central pole body is the appearance of puckers in the center of the the meiosis I spindle, followed by the pinching out from the spindle of a disc or ring of microtubules that becomes the central pole body. The manner in which the central spindle pole body forms suggests the involvement of a microtubule motor. If so, the motor involved is likely to be different from Ncd (Nonclaret disjunctional), since loss of Ncd function does not seem to prevent its formation. Following the formation of the central spindle pole body, the microtubules arrayed to either side of the central body narrow into poles, forming the mature meiosis II spindle. The central poles become more tapered during progression through meiosis II, and the central spindle pole body also changes in morphology: the disc or ring becomes asterlike, then enlarges into a ring that lies between the two central telophase II nuclei (Endow, 1998).

Localization of gamma-tubulin to the meiosis II spindle is dependent on the microtubule motor protein, Ncd. Absence of Ncd results in loss of gamma-tubulin localization to the spindle and destabilization of microtubules in the central region of the spindle. Likewise, during meiosis I, the minus-end motility of Ncd and its crosslinking activity are probably needed to focus microtubules into spindle poles for the correct functioning of meiosis I. Assembly of the anastral:astral meiosis II spindle probably involves rapid reassortment of microtubule plus and minus ends in the center of the meiosis I spindle. This can be accounted for by a model that also accounts for the loss of gamma-tubulin localization to the spindle and destabilization of microtubules in the absence of Ncd (Endow, 1998).

A model for assembly of the Drosophila oocyte meiosis II spindle is suggested: gamma-Tubulin is first recruited or relocalized, possibly as gamma-TuRC, to the midbody of the meiosis I spindle, where it functions to nucleate microtubules for formation of the meiosis II central spondle poles. The loss of gamma-tubulin localization to the spindle in the absence of Ncd suggests that the Ncd motor serves to recruit or anchor gamma-tubulin to the center of the spindle. The Ncd motor would then stabilize newly nucleated microtubule minus ends and focus the microtubules into poles. The unstabilized plus ends of the microtubules in the center of the spindle (remaining from meiosis I) would undergo rapid depolymerization as a consequence of dynamic instability. Stabilization of the newly nucleated microtubule minus ends and depolymerization of the plus ends would cause a rapid sorting out of the microtubules in the center of the meiosis I spindle, replacing microtubule plus ends with minus ends. The distal poles of the meiosis II spindle would be retained from the meiosis I spindle and maintained by the same forces that originally formed them: the crosslinking activity and minus-end movement of Ncd along spindle microtubules (Endow, 1998).

The centrosome-nucleus complex and microtubule organization in the Drosophila oocyte

Molecular motors transport the axis-determining mRNAs oskar, bicoid and gurken along microtubules (MTs) in the Drosophila oocyte. However, it remains unclear how the underlying MT network is organized and how this transport takes place. A centriole-containing centrosome has been detected close to the oocyte nucleus. Remarkably, the centrosomal components, gamma-tubulin and Drosophila Pericentrin-like protein also strongly accumulate at the periphery of this nucleus. MT polymerization after cold-induced disassembly in wild type and in gurken mutants suggests that in the oocyte the centrosome-nucleus complex is an active center of MT polymerization. The MT network comprises two perpendicular MT subsets that undergo dynamic rearrangements during oogenesis. This MT reorganization parallels the successive steps in localization of gurken and oskar mRNAs. It is proposed that in addition to a highly polarized microtubule scaffold specified by the cortex oocyte, the repositioning of the nucleus and its tightly associated centrosome could control MT reorganization and, hence, oocyte polarization (Januschke, 2006).

Both the nature and the localization of the MTOC beyond stage 6 of Drosophila oogenesis have not yet been clarified. Up to stage 6, gamma-tubulin has been shown to closely associate with the nucleus at the posterior of the oocyte. In addition, electron microscopy studies have demonstrated the presence of centrioles close to the oocyte nucleus up to stage 4. Thus, until stage 6, the centrosome associates with the nucleus at the posterior of the oocyte. In Drosophila females, meiosis takes place in the absence of centrosomes. It has therefore been speculated that, at stage 6, centrosome organization changes, involving the disappearance of centrioles and the generation of MTs from a diffuse organizing center. To better understand this process, the distribution of gamma-tubulin in the oocyte was re-investigated. Before repolarization of the MT cytoskeleton, it was found that gammaTub23C and gammaTub37C localize in a layer around the nucleus, with an enrichment at the posterior pole of the oocyte. This is in agreement with the location of the MTOC at this stage. After repolarization of the MT cytoskeleton, both gamma-tubulin isoforms remain located in a perinuclear manner. Interestingly, gammaTub37C, but not gammaTub23C, labels a small body close to the oocyte nucleus. In addition, gammaTub37C and gammaTub23C also exhibit differential expression patterns in embryos: gammaTub37C is located with the centrosomes of mitotic cells, whereas gammaTub23C is not. Thus, gamma-tubulin is distributed in close association with the nucleus periphery and possibly on a centrosome-like structure. Pericentrin/AKAP450 is another major component of the centrosome. Green fluorescence protein (GFP) fusion of the C-terminal part of Pericentrin/AKAP450 and its Drosophila homolog pericentrin-like protein (D-PLP) have been shown to localize to the centrosomes respectively in cultured human cells, Drosophila embryos and spermatocytes. Using the UAS/Gal4 system, GFP-cter-D-PLP was specifically expressed in the germline and a bright dot was detected in the vicinity of the nucleus before and after nuclear migration. GFP-cter-D-PLP was also detected in all germline nuclei, as has been observed previously. From stage 7 onward, the bright dot remained in the immediate vicinity of the oocyte nucleus (<1 µm distance). Furthermore, both GFP-cter-D-PLP and gammaTub37C co-localize to this discrete body, indicating that this structure could correspond to a centrosome. In G2 centriole, tubulin is highly polyglutamylated. The ID5 antibody labels basal bodies and centrioles in several species. Using this antibody, a dot was detected close to the nucleus throughout oogenesis that remained detectable up to stage 10A. This suggests that the dot represents a centriole-containing centrosome. Indeed, using electron microscopy, two to possibly four centrioles were clearly detected closely associated with the nucleus in stage 9 oocytes. This demonstrates the existence of centrioles associated with the nucleus at least up to stage 9. MT fibers emanating from those centrioles could not be unambiguously detected. Then the link between centrosome and nucleus was examined using colchicine. In flies fed with colchicine, MTs in the germline were completely depolymerized, and the oocyte nucleus was mispositioned. In the oocyte, it was observed that the nucleus and the centrosome were significantly separated, the distance between them increasing during oocyte growth. In a few cases, it was noticed that the nucleus could reach the anterior cortex without the centrosome; however, a centrosome was never observed at the anterior without the nucleus. It is concluded that the close localization of the centriole-containing centrosome to the nucleus depends on MTs (Januschke, 2006).

The structure of the MT network during mid-oogenesis is dynamic. At stage 7, MTs are visible as a mesh at the anterior cortex. Later, at stage 10, MT bundles are observed that promote cytoplasmic streaming. In-between MT distribution has been described as an AP gradient. However, high-resolution images of oocyte MTs are lacking. Therefore, a protocol frequently used to increase the detection of the MT cytoskeleton in cell culture was modified for the Drosophila egg chamber to characterize MT organization in the oocyte during the crucial period in which bcd, grk and osk mRNAs are localized. MTs were detected throughout oogenesis using alpha-Tubulin but also with a Kinesin heavy chain antibody (alpha-Khc), which revealed the MT array and its complexity in unprecedented definition. It was noticed that the range of detected details was increased and more reproducible with alpha-Khc antibody. To control the specificity of Khc detection, germline and follicle cell mutant clones were generated homozygous for khc7.288. In such mutant cells, no Khc was detected, indicating that the detection is specific. Labeling with antibodies directed against aromatic C-terminal amino acid residues (Tyr or Phe) of alpha-tubulin and against Khc largely overlapped. This confirmed that the structures revealed by Khc were MTs. A Khc fraction was also detected at the posterior of the oocyte. That Khc accumulates along MTs may be due to permeabilization before fixation, which could cause rigor binding of Khc to MTs. This detection procedure may also permit the extraction of a soluble pool of Khc and reveal the remaining fraction distributed along the MTs. With this detection procedure, Khc revealed by Kinesin-ßgal exhibited a more restricted distribution compared with alpha-Khc antibody. This is probably due to the substitution of the C-terminal part of Khc by the ß-galactosidase in the reporter construct, impairing the recycling of the chimeric Kinesin motor leading to its accumulation exclusively at the posterior. With this detection method, the MT minus-end marker, Nod-ßgal, was detected in the antero-dorsal corner above the oocyte nucleus as well as in the opposite antero-ventral corner. Moreover, localized determinants such as Osk and Grk were correctly positioned in the oocyte (Januschke, 2006).

To confirm that the detection method does not alter MT organization, MT distribution was analyzed in follicle cells, which should be sensitive to the extraction procedure, since they are more directly exposed than the oocyte. MT distribution in different follicle cell types was unchanged, when comparing living and fixed egg chambers. The main body follicle cell MTs seemed unchanged. Main body follicle cell MTs have been shown to be highly stable, and might therefore reflect the sensitivity of the protocol with limitations. Nevertheless, stretched follicle cells showed strikingly similar MT patterns in living and fixed conditions as well. Apicalbasal polarity was not affected in follicle cells, as demonstrated by the correct apical localization of atypical protein kinase C. Importantly, the MT distribution of living egg chambers expressing GFP-alpha-Tubulin at stage 7 and stage 9 was similar to the one observed using anti-alpha-Tubulin and Khc antibodies. Therefore it seems that the fixation conditions preserve the wild-type MT organization and that Khc can be suitable to label bulk MTs (Januschke, 2006).

When fixed wild-type oocytes were analyzed by confocal microscopy, MT organization in the oocyte appeared unchanged from stage 2 to stage 6. With stage 7, MT organization was modified and two MT subsets became apparent. This organization was more evident at stage 8. A first subset consisted of cortical MTs oriented along the dorso-ventral (DV) axis parallel to the oocyte nurse cell border, and juxtaposed to the lateral cortices, wrapping the oocyte from stage 7 to 9. At least some MT bundles of this subset could be traced back to the oocyte nucleus. The DV orientation of MT bundles, depicted as black fibers in the schematic representations, was highly reproducible for all stages and persisted throughout mid-oogenesis (Januschke, 2006).

A second MT subset was present in the center of the oocyte. Although there was some variability in the patterns observed, it was found that each developmental stage showed a characteristic MT distribution. During stage 6, MTs from this subset were cortical and extended from the nucleus at the posterior to the anterior cortex, compact bundles of MTs formed a circle-like structure resembling a diaphragm. This subset was formed by long MT bundles that extended (once or more) along the entire cortex. By stage 8, the oocyte had considerably grown and individual MT bundles were therefore easier to track. MT bundles emanated from the anterior and the nucleus to point toward the posterior. MTs extended again along the entire cortex, after which they turned to the central cytoplasm. This, in turn, generated free MT (plus) ends in the center of the oocyte. By stage 9, the central MT network was clearly oriented along the oocyte AP axis. One or two thick MT bundles extended from the anterior, pointing toward the posterior pole. These bundles formed a structure resembling a horseshoe, with its open side facing the posterior. Importantly, both subsets could also be detected in living egg chambers, as shown for the DV subset and the AP subset. Thus, MTs show strong rearrangements throughout mid-oogenesis, which results in two perpendicular MT arrays reflecting the two axes of the oocyte (Januschke, 2006). An ex-vivo assay was developed to localize MT nucleation sites by dissecting ovaries and placing them on ice for 30 minutes. This treatment resulted in complete depolymerization of MTs. When allowed to recover at 25°C for 30 minutes, MT distribution could be re-established to the wild-type situation, in which both the cortical and the central subsets of MTs were detectable. gamma-Tubulin distribution was not affected by cold-induced MT depolymerization. When short periods of regrowth were analyzed, MT nucleation appeared limited to the close vicinity of the nucleus and was often asymmetric, suggesting a centrosome-associated nucleation activity. MT regrowth appeared to be stepwise, since after 15 minutes only the DV cortical subset was established. MTs clustered around the oocyte nucleus and aligned along the cortex in the DV direction. The cortical location of these fibers was clearly revealed by the presence of Khc-positive dots at either the dorsal or the ventral side. This indicates that the DV MT subset is the first to regrow. The regrowth experiment was repeated using colchicine. After the drug was washed out, MT repolymerization was observed at the oocyte nucleus. Taken together, these results indicate that, at least with the detection method that was used, the oocyte nucleus and its immediate surroundings have the capacity to nucleate MTs (Januschke, 2006).

To test whether the centrosome-nucleus complex could direct the repolarization of the MT network, how MTs distribute in grk mutant oocytes was examined. In this mutant, the nucleus frequently remains at the posterior of the oocyte due to a failure in the signaling cascade that induces the repolarization of the cytoskeleton. In grk mutant oocytes similar in size to wild-type stage 8, the MT distribution was dramatically affected. Specifically, MT organization appeared completely reversed compared with wild type, in which the nucleus is at the anterior and MT plus-ends are located toward the posterior at stage 8. In slightly older oocytes, MTs remain stretched out along the cortex from the posterior toward the anterior, where they fold back to the center of the oocyte. MT ends in the center are most probably plus-ends, since the pool of Khc (localized at the posterior of wild-type oocytes, co-localizes with Kinesin-ßGal to the center of the oocyte, between the flanking MT ends. Interestingly, MT distribution in grk oocytes was strikingly similar to MTs of wild-type egg chambers before the migration of the oocyte nucleus. Likewise, the centrosome, as revealed by gamma-tubulin, which is found at the posterior of stage 6 wild-type oocytes, stays at the posterior in grk mutants. Thus, in grk mutants, distribution of MT and MTOC seemed similar to their distribution in wild-type stage 6 (Januschke, 2006).

grk mutant oocytes, having mispositioned nuclei, provide an ideal basis to test the MT nucleating capacity of the centrosome-nucleus complex using the cold-shock assay. After cold-shock treatment of grk oocytes, complete MT depolymerization was checked for. As in the wild type, during the initial period of recovery at 25°C, MT polymerization took place only in the immediate vicinity of the mispositioned oocyte nucleus. Therefore, as in wild-type oocytes, MT nucleation is often asymmetric and restricted to the area surrounding the nucleus. This result strengthens the possibility that the centrosome-nucleus complex is an active MTOC (Januschke, 2006).

Thus, in the Drosophila oocyte a centriole-containing centrosome is present in close association with the nucleus, which itself is covered by PCM components until late in oogenesis. In addition, MTs can nucleate from this centrosome-nucleus complex. The MTs appear to form two orthogonal MT populations that develop through several steps during mid-oogenesis. It is proposed that the migration of the nucleus in the oocyte could control the reorganization of the MT network (Januschke, 2006).

In region 2 of the germarium, nurse cell centrosomes migrate toward the oocyte. Later, in region 3, these centriole-containing centrosomes become located as an aggregate between the oocyte nucleus and the follicle cell border. Pericentriolar material closely associated with the oocyte nucleus can be clearly detected until stage 6 with several centrosomal markers, such as gamma-tubulin, Centrosomin and D-Tacc. From stage 4 onward, the fate of the centriole cluster has been unknown. This study shows that both gammaTub37C and gammaTub23C are localized in a perinuclear manner throughout oogenesis. gammaTub37C highlights a discrete body close to the nucleus. This body is similarly detected by the centrosomal marker D-PLP and by a specific antibody for polyglutamylated Tubulin, which detects centrioles. Consistent with this, two to possibly four centrioles were detected in the immediate vicinity of the nucleus in stage 9 oocytes. This result demonstrates that at least until stage 9, a centriole-containing centrosome is present in the oocyte. Currently, it is not known whether they are still present at the onset of meiosis I during stage 13, since it has previously been proposed that the meiotic spindle is achieved without centrosomes. During skeletal muscle morphogenesis, myotube centrosomes dissociate from their nuclei, centrioles disappear and the centrosomal matrix is redistributed to the nucleus periphery. Similarly, during oogenesis, centrioles from nurse cell centrosomes may disappear. However, their pericentriolar material may relocate to the oocyte nucleus periphery. This would explain the specific enrichment of the oocyte nucleus with perinuclear MTOC material. The only centrosome remaining associated with a nucleus is that of the oocyte. Furthermore, the structure of this centrosome remains intact. It is concluded that the four centrioles found close to the nucleus in stage 9 may correspond to the initial oocyte centrosome in the duplication phase observed in G2 (Januschke, 2006).

Effects of Mutation or Deletion

In Drosophila, gammaTubulin is required for the structure as well as the function of microtubule organizing centres (MTOCs). This conclusion is based on the identification and phenotypic characterization of a mutant allele of the gamma-tubulin gene located at region 23C of the polytene chromosome map. This mutation, termed gamma-tub23CPl, is caused by the insertion of a P-element within the 5' untranslated leader of the gamma-tubulin transcript. Northern and Western analysis show that gamma-tub23CPl is either a null or a very severe hypomorph as no gamma-tubulin mRNA or protein can be detected in mutant individuals. Visualization of DNA, MTOCs and microtubules by confocal laser scanning microscopy of cells from individuals homozygous for gamma-tub23CPl reveals a series of phenotypic abnormalities. Some of these are similar to those observed after disruption of gamma-tubulin function in other organisms, including mitotic arrest and a dramatic decrease in the number of microtubules. Mutation in this gene also results in highly abnormal MTOCs showing a variety of shapes and sizes never observed in wild type cells. These results show that gammaTubulin is required for both structural and functional roles in the MTOCs (Sunkel, 1995).

Differentiation of the Drosophila oocyte takes place in a cyst of 16 interconnected germ cells and is dependent on a network of microtubules that becomes polarized as differentiation progresses (polarization). How the microtubule network polarizes was investigated using a GFP-tubulin construct that allows germ-cell microtubules to be visualized with greater sensitivity than in previous studies. Unexpectedly, microtubules are seen to associate with the fusome, an asymmetric germline-specific organelle, which elaborates as cysts form and undergoes complex changes during cyst polarization. This fusome-microtubule association occurs periodically during late interphases of cyst divisions and then continuously in 16-cell cysts that have entered meiotic prophase. As meiotic cysts move through the germarium, microtubule minus ends progressively focus towards the center of the fusome, as visualized using a NOD-lacZ marker. During this same period, discrete foci rich in gamma tubulin that very probably correspond to migrating cystocyte centrosomes also associate with the fusome, first on the fusome arms and then in its center, subsequently moving into the differentiating oocyte. The fusome is required for this complex process, because microtubule network organization and polarization are disrupted in hts1 mutant cysts, which lack fusomes. These results suggest that the fusome, a specialized membrane-skeletal structure, that arises in early germ cells, plays a crucial role in polarizing 16-cell cysts, at least in part by interacting with microtubules and centrosomes (Grieder, 2000).

Polarity within germline cysts arises at two distinct times. First, polarizing forces build the asymmetric structure of the cyst in region 1 and are dependent on the fusome. Later, polarizing forces operate in fully formed 16-cell cysts to re-organize and polarize the MT network towards the differentiating oocyte. Since the oocyte is invariably one of the four-ring canal cells, early asymmetries must be preserved and re-used within polarizing 16-cell cysts; however, the mechanisms responsible for preserving and interpreting early polarity information have remained unclear. The fusome is required to organize a complex and dynamic MT network both in dividing cysts of region 1 and also in polarizing 16-cell cysts. The experiments reported here strongly suggest that a persistent asymmetry within the fusome stores early polarity information for use in fully formed cysts. Moreover, this polarity is likely read out through an action of the fusome on microtubule organization. In the absence of a fusome, microtubule polarity in meiotic cysts is abolished and oocytes do not form (Grieder, 2000).

The requirement of a fusome to organize MTs and polarize 16-cell cysts provides the strongest argument that the associations observed between MTs and the fusome are functionally important. However, it could be argued that the MT organization in hts mutants is disrupted by a fusome-independent mechanism. A processed fragment of Hts protein is incorporated into maturing ring canals in region 2b, raising the possibility that ring canals, rather than the fusome, mediate MT polarization. This interpretation, however, is not supported by experimental observations. The MT network associates with the fusome and not the ring canals, and this association is detected in late interphase of cyst divisions and in meiotic cysts in region 2a, long before Hts proteins are incorporated into ring canals. All these associations, not just those in region 2b, are disrupted in hts mutants. Consequently, the fusome rather than the ring canals is responsible for organizing MTs during early germ-cell development (Grieder, 2000).

Genetic studies of three molecules that mediate transport along microtubules provide further evidence for a connection to the fusome. Mutations in cytoplasmic DHC64C disrupt cyst production and block production of a normally structured fusome, at least in part by disrupting the normal orientation of the mitotic spindle with the fusome during cyst divisions. Unfortunately, it has remained unclear whether DHC64C is localized to the centrosomes and the fusome during prophase of meiotic cysts. It is, however, tempting to speculate that DHC64C plays a critical role in organizing the MTs of polarizing cysts, as there is a genetic requirement for DHC64C in oocyte formation and DHC64C staining is lost in hts mutant cysts. In addition, Drosophila Lissencephaly-1 is involved in MT dynamics. LIS1 mutants have defects not only in MT organization but also in fusome formation. Finally, mutations in the kinesin-like protein KLP61F interfere with the production of a normal fusome in Drosophila males. Whether these last two proteins have a specific role during polarization or only during cyst formation remains unclear (Grieder, 2000).

These observations reveal that a novel mechanism, directed centrosome migration, is likely to be involved in cyst polarization. Foci rich in gamma tubulin move onto and along the fusome arms in region 2a and 2b cysts, indicating that the nurse-cell centrosomes migrate along the fusome on their way to the oocyte. The total number of gamma tubulin foci in region 2a cysts range between 13 and 20, consistent with the number expected if most of the cystocyte centrosomes were labeled. The developmental changes in the timing and the approximate number of gamma-tubulin foci are indistinguishable from the behavior of migrating centrioles. The association of the fusome with this process of directed movement provides further evidence that the fusome is itself a polarized structure, not only during its formation in region 1, but also in region 2 cysts. It will be important to look for specific molecules that link centrosomes to the fusome and move them in a directional manner in response to fusome polarity (Grieder, 2000).

Previously, only the movement of centrioles, not centrosomes, was reported. It has been suggested that the migrating centrioles are inactive and break down in the oocyte so that centriolar constituents can be re-used in the early embryo. The gamma-tubulin staining observed argues that the migrating centrioles are accompanied by peri-centriolar material and can actively nucleate microtubules. The preferential accumulation of active centrosomes in the central region of the fusome would probably contribute to polarizing the MT network. Later movements of the centrosomes probably contribute to subsequent stages of polarization. These include the focusing of the MT minus ends within a single cyst cell, and the movement of MTs to the posterior of the oocyte. The existence of 13-20 actively migrating centrosomes is not consistent with the suggestion that MTOC formation and oocyte determination are controlled by the selective inheritance of a single active centrosome (Grieder, 2000 and references therein).

At least some of the microtubule organizing activity of the fusome is not due to associated centrosomes. During late interphase of the cystocyte divisions, the main part of the fusome strongly interacts with microtubules. Many MTs and/or MT bundles exit the fusome at this time, and it appears unlikely from their number and distribution that associated centrioles could account for all this activity. Even though many discrete gamma-tubulin spots/centrosomes were localized on or close to the fusome arms in region 2a, the overall MT polarity is not uniformly oriented towards the fusome, based on the NOD-lacZ staining. Consequently, the fusome may transiently acquire the ability to bind or bundle MTs, rather than nucleating them (Grieder, 2000).

At other times, the fusome might interact directly with MT minus ends. Fusomal material may acquire the ability to nucleate MT minus ends by associating with peri-centriolar material. The fusome in polarizing cysts appears most likely to exhibit such an activity, since MT-fusome interaction during polarization involves MT minus end interactions. Alternatively, the fusome might interact with motor proteins that bridge it to MTs; these proteins would specifically interact with minus ends. Polarization of the MT network into the differentiating oocyte could involve both minus end interaction as well as bundling. Both of these mechanisms would probably be amplified through MT-MT interactions outside the fusome (Grieder, 2000).

Previously, it has been unclear when and if the fusome contained polymerized microtubules. The retention of such MTs has been proposed to store polarity information from the fusome for later use in developing 16-cell cysts. GFP-alpha-tubulin staining does reveal MTs that run parallel to the fusome in region 2b cysts. However, there is no evidence for such MTs in aligning cysts within region 2a, or within the main body of the fusome in early interphase of the cystocyte divisions. Consequently, polymerized MTs within the fusome that persist from the time of cyst formation do not appear to be the basis of the fusomal polarity (Grieder, 2000).

The use of a highly sensitive GFP-alpha-tubulin fusion protein reveals a more detailed picture than previously available of the dynamic process of MT polarization in developing 16-cell cysts. At the beginning of the polarization process in oblique meiotic cysts, MTs extend equally into all cystocytes and are embedded in the fusome. Subsequently, MT minus ends coalesce towards the central region of the fusome, which resides within both four-ring canal cells and their immediate neighbors. Gradually, the MT network continues to polarize until it becomes clearly focused in a single cell, the future oocyte, late in region 2b. Thus, an organized microtubule cytoskeleton does not arise spontaneously within region 2a cysts in response to the appearance of a new MTOC in one of the cyst cells. Rather, the MT network polarizes progressively over an extended period of time and the new MTOC develops gradually (Grieder, 2000).

These observations clarify the likely relationship between cyst polarization and the asymmetric accumulation of specific molecules. The exact stage at which specific RNAs and proteins accumulate has remained unclear in previous studies, because reagents differing in sensitivity were often used. Only after a significant fraction of the MT minus ends become concentrated would mRNAs and proteins whose transport is MT dependent be able to accumulate in their vicinity. MT minus ends begin to cluster in mid- to late-region 2a cysts; it is proposed that this event determines when specific molecules begin to accumulate unequally. This is consistent with the observation that orb, hts and probably other mRNAs become clearly localized within a small region of the cyst as early as the middle of region 2a in some germaria (Grieder, 2000).

The origin of cellular asymmetry is being studied in diverse systems, including the formation of yeast buds, the development of embryonic axes in C. elegans and Drosophila embryos, the segregation of Drosophila neuroblasts and the polarization of epithelial layers. In all these systems, there is evidence that the microtubule cytoskeleton interacts with other cytoskeletal components, particularly actin, to orient divisions and mediate intracellular asymmetry. For example, localization of the MT-orienting KAR9 protein in yeast depends on actin. The asymmetric distribution of proteins after the first division of the C. elegans embryo requires actin, and some localized gene products such as PAR1 appear to act on microtubules. In at least one case, the requirement for actin in spindle orientation exists during only a portion of the cell cycle. The results presented here indicate that Drosophila germline cysts provide another system where the development of polarity depends on interactions between the microtubule cytoskeleton and another cytoskeletal system, the fusome. The fusome arises early in germ cell development at the time germ cells migrate toward the gonadal mesoderm, and subsequently is often located near the centrioles. Interactions, between the fusome and the MT cytoskeleton may take place in some form during much of germ-cell development, and be elaborated as cysts form and polarize. Further study of the molecular basis of fusome-microtubule interactions is likely to advance an understanding of many aspects of cell and tissue polarization (Grieder, 2000).

To assess the role of gamma-tubulin in spindle assembly in vivo, meiosis progression has been followed by immunofluorescence and time-lapse video microscopy in gammaTub23CPI mutant spermatocytes. Centrosomes associate with large numbers of astral microtubules even though gamma-tubulin is severely depleted; bipolar meiotic spindles are never assembled; and later in meiosis, the microtubules get organized into a conical structure that is never observed in wild-type cells. Several lines of evidence suggest that these cones may be related to wild-type central spindles: (1) they are assembled midway through meiosis and elongate during anaphase; (2) they are constricted during late meiosis, giving rise to a pointed end similar to those that form in each half of the wild-type spindle midzone; (3) Klp3A and Polo, two markers of the wild-type central spindle are also found around the pointed end of the mutant cones, and (4) ectopic cytokinesis furrows are often formed at the distal end of the cone. These results suggest that microtubule polymerization or stabilization from the centrosome may be possible in a gamma-tubulin-independent manner in Drosophila spermatocytes. However, gamma-tubulin seems to be essential for spindle assembly in these cells. Finally, these results show that at least part of the central spindle and constriction-ring assembly machinery can operate on microtubule bundles that are not organized as bipolar spindles (Sampaio, 2001).

Two gamma-tubulin isoforms have been identified in Drosophila, gammaTub37C and gammaTub23C. gammaTub37C is largely restricted to the female germ line and early stages of embryogenesis. The gammaTUB23C isoform, in contrast, is expressed in a variety of tissues in both sexes and is the only isoform present in testes. Genetic, biochemical, and cytological data suggest that the gammaTub23CPI mutant allele leads to a very severe loss of function of the gammaTub23C gene and thus to a severe reduction of the levels of gamma-tubulin in mutant testes (Sampaio, 2001).

At the onset of meiosis, the two asters segregate normally in gammaTub23CPI mutant spermatocytes, but soon afterwards, they collapse back together again. As meiosis proceeds, the normal figures found in control cells are never observed in gammaTub23CPI spermatocytes. Instead, the microtubules form cone-shaped structures, one per cell, with a pointed end and the two asters fused or very close to each other at the base. Such figures have not been reported before in other meiotic mutants in Drosophila. gammaTub23CPI early spermatid cysts contain only 16 cells, and each spermatid has several nuclei of different sizes associated with a single large nebenkern, revealing the failure of the two meiotic divisions. Around 75% of these cysts contain small anuclear and anastral cell fragments that carry a small nebenkern, suggesting that some highly asymmetric cytokinesis takes place in these cells (Sampaio, 2001).

To further investigate the organization of microtubules in gammaTub23CPI meiotic spermatocytes, fixed preparations were stained with antibodies against alpha-tubulin. At prophase, a pair of well-defined astral arrays can be observed both in wild-type and gammaTub23CPI mutant spermatocytes. By the time of prometaphase/metaphase, a considerable number of microtubules are present in gammaTub23CPI mutant spermatocytes, but they only form a disordered mesh that bears no resemblance to the bipolar spindles found at this stage in control spermatocytes. As meiosis proceeds, the microtubules found in the mutant spermatocytes are organized into cones. With very rare exceptions, the chromosomes and centrosomes are located at the base of the cone (Sampaio, 2001).

The abundance of microtubules in gammaTub23CPI spermatocytes is quite remarkable given the very low gamma-tubulin levels observed in these cells. If the leaky function provided by this mutant allele is able to sustain the observed levels of microtubule polymerization, gamma-tubulin must be present in vast excess in wild-type spermatocytes. Alternatively, microtubule polymerization and stabilization at the centrosome may not be completely dependent upon gamma-tubulin in these cells. This interpretation is also supported by previous work carried out in Drosophila, Caenorhabditis elegans, and Schizosaccharomyces pombe (Sampaio, 2001).

However, despite the presence of a significant number of microtubules, bipolar spindles are never assembled in gammaTub23CPI mutant spermatocytes. This is a surprising result given that spindle self-assembly through a centrosome-independent pathway has been reported in several experimental models, including Drosophila neuroblasts and spermatocytes, in vitro. The reasons for this failure to organize the meiotic spindle are not understood. A simple explanation could be the presence of impaired, but partially functional, centrosomes that may still function as the major MTOCs (microtubule organizing centers) in these cells, thus overriding the organizing activity of the acentrosomal spindle assembly pathway. This hypothesis is not supported by the absence of organized spindles in gammaTub23CPI;asl double-mutant spermatocytes. However, some centrosomal function may still remain in the double mutant. In this regard, it would be very interesting to investigate the phenotype of cnn gammaTub23CPI double-mutant spermatocytes. An alternative interpretation could be that the dynamic properties of the microtubules present in gammaTub23CPI cells are such that they no longer serve as good substrates for the organizing activities of the molecular motors involved in the acentrosomal spindle assembly pathway (Sampaio, 2001).

To get a better understanding of the process of assembly of the cones found in gammaTub23CPI spermatocytes, meiosis was followed in these cells by time-lapse microscopy. At late prophase, the two mutant asters that had previously migrated apart start to approach each other, and the remains of the nuclear envelope become deformed. Similar results have been observed in C. elegans embryos in which, following gammaTub inactivation by RNAi, the two asters that had initially segregated collapse back together again. As meiosis proceeds in the mutant cells, the condensed bivalents are clearly visible, and a large number of microtubules is revealed by their association with phase-dark membranes. At this stage, most microtubules are sorted into two populations that emanate from each aster. Their distal ends can be seen moving along the cell membrane, becoming clustered at one side of the cell. Coinciding with the separation of the homolog univalents, and thus at a stage similar to anaphase, the microtubule array found in the mutant starts to elongate and continues to do so until the new nuclear envelopes are formed. Finally, this microtubule network is disassembled at the end of meiosis I. Thus, the time of organization and elongation of the cone found in gammaTub23CPI spermatocytes corresponds with the timing of organization and elongation of the wild-type central spindle (Sampaio, 2001).

Given the similarities in the dynamics of assembly and elongation of mutant cones and wild-type central spindles, the presence of common molecular components was investigated. To this end, the localization of the wild-type central spindle markers Polo and Klp3A was studied in the mutant cones. Immediately before nuclear envelope breakdown, the V-shaped centrosomes characteristic of these cells are strongly labeled by a functional GFP-Polo fusion that, at this stage, also very distinctively labels the nucleolus. Two pairs of centrosomes can be seen at nearly opposite sides of the nucleus, both in wild-type and in gammaTub23CPI mutant spermatocytes. However, as meiosis proceeds in the mutant, the centrosomes get closer to each other and the spindle fails to become organized. When the microtubule cone is formed, the GFP-Polo fusion strongly accumulates at the pointed end, just as it does in the wild-type spindle midzone. It also labels the centrosomes in both wild-type and mutant cells. During cytokinesis, GFP-Polo colocalizes with the constriction ring in the wild-type. In those instances in which highly asymmetric cytokinesis take place in the mutant cells, the GFP-Polo fusion is localized near the furrow. Similar results were obtained with Klp3A. In wild-type spermatocytes, the kinesin-like protein Klp3A accumulates in the central spindle midzone. In gammaTub23CPI mutant spermatocytes, Klp3A is found at the pointed end of the cone. Therefore, at least part of the machinery involved in the assembly of the constriction ring may also be used to organize the pointed end of the cone (Sampaio, 2001).

Therefore, the cone-shaped microtubule arrays found in gammaTub23CPI mutant spermatocytes bear some striking similarities to wild-type central spindles: (1) they are assembled midway through meiosis and elongate soon afterwards, just as wild-type central spindles do during anaphase B; (2) they are constricted during late meiosis, giving rise to a pointed end similar to those that form in each half of the wild-type central spindle during telophase; (3) Klp3A and Polo, two of the proteins localized in the wild-type central spindle midzone that are required for the recruitment of several components of the contractile ring, are also found around the distal pointed end of the mutant cones. Also, highly asymmetric cytokinesis can occur within gammaTub23CPI mutant cells at the distal end of the cone (Sampaio, 2001).

In summary, the depletion of gamma-tubulin caused by the gammaTub23CPI mutant allele does not prevent microtubule polymerization or stabilization, abolishes spindle assembly, and allows for the activity of the mechanisms that organize central spindles (Sampaio, 2001).

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

The assembly of the mitotic spindle after depletion of the major gamma-tubulin isotype by RNA-mediated interference was assessed in the Drosophila S2 cell line. Depletion of gamma-tubulin has no significant effect on the cytoskeletal microtubules during interphase. However, it promotes an increase in the mitotic index, resulting mainly in monopolar and, to a lesser extent, asymmetrical bipolar prometaphases lacking astral microtubules. This mitotic accumulation coincides with the activation of the mitotic checkpoint. Immunostaining with an anti-Abnormal spindle (Asp) antibody revealed that the spindle poles, which were always devoid of gamma-tubulin, were unfocused and organized into sub-spindles. Despite the marked depletion of gamma-tubulin, the pericentriolar proteins CP190 and centrosomin were recruited to the spindle pole(s), where they formed three or four dots, suggesting the presence of several centrioles. Electron microscopic reconstructions demonstrated that most of the monopolar spindles exhibited three or four centrioles, indicating centriole duplication with a failure in the separation process. Most of the centrioles were shortened, suggesting a role for gamma-tubulin in centriole morphogenesis. Moreover, in contrast to metaphases observed in control cells, in which the spindle microtubules radiate from the pericentriolar material, in gamma-tubulin-depleted cells, microtubule assembly still occurs at the poles but involves the elongation of centriolar microtubule triplets. These results demonstrate that, after depletion of gamma-tubulin, the pericentriolar material is unable to promote efficient microtubule nucleation. They point to an alternative mechanism of centrosomal microtubule assembly that contributes to the formation of abnormal, albeit partially functional, mitotic spindles (Raynaud-Messina, 2004).

This study used RNAi to deplete the major gamma-tubulin isotype significantly in cultured Drosophila S2 cells. Only a few cells escaped this treatment, because less than 0.7% of all mitotic cells presented a bipolar morphology with a positive 23C gamma-tubulin signal at both poles. Depletion of 23C gamma-tubulin caused S2 cells to accumulate in mitosis, with an active mitotic checkpoint. They exhibited abnormal prometaphase stages, characterized by unseparated condensed chromatids and spindle poles with supernumerary short centrioles. The emergence of a characteristic phenotype (i.e. monopolar prometaphase figures) prompted an investigation of the mechanism of spindle organization in 23C-gamma-tubulin-depleted cells. This investigation revealed that, under gamma-tubulin-deficient conditions, the pericentriolar material is unable to efficiently promote microtubule nucleation. These observations point to the involvement of a previously undescribed centrosomal mechanism of microtubule assembly that operates via elongation of the distal centriolar extremities (Raynaud-Messina, 2004).

After depletion of 23C gamma-tubulin, the duplicated centrioles were present at the one pole of the monopolar prometaphase figures and at only one of the two poles in at least half of the bipolar figures, indicating a segregation failure. This phenomenon has also been suggested to occur in Drosophila Dgrip91 mutants. Observations made on mammalian cells have demonstrated that the relative positions of the two centriole pairs depend partly on the dynamics of the microtubule cytoskeleton. Hence, after gamma-tubulin depletion, it is likely that the microtubule subnetwork involved in the separation of the centriole pairs undergoes modifications in either its dynamics or in its density. Despite the lack of ultrastructural studies excluding morphogenetic and/or separation defects, basal-body duplication has been reported to be impaired after silencing of the gamma-tubulin-encoding genes in Paramecium. This discrepancy with the observations could result from differences in the timing of centriole and basal-body duplication, and the many rounds of basal-body duplication in Paramecium leading to many basal bodies. Thus, it is possible that the critical concentration of gamma-tubulin required for centriole duplication is lower than the one necessary for microtubule assembly by the pericentriolar material. In this regard, it is possible that the small amount of the 37CD gamma-tubulin isotype constitutively associated with the centrosomal fraction plays a role in centriole duplication. However, it is unlikely that this low-abundance gamma-tubulin isotype plays an important role in the nucleation of mitotic microtubules, as suggested by the absence of microtubules issuing directly from the pericentriolar material. After 23C gamma-tubulin depletion, the 37CD isotype was not affected; its expression level was maintained and it retained its polar localization. Moreover, under the experimental conditions used, the function of this minor gamma-tubulin isotype could not be determined by RNAi, despite the use of two distinct dsRNAs. The poor susceptibility of the 37CD gamma-tubulin isoform to the RNAi strategy might stem from its specific properties in Drosophila cells, such as its low concentration, its absence from the cytosolic fraction and/or a possible slow turnover (Raynaud-Messina, 2004).

In 23C gamma-tubulin-depleted S2 cells, the centrioles were often shorter than in the control cells. The presence of a short centriole among normal sized centrioles has been noticed in abnormal metaphases of neuroblasts from the Drosophila Dgrip91 mutants. Centrioles are generally believed to be very stable organelles. However, in Physarum amoebae, a transient decrease in the length of the parental centrioles occurs in mitosis, during the assembly of the daughter centrioles. If the gamma-tubulin present in the centriole and basal-body lumen interacts with the centriolar microtubules, its depletion might well alter centriole morphogenesis, possibly by an action on centriolar microtubule dynamics. Indeed, a role of gamma-tubulin in microtubule dynamics and stability has been suggested in Aspergillus, Caenorhabditis, as well as in mammalian cells, where gamma-tubulin interacts with the stable kinetochore microtubules. gamma-Tubulin-depleted Drosophila S2 cells mostly assembled half-spindles, resulting in monopolar metaphases with a normal overall microtubule orientation and a 13-protofilament structure. The presence of pericentriolar material was confirmed by electron microscopy and by the recruitment of the centrosomal proteins CP190 and centrosomin, which appeared to be independent of the presence of gamma-tubulin. Microtubule reassembly after cold treatment suggested that the pole retained a nucleation activity. However, consistent with the severe depletion of the main centrosomal gamma-tubulin, most of the spindle microtubules were not assembled from the pericentriolar material, which accounted for the absence of astral microtubules. Nucleation resulted at least partially from elongation of the distal extremity of the microtubules of the four centrioles. This process differs from the formation of primary cilia observed in several interphase animal cells. In mitotic 23C-gamma-tubulin-depleted S2 cells, the microtubules produced by the centrioles fail to form the characteristic axonemal structure. They are not surrounded by a membrane and are not associated in microtubule doublets as in typical primary cilia but rather diverge in the cytoplasm, giving rise to a few sub-spindles. Moreover, they did not arise from the dispersion of the ensheathing membrane of a primary cilium, as has been reported in cells undergoing early mitosis, because primary cilia were not observed in either control or treated interphase cells. It is likely that the distal extremity of the centriolar microtubules is not blocked by regulatory proteins and can assemble alpha-tubulin heterodimers, as observed in vitro with isolated centrioles. Unless triggered by the specific axonemal program, during normal mitosis, this elongation process remains exceptional. Observations suggest that this atypical elongation pathway is normally overridden by the efficiency of nucleation from the high number of gamma-tubulin nucleation sites present in the pericentriolar material. The failure of centriole separation and elongation of the centriolar microtubules that was observed is consistent with the formation of monopolar spindles. However, this assembly pathway might only give rise to a limited number of spindle microtubules, unless microtubules detach from the distal ends of the centriole. Alternatively, the presence of additional microtubule bundles on the chromosome face opposite the main pole suggests that microtubules might also be nucleated at the kinetochores. In contrast to the microtubule assembly pathway predominantly mediated by the pericentriolar gamma-tubulin, the results suggest that, below a critical concentration of gamma-tubulin, other assembly mechanisms could contribute to spindle formation. These alternative mechanisms are unable to generate a fully functional mitotic apparatus in most cases (Raynaud-Messina, 2004).

Taken together, the results demonstrate the inefficiency of the pericentriolar material in promoting microtubule nucleation after depletion of gamma-tubulin. They point to an alternative centrosome-driven mechanism of microtubule assembly that might contribute to the formation of abnormal, albeit partially functional, mitotic spindles (Raynaud-Messina, 2004).

γTub23C interacts genetically with brahma chromatin-remodeling complexes in Drosophila melanogaster

The brahma gene encodes the catalytic subunit of the Drosophila BRM chromatin-remodeling complexes. Screening for mutations that interact with brahma, the dominant-negative Pearl-2 allele of γTub23C was isolated. γTub23C encodes one of the two γ-tubulin isoforms in Drosophila and is essential for zygotic viability and normal adult patterning. γ-Tubulin is a subunit of microtubule organizer complexes. This study shows that mutations in lethal (1) discs degenerate 4, which encodes the Grip91 subunit of microtubule organizer complexes, suppress the recessive lethality and the imaginal phenotypes caused by γTub23C mutations. The genetic interactions between γTub23C and chromatin-remodeling mutations suggest that γ-tubulin might have a role in regulating gene expression (Vázquez, 2008).

Proteins identified as part of the eukaryotic cytoskeleton may have more direct roles in transcriptional regulation than originally thought. Actin and actin-related proteins (ARPs) are found in BRM complexes from yeast to humans, including the BRM complexes in Drosophila. The function of actin and ARPs in these complexes is not well understood. Some ARPs interact with DNA-bending proteins and with histones and it was proposed that they facilitate chromatin architecture and interactions between complexes or function as histone chaperones. Actin is also part of preinitiation complexes and is necessary for transcription by RNA polymerases I, II, and III. The α- and/or β-tubulins are also found with a subset of trithorax-group proteins in the mammalian ASCOM complex (Activating signal cointegrator 2, Asc2 complex), which is required for transactivation by nuclear receptors, and in a histone H2A deubiquitinase complex. γ-Tubulin is essential for microtubule function, but unlike α- and β-tubulin, it is not a component of microtubules. Rather, it is located at microtubule-organizing centers (MTOCs) and functions in the initiation of microtubule nucleation and in the establishment of microtubule polarity. γ-Tubulin contributes to the proper formation of mitotic spindles and cytoplasmic microtubular arrays. There are critical cytoskeletal and nuclear envelope connections, linking, for example, MTOCs to the nuclear lamina. In addtion, γ-tubulin has been proposed to have microtubule- and/or centrosome-independent function(s) in mitosis or spindle assembly checkpoints (Vázquez, 2008).

Drosophila embryonic γ-tubulin exists in two related complexes: a large complex similar to the Xenopus γTuRC (γ-tubulin ring complex) (36.9S, ~2000 kDa) and a small soluble complex called γTuSC (γ-tubulin small complex) (8.5S ~240 kDa). The Drosophila γTuRC consists of approximately eight polypeptides, including γ-tubulin, Grip163, Grip128, Grip91, Grip84, Grip75, and GP71WD. The γTuRC has a lockwasher-like structure and a cap at one of the ends of the complex. The Drosophila γTuSC is a tetramer of two γ-tubulin molecules and one molecule each of Grip91 and Grip84. Several γTuSCs form the γTuRC lockwasher region. The other Grips (Grip163, 128, and 75) form the cap (Vázquez, 2008).

Drosophila is the only metazoan in which the genes encoding subunits of the γTuSC and γTuRC complexes have been functionally studied using genetic approaches. Null mutations in dd4 (which encodes Grip91) and in Grip84 are lethal and display defects in spindle assembly (Barbosa, 2003; Colombié, 2006), while null mutations in Grip128 and Grip75 are viable, but sterile (Vázquez, 2008).

In Drosophila there are two γ-tubulin genes, γTub23C and γTub37C. They encode very similar (but not identical) proteins, but they have different expression patterns and mutant phenotypes. γTub37C is largely restricted to the female germline and early stages of embryogenesis. It is required for bicoid (bcd) mRNA localization at mid-oogenesis, female meiosis, and nuclear proliferation. In syncytial embryos, γTub23C is in the soluble small γTuSC fraction and is absent at the centrosome. At this stage, γTub37C is found in both γTuSC and γTuRC fractions. It is localized at the centrosome and over the spindle regions. γTub37C mutants are female sterile (Vázquez, 2008).

The γTub23C isoform is expressed in a variety of tissues in both sexes, including larval brains and imaginal discs, and it is required for somatic mitotic divisions. It is also expressed in ovaries and is the only isoform expressed in testes. γTub23C is required for meiosis in males and for spermatogenesis (Vázquez, 2008).

The γTub23CPl-2 mutation was isolated in a mutant screen designed to identify genes that interact with brm in wing development. In addition to showing genetic interactions with brm, γTub23CPl-2 mutants are homozygous lethal, while the heterozygotes have defects in imaginal eye and wing development. γTub23CPl-2 is a dominant-negative mutation and l(2)23Ce alleles are loss-of-function mutations in γTub23C with recessive phenotypes similar to the dominant phenotypes of γTub23CPl-2. γTub23C has 30% identity to α- and β-tubulins, which are structural components of microtubules. It is known which parts of the β-tubulin protein are involved in autoregulation for translation and for binding and hydrolysis of GTP. The γ-tubulin protein shares some of these regions with β-tubulin. The γTub23C mutations characterized in this work do not map to any of these known regions, with the exception of the truncated form in the γTub23CA15-2 allele. This suggests that the proteins synthesized from the γTub23CA14-9, γTub23CA6-2, γTub23CPl-2, and γTub23Cbmps1 alleles might affect other γ-tubulin functions (Vázquez, 2008).

It was a surprise to identify a dd4 allele with no discernible phenotype except the suppression of some γTub23C mutant phenotypes (including zygotic lethality). Since dd4 encodes Grip91, a protein that physically interacts with γ-tubulin, it is believed that the genetic interactions have important implications (Vázquez, 2008).

Grip91, Grip84, and γ-tubulin form the lockwasher region of γTuRC and γTuSC complexes. Grip91 and Grip84 (or their orthologs in yeast and humans) interact with each other and with γ-tubulin. The interactions between Grip91 and γ-tubulin facilitate binding of GTP to γ-tubulin. Grip91 is required for correct bipolar spindle assembly during mitosis and male meiosis and it helps to locate γ-tubulin to the centrosome (Vázquez, 2008).

Grip91 is an essential protein encoded by the dd4 gene. Semilethal alleles have held-up wings and other imaginal defects and are male sterile. The dd4su(Pl) allele is unusual in that it has no defects in viability, fertility, or developmental patterning. Its only phenotype is the suppression of class I (but not class II) genotypes of γTub23C (Vázquez, 2008).

What is the significance of the two types of γTub23C alleles from the functional point of view? The defects produced by suppressible alleles may involve γTuSC and/or γTuRC functions, while the defects produced by nonsuppressible alleles may involve γTub23C functions independent of the γTuSC and γTuRC complexes. It is also possible that different mutant proteins, although in some cases retaining partial activity, may affect other different functions of γTub23C. Some of these other functions may require Grip91 (and possibly the integrity of γTuRC and/or γTuSC complexes) and some may not. Such functions could affect the assembly of the γTuSC and/or γTuRC complexes, the transport of the complex(es) to subcellular compartments, and/or the relationships of γTub23C with other proteins involved in microtubule-independent processes. It is believed that the new alleles of γTub23C and dd4 that have been characterized can help to test the current structural models of γTuRC and γTuSC complexes proposed in biochemical and crystallographic studies (Vázquez, 2008).

Recent work shows that γ-tubulin has a microtubule-independent role in establishing or maintaining a mitotic checkpoint block (Prigozhina, 2004) and that γTuRCs proteins may have a centrosome-independent role in the spindle assembly checkpoint. For this latter function, γ-tubulin is probably in a complex associated with Cdc20 and BubR1 kinases (Muller, 2006). This study found that the genetic interactions between γTub23C and Brm are caused not by reduced γTub23C transcription, but more probably by the presence of defective γ-tubulin proteins. This suggests roles for γ-tubulin in transcription and/or chromatin remodeling. This is further supported by the recent description of interactions between Pericentrin (an integral centrosomal component) and CHD3, a Brm-related protein in the NuRD chromatin-remodeling complex (Vázquez, 2008).

A centrosome-independent role for gamma-TuRC proteins in the spindle assembly checkpoint.

The spindle assembly checkpoint guards the fidelity of chromosome segregation. It requires the close cooperation of cell cycle regulatory proteins and cytoskeletal elements to sense spindle integrity. The role of the centrosome, the organizing center of the microtubule cytoskeleton, in the spindle checkpoint is unclear. This study found that the molecular requirements for a functional spindle checkpoint included components of the large gamma-tubulin ring complex (gamma-TuRC). However, their localization at the centrosome and centrosome integrity are not essential for this function. Thus, the spindle checkpoint can be activated at the level of microtubule nucleation (Müller, 2006).

Comments on 'A centrosome-independent role for gamma-TuRC proteins in the spindle assembly checkpoint'

Müller (2006) showed that inhibition of the gamma-tubulin ring complex (gamma-TuRC) activates the spindle assembly checkpoint (SAC), which led them to suggest that gamma-TuRC proteins play molecular roles in SAC activation. Because gamma-TuRC inhibition leads to pleiotropic spindle defects, which are well known to activate kinetochore-derived checkpoint signaling, this conclusion might be premature (Taylor, 2007).

Müller (2006) proposed a role for microtubule nucleation in mitotic checkpoint signaling. However, their observations of spindle defects and mitotic delay after depletion of gamma-tubulin ring complex (gamma-TuRC) components are fully consistent with activation of the established pathway of checkpoint signaling in response to incomplete or unstable interactions between kinetochores of mitotic chromosomes and spindle microtubules (Weaver, 2007).


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

date revised: 2 December 2018

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