Induction of centrosome injury, multipolar spindles and multipolar division in cultured V79 cells exposed to dimethylarsinic acid: role for microtubules in centrosome dynamics

Role for microtubules in the induction of multiple microtubule organizing centers (MTOCs) and multipolar spindles by dimethylarsinic acid (DMAA), a methylated derivative of inorganic arsenics, was investigated with respect to the effects of microtubule disruption and reorganization. DMAA induced multiple signals of gamma-tubulin, a well-characterized component of MTOCs in the centrosome, in a manner specific to mitotic cells. The multiple signals of gamma-tubulin were co-localized with multipolar spindles caused by DMAA. Disruption of microtubules by nocodazole (NOZ) suppressed the appearance of centrosome injury caused by DMAA while disorganization of actin microfilaments by cytochalasin D did not. Post-treatment incubation of cells in which multiple signals of gamma-tubulin caused by DMAA had been coalesced to one or two dots by NOZ caused the reappearance of mitotic cells with multiple signals of gamma-tubulin, in conjunction with reorganization of the microtubules. These results suggest a role for microtubules in the dynamic behavior of the mitotic centrosome. DMAA induced aberrant cytokinesis, such as tripolar and quadripolar division, in a concentration-dependent manner. These results, together with the findings of earlier studies, suggest that the centrosome is the primary target for the induction of multipolar spindles by DMAA and the resultant induction of multinucleation and multipolar division.     

Making microtubules and mitotic spindles in cells without functional centrosomes

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

CDK5RAP2 is a pericentriolar protein that functions in centrosomal attachment of the gamma-tubulin ring complex

Microtubule nucleation and organization by the centrosome require gamma-tubulin, a protein that exists in a macromolecular complex called the gamma-tubulin ring complex (gammaTuRC). We report characterization of CDK5RAP2, a novel centrosomal protein whose mutations have been linked to autosomal recessive primary microcephaly. In somatic cells, CDK5RAP2 localizes throughout the pericentriolar material in all stages of the cell cycle. When overexpressed, CDK5RAP2 assembled a subset of centrosomal proteins including gamma-tubulin onto the centrosomes or under the microtubule-disrupting conditions into microtubule-nucleating clusters in the cytoplasm. CDK5RAP2 associates with the gammaTuRC via a short conserved sequence present in several related proteins found in a range of organisms from fungi to mammals. The binding of CDK5RAP2 is required for gammaTuRC attachment to the centrosome but not for gammaTuRC assembly. Perturbing CDK5RAP2 function delocalized gamma-tubulin from the centrosomes and inhibited centrosomal microtubule nucleation, thus leading to disorganization of interphase microtubule arrays and formation of anastral mitotic spindles. Together, CDK5RAP2 is a pericentriolar structural component that functions in gammaTuRC attachment and therefore in the microtubule organizing function of the centrosome. Our findings suggest that centrosome malfunction due to the CDK5RAP2 mutations may underlie autosomal recessive primary microcephaly.     

GSK-3beta regulates proper mitotic spindle formation in cooperation with a component of the gamma-tubulin ring complex, GCP5

Glycogen synthase kinase-3beta (GSK-3beta) is known to play a role in the regulation of the dynamics of microtubule networks in cells. Here we show the role of GSK-3beta in the proper formation of the mitotic spindles through an interaction with GCP5, a component of the gamma-tubulin ring complex (gammaTuRC). GCP5 bound directly to GSK-3beta in vitro, and their interaction was also observed in intact cells at endogenous levels. Depletion of GCP5 dramatically reduced the GCP2 and gamma-tubulin in the gammaTuRC fraction of sucrose density gradients and disrupted gamma-tubulin localization to the spindle poles in mitotic cells. GCP5 appears to be required for the formation or stability of gammaTuRC and the recruitment of gamma-tubulin to the spindle poles. A GSK-3 inhibitor not only led to the accumulation of gamma-tubulin and GCP5 at the spindle poles but also enhanced microtubule nucleation activity at the spindle poles. Depletion of GCP5 rescued this disrupted organization of spindle poles observed in cells treated with the GSK-3 inhibitor. Furthermore, the inhibition of GSK-3 enhanced the binding of gammaTuRC to the centrosome isolated from mitotic cells in vitro. Our findings suggest that GSK-3beta regulates the localization of gammaTuRC, including GCP5, to the spindle poles, thereby controlling the formation of proper mitotic spindles.     

Gamma-tubulin distribution in interphase and mitotic cells upon stabilization and depolymerization of microtubules

Indirect immunofluorescence and digital videomicroscopy were used to study gamma-tubulin distribution in normal mitotic and interphase HeLa cells and after their treatment with microtubule-stabilizing (taxol) and depolymerizing (nocodazole) drugs. In interphase HeLa cells, the affinity-purified antibodies against gamma-tubulin and monoclonal antibodies against acetylated tubulin stain one or two neighboring dots, centrioles. The gamma-tubulin content in two centrioles from the same cell differs insignificantly. Mitotic poles contain fourfold amount of gamma-tubulin as compared with the centrioles in interphase. The effect of nocodazole (5 microg/ml) on interphase cells resulted in lowering the amount of gamma-tubulin in the centrosome, and in 24 h it was reduced by half. Treatment with nocodazole for 2 h caused a fourfold decrease in the gamma-tubulin content in mitotic poles. Besides, the mitotic poles were unevenly stained, the fluorescence intensity in the center was lower than at the periphery. Upon treatment with taxol (10 microg/ml), the gamma-tubulin content in the interphase cell centrosome first decreased, then increased, and in 24 h it doubled as compared with control. In the latter case, bright dots appeared in the cell cytoplasm along the microtubule bundles. However, after 24 h treatment with taxol, the total amount of intracellular gamma-tubulin did not change. Treatment with taxol for 2-4 h halved the gamma-tubulin content in the centrosome as compared with normal mitosis. In some cells, antibodies against gamma-tubulin revealed up to four microtubule convergence foci. Other numerous microtubule convergence foci were not stained. Thus, the existence of at least three gamma-tubulin pools is suggested: (1) constitutive gamma-tubulin permanently associated with centrioles irrespective of the cell cycle stage and of their ability to serve as microtubule organizing centers; (2) gamma-tubulin unstably associated with the centrosome only during mitosis; (3) cytoplasmic gamma-tubulin that can bind to stable microtubules.     

Maternal gamma (gamma)-tubulin is involved in microtubule reorganization during bovine fertilization and parthenogenesis

In this study, gamma-tubulin distribution was determined chronologically in conjunction with microtubule dynamics during bovine fertilization and parthenogenesis. In unfertilized bovine oocytes, gamma-tubulin was identified in the cytoplasm, mainly in the cortex and concentrated in the meiotic spindle. Following sperm penetration, gamma-tubulin in the cytoplasm was recruited by a sperm component. During pronuclear apposition, gamma-tubulin was localized as spots at the spindle poles. gamma-tubulin spots were observed in blastomeres of embryos cleaved in vitro. Following electrical stimulation, gamma-tubulin and microtubule matrix were noted in oocyte cortex. In the late pronuclear stage, considerably less gamma-tubulin and microtubules were detected in the cytoplasm. At the mitotic metaphase of parthenotes, gamma-tubulin was recruited to the condensed chromatin and concentrated in the spindle. The gamma-tubulin spots were not detected until the 8-cell stage of parthenotes. This suggests that maternal gamma-tubulin is recruited by a sperm component to reconstitute the zygotic centrosome. In the absence of sperm components, the cell cycle-related assembly of gamma-tubulin organizes microtubule nucleation for positioning the pronucleus and spindle protein of mitotic metaphase during the first cell cycle of bovine parthenotes.     

Human gamma-tubulin functions in fission yeast

gamma-Tubulin is a phylogenetically conserved component of microtubule- organizing centers that is essential for viability and microtubule function. To examine the functional conservation of gamma-tubulin, we have tested the ability of human gamma-tubulin to function in the fission yeast Schizosaccharomyces pombe. We have found that expression of a human gamma-tubulin cDNA restores viability and a near-normal growth rate to cells of S. pombe lacking endogenous gamma-tubulin. Immunofluorescence microscopy showed that these cells contained normal mitotic spindles and interphase microtubule arrays, and that human gamma-tubulin, like S. pombe gamma-tubulin, localized to spindle pole bodies, the fungal microtubule-organizing centers. These results demonstrate that human gamma-tubulin functions in fission yeast, and they suggest that in spite of the great morphological differences between the microtubule-organizing centers of humans and fission yeasts, gamma-tubulin is likely to perform the same tasks in both. They suggest, moreover, that the proteins that interact with gamma-tubulin, including, most obviously, microtubule-organizing center proteins, must also be conserved. We have also found that a fivefold overexpression of S. pombe gamma-tubulin causes no reduction in growth rates or alteration of microtubule organization. We hypothesize that the excess gamma-tubulin is maintained in the cytoplasm in a form incapable of nucleating microtubule assembly. Finally, we have found that expression of human gamma-tubulin or overexpression of S. pombe gamma-tubulin causes no significant alteration of resistance to the antimicrotubule agents benomyl, thiabendazole and nocodazole.     

Regulation of tubulin synthesis and cell cycle progression in mammalian cells by gamma-tubulin-mediated microtubule nucleation

We have previously shown that gamma-tubulin, the third member of the tubulin family that functions in microtubule nucleation, when overexpressed, accumulates throughout the cytoplasm and forms numerous ectopic microtubule nucleation sites in mammalian cells (Shu and Joshi [1995] J. Cell. Biol. 130:1137-1147). We now show that overexpression of gamma-tubulin differentially upregulates the synthesis of alpha- and beta-tubulins in mammalian cells. Surprisingly, despite a dramatic increase in the level of gamma-tubulin protein in transfected cells, there is no obvious alteration in the level of endogenous gamma-tubulin mRNA, suggesting that synthesis of gamma-tubulin might employ a regulatory mechanism other than the autoregulatory pathway shared by alpha- and beta-tubulins. Interestingly, a significant number of mammalian cells transfected with gamma-tubulin fail to form normal bipolar mitotic spindle during mitosis; instead, numerous microtubules occur in the cytoplasm intermingled with the condensed chromosomes. In addition, they reduplicate their DNA after an abnormal mitotic exit. These results thus suggest that the number of microtubule nucleation sites, or even gamma-tubulin itself, might play an important role in the regulation of tubulin synthesis as well as cell cycle progression.     

Gamma-tubulin at ten: progress and prospects

The existence of gamma-tubulin was first reported approximately ten years ago, and it is appropriate to review the progress that has been made in gamma-tubulin research and to discuss some of the unanswered questions about gamma-tubulin function. gamma-Tubulin is ubiquitous in eukaryotes and is generally quite conserved. Two highly divergent gamma-tubulins have been discovered, however, one in Saccharomyces cerevisiae and one in Caenorhabditis elegans. Several organisms have two gamma-tubulin genes. In Drosophila melanogaster, the two gamma-tubulins differ significantly in sequence and expression pattern. In other organisms the two gamma-tubulins are almost identical and expression patterns have not been determined. gamma-Tubulin is located at microtubule organizing centers in many organisms, and it is also frequently associated with the mitotic spindle. gamma-Tubulin is essential for the formation of functional mitotic spindles in all organisms that have been examined to date. In animal cells, complexes containing gamma-tubulin are located at microtubule organizing centers where they nucleate the assembly of microtubules. In spite of the considerable progress that has been made in gamma-tubulin research important questions remain to be answered. The exact mechanisms of microtubule nucleation by gamma-tubulin complexes remain to be resolved as do the mechanisms by which microtubule nucleation from gamma-tubulin complexes is regulated. Finally, there is evidence that gamma-tubulin has important functions in addition to microtubule nucleation, and these functions are just beginning to be investigated.     

NEDD1-dependent recruitment of the gamma-tubulin ring complex to the centrosome is necessary for centriole duplication and spindle assembly

The centrosome is the major microtubule organizing structure in somatic cells. Centrosomal microtubule nucleation depends on the protein gamma-tubulin. In mammals, gamma-tubulin associates with additional proteins into a large complex, the gamma-tubulin ring complex (gammaTuRC). We characterize NEDD1, a centrosomal protein that associates with gammaTuRCs. We show that the majority of gammaTuRCs assemble even after NEDD1 depletion but require NEDD1 for centrosomal targeting. In contrast, NEDD1 can target to the centrosome in the absence of gamma-tubulin. NEDD1-depleted cells show defects in centrosomal microtubule nucleation and form aberrant mitotic spindles with poorly separated poles. Similar spindle defects are obtained by overexpression of a fusion protein of GFP tagged to the carboxy-terminal half of NEDD1, which mediates binding to gammaTuRCs. Further, we show that depletion of NEDD1 inhibits centriole duplication, as does depletion of gamma-tubulin. Our data suggest that centriole duplication requires NEDD1-dependent recruitment of gamma-tubulin to the centrosome.     

Gamma-tubulin and MTOCs in Paramecium

The characterization of the two Paramecium gamma-tubulin genes, gammaPT1 and gammaPT2, allowed us to raise Paramecium-specific antibodies, directed against their most divergent carboxy-terminal peptide and to analyze the localization and dynamics of gamma-tubulin throughout the cell cycle. As in other cell types, a large proportion of the protein was found to be cytosolic, but in contrast to the general situation, gamma-tubulin was found to be permanently associated to four types of sites: basal bodies, the micronuclear compartment--within which mitotic and meiotic spindles develop without membrane breakdown, the pores of the contractile vacuoles and the cytoproct which are cortical microtubular organelles fulfilling excretory functions. In addition, a transient site of gamma-tubulin and microtubule assembly was observed at the site of nuclear exchange during conjugation. This complexity accounts for the nucleation of most of the numerous and diverse microtubule arrays present in Paramecium. The sites and mode of nucleation of the microtubule bundles formed in the macronuclear compartment during division remain unclear. These observations lead us to discuss the relationships between microtubules, gamma-tubulin and MTOCs.     

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

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

Microtubule-dependent microtubule nucleation based on recruitment of gamma-tubulin in higher plants

Despite the absence of a conspicuous microtubule-organizing centre, microtubules in plant cells at interphase are present in the cell cortex as a well oriented array. A recent report suggests that microtubule nucleation sites for the array are capable of associating with and dissociating from the cortex. Here, we show that nucleation requires extant cortical microtubules, onto which cytosolic gamma-tubulin is recruited. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain gamma-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting gamma-tubulin function with a specific antibody. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated. However, when the membrane is exposed to a cytosolic extract, gamma-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic gamma-tubulin complex shuttles between the cytoplasm and the side of a cortical microtubule, and has nucleation activity only when bound to the microtubule.     

Localization of gamma-tubulin in interphase and mitotic cells of a unicellular eukaryote, Giardia intestinalis

Giardia intestinalis, a bi-nucleated amitochondrial flagellate, possesses a complex cytoskeleton based on several microtubular systems (flagella, adhesive disk, median body, funis, mitotic spindles). MTOCs of the individual systems have not been fully defined. By using monoclonal antibodies against a conserved synthetic peptide from the C-terminus of human gamma-tubulin we investigated occurrence and distribution of gamma-tubulin in interphase and mitotic Giardia cells. On the immunoblots of Giardia cytoskeletal extracts the antibodies bound to a single polypeptide of approximately 50 kDa. Immunostaining of the interphase cell demonstrated gamma-tubulin as four bright spots at the basis of four out of eight flagella. Gamma-tubulin label was associated with perikinetosomal areas of the ventral and posterolateral pairs of flagella which are formed de novo during cell division. Basal body regions of the anterolateral and caudal pairs of flagella which persist during the division and are integrated into the flagellar systems of the daughter cells did not show gamma-tubulin staining. At early mitosis, gamma-tubulin spots disappeared reappearing again at late mitosis in accord with reorientation of parent flagella and reorganization of flagellar apparatus during cell division. The antibody-detectable gamma-tubulin epitope was absent at the poles of both mitotic spindles. Albendazole-treated Giardia, in which spindle assembly was completely inhibited, showed the same gamma-tubulin staining pattern thus confirming that the fluorescent label is exclusively located in the basal body regions. Our results point to a role of gamma-tubulin in nucleation of microtubules of newly formed flagella and indicate unusual mitotic spindle assembly. Moreover, the demonstration of gamma-tubulin in Giardia shows ubiquity of this protein through the evolutionary history of eukaryotes.     

Structure-effect relationship in the induction of mitotic phase-specific abnormality of centrosome integrity and multipolar spindles by steroidal estrogens and their derivatives in cultured mammalian cells

In order to determine the structure-effect relationship in the induction of centrosome disintegrity (abnormality of gamma-tubulin signals) and multipolar spindles in a cultured fibroblast cell line V79 by steroidal estrogens, the activities of various estrogens and their derivatives were investigated. Induction of centrosome disintegrity by estrogens was specific in cells in the mitotic phase and was not observed in interphase cells. The centrosome disintegrity induced 24 h after exposure to estrogens was accompanied by the appearance of multinucleated cells, but the microtubule network was organized. The rank order of potency of estrogens in inducing mitotic phase-specific centrosome disintegrity and multipolar spindles was as follows: 2-methoxyestradiol>dihydroequilin 3-methyl ether=equilin 3-methyl ether>17alpha-estradiol>17beta-estradiol 3-methyl ether=17beta-estradiol>dihydroequilin>estrone 3-methyl ether. Equilin and estrone were not effective in causing centrosome disintegrity. These results suggest that the 17-hydroxyl group, irrespective of whether it is the sterically alpha or beta form, is necessary for estradiol and dihydroequilin to cause centrosome disintegrity and that O-methylation at the C-3 position was effective for equilin and dihydroequilin in enhancing the centrosome abnormality. 2-Methoxyestradiol was the most potent inducer of the centrosome disintegrity among the tested compounds and caused the induction of multiple signals of gamma-tubulin, including more than five signals.     

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

Gamma-tubulin, a protein critical for microtubule assembly, functions within multiprotein complexes. However, little is known about the respective role of gamma-tubulin partners in metazoans. For the first time in a multicellular organism, we have investigated the function of Dgrip84, the Drosophila orthologue of the Saccharomyces cerevisiae gamma-tubulin-associated protein Spc97p. Mutant analysis shows that Dgrip84 is essential for viability. Its depletion promotes a moderate increase in the mitotic index, correlated with the appearance of monopolar or unpolarized spindles, impairment of centrosome maturation, and increase of polyploid nuclei. This in vivo study is strengthened by an RNA interference approach in cultured S2 cells. Electron microscopy analysis suggests that monopolar spindles might result from a failure of centrosome separation and an unusual microtubule assembly pathway via centriolar triplets. Moreover, we point to an involvement of Dgrip84 in the spindle checkpoint regulation and in the maintenance of interphase microtubule dynamics. Dgrip84 also seems essential for male meiosis, ensuring spindle bipolarity and correct completion of cytokinesis. These data sustain that Dgrip84 is required in some aspects of microtubule dynamics and organization both in interphase and mitosis. The nature of a minimal gamma-tubulin complex necessary for proper microtubule organization in the metazoans is discussed.     

hTPX2 is required for normal spindle morphology and centrosome integrity during vertebrate cell division

Bipolar spindle formation is essential for the accurate segregation of genetic material during cell division. Although centrosomes influence the number of spindle poles during mitosis, motor and non-motor microtubule-associated proteins (MAPs) also play key roles in determining spindle morphology. TPX2 is a novel MAP also characterized in Xenopus cell-free extracts. To examine hTPX2 (human TPX2) function in human cells, we used siRNA to knock-down its expression and found that cells lacking hTPX2 arrest in mitosis with multipolar spindles. NuMA, gamma-tubulin, and centrin localize to each pole, and nocodazole treatment of cells lacking hTPX2 demonstrates that the localization of gamma-tubulin to multiple spindle poles requires intact microtubules. Furthermore, we show that the formation of monopolar microtubule arrays in human cell extracts does not require hTPX2, demonstrating that the mechanism by which hTPX2 promotes spindle bipolarity is independent of activities focusing microtubule minus ends at spindle poles. Finally, inhibition of the kinesin Eg5 in hTPX2-depleted cells leads to monopolar spindles, indicating that Eg5 function is necessary for multipolar spindle formation in the absence of hTPX2. Our observations reveal a structural role for hTPX2 in spindles and provide evidence for a balance between microtubule-based motor forces and structural spindle components.     

Gamma-tubulin in barley and tobacco: sequence relationship and RNA expression patterns in developing leaves during mitosis and post-mitotic growth

gamma-Tubulin is typically associated with microtubule organising centres, such as the centrosome, and appears to mediate microtubule nucleation. Centrosomes are usually not found in higher plants, but active genes homologous to gamma-tubulin have been identified in the plant kingdom, including the angiosperms Arabidopsis, maize and rice. We have isolated and characterised gamma-tubulin cDNA sequences of two further angiosperm species, barley and tobacco. Sequence comparison revealed a phylogenetic tree with distinct clusters corresponding to the systematic position of the species. Furthermore, domains, thought to be exposed in the folded protein and to be candidates for interaction with associated, nucleation-site related proteins, exhibited motifs highly specific of multicellular plants. Strong expression of the gamma-tubulin genes, as determined by Northern blotting, correlated with mitotic activity. Expression dropped distinctly when mitotic activity ceased. Thus, in post-mitotic tissues that showed intricate reshuffling of cortical microtubule arrays related to cell shaping only very low gamma-tubulin steady-state RNA levels were found, contrasting with the situation for alpha-tubulin. The findings indicate that gamma-tubulin expression in plants may be more tightly linked to mitosis, although there is some gamma-tubulin expression at the RNA level even after mitosis. It follows that the post-mitotic changes in microtubular arrays may be less dependent on concurrent gamma-tubulin RNA expression than mitotic cells.     

The abnormal spindle protein is required for germ cell mitosis and oocyte differentiation during Drosophila oogenesis

In this study, we present evidence that the asp function is required in oogenesis for germline cell divisions as well as for cyst polarity and oocyte differentiation. Consistent with previously described roles in spindle organization during Drosophila meiosis and mitosis, asp mutation leads to severe defects in spindle microtubule organization within the germarium. The mitotic spindles of the mutant cystocytes are composed by wavy microtubules and have abnormal poles that often lack gamma-tubulin. The fusome structure is also compromised. In the absence of asp function, the cystocyte divisions fail resulting in egg chamber with fewer than 16 germ cells. Moreover, the microtubule network within the developing germline cysts may assemble incorrectly in turn affecting the microtubule based transport of the specific determinants that is required during mid-oogenesis for the oocyte differentiation program.     

Drosophila melanogaster gamma-TuRC is dispensable for targeting gamma-tubulin to the centrosome and microtubule nucleation

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