The role of Xgrip210 in gamma-tubulin ring complex assembly and centrosome recruitment

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

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

The gamma-tubulin ring complex (gammaTuRC) is a protein complex of relative molecular mass approximately 2.2 x 10(6) that nucleates microtubules at the centrosome. Here we use electron-microscopic tomography and metal shadowing to examine the structure of isolated Drosophila gammaTuRCs and the ends of microtubules nucleated by gammaTuRCs and by centrosomes. We show that the gammaTuRC is a lockwasher-like structure made up of repeating subunits, topped asymmetrically with a cap. A similar capped ring is also visible at one end of microtubules grown from isolated gammaTuRCs and from centrosomes. Antibodies against gamma-tubulin label microtubule ends, but not walls, in centrosomes. These data are consistent with a template-mediated mechanism for microtubule nucleation by the gammaTuRC.     

CDK5RAP2 stimulates microtubule nucleation by the gamma-tubulin ring complex

CDK5RAP2 is a human microcephaly protein that contains a γ-tubulin complex (γ-TuC)-binding domain conserved in Drosophila melanogaster centrosomin and Schizosaccharomyces pombe Mto1p and Pcp1p, which are γ-TuC-tethering proteins. In this study, we show that this domain within CDK5RAP2 associates with the γ-tubulin ring complex (γ-TuRC) to stimulate its microtubule-nucleating activity and is therefore referred to as the γ-TuRC-mediated nucleation activator (γ-TuNA). γ-TuNA but not its γ-TuC-binding-deficient mutant stimulates microtubule nucleation by purified γ-TuRC in vitro and induces extensive, γ-TuRC-dependent nucleation of microtubules in a microtubule regrowth assay. γ-TuRC bound to γ-TuNA contains NME7, FAM128A/B, and actin in addition to γ-tubulin and GCP2-6. RNA interference-mediated depletion of CDK5RAP2 impairs both centrosomal and acentrosomal microtubule nucleation, although γ-TuRC assembly is unaffected. Collectively, these results suggest that the γ-TuNA found in CDK5RAP2 has regulatory functions in γ-TuRC-mediated microtubule nucleation.     

A new function for the gamma-tubulin ring complex as a microtubule minus-end cap

Microtubule nucleation from centrosomes involves a lockwasher-shaped protein complex containing gamma-tubulin, named the gamma-tubulin ring complex (gammaTuRC). Here we investigate the mechanism by which the gammaTuRC nucleates microtubules, using a direct labelling method to visualize the behaviour of individual gammaTuRCs. A fluorescently-labelled version of the gammaTuRC binds to the minus ends of microtubules nucleated in vitro. Both gammaTuRC-mediated nucleation and binding of the gammaTuRC to preformed microtubules block further minus-end growth and prevent microtubule depolymerization. The gammaTuRC therefore acts as a minus-end-capping protein, as confirmed by electron-microscopic examination of gold-labelled gammaTuRCs. These data support a nucleation model for gammaTuRC function that involves capping of microtubules.     

Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex

Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC). In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors gamma-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC.     

Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules

gamma-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1- 12). Here, we report the purification and characterization of both complexes that we name gamma-tubulin small complex (gammaTuSC; approximately 280,000 D) and Drosophila gammaTuRC ( approximately 2,200,000 D). In addition to gamma-tubulin, the gammaTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The gammaTuSC is a structural subunit of the gammaTuRC, a larger complex containing about six additional polypeptides. Like the gammaTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), the Drosophila gammaTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with approximately 13 radially arranged structural repeats. The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.     

Reconstitution and characterization of budding yeast gamma-tubulin complex

Nucleation of microtubules is central to assembly of the mitotic spindle, which is required for each cell division. gamma-Tubulin is a universal component essential for microtubule nucleation from centrosomes. To elucidate the mechanism of microtubule nucleation in budding yeast we reconstituted and characterized the yeast gamma-tubulin complex (Tub4p complex) produced in insect cells. The recombinant complex has the same sedimentation coefficient (11.6 S) as the native complex in yeast cell extracts and contains one molecule of Spc97p, one molecule of Spc98p, and two molecules of Tub4p. The reconstituted Tub4p complex binds preformed microtubules and has a low nucleating activity, allowing us to begin a detailed analysis of conditions that enhance this nucleating activity. We tested whether binding of the recombinant Tub4p complex to the spindle pole body docking protein Spc110p affects its nucleating activity. The solubility of recombinant Spc110p in insect cells is improved by coexpression with yeast calmodulin (Cmd1p). The Spc110p/Cmd1p complex has a small sedimentation coefficient (4.2 S) and a large Stokes radius (14.3 nm), indicative of an elongated structure. The Tub4p complex binds Spc110p/Cmd1p via Spc98p and the K(d) for binding is 150 nM. The low nucleation activity of the Tub4p complex is not enhanced when it is bound to Spc110p/Cmd1p, suggesting that it requires additional components or modifications to achieve robust activity. Finally, we report the identification of a large 22 S Tub4p complex in yeast extract that contains multimers of Spc97p similar to gamma-tubulin ring complexes found in higher eukaryotic cells.     

Gamma-tubulin37C and gamma-tubulin ring complex protein 75 are essential for bicoid RNA localization during drosophila oogenesis

bicoid (bcd) RNA localization requires the activity of exuperantia and swallow at sequential steps of oogenesis and is microtubule dependent. In a genetic screen, we identified two novel genes essential for bcd RNA localization. They encode maternal gamma-Tubulin37C (gammaTub37C) and gamma-tubulin ring complex protein 75 (Dgrip75), both of which are gamma-tubulin ring complex components. Mutations in these genes specifically affect bcd RNA localization, whereas other microtubule-dependent processes during oogenesis are not impaired. This provides direct evidence that a subset of microtubules organized by the gamma-tubulin ring complex is essential for localization of bcd RNA. At stage 10b, we find gammaTub37C and Dgrip75 anteriorly concentrated and propose the formation of a microtubule-organizing center at the anterior pole of the oocyte.     

Characterization of gamma-tubulin filaments in mammalian cells

Overexpression of γ-tubulin leads to the formation of filaments, but nothing is known about such filaments with regard to possible presence in cells, structure and probable dynamics. Here, we used mammalian cell lines to investigate the ability of γ-tubulin to form filaments. We found that γ-tubulin produces fibers called γ-tubules in a GTP-dependent manner and that γ-tubules are made up of pericentrin and the γ-tubulin complex proteins 2, 3, 5 and 6. Furthermore, we noted that the number of cells with cytosolic γ-tubules is increased in non-dividing cells. Our experiments showed that γ-tubules are polar structures that have a low regrowth rate compared to microtubules. Also, we observed that γ-tubules were disassembled by treatment with cold, colcemid, citral dimethyl acetal, dimethyl fumarate or mutation of γ-tubulin GTPase domain, but were increased in number by treatment with taxol or by stable expression of the γ-tubulin^1–333 GTPase domain. Our results demonstrate that γ-tubulin forms filaments, and such assembly is facilitated by the GTPase domain of γ-tubulin.     

Differential properties of the two Drosophila gamma-tubulin isotypes.

The functional significance of distinct gamma-tubulins in several unrelated eukaryotes remains an enigma due to the difficulties to investigate this question experimentally. Using specific nucleotidic and immunological probes, we have demonstrated that the two divergent Drosophila gamma-tubulins, gamma-tub23C and gamma-tub37CD, are expressed in cultured cells. Gamma-tub37CD is constantly detected at the centrosome and absent in the mitotic spindle, while gamma-tub23C is extensively recruited to the centrosome during mitosis and relocalizes in the mitotic spindle. The two gamma-tubulins exhibit distinct biochemical properties. Gamma-tub23C is present in the soluble gamma-tubulin small complexes (10S) and gamma-tubulin big complexes (35S) and is loosely associated to the cytoskeleton. In contrast, gamma-tub37CD is undetectable in the soluble fraction and exhibits a tight binding to the centrosome. Syncytial embryos also contain the two gamma-tubulin isotypes, which are differentially recruited at the centrosome. Gamma-tub23C is present in the 10S soluble complexes only, while y-tub37CD is contained in the two soluble complexes and is recruited at the centrosome where it exhibits an heterogeneous binding. These results demonstrated an heterogeneity of the two Drosophila gamma-tubulin isotypes both in the cytoskeletal and the soluble fractions. They suggest the direct implication of the 35S complex in the centrosomal recruitment of gamma-tubulin and a conditional functional redundancy between the two gamma-tubulins.     

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.     

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.     

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.     

Disassembly and reconstitution of yeast 60S ribosomal subunits

Summary Yeast 60S ribosomal subunits have been dissociated by reversible modification with dimethylmaleic anhydride. Treatment with 40 mol reagent/ml releases 35% of the protein, producing core particles inactive in polyphenylalanine synthesis, which are totally or highly deficient in 17 different proteins. This preparation of residual particles recovers 45% of the original activity upon incubation with the released proteins. The reconstituted particles can be isolated by centrifugation without loss of activity, having the protein composition of the original subunits.Abbreviations DMMA Dimethylmaleic Anhydride     

Molecular analysis of the cytosolic Dictyostelium gamma-tubulin complex

gamma-Tubulin plays an essential role in microtubule nucleation and organization and occurs, besides its centrosomal localization, in the cytosol, where it forms soluble complexes with other proteins. We investigated the size and composition of gamma-tubulin complexes in Dictyostelium, using a mutant cell line in which the endogenous copy of the gamma-tubulin gene had been replaced by a tagged version. Dictyostelium gamma-tubulin complexes were generally much smaller than the large gamma-tubulin ring complexes found in higher organisms. The stability of the small Dictyostelium gamma-tubulin complexes depended strongly on the purification conditions, with a striking stabilization of the complexes under high salt conditions. Furthermore, we cloned the Dictyostelium homolog of Spc97 and an almost complete sequence of the Dictyostelium homolog of Spc98, which are both components of gamma-tubulin complexes in other organisms. Both proteins localize to the centrosome in Dictyostelium throughout the cell cycle and are also present in a cytosolic pool. We could show that the prevailing small complex present in Dictyostelium consists of DdSpc98 and gamma-tubulin, whereas DdSpc97 does not associate. Dictyostelium is thus the first organism investigated so far where the three proteins do not interact stably in the cytosol.     

Noncore components of the fission yeast gamma-tubulin complex

Relatively little is known about the in vivo function of individual components of the eukaryotic gamma-tubulin complex (gamma-TuC). We identified three genes, gfh1+, mod21+, and mod22+, in a screen for fission yeast mutants affecting microtubule organization. gfh1+ is a previously characterized gamma-TuC protein weakly similar to human gamma-TuC subunit GCP4, whereas mod21+ is novel and shows weak similarity to human gamma-TuC subunit GCP5. We show that mod21p is a bona fide gamma-TuC protein and that, like gfh1Delta mutants, mod21Delta mutants are viable. We find that gfh1Delta and mod21Delta mutants have qualitatively normal microtubule nucleation from all types of microtubule-organizing centers (MTOCs) in vivo but quantitatively reduced nucleation from interphase MTOCs, and this is exacerbated by mutations in mod22+. Simultaneous deletion of gfh1p, mod21p, and alp16p, a third nonessential gamma-TuC protein, does not lead to additive defects, suggesting that all three proteins contribute to a single function. Coimmunoprecipitation experiments suggest that gfh1p and alp16p are codependent for association with a small "core" gamma-TuC, whereas mod21p is more peripherally associated, and that gfh1p and mod21p may form a subcomplex independently of the small gamma-TuC. Interestingly, sucrose gradient analysis suggests that the major form of the gamma-TuC in fission yeast may be a small complex. We propose that gfh1p, mod21p, and alp16 act as facultative "noncore" components of the fission yeast gamma-TuC and enhance its microtubule-nucleating ability.     

Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex.

This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined. The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity. Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe-2S] cluster, and exhibits a typical flavodoxin absorption spectrum. Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected. C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe-2S] cluster. The cluster is very sensitive to oxygen damage. Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified. Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen.     

Characterization of new regulatory elements within the Drosophila bithorax complex

The homeotic Abdominal-B (Abd-B) gene expression depends on a modular cis-regulatory region divided into discrete functional domains (iab) that control the expression of the gene in a particular segment of the fly. These domains contain regulatory elements implicated in both initiation and maintenance of homeotic gene expression and elements that separate the different domains. In this paper we have performed an extensive analysis of the iab-6 regulatory region, which regulates Abd-B expression at abdominal segment A6 (PS11), and we have characterized two new polycomb response elements (PREs) within this domain. We report that PREs at Abd-B cis-regulatory domains present a particular chromatin structure which is nuclease accessible all along Drosophila development and both in active and repressed states. We also show that one of these regions contains a dCTCF and CP190 dependent activity in transgenic enhancer-blocking assays, suggesting that it corresponds to the Fab-6 boundary element of the Drosophila bithorax complex.     

Partial reconstitution of 60S ribosomal subunits from yeast

Summary Ribosomal 60S subunits active in polyphenylalanine synthesis can be reconstituted from core particles lacking 20–40% of the total protein. These core particles were obtained by treatment of yeast 60S subunits with dimethylmaleic anhydride, a reagent for protein amino groups. Upon reconstitution a complementary amount of split proteins is incorporated into the ribosomal particles, which have the sedimentation coefficient of the original subunits. Ribosomal protein fractions obtained by extraction with 1.25 M NH₄Cl, 4 M LiCl, 7 M LiCl, or 67% acetic acid, are much less efficient in the reconstitution of active subunits from these core particles than the corresponding released fraction prepared with dimethylmaleic anhydride. Attempts to reconstitute active subunits from protein-deficient particles obtained with 1.25 M NH₄Cl plus different preparations of ribosomal proteins, including the fraction released with dimethylmaleic anhydride, were unsuccessful. Therefore, under our conditions, of the disassembly procedures assayed only dimethylmaleic anhydride allows partial reconstitution of active 60S subunits.Abbreviation DMMA dimethylmaleic anhydride