The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Harish C Joshi - One of the best experts on this subject based on the ideXlab platform.
-
Gamma Tubulin and microtubule nucleation in mammalian cells
Methods in Cell Biology, 2001Co-Authors: Harish C Joshi, Jun ZhouAbstract:Publisher Summary This chapter describes methods to study the function of γ-Tubulin in mammalian cells. Antibody interference is a method often used to study the properties of proteins, using antibodies that react with the protein of interest and block its function. This approach can also be used to investigate the function of γ-Tubulin in the nucleation of microtubule assembly in mammalian cells. The γ-Tubulin antibody can be generated to conserved regions of this protein, and then the antibody can be incubated with permeabilized cells to study γ-Tubulin function in vitro or injected into cultured mammalian cells to study γ-Tubulin function in vivo. Expression of exogenous γ-Tubulin is another important approach to study the function of γ-Tubulin in microtubule nucleation in vivo in mammalian cells. To doso, a γ-Tubulin expression vector with a strong promoter needs to be constructed and, on transfection, the promoter in the vector can efficiently drive the expression of γ-Tubulin in mammalian cells. Consequently, γ-Tubulin and its function in microtubule nucleation can be investigated.
-
centrosome reduction during mouse spermiogenesis
Developmental Biology, 1998Co-Authors: G Manandhar, Tim Stearns, Harish C Joshi, Peter Sutovsky, Gerald SchattenAbstract:The sperm does not contribute the centrosome during murine fertilization. To determine the manner in which a functional centrosome is reduced, we have studied centrosome degeneration during spermiogenesis of mice. The round spermatids display normal centrosomes consisting of a pair of centrioles along with Gamma-Tubulin containing foci. However, they do not seem to organize microtubules. Elongating spermatids display Gamma-Tubulin spots in the neck region, while microtubules are organized from the perinuclear ring as the manchette. Electron microscopic studies using immunogold labeling revealed that Gamma-Tubulin is mainly localized in the centriolar adjunct from which an aster of microtubules emanates. Microtubules repolymerized randomly in the cytoplasm after nocodazole treatment and reversal. Gamma-Tubulin dissociates from the neck region and is discarded in the residual bodies during spermiation. The distal centriole degenerates during testicular stage of spermiogenesis, while the proximal centriole is lost during epididymal stage. Loss of centrosomal protein and centrioles in mouse sperm further confirm the maternal inheritance of centrosome during murine fertilization.
-
microtubule organization and distribution of Gamma Tubulin in male meiosis of lepidoptera
Molecular Reproduction and Development, 1996Co-Authors: Klaus Werner Wolf, Harish C JoshiAbstract:Meiotic spindles in males of higher Lepidotera are unusual in that the bulk of the spindle micro-tubules (MTs) ends about halfway between the equatorial plate and the centrosomes in metaphase. It appears worthwhile to determine how the MTs are nucleated, while their pole proximal ends are distant from the centrosomes. To this end, spermatocytes of Phragmatobia fuliginosa (Arctiidae), collected in the field, were double-labeled with antibodies to beta- and Gamma-Tubulin. The former antibody reveals the entire microtubular cytoskeleton, and the latter is directed against a newly-discovered tublin isoform that is prevalent in microtubule-organizing centers (MTOCs). The immunocytochemical work was supplemented by a fine structural analysis of MTOCs and spindles. Gamma-Tubulin was clearly detected at the spindle poles, and prominent microtubular asters originated from these sites. Additionally, MT arrays at both sides of the equatorial plate in metaphase spermatocytes contained Gamma-Tubulin. The staining persisted in late anaphase, when kinetochore MTs are depolymerized. This indicates that at least nonkinetochore MTs contain Gamma-Tubulin. The analysis of ultrathin sections through spindles revealed large amounts of pericentriolar material at the spindles poles, in prometaphase through anaphase. The spindle MTs appeared as regular, straight elements in longitudinal sections. We assume that Gamma-Tubulin is located at the pole proximal ends of the MTs and/or is associated with the spindle MTs throughout their lengths. In order to distinguish between these possibilities, testes of Ephestia kuehniella (Pyralidae), a laboratory species, were cold-treated prior to double-labeling with antibodies to beta- and Gamma-Tubulin. The treatment was expected to depolymerize MTs. Astral MTs, which were nucleated end-on by Gamma-Tubulin-containing material, indeed depolymerized. In contrast, the Gamma-Tubulin-containing spindle MTs persisted. It is, therefore, conceivable that Gamma-Tubulin is associated with MTs throughout their lengths in male meiosis of Lepidoptera species. It is plausible that this association stabilizes the MTs against cold-induced disassembly.
-
distribution of Gamma Tubulin differs in primary and secondary oocytes of ephestia kuehniella pyralidae lepidoptera
Molecular Reproduction and Development, 1996Co-Authors: Klaus Werner Wolf, Harish C JoshiAbstract:In a previous study, barrel-shaped spindles were found in metaphase I oocytes of Ephestia kuehniella (Pyralidae, Lepidoptera). Aster microtubules (MTs) were missing (Wolf, 1993 : Cell Motil Cytoskeleton 24 :200-204). This points to an acentriolar organization of the spindle apparatus. The present study was aimed at the question of whether Gamma-Tubulin, a newly detected member of the Tubulin superfamily that has often been identified in microtubule-organizing centers, plays a role in the nucleation of MTs in meiotic spindles of the moth. To this end, the distribution of Gamma-Tubulin was examined in oocytes of E. kuehniella using an antibody against Gamma-Tubulin in combination with indirect immunofluorescence. The antibody evenly decorated spindle MTs in metaphase I oocytes of the moth. Enhanced staining of the spindle poles was not detectable. In subsequent stages of meiosis, Gamma-Tubulin was gradually lost from spindle MTs and was then found at the surface of the so-called elimination chromatin. Female meiosis in Lepidoptera is achiasmatic. The elimination chromatin, i.e., modified and persisting synaptonemal complexes, is believed to keep homologous chromosomes linked until the onset of anaphase I. In meiosis I of female Lepidoptera, the elimination chromatin persists at the spindle equator between the segregating chromatin masses. It is plausible to assume that Gamma-Tubulin is involved in spindle organization in the absence of canonical centrosomes. In MTs of metaphase II spindles of E. kuehniella, Gamma-Tubulin was no longer detectable with our immunological approach. This points to a far-reaching change in spindle organization during transition from meiosis I to meiosis II.
-
Gamma Tubulin is a minus end specific microtubule binding protein
Journal of Cell Biology, 1995Co-Authors: Harish C JoshiAbstract:The role of microtubules in mediating chromosome segregation during mitosis is well-recognized. In addition, interphase cells depend upon a radial and uniform orientation of microtubules, which are intrinsically asymmetric polymers, for the directional transport of many cytoplasmic components and for the maintenance of the structural integrity of certain organelles. The slow growing minus ends of microtubules are linked to the centrosome ensuring extension of the fast growing plus ends toward the cell periphery. However, the molecular mechanism of this linkage is not clear. One hypothesis is that Gamma-Tubulin, located at the centrosome, binds to the minus ends of microtubules. To test this model, we synthesized radiolabeled Gamma-Tubulin in vitro. We demonstrate here biochemically a specific, saturable, and tight (Kd = 10(-10) M) interaction of Gamma-Tubulin and microtubule ends with a stoichiometry of 12.6 +/- 4.9 molecules of Gamma-Tubulin per microtubule. In addition, we designed an in vitro assay to visualize Gamma-Tubulin at the minus ends of axonemal microtubules. These data show that Gamma-Tubulin represents the first protein to bind microtubule minus ends and might be responsible for mediating the link between microtubules and the centrosome.
Michel Wright - One of the best experts on this subject based on the ideXlab platform.
-
The Drosophila Gamma-Tubulin small complex subunit Dgrip84 is required for structural and functional integrity of the spindle apparatus.
Molecular Biology of the Cell, 2006Co-Authors: Nathalie Colombié, Andre Moisand, Michel Wright, Paula Sampaio, Christel Vérollet, Claudio Sunkel, Henri-marc Bourbon, Brigitte Raynaud-messinaAbstract: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.
-
DNA damage induce Gamma-Tubulin-RAD51 nuclear complexes in mammalian cells.
Oncogene, 2005Co-Authors: Claire Lesca, Michel Wright, Brigitte Raynaud-messina, Maryse Germanier, Carole Pichereaux, Chantal Etievant, Stéphane Emond, Odile Burlet-schiltz, Bernard Monsarrat, Martine DefaisAbstract:Rad51 protein plays an essential role in recombination repair of DNA double-strand breaks and DNA crosslinking adducts. It is part of complexes which can vary with the stage of the cell cycle and the nature of the DNA lesions. During a search for Rad51-associated proteins in CHO nuclear extracts of S-phase cells by mass spectrometry of proteins immunoprecipitated with Rad51 antibodies, we identified a centrosomal protein, γ-Tubulin. This association was confirmed by the reverse immunoprecipitation with γ-Tubulin antibodies. Both proteins copurified from HeLa cells nuclear extracts following a tandem affinity purification of double-tagged Rad51. Immunofluorescence analysis showed colocalization of both Rad51 and γ-Tubulin in discrete foci in mammalian cell nuclei. The number of colocalized foci and their overlapping area increased in the presence of DNA damage produced by genotoxic treatments either during S phase or in exponentially growing cells. These variations did not result from an overall stress because microtubule cytoskeleton poisons devoid of direct interactions with DNA, such as taxol or colcemid, did not lead to an increase of this association. The recruitment of Rad51 and γ-Tubulin in the same nuclear complex suggests a link between DNA recombination repair and the centrosome function during the cell cycle.
-
Gamma-Tubulin and MTOCs in Paramecium.
Protist, 2003Co-Authors: Catherine Klotz, Michel Wright, Françoise Rulz, Nicole Garreau De Loubresse, Pascale Dupuid-williams, Janine BeissonAbstract:Summary The characterization of the two Paramecium γ-Tubulin genes, γPT1 and γPT2, allowed us to raise Paramecium-specific antibodies, directed against their most divergent carboxy-terminal peptide and to analyze the localization and dynamics of γ-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, γ-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 γ-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, γ-Tubulin and MTOCs.
-
protein complexes containing Gamma Tubulin are present in mammalian brain microtubule protein preparations
Cytoskeleton, 1997Co-Authors: Claire Detraves, Isabelle Lajoiemazenc, Monique Julian, Honore Mazarguil, Brigitte Raynaudmessina, Michel WrightAbstract:The presence of Gamma-Tubulin in microtubule preparations, obtained by disassembly/ assembly cycles at 0degreesC/37degreesC from the brain of several mammals, is demonstrated by immunoblotting with specific antibodies directed against three distinct regions of the protein. In contrast Gamma-Tubulin was absent from pure Tubulin obtained by chromatography on phosphocellulose, but was retained on the column with the other microtubule-associated proteins. A large part of the Gamma-Tubulin was present in cold stable material remaining after microtubule disassembly at OdegreesC and was partially solubilized using high salt, thus preventing its purification by the usual assembly/disassembly procedure used for alpha/beta-Tubulin heterodimers. Brain Gamma-Tubulin was purified by affinity chromatography with Gamma-Tubulin antibodies raised against its carboxyl terminal region. Purified Gamma-Tubulin consisted of at least two polypeptides present in equal quantities and exhibiting a pI of 6.5 and 6.6, respectively. It was associated with the alpha/beta-Tubulin heterodimer and with at least five other polypeptides of 75, 105, 130, 195, and 250 kDa. With the exception of the 250 kDa polypeptide, all of these proteins seem to be present in Gamma-Tubulin complexes isolated from Xenopus eggs. But, in contrast with Xenopus egg complexes, brain complexes exhibited a considerable heterogeneity of their apparent masses and composition in sucrose gradient centrifugation, in agreement with the absence of an homogeneous structure in electron microscopy. Despite this heterogeneity, Gamma-Tubulin complexes bind quantitatively to microtubule extremities. The possibility to further use mammalian brain Gamma-Tubulin and some of its associated proteins in biochemical and pharmacological experiments is of interest since brain microtubule protein preparations have been extensively used for studying both microtubule dynamics and the activity of microtubule poisons.
-
a single Gamma Tubulin gene and mrna but two Gamma Tubulin polypeptides differing by their binding to the spindle pole organizing centres
Journal of Cell Science, 1996Co-Authors: Isabelle Lajoiemazenc, Yvette Tollon, Claire Detraves, Monique Julian, Michel Wright, Victor Rotaru, Michele Gares, Catherine Jean, Brigitte RaynaudmessinaAbstract:Cells of eukaryotic organisms exhibit microtubules with various functions during the different developmental stages. The identification of multiple forms of α- and βTubulins had raised the question of their possible physiological roles. In the myxomycete Physarum polycephalum a complex polymorphism for α- and β-Tubulins has been correlated with a specific developmental expression pattern. Here, we have investigated the potential heterogeneity of γTubulin in this organism. A single gene, with 3 introns and 4 exons, and a single mRNA coding for γ-Tubulin were detected. They coded for a polypeptide of 454 amino acids, with a predicted molecular mass of 50,674, which presented 64-76% identity with other γ-Tubulins. However, immunological studies identified two γ-Tubulin polypeptides, both present in the two developmental stages of the organism, uninucleate amoebae and multinucleate plasmodia. The two γ-Tubulins, called γs- and γf-Tubulin for slow and fast electrophoretic mobility, exhibited apparent molecular masses of 52,000 and 50,000, respectively. They were recognized by two antibodies (R70 and JH46) raised against two distinct conserved sequences of γ-Tubulins. They were present both in the preparations of amoebal centrosomes possessing two centrioles and in the preparations of plasmodial nuclear metaphases devoid of structurally distinct polar structures. These two γ-Tubulins exhibited different sedimentation properties as shown by ultracentrifugation and sedimentation in sucrose gradients. Moreover, γsTubulin was tightly bound to microtubule organizing centers (MTOCs) while γf-Tubulin was loosely associated with these structures. This first demonstration of the presence of two γ-Tubulins with distinct properties in the same MTOC suggests a more complex physiological role than previously assumed. SUMMARY
Elmar Schiebel - One of the best experts on this subject based on the ideXlab platform.
-
the Gamma Tubulin ring complex deciphering the molecular organization and assembly mechanism of a major vertebrate microtubule nucleator
BioEssays, 2021Co-Authors: Anna Bohler, Bram J A Vermeulen, Martin Wurtz, Erik Zupa, Stefan Pfeffer, Elmar SchiebelAbstract:Microtubules are protein cylinders with functions in cell motility, signal sensing, cell organization, intracellular transport, and chromosome segregation. One of the key properties of microtubules is their dynamic architecture, allowing them to grow and shrink in length by adding or removing copies of their basic subunit, the heterodimer αβ-Tubulin. In higher eukaryotes, de novo assembly of microtubules from αβ-Tubulin is initiated by a 2 MDa multi-subunit complex, the Gamma-Tubulin ring complex (γ-TuRC). For many years, the structure of the γ-TuRC and the function of its subunits remained enigmatic, although structural data from the much simpler yeast counterpart, the γ-Tubulin small complex (γ-TuSC), were available. Two recent breakthroughs in the field, high-resolution structural analysis and recombinant reconstitution of the complex, have revolutionized our knowledge about the architecture and function of the γ-TuRC and will form the basis for addressing outstanding questions about biogenesis and regulation of this essential microtubule organizer.
-
spc98p directs the yeast γ Tubulin complex into the nucleus and is subject to cell cycle dependent phosphorylation on the nuclear side of the spindle pole body
Molecular Biology of the Cell, 1998Co-Authors: Gislene Pereira, Michael Knop, Elmar SchiebelAbstract:: In the yeast Saccharomyces cerevisiae, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope. Microtubule organization requires the Gamma-Tubulin complex containing the Gamma-Tubulin Tub4p, Spc98p, and Spc97p. The Tub4p complex is associated with cytoplasmic and nuclear substructures of the SPB, which organize the cytoplasmic and nuclear microtubules. Here we present evidence that the Tub4p complex assembles in the cytoplasm and then either binds to the cytoplasmic side of the SPB or is imported into the nucleus followed by binding to the nuclear side of the SPB. Nuclear import of the Tub4p complex is mediated by the essential nuclear localization sequence of Spc98p. Our studies also indicate that Spc98p in the Tub4p complex is phosphorylated at the nuclear, but not at the cytoplasmic, side of the SPB. This phosphorylation is cell cycle dependent and occurs after SPB duplication and nucleation of microtubules by the new SPB and therefore may have a role in mitotic spindle function. In addition, activation of the mitotic checkpoint stimulates Spc98p phosphorylation. The kinase Mps1p, which functions in SPB duplication and mitotic checkpoint control, seems to be involved in Spc98p phosphorylation. Our results also suggest that the nuclear and cytoplasmic Tub4p complexes are regulated differently.
-
a novel protein complex promoting formation of functional alpha and Gamma Tubulin
The EMBO Journal, 1998Co-Authors: Silke Geissler, Katja Siegers, Elmar SchiebelAbstract:We describe the identification of GIM1/YKE2, GIM2/PAC10, GIM3, GIM4 and GIM5 in a screen for mutants that are synthetically lethal with tub4-1, encoding a mutated yeast Gamma-Tubulin. The cytoplasmic Gim proteins encoded by these GIM genes are present in common complexes as judged by co-immunoprecipitation and gel filtration experiments. The disruption of any of these genes results in similar phenotypes: the gim null mutants are synthetically lethal with tub4-1 and super-sensitive towards the microtubule-depolymerizing drug benomyl. All except Deltagim4 are cold-sensitive and their microtubules disassemble at 14 degrees C. The Gim proteins have one function related to alpha-Tubulin and another to Tub4p, supported by the finding that the benomyl super-sensitivity is caused by a reduced level of alpha-Tubulin while the synthetic lethality with tub4-1 is not. In addition, GIM1/YKE2 genetically interacts with two distinct classes of genes, one of which is involved in Tubulin folding and the other in microtubule nucleation. We show that the Gim proteins are important for Tub4p function and bind to overproduced Tub4p. The mammalian homologues of GIM1/YKE2 and GIM2/PAC10 rescue the synthetically lethal phenotype with tub4-1 as well as the cold-sensitivity and benomyl super-sensitivity of the yeast deletion mutants. We suggest that the Gim proteins form a protein complex that promotes formation of functional alpha- and Gamma-Tubulin.
-
the spindle pole body component spc98p interacts with the Gamma Tubulin like tub4p of saccharomyces cerevisiae at the sites of microtubule attachment
The EMBO Journal, 1996Co-Authors: Silke Geissler, Gislene Pereira, Anne Spang, Michael Knop, Sylvie Soues, John Kilmartin, Elmar SchiebelAbstract:Tub4p is a novel Tubulin found in Saccharomyces cerevisiae. It most resembles Gamma-Tubulin and, like it, is localized to the yeast microtubule organizing centre, the spindle pole body (SPB). In this paper we report the identification of SPC98 as a dosage-dependent suppressor of the conditional lethal tub4-1 allele. SPC98 encodes an SPB component of 98 kDa which is identical to the previously described 90 kDa SPB protein. Strong overexpression of SPC98 is toxic, causing cells to arrest with a large bud, defective microtubule structures, undivided nucleus and replicated DNA. The toxicity of SPC98 overexpression was relieved by co-overexpression of TUB4. Further evidence for an interaction between Tub4p and Spc98p came from the synthetic toxicity of tub4-1 and spc98-1 alleles, the dosage-dependent suppression of spc98-4 by TUB4, the binding of Tub4p to Spc98p in the two-hybrid system and the co-immunoprecipitation of Tub4p and Spc98p. In addition, Spc98-1p is defective in its interaction with Tub4p in the two-hybrid system. We suggest a model in which Tub4p and Spc98p form a complex involved in microtubule organization by the SPB.
-
Gamma Tubulin like tub4p of saccharomyces cerevisiae is associated with the spindle pole body substructures that organize microtubules and is required for mitotic spindle formation
Journal of Cell Biology, 1996Co-Authors: Anne Spang, Silke Geissler, Katrin Grein, Elmar SchiebelAbstract:Tub4p is a novel Tubulin in Saccharomyces cerevisiae that most closely resembles Gamma-Tubulin. We report in this manuscript that the essential Tub4p is associated with the inner and outer plaques of the yeast microtubule organizing center, the spindle pole body (SPB). These SPB substructures are involved in the attachment of the nuclear and cytoplasmic microtubules, respectively (Byers, B., and L. Goetsch. 1975. J. Bacteriol. 124:511-523). Study of a temperature sensitive tub4-1 allele revealed that TUB4 has essential functions in microtubule organization. Remarkably, SPB duplication and separation are not impaired in tub4-1 cells incubated at the nonpermissive temperature. However, SPBs from such cells contain less or misdirected nuclear microtubules. Further analysis revealed that tub4-1 cells are able to assemble a short bipolar spindle, suggesting that the defect in microtubule organization occurs after spindle formation. A role of Tub4p in microtubule organization is further suggested by an increase in chromosome loss in tub4-1 cells. In addition, cell cycle arrest and survival of tub4-1 cells is dependent on the mitotic checkpoint control gene BUB2 (Hoyt, M.A., L. Totis, B.T. Roberts. 1991. Cell. 66:507-517), one of the cell's monitors of spindle integrity.
Berl R Oakley - One of the best experts on this subject based on the ideXlab platform.
-
Gamma Tubulin in plant cells
Methods in Cell Biology, 2001Co-Authors: Yulia Ovechkina, Berl R OakleyAbstract:Publisher Summary Substantial progress has been made in cloning of plant γ-Tubulin genes and immunolocalization of γ-Tubulin. This chapter describes microtubule arrays in plants and discusses the importance of γ-Tubulin in plants. The chapter discusses methods for cloning γ-Tubulin genes in plants and methods for localizing γ-Tubulin in plant cells. In view of the importance of γ-Tubulin in mitotic spindle formation in fungi and higher organisms, it is likely that γ-Tubulin plays an important role in nucleation and organization of microtubule arrays in plants. A number of plant γ-Tubulin cDNAs have been cloned and sequenced. The high degree of conservation of plant γ-Tubulins makes cloning plant γ-Tubulin genes and cDNAs relatively straightforward. One approach that has been successful is to use a γ-Tubulin cDNA from another organism to probe a library at low stringency. A second approach has been to use the polymerase chain reaction (PCR) to generate specific probes that can be used to probe libraries at high stringencies. This approach takes advantage of the fact that there are regions of γ-Tubulin in which the amino acid sequence is highly conserved among phylogenetically diverse organisms.
-
Gamma Tubulin at ten progress and prospects
Cell Structure and Function, 1999Co-Authors: Berl R Oakley, Yassmine N AkkariAbstract:The existence of γ-Tubulin was first reported approximately ten years ago, and it is appropriate to review the progress that has been made in γ-Tubulin research and to discuss some of the unanswered questions about γ-Tubulin function. γ-Tubulin is ubiquitous in eukaryotes and is generally quite conserved. Two highly divergent γ-Tubulins have been discovered, however, one in Saccharomyces cerevisiae and one in Caenorhabditis elegans. Several organisms have two γ-Tubulin genes. In Drosophila melanogaster, the two γ-Tubulins differ significantly in sequence and expression pattern. In other organisms the two γ-Tubulins are almost identical and expression patterns have not been determined. γ-Tubulin is located at microtubule organizing centers in many organisms, and it is also frequently associated with the mitotic spindle. γ-Tubulin is essential for the formation of functional mitotic spindles in all organisms that have been examined to date. In animal cells, complexes containing γ-Tubulin are located at microtubule organizing centers where they nucleate the assembly of microtubles. In spite of the considerable progress that has been made in γ-Tubulin research important questions remain to be answered. The exact mechanisms of microtubule nucleation by γ-Tubulin complexes remain to be resolved as do the mechanisms by which microtubule nucleation from γ-Tubulin complexes is regulated. Finally, there is evidence that γ-Tubulin has important functions in addition to microtubule nucleation, and these functions are just beginning to be investigated.
-
the role of Gamma Tubulin in mitotic spindle formation and cell cycle progression in aspergillus nidulans
Journal of Cell Science, 1997Co-Authors: Mary Ann Martin, Stephen A Osmani, Berl R OakleyAbstract:Gamma-Tubulin has been hypothesized to be essential for the nucleation of the assembly of mitotic spindle microtubules, but some recent results suggest that this may not be the case. To clarify the role of Gamma-Tubulin in microtubule assembly and cell-cycle progression, we have developed a novel variation of the gene disruption/heterokaryon rescue technique of Aspergillus nidulans. We have used temperature-sensitive cell-cycle mutations to synchronize germlings carrying a Gamma-Tubulin disruption and observe the phenotypes caused by the disruption in the first cell cycle after germination. Our results indicate that Gamma-Tubulin is absolutely required for the assembly of mitotic spindle microtubules, a finding that supports the hypothesis that Gamma-Tubulin is involved in spindle microtubule nucleation. In the absence of functional Gamma-Tubulin, nuclei are blocked with condensed chromosomes for about the length of one cell cycle before chromatin decondenses without nuclear division. Our results indicate that Gamma-Tubulin is not essential for progression from G1 to G2, for entry into mitosis nor for spindle pole body replication. It is also not required for reactivity of spindle pole bodies with the MPM-2 antibody which recognizes a phosphoepitope important to mitotic spindle formation. Finally, it does not appear to be absolutely required for cytoplasmic microtubule assembly but may play a role in the formation of normal cytoplasmic microtubule arrays.
-
human Gamma Tubulin functions in fission yeast
Journal of Cell Biology, 1994Co-Authors: Tetsuya Horio, Berl R OakleyAbstract: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.
-
the fission yeast Gamma Tubulin is essential for mitosis and is localized at microtubule organizing centers
Journal of Cell Science, 1991Co-Authors: Tetsuya Horio, Berl R Oakley, Satoru Uzawa, M K Jung, Kenji Tanaka, Mitsuhiro YanagidaAbstract:Gamma-Tubulin exists in fission yeast as the product of an essential gene, encoding a 446 amino acid protein that is 77.3% identical to Aspergillus nidulans Gamma-Tubulin. The gene disruption caused cell lethality, displaying condensed, undivided chromosomes with aberrant spindle structures. Anti-Gamma-Tubulin staining showed that Gamma-Tubulin is located, throughout the wild-type cell cycle, at the spindle pole bodies (SPBs), indicating that Gamma-Tubulin associates with interphase SPB in the absence of microtubules. In addition, anti-Gamma-Tubulin immunofluorescence staining revealed cytoplasmic, cell-equatorial putative MTOCs (microtubule organizing centers), which appear only during mitotic telophase and cytokinesis, and are located at the centers for the new cytoplasmic microtubule arrays of the two daughter cells. In the multiple-SPB mutant cut1-cdc11, anti-Gamma-Tubulin antibodies revealed many dots on the periphery of the nucleus. These results confirm that Gamma-Tubulin is an important member of the Tubulin superfamily, suggest that it may be a universal component of MTOCs, and are consistent with a role for Gamma-Tubulin in controlling microtubule formation in vivo.
Yixian Zheng - One of the best experts on this subject based on the ideXlab platform.
-
purification and reconstitution of drosophila Gamma Tubulin complexes
Methods in Cell Biology, 2001Co-Authors: Ruwanthi N Gunawardane, Karen Oegema, Yixian Zheng, Christiane WieseAbstract:Publisher Summary This chapter discusses the purification and reconstitution of Drosophila γ-Tubulin complexes. To clone the individual Xenopus and Drosophila γTuRC subunits there are two approaches: (1) raising mouse polyclonal antibodies against purified γTuRCs and (2) obtaining protein sequences for individual γTuRC subunits. To date, four of the Drosophila γTuRC subunits are cloned, sequenced, and characterized. The isolation of γTuRC from Drosophila embryos involves an affinity purification step, using antipeptide antibodies specific for the C-terminal 17 amino acids of Drosophila γ-Tubulin. An advantage of using Drosophila extracts is that once fly population cages are established, embryos are readily available in large quantities and flies do not require hormone injections. Another advantage is that Drosophila embryo extracts are less variable in quality than Xenopus egg extracts. Although the γ-Tubulin-containing complexes—namely, γTuRC and γTuSC—can be purified from Drosophila embryos with relative ease, a system is developed that would allow to express the Dgrip proteins individually and in various combinations to study the organization and function of the γ-Tubulin complexes biochemically. The chapter describes methods used to culture and infect the Sf9 cells and focuses on the general methods used to construct and assay the baculovirus.
-
Gamma Tubulin complexes and their role in microtubule nucleation
Current Topics in Developmental Biology, 1999Co-Authors: Ruwanthi N Gunawardane, Christiane Wiese, Sofia B Lizarraga, Andrew Wilde, Yixian ZhengAbstract:Publisher Summary This chapter discusses the purification and characterization of γ-Tubulin ring complex (γTuRC) and γ-Tubulin small complex (γTuSC) and the function of γTuRC at the centrosome. The proposed mechanism of γTuRC-mediated microtubule nucleation is also reviewed. The identification of γ-Tubulin and its essential role in microtubule nucleation is a major breakthrough in the study of microtubules and centrosomes. The subsequent purification of γTuRC provides the much-needed tool to investigate the role of γ-Tubulin in microtubule nucleation and centrosome assembly. γTuRC is essential for both microtubule nucleation and formation of a functional centrosome. Further characterization of the γTuRC components helps to study the assembly of γTuRC and its recruitment to the centrosome. It provides an understanding of centrosome assembly, which is an essential process for proper cell division. The mechanism of microtubule nucleation by γ-Tubulin comes from both biochemical and structural studies. γ-Tubulin exists as a complex with other proteins inside the cell; in certain cell types, multiple γ-Tubulin complexes of different sizes co-exist. Antibody affinity production of γ-Tubulin complexes from Drosophila embryos and Xenopus egg extracts is diagrammatically represented in the chapter.
-
recruitment of the Gamma Tubulin ring complex to drosophila salt stripped centrosome scaffolds
Journal of Cell Biology, 1998Co-Authors: Michelle Moritz, Bruce Alberts, Yixian Zheng, Karen OegemaAbstract:Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and Gamma-Tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the Gamma-Tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, Gamma-Tubulin is found in two distinct protein complexes of 240,000 and approximately 3,000,000 D. The larger complex, which is analogous to the Xenopus Gamma-Tubulin ring complex (GammaTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the GammaTuRC is required for microtubule nucleation at the centrosome.
-
nucleation of microtubule assembly by a Gamma Tubulin containing ring complex
Nature, 1995Co-Authors: Yixian Zheng, Bruce Alberts, Mei Lie Wong, Timothy J MitchisonAbstract:The highly conserved protein Gamma-Tubulin is required for microtubule nucleation in vivo. When viewed in the electron microscope, a highly purified Gamma-Tubulin complex from Xenopus consisting of at least seven different proteins is seen to have an open ring structure. This complex acts as an active microtubule-nucleating unit which can cap the minus ends of microtubules in vitro.
-
γ Tubulin is present in drosophila melanogaster and homo sapiens and is associated with the centrosome
Cell, 1991Co-Authors: Yixian Zheng, Katherine M Jung, Berl R OakleyAbstract:The mipA gene of A. nidulans encodes a newly discovered member of the Tubulin superfamily of proteins, Gamma-Tubulin. In A. nidulans, Gamma-Tubulin is essential for nuclear division and microtubule assembly and is associated with the spindle pole body, the fungal microtubule organizing center. By low stringency hybridizations we have cloned cDNAs from D. melanogaster and H. sapiens, the predicted products of which share more than 66% amino acid identity with A. nidulans Gamma-Tubulin. Gamma-Tubulin-specific antibodies stained centrosomes of Drosophila, human, and mouse cell lines. Staining was most intense in prophase through metaphase when microtubule assembly from centrosomes was maximal. These results demonstrate that Gamma-Tubulin genes are present and expressed in humans and flies; they suggest that Gamma-Tubulin may be a universal component of microtubule organizing centers; and they are consistent with an earlier hypothesis that Gamma-Tubulin is a minus-end nucleator of microtubule assembly.
