The fission yeast gamma-tubulin is essential for mitosis and is localized at microtubule organizing centers

1991 ◽  
Vol 99 (4) ◽  
pp. 693-700 ◽  
Author(s):  
T. Horio ◽  
S. Uzawa ◽  
M.K. Jung ◽  
B.R. Oakley ◽  
K. Tanaka ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Essential Gene ◽  
Spindle Pole ◽  
Gene Encoding ◽  
Cytoplasmic Microtubule ◽  
Microtubule Organizing Centers ◽  
Tubulin Antibodies ◽  
Gamma Tubulin ◽  
Tubulin Immunofluorescence

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.

scholarly journals Human gamma-tubulin functions in fission yeast.

1994 ◽  
Vol 126 (6) ◽  
pp. 1465-1473 ◽  
Author(s):  
T Horio ◽  
B R Oakley
Keyword(s):  
Fission Yeast ◽  
Normal Growth ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Microtubule Organizing Center ◽  
Microtubule Organization ◽  
Microtubule Organizing Centers ◽  
Functional Conservation ◽  
Gamma Tubulin ◽  
Tubulin Immunofluorescence

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.

scholarly journals In vitro microtubule-nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe: cell cycle-dependent activation in xenopus cell-free extracts

1992 ◽  
Vol 117 (5) ◽  
pp. 1055-1066 ◽  
Author(s):  
H Masuda ◽  
M Sevik ◽  
WZ Cande
Keyword(s):  
Fission Yeast ◽  
Spindle Pole ◽  
Protein Kinase Inhibitors ◽  
Cyclin B ◽  
Microtubule Organizing Center ◽  
Cdc2 Kinase ◽  
Gamma Tubulin ◽  
In Cells

The spindle pole body (SPB) is the equivalent of the centrosome in fission yeast. In vivo it nucleates microtubules (MTs) during mitosis, but, unlike animal centrosomes, does not act as a microtubule organizing center (MTOC) during interphase. We have studied the MT-nucleating activity of SPBs in vitro and have found that SPBs in permeabilized cells retain in vivo characteristics. SPBs in cells permeabilized during mitosis can nucleate MTs, and are recognized by two antibodies: anti-gamma-tubulin and MPM-2 which recognizes phosphoepitopes. SPBs in cells permeabilized during interphase cannot nucleate MTs and are only recognized by anti-gamma-tubulin. Interphase SPBs which cannot nucleate can be converted to a nucleation competent state by incubation in cytostatic factor (CSF)-arrested Xenopus egg extracts. After incubation, they are recognized by MPM-2, and can nucleate MTs. The conversion does not occur in Xenopus interphase extract, but occurs in Xenopus interphase extract driven into mitosis by preincubation with exogenous cyclin B. The conversion is ATP dependent and inhibited by protein kinase inhibitors and alkaline phosphatase. Purified, active, cdc2 kinase/cyclin B complex in itself is not effective for activation of MT nucleation, although some interphase SPBs are now stained with MPM-2. These results suggest that the ability of SPBs in vitro to nucleate MTs after exposure to CSF-arrested extracts is activated through a downstream pathway which is regulated by cdc2 kinase.

scholarly journals Mobility, Microtubule Nucleation and Structure of Microtubule-organizing Centers in Multinucleated Hyphae ofAshbya gossypii

2010 ◽  
Vol 21 (1) ◽  
pp. 18-28 ◽  
Author(s):  
Claudia Lang ◽  
Sandrine Grava ◽  
Tineke van den Hoorn ◽  
Rhonda Trimble ◽  
Peter Philippsen ◽  
...  
Keyword(s):  
Spindle Pole Body ◽  
Hyphal Growth ◽  
Spindle Pole ◽  
Microtubule Organizing Center ◽  
Microtubule Nucleation ◽  
Cytoplasmic Microtubule ◽  
Organizing Center ◽  
Cytoplasmic Side ◽  
Independent Mode

We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 μm/min), rotations (up to 180° in 30 s), and long-range nuclear bypassing (up to 9 μm/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis–oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.

scholarly journals Fission Yeast Mto1 Regulates Diversity of Cytoplasmic Microtubule Organizing Centers

2010 ◽  
Vol 20 (21) ◽  
pp. 1959-1965 ◽  
Author(s):  
Itaru Samejima ◽  
Victoria J. Miller ◽  
Sergio A. Rincon ◽  
Kenneth E. Sawin
Keyword(s):  
Fission Yeast ◽  
Cytoplasmic Microtubule ◽  
Microtubule Organizing Centers

Cytoplasmic microtubular system implicated in de novo formation of a Rabl-like orientation of chromosomes in fission yeast

2001 ◽  
Vol 114 (13) ◽  
pp. 2427-2435 ◽  
Author(s):  
Bunshiro Goto ◽  
Koei Okazaki ◽  
Osami Niwa
Keyword(s):  
Fission Yeast ◽  
De Novo ◽  
Nuclear Periphery ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Limited Area ◽  
Cytoplasmic Microtubule ◽  
Random Arrangement ◽  
Triplex Structure ◽  
Microtubular System

Chromosomes are not packed randomly in the nucleus. The Rabl orientation is an example of the non-random arrangement of chromosomes, centromeres are grouped in a limited area near the nuclear periphery and telomeres are located apart from centromeres. This orientation is established during mitosis and maintained through subsequent interphase in a range of species. We report that a Rabl-like configuration can be formed de novo without a preceding mitosis during the transition from the sexual phase to the vegetative phase of the life cycle in fission yeast. In this process, each of the dispersed centromeres is often associated with a novel Sad1-containing body that is contacting a cytoplasmic microtubule laterally (Sad1 is a component of the spindle pole body (SPB)). The Sad1-containing body was colocalized with other known SPB components, Kms1 and Spo15 but not with Cut12, indicating that it represents a novel SPB-related complex. The existence of the triplex structure (centromere-microtubule-Sad1 body) suggests that the clustering of centromeres is controlled by a cytoplasmic microtubular system. Accordingly, when microtubules are destabilized, clustering is markedly reduced.

The role of gamma-tubulin in mitotic spindle formation and cell cycle progression in Aspergillus nidulans

1997 ◽  
Vol 110 (5) ◽  
pp. 623-633 ◽  
Author(s):  
M.A. Martin ◽  
S.A. Osmani ◽  
B.R. Oakley
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Cycle Progression ◽  
Spindle Microtubules ◽  
Gamma Tubulin

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.

scholarly journals Stu2p: A Microtubule-Binding Protein that Is an Essential Component of the Yeast Spindle Pole Body

1997 ◽  
Vol 139 (5) ◽  
pp. 1271-1280 ◽  
Author(s):  
Peijing Jeremy Wang ◽  
Tim C. Huffaker
Keyword(s):  
Essential Gene ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cold Sensitivity ◽  
Gene Encoding ◽  
Microtubule Binding ◽  
Amino Acid Region ◽  
Pole Body ◽  
Spindle Function

Previously we isolated tub2-423, a cold-sensitive allele of the Saccharomyces cerevisiae gene encoding β-tubulin that confers a defect in mitotic spindle function. In an attempt to identify additional proteins that are important for spindle function, we screened for suppressors of the cold sensitivity of tub2-423 and obtained two alleles of a novel gene, STU2. STU2 is an essential gene and encodes a protein whose sequence is similar to proteins identified in a variety of organisms. Stu2p localizes primarily to the spindle pole body (SPB) and to a lesser extent along spindle microtubules. Localization to the SPB is not dependent on the presence of microtubules, indicating that Stu2p is an integral component of the SPB. Stu2p also binds microtubules in vitro. We have localized the microtubule-binding domain of Stu2p to a highly basic 100-amino acid region. This region contains two imperfect repeats; both repeats appear to contribute to microtubule binding to similar extents. These results suggest that Stu2p may play a role in the attachment, organization, and/or dynamics of microtubule ends at the SPB.

scholarly journals β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

2002 ◽  
Vol 13 (8) ◽  
pp. 2919-2932 ◽  
Author(s):  
Mohan L. Gupta ◽  
Claudia J. Bode ◽  
Douglas A. Thrower ◽  
Chad G. Pearson ◽  
Kathy A. Suprenant ◽  
...  
Keyword(s):  
Essential Gene ◽  
Dynamic Properties ◽  
Microtubule Dynamics ◽  
Cytoplasmic Microtubule ◽  
Bud Emergence ◽  
Spindle Elongation ◽  
Mutant Cells ◽  
Β Tubulin

Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast β-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules. The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells. The single microtubule effectively located the bud site before bud emergence. Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis. Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants. The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth. The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function.

scholarly journals A Fourth Component of the Fission Yeast γ-Tubulin Complex, Alp16, Is Required for Cytoplasmic Microtubule Integrity and Becomes Indispensable When γ-Tubulin Function Is Compromised

2002 ◽  
Vol 13 (7) ◽  
pp. 2360-2373 ◽  
Author(s):  
Akiko Fujita ◽  
Leah Vardy ◽  
Miguel Angel Garcia ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Cytoplasmic Microtubule ◽  
Multicopy Suppressor ◽  
Large Complex ◽  
Synthetically Lethal ◽  
And Function

γ-Tubulin functions as a multiprotein complex, called the γ-tubulin complex (γ-TuC), and composes the microtubule organizing center (MTOC). Fission yeast Alp4 and Alp6 are homologues of two conserved γ-TuC proteins, hGCP2 and hGCP3, respectively. We isolated a novel gene, alp16 + , as a multicopy suppressor of temperature-sensitive alp6-719mutants. alp16 + encodes a 759-amino-acid protein with two conserved regions found in all other members of γ-TuC components. In addition, Alp16 contains an additional motif, which shows homology to hGCP6/Xgrip210. Gene disruption shows that alp16 + is not essential for cell viability. However, alp16 deletion displays abnormally long cytoplasmic microtubules, which curve around the cell tip. Furthermore, alp16-deleted mutants are hypersensitive to microtubule-depolymerizing drugs and synthetically lethal with either temperature-sensitive alp4-225,alp4-1891, or alp6-719 mutants. Overproduction of Alp16 is lethal, with defective phenotypes very similar to loss of Alp4 or Alp6. Alp16 localizes to the spindle pole body throughout the cell cycle and to the equatorial MTOC at postanaphase. Alp16 coimmunoprecipitates with γ-tubulin and cosediments with the γ-TuC in a large complex (>20 S). Alp16 is, however, not required for the formation of this large complex. We discuss evolutional conservation and divergence of structure and function of the γ-TuC between yeast and higher eukaryotes.

Gamma-tubulin reorganization during mouse fertilization and early development

1993 ◽  
Vol 104 (2) ◽  
pp. 383-389 ◽  
Author(s):  
M.J. Palacios ◽  
H.C. Joshi ◽  
C. Simerly ◽  
G. Schatten
Keyword(s):  
Early Development ◽  
Basal Body ◽  
Spindle Pole ◽  
Meiotic Spindle ◽  
Cytoplasmic Microtubule ◽  
Mouse Oocytes ◽  
Spindle Pole Bodies ◽  
Preimplantation Stage ◽  
Pericentriolar Material ◽  
Gamma Tubulin

gamma-Tubulin, a component of spindle pole bodies in fungal cells and pericentriolar material in vertebrate cells, is thought to play a role in the nucleation of microtubule growth and to define their polarity. In contrast to the adult somatic cells, microtubules are nucleated in the absence of centrioles in mammalian oocytes and early embryos. By studying acentriolar mouse oocytes and their early development following fertilization, we show that gamma-tubulin antibody crossreacts with a 50,000 M(r) protein in unfertilized mouse oocytes and demonstrate that gamma-tubulin distribution is rearranged dramatically during fertilization. In unfertilized mouse oocytes, gamma-tubulin is concentrated in the broad spindle poles of meiotic spindle (MII) and as the distinct foci which form the centers of the cytoplasmic microtubule asters (cytasters). The integrity of these gamma-tubulin foci and their cytoplasmic location is maintained during the drug- or cold-induced depolymerization of microtubules. gamma-Tubulin is also found in the basal body of the mouse sperm. During fertilization, the gamma-tubulin is found at the cytastral centers as well as in the incorporated sperm basal body complex, and the gamma-tubulin foci coalesce at the perinuclear microtubule organizing regions of the two pronuclei at the first mitotic prophase. During mitosis, gamma-tubulin is found associated with broad bands that form the poles of the first mitotic spindle. By the late preimplantation stage, when newly generated centrioles have been reported to arise, gamma-tubulin remains localized at the centrosome of mitotic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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