scholarly journals Spc98p and Spc97p of the yeast gamma -tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p

1997 ◽  
Vol 16 (23) ◽  
pp. 6985-6995 ◽  
Author(s):  
M. Knop
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Pole Body ◽  
Gamma Tubulin

Identification of an Spc110p-related protein in vertebrates

1997 ◽  
Vol 110 (20) ◽  
pp. 2533-2545 ◽  
Author(s):  
A.M. Tassin ◽  
C. Celati ◽  
M. Paintrand ◽  
M. Bornens
Keyword(s):  
Mammalian Cells ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Partial Purification ◽  
Related Protein ◽  
Microtubule Nucleation ◽  
Pole Body ◽  
Egg Extract ◽  
Ring Complex ◽  
Gamma Tubulin

Although varying in size and complexity, centrosomes have conserved functions throughout the evolutionary range of eukaryotes, and thus may display conserved components. In this work, we took advantage of the recent advances in the isolation of the budding yeast spindle pole body, the development of specific immunological probes and the molecular characterisation of genes involved in spindle pole body duplication or assembly. Screening a monoclonal antibody library against Saccharomyces cerevisiae spindle pole body components, we found that two monoclonal antibodies, directed against two different parts of the yeast Spc110p, decorate the centrosome from mammalian cells in an asymmetrical manner. Western blot experiments identified a 100 kDa protein specifically enriched in centrosome preparations from human cells. This protein is phosphorylated during mitosis and is tightly associated with the centrosome: only denaturing conditions such as 8 M urea were able to solubilise it. Purified immunoglobulins directed against Spc110p inhibit microtubule nucleation on isolated human centrosomes, using brain phosphocellulose-tubulin or Xenopus egg extract tubulin. This result suggested that the centrosomal 100 kDa protein could be involved in a microtubule nucleation complex. To test this hypothesis, we turned to Xenopus species, in which mAb anti-Spc110p decorated centrosomes from somatic cells and identified a 116 kDa protein in egg extract. We performed a partial purification of the gamma-tubulin-ring complex from egg extract. Sucrose gradient sedimentation, immunoprecipitation and native gels demonstrated that the Xenopus 116 kDa protein and gamma-tubulin were found in the same complex. Altogether, these results suggest the existence of an yeast Spc110-related protein in vertebrate centrosomes which is involved in microtubule nucleation.

Assembly and dynamics of an anastral:astral spindle: the meiosis II spindle of Drosophila oocytes

1998 ◽  
Vol 111 (17) ◽  
pp. 2487-2495 ◽  
Author(s):  
S.A. Endow ◽  
D.J. Komma
Keyword(s):  
De Novo ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
The Body ◽  
Central Spindle ◽  
Spindle Poles ◽  
Pole Body ◽  
Microtubule Motor ◽  
Gamma Tubulin ◽  
Meiosis I

The meiosis II spindle of Drosophila oocytes is distinctive in structure, consisting of two tandem spindles with anastral distal poles and an aster-associated spindle pole body between the central poles. Assembly of the anastral:astral meiosis II spindle occurs by reorganization of the meiosis I spindle, without breakdown of the meiosis I spindle. The unusual disk- or ring-shaped central spindle pole body forms de novo in the center of the elongated meiosis I spindle, followed by formation of the central spindle poles. gamma-Tubulin transiently localizes to the central spindle pole body, implying that the body acts as a microtubule nucleating center for assembly of the central poles. Localization of gamma-tubulin to the meiosis II spindle is dependent on the microtubule motor protein, Nonclaret disjunctional (Ncd). Absence of Ncd results in loss of gamma-tubulin localization to the spindle and destabilization of microtubules in the central region of the spindle. Assembly of the anastral:astral meiosis II spindle probably involves rapid reassortment of microtubule plus and minus ends in the center of the meiosis I spindle - this can be accounted for by a model that also accounts for the loss of gamma-tubulin localization to the spindle and destabilization of microtubules in the absence of Ncd.

Role of gamma-tubulin in mitosis-specific microtubule nucleation from the Schizosaccharomyces pombe spindle pole body

1996 ◽  
Vol 109 (1) ◽  
pp. 165-177 ◽  
Author(s):  
H. Masuda ◽  
T. Shibata
Keyword(s):  
Protein Kinase ◽  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Nucleation ◽  
Amino Terminal ◽  
Pole Body ◽  
Gamma Tubulin ◽  
Mitotic Spindle Pole

The ability of the Schizosacchromyces pombe spindle pole body to nucleate microtubules is activated at the onset of mitosis for forming a mitotic spindle, but it is inactivated during interphase. We have previously developed an in vitro assay for studying the molecular mechanism of spindle pole body activation using permeabilized interphase S. pombe cells and Xenopus mitotic extracts. We have shown that the interphase spindle pole body is activated indirectly by p34cdc2 protein kinase in Xenopus mitotic extracts. In this study we examined the role of gamma-tubulin, a component of both interphase and mitotic spindle pole body, in formation of the microtubule nucleating complex at the mitotic spindle pole body. A polyclonal antibody specific to S. pombe gamma-tubulin inhibited both activation of the interphase spindle pole body and microtubule nucleation from the mitotic spindle pole body. Addition of bacterially expressed S. pombe gamma-tubulin or its amino-terminal fragments to Xenopus mitotic extracts inhibited spindle pole body activation. Affinity chromatography of partially fractionated Xenopus mitotic extracts with the amino-terminal fragment of S. pombe gamma-tubulin showed that fractions bound to the fragment supported the activation. The fractions did not contain Xenopus gamma-tubulin, showing that activation of the spindle pole body is not due to recruitment of Xenopus gamma-tubulin to the spindle pole body. The spindle pole body activation occurred in extracts depleted of p34cdc2 protein kinase or MAP kinase. The activity of the fractions bound to the fragment was inhibited by a protein kinase inhibitor, staurosporine. These results suggest that S. pombe gamma-tubulin is a component of the microtubule nucleating complex, and that the function of proteins that interact with gamma-tubulin is required for activation of the spindle pole body. We present possible models for the activation that convert the immature microtubule nucleating complex at interphase into the mature microtubule nucleating complex at mitosis.

scholarly journals 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.

1996 ◽  
Vol 134 (2) ◽  
pp. 429-441 ◽  
Author(s):  
A Spang ◽  
S Geissler ◽  
K Grein ◽  
E Schiebel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Checkpoint Control ◽  
Microtubule Organizing Center ◽  
Microtubule Organization ◽  
Spindle Formation ◽  
Pole Body ◽  
Gamma Tubulin

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.

scholarly journals Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function.

1996 ◽  
Vol 134 (2) ◽  
pp. 443-454 ◽  
Author(s):  
L G Marschall ◽  
R L Jeng ◽  
J Mulholland ◽  
T Stearns
Keyword(s):  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Dependent Manner ◽  
Protein Fusion ◽  
Microtubule Organizing Center ◽  
Pole Body ◽  
Monopolar Spindle ◽  
Organizing Center ◽  
Gamma Tubulin

gamma-Tubulin is a conserved component of microtubule-organizing centers and is thought to be involved in microtubule nucleation. A recently discovered Saccharomyces cerevisiae gene (TUB4) encodes a tubulin that is related to, but divergent from, gamma-tubulins. TUB4 is essential for cell viability, and epitope-tagged Tub4 protein (Tub4p) is localized to the spindle pole body (Sobel, S.G., and M. Snyder. 1995.J. Cell Biol. 131:1775-1788). We have characterized the expression of TUB4, the association of Tub4p with the spindle pole body, and its role in microtubule organization. Tub4p is a minor protein in the cell, and expression of TUB4 is regulated in a cell cycle-dependent manner. Wild-type Tub4p is localized to the spindle pole body, and a Tub4p-green fluorescent protein fusion is able to associate with a preexisting spindle pole body, suggesting that there is dynamic exchange between cytoplasmic and spindle pole body forms of Tub4p. Perturbation of Tub4p function, either by conditional mutation or by depletion of the protein, results in spindle as well as spindle pole body defects, but does not eliminate the ability of microtubules to regrow from, or remain attached to, the spindle pole body. The spindle pole bodies in tub4 mutant cells duplicate but do not separate, resulting in a monopolar spindle. EM revealed that one spindle pole body of the duplicated pair appears to be defective for the nucleation of microtubules. These results offer insight into the role of gamma-tubulin in microtubule-organizing center function.

scholarly journals The spindle pole body of Schizosaccharomyces pombe enters and leaves the nuclear envelope as the cell cycle proceeds.

1997 ◽  
Vol 8 (8) ◽  
pp. 1461-1479 ◽  
Author(s):  
R Ding ◽  
R R West ◽  
D M Morphew ◽  
B R Oakley ◽  
J R McIntosh
Keyword(s):  
Nuclear Envelope ◽  
Schizosaccharomyces Pombe ◽  
Mitotic Spindle ◽  
Amorphous Material ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Pole Body ◽  
G2 Phase ◽  
Gamma Tubulin ◽  
Small Bundle

The cycle of spindle pole body (SPB) duplication, differentiation, and segregation in Schizosaccharomyces pombe is different from that in some other yeasts. Like the centrosome of vertebrate cells, the SPB of S. pombe spends most of interphase in the cytoplasm, immediately next to the nuclear envelope. Some gamma-tubulin is localized on the SPB, suggesting that it plays a role in the organization of interphase microtubules (MTs), and serial sections demonstrate that some interphase MTs end on or very near to the SPB. gamma-Tubulin is also found on osmiophilic material that lies near the inner surface of the nuclear envelope, immediately adjacent to the SPB, even though there are no MTs in the interphase nucleus. Apparently, the MT initiation activities of gamma-tubulin in S. pombe are regulated. The SPB duplicates in the cytoplasm during late G2 phase, and the two resulting structures are connected by a darkly staining bridge until the mitotic spindle forms. As the cell enters mitosis, the nuclear envelope invaginates beside the SPB, forming a pocket of cytoplasm that accumulates dark amorphous material. The nuclear envelope then opens to form a fenestra, and the duplicated SPB settles into it. Each part of the SPB initiates intranuclear MTs, and then the two structures separate to lie in distinct fenestrae as a bipolar spindle forms. Through metaphase, the SPBs remain in their fenestrae, bound to the polar ends of spindle MTs; at about this time, a small bundle of cytoplasmic MTs forms in association with each SPB. These MTs are situated with one end near to, but not on, the SPBs, and they project into the cytoplasm at an orientation that is oblique to the simple axis. As anaphase proceeds, the nuclear fenestrae close, and the SPBs are extruded back into the cytoplasm. These observations define new fields of enquiry about the control of SPB duplication and the dynamics of the nuclear envelope.

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