scholarly journals The calcium-binding protein cell division cycle 31 of Saccharomyces cerevisiae is a component of the half bridge of the spindle pole body.

1993 ◽  
Vol 123 (2) ◽  
pp. 405-416 ◽  
Author(s):  
A Spang ◽  
I Courtney ◽  
U Fackler ◽  
M Matzner ◽  
E Schiebel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Calcium Binding ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Nonpermissive Temperature ◽  
Whole Cells ◽  
Pole Body ◽  
Associated Proteins ◽  
Protein Cell ◽  
Cytoplasmic Side

Cdc31 mutants of Saccharomyces cerevisiae arrest at the nonpermissive temperature with large buds, G2 DNA content and, a single, abnormally large spindle pole body (SPB) (Byers, B. 1981. Molecular Genetics in Yeast. Alfred Benzon Symposium. 16:119-133). In this report, we show that the CDC31 gene product is essential for cell viability. We demonstrate that purified CDC31 protein binds Ca2+ and that this binding is highly specific. Taken together, three lines of evidence indicate that CDC31 is a component of the SPB. First, CDC31 cofractionates with enriched preparations of SPBs. Second, immunofluorescence staining indicates that CDC31 colocalizes with a known SPB component. Third, immunoelectron microscopy with whole cells and with isolated SPBs reveals that CDC31 is localized to the half bridge of the SPB, which lies immediately adjacent to the SPB plaques. CDC31 was detected mainly at the cytoplasmic side of the half bridge and, therefore, defines a further substructure of the SPB. We suggest that CDC31 is a member of a family of calcium-binding, centrosome-associated proteins from a phylogenetically diverse group of organisms.

scholarly journals Characterization of spindle pole body duplication reveals a regulatory role for nuclear pore complexes

2017 ◽  
Vol 216 (8) ◽  
pp. 2425-2442 ◽  
Author(s):  
Diana Rüthnick ◽  
Annett Neuner ◽  
Franziska Dietrich ◽  
Daniel Kirrmaier ◽  
Ulrike Engel ◽  
...  
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Pole Body ◽  
Associated Proteins ◽  
Pore Complexes ◽  
Open Question ◽  
Cytoplasmic Side

The spindle pole body (SPB) of budding yeast duplicates once per cell cycle. In G1, the satellite, an SPB precursor, assembles next to the mother SPB (mSPB) on the cytoplasmic side of the nuclear envelope (NE). How the growing satellite subsequently inserts into the NE is an open question. To address this, we have uncoupled satellite growth from NE insertion. We show that the bridge structure that separates the mSPB from the satellite is a distance holder that prevents deleterious fusion of both structures. Binding of the γ-tubulin receptor Spc110 to the central plaque from within the nucleus is important for NE insertion of the new SPB. Moreover, we provide evidence that a nuclear pore complex associates with the duplicating SPB and helps to insert the SPB into the NE. After SPB insertion, membrane-associated proteins including the conserved Ndc1 encircle the SPB and retain it within the NE. Thus, uncoupling SPB growth from NE insertion unmasks functions of the duplication machinery.

Ady3p Links Spindle Pole Body Function to Spore Wall Synthesis in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1439-1450
Author(s):  
Mark E Nickas ◽  
Aaron M Neiman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Spindle Pole Body ◽  
Leading Edge ◽  
Spindle Pole ◽  
Spore Wall ◽  
Pole Body ◽  
Prospore Membrane ◽  
Sporulation Efficiency ◽  
Spore Walls

Abstract Spore formation in Saccharomyces cerevisiae requires the de novo synthesis of prospore membranes and spore walls. Ady3p has been identified as an interaction partner for Mpc70p/Spo21p, a meiosis-specific component of the outer plaque of the spindle pole body (SPB) that is required for prospore membrane formation, and for Don1p, which forms a ring-like structure at the leading edge of the prospore membrane during meiosis II. ADY3 expression has been shown to be induced in midsporulation. We report here that Ady3p interacts with additional components of the outer and central plaques of the SPB in the two-hybrid assay. Cells that lack ADY3 display a decrease in sporulation efficiency, and most ady3Δ/ady3Δ asci that do form contain fewer than four spores. The sporulation defect in ady3Δ/ady3Δ cells is due to a failure to synthesize spore wall polymers. Ady3p forms ring-like structures around meiosis II spindles that colocalize with those formed by Don1p, and Don1p rings are absent during meiosis II in ady3Δ/ady3Δ cells. In mpc70Δ/mpc70Δ cells, Ady3p remains associated with SPBs during meiosis II. Our results suggest that Ady3p mediates assembly of the Don1p-containing structure at the leading edge of the prospore membrane via interaction with components of the SPB and that this structure is involved in spore wall formation.

The influence of the microtubule inhibitor, methyl benzimidazol-2-yl-carbamate (MBC) on nuclear division and the cell cycle in Saccharomyces cerevisiae

1980 ◽  
Vol 46 (1) ◽  
pp. 341-352
Author(s):  
R.A. Quinlan ◽  
C.I. Pogson ◽  
K. Gull
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Ultrastructural Examination ◽  
Microtubule Inhibitor ◽  
Microtubule Polymerization ◽  
Pole Body ◽  
Outer Component ◽  
Spindle Microtubules

Methyl benzimidazol-2-yl-carbamate (MBC), at a concentration of 100 microM, has a pronounced effect on the growth of Saccharomyces cerevisiae, resulting in the accumulation of cells as large doublets. We have determined a specific execution point for the effect of MBC on the yeast cell cycle, and have shown that this execution point is between the cycle events of spindle pole body duplication and spindle pole body separation. An ultrastructural examination of the MBC-treated cells revealed the absence of cytoplasmic and spindle microtubules. MBC treatment also produced an altered spindle pole body morphology, causing the disappearance of the outer component. Nuclear size was also markedly increased in the MBC-induced doublet cells, although the septa were completely absent from these doublet cells. It is proposed that MBC inhibits microtubule polymerization, rather than causing the depolymerization of stable microtubules.

scholarly journals 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

1998 ◽  
Vol 9 (4) ◽  
pp. 775-793 ◽  
Author(s):  
Gislene Pereira ◽  
Michael Knop ◽  
Elmar Schiebel
Keyword(s):  
Cell Cycle ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Nuclear Localization Sequence ◽  
Checkpoint Control ◽  
Yeast Saccharomyces Cerevisiae ◽  
Pole Body ◽  
Cycle Dependent ◽  
Cytoplasmic Side

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 γ-tubulin complex containing the γ-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.

scholarly journals Toxic effects of excess cloned centromeres.

1986 ◽  
Vol 6 (6) ◽  
pp. 2213-2222 ◽  
Author(s):  
B Futcher ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Copy Number ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Host Cells ◽  
Selectable Markers ◽  
Pole Body ◽  
Copy Numbers ◽  
Yeast Plasmids

Plasmids carrying a Saccharomyces cerevisiae centromere have a copy number of one or two, whereas other yeast plasmids have high copy numbers. The number of CEN plasmids per yeast cell was made artificially high by transforming cells simultaneously with several different CEN plasmids carrying different, independently selectable markers. Some host cells carried five different CEN plasmids and an average total of 13 extra copies of CEN3. Several effects were noted. The copy number of each plasmid was unexpectedly high. The plasmids were mutually unstable. Cultures contained many dead cells. The viable host cells grew more slowly than control cells, even in nonselective medium. There was a pause in the cell cycle at or just before mitosis. We conclude that an excess of centromeres is toxic and that the copy number of centromere plasmids is low partly because of selection against cells carrying multiple centromere plasmids. The toxicity may be caused by competition between the centromeres for some factor present in limiting quantities, e.g., centromere-binding proteins, microtubules, or space on the spindle pole body.

scholarly journals Role of calmodulin and Spc110p interaction in the proper assembly of spindle pole body compenents.

1996 ◽  
Vol 133 (1) ◽  
pp. 111-124 ◽  
Author(s):  
H A Sundberg ◽  
L Goetsch ◽  
B Byers ◽  
T N Davis
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Immunofluorescent Staining ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Nonpermissive Temperature ◽  
Carboxy Terminus ◽  
Calmodulin Binding ◽  
Pole Body ◽  
Mutant Cells

Previously we demonstrated that calmodulin binds to the carboxy terminus of Spc110p, an essential component of the Saccharomyces cerevisiae spindle pole body (SPB), and that this interaction is required for chromosome segregation. Immunoelectron microscopy presented here shows that calmodulin and thus the carboxy terminus of Spc110p localize to the central plaque. We created temperature-sensitive SPC110 mutations by combining PCR mutagenesis with a plasmid shuffle strategy. The temperature-sensitive allele spc110-220 differs from wild type at two sites. The cysteine 911 to arginine mutation resides in the calmodulin-binding site and alone confers a temperature-sensitive phenotype. Calmodulin overproduction suppresses the temperature sensitivity of spc110-220. Furthermore, calmodulin levels at the SPB decrease in the mutant cells at the restrictive temperature. Thus, calmodulin binding to Spc110-220p is defective at the nonpermissive temperature. Synchronized mutant cells incubated at the nonpermissive temperature arrest as large budded cells with a G2 content of DNA and suffer considerable lethality. Immunofluorescent staining demonstrates failure of nuclear DNA segregation and breakage of many spindles. Electron microscopy reveals an aberrant nuclear structure, the intranuclear microtubule organizer (IMO), that differs from a SPB but serves as a center of microtubule organization. The IMO appears during nascent SPB formation and disappears after SPB separation. The IMO contains both the 90-kD and the mutant 110-kD SPB components. Our results suggest that disruption of the calmodulin Spc110p interaction leads to the aberrant assembly of SPB components into the IMO, which in turn perturbs spindle formation.

scholarly journals Conservative Duplication of Spindle Poles during Meiosis in Saccharomyces cerevisiae

2001 ◽  
Vol 183 (7) ◽  
pp. 2372-2375 ◽  
Author(s):  
Andreas Wesp ◽  
Susanne Prinz ◽  
Gerald R. Fink
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Spore Formation ◽  
Wild Type ◽  
Body Distribution ◽  
Spindle Poles ◽  
Pole Body ◽  
Spindle Pole Bodies ◽  
Type Strains

ABSTRACT During sporulation in diploid Saccharomyces cerevisiae, spindle pole bodies acquire the so-called meiotic plaque, a prerequisite for spore formation. Mpc70p is a component of the meiotic plaque and is thus essential for spore formation. We show here that MPC70/mpc70 heterozygous strains most often produce two spores instead of four and that these spores are always nonsisters. In wild-type strains, Mpc70p localizes to all four spindle pole bodies, whereas in MPC70/mpc70 strains Mpc70p localizes to only two of the four spindle pole bodies, and these are always nonsisters. Our data can be explained by conservative spindle pole body distribution in which the two newly synthesized meiosis II spindle pole bodies of MPC70/mpc70 strains lack Mpc70p.

scholarly journals Nuf2, a spindle pole body-associated protein required for nuclear division in yeast.

1994 ◽  
Vol 125 (4) ◽  
pp. 853-866 ◽  
Author(s):  
M A Osborne ◽  
G Schlenstedt ◽  
T Jinks ◽  
P A Silver
Keyword(s):  
Nuclear Division ◽  
Spindle Pole Body ◽  
Coiled Coil ◽  
Temperature Shift ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Culture Cells ◽  
Pole Body ◽  
Associated Proteins

The NUF2 gene of the yeast Saccharomyces cerevisiae encodes an essential 53-kd protein with a high content of potential coiled-coil structure similar to myosin. Nuf2 is associated with the spindle pole body (SPB) as determined by coimmunofluorescence with known SPB proteins. Nuf2 appears to be localized to the intranuclear region and is a candidate for a protein involved in SPB separation. The nuclear association of Nuf2 can be disrupted, in part, by 1 M salt but not by the detergent Triton X-100. All Nuf2 can be removed from nuclei by 8 M urea extraction. In this regard, Nuf2 is similar to other SPB-associated proteins including Nufl/SPC110, also a coiled-coil protein. Temperature-sensitive alleles of NUF2 were generated within the coiled-coil region of Nuf2 and such NUF2 mutant cells rapidly arrest after temperature shift with a single undivided or partially divided nucleus in the bud neck, a shortened mitotic spindle and their DNA fully replicated. In sum, Nuf2 is a protein associated with the SPB that is critical for nuclear division. Anti-Nuf2 antibodies also recognize a mammalian 73-kd protein and display centrosome staining of mammalian tissue culture cells suggesting the presence of a protein with similar function.

scholarly journals The Cyclin-dependent Kinase Cdc28p Regulates Multiple Aspects of Kar9p Function in Yeast

2007 ◽  
Vol 18 (4) ◽  
pp. 1187-1202 ◽  
Author(s):  
Jeffrey K. Moore ◽  
Rita K. Miller
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cyclin Dependent Kinase ◽  
Hybrid Analysis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Microtubule Associated Proteins ◽  
Pole Body ◽  
Associated Proteins ◽  
Cytoplasmic Microtubules ◽  
Two Hybrid

During mitosis in the yeast Saccharomyces cerevisiae, Kar9p directs one spindle pole body (SPB) toward the incipient daughter cell by linking the associated set of cytoplasmic microtubules (cMTs) to the polarized actin network on the bud cortex. The asymmetric localization of Kar9p to one SPB and attached cMTs is dependent on its interactions with microtubule-associated proteins and is regulated by the yeast Cdk1 Cdc28p. Two phosphorylation sites in Kar9p were previously identified. Here, we propose that the two sites are likely to govern Kar9p function through separate mechanisms, each involving a distinct cyclin. In the first mechanism, phosphorylation at serine 496 recruits Kar9p to one SPB. A phosphomimetic mutation at serine 496 bypasses the requirement of BIK1 and CLB5 in generating Kar9p asymmetry. In the second mechanism, Clb4p may target serine 197 of Kar9p for phosphorylation. This modification is required for Kar9p to direct cMTs to the bud. Two-hybrid analysis suggests that this phosphorylation may attenuate the interaction between Kar9p and the XMAP215-homologue Stu2p. We propose that phosphorylation at serine 197 regulates the release of Kar9p from Stu2p at the SPB, either to clear it from the mother-SPB or to allow it to travel to the plus end.

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