scholarly journals Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae.

1984 ◽  
Vol 98 (3) ◽  
pp. 934-945 ◽  
Author(s):  
A E Adams ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Spindle Pole Body ◽  
Neck Region ◽  
Spindle Pole ◽  
Wild Type ◽  
Active Growth ◽  
Yeast Saccharomyces Cerevisiae ◽  
The One ◽  
Relationship Of

The distribution of actin in wild-type cells and in morphogenetic mutants of the budding yeast Saccharomyces cerevisiae was explored by staining cells with fluorochrome-labeled phallotoxins after fixing and permeabilizing the cells by several methods. The actin appeared to be localized in a set of cortical spots or patches, as well as in a network of cytoplasmic fibers. Bundles of filaments that may possibly correspond to the fibers visualized by fluorescence were observed with the electron microscope. The putative actin spots were concentrated in small and medium-sized buds and at what were apparently the sites of incipient bud formation on unbudded cells, whereas the putative actin fibers were generally oriented along the long axes of the mother-bud pairs. In several morphogenetic mutants that form multiple, abnormally elongated buds, the actin patches were conspicuously clustered at the tips of most buds, and actin fibers were clearly oriented along the long axes of the buds. There was a strong correlation between the occurrence of active growth at particular bud tips and clustering of actin spots at those same tips. Near the end of the cell cycle in wild-type cells, actin appeared to concentrate (as a cluster of spots or a band) in the neck region connecting the mother cell to its bud. Observations made using indirect immunofluorescence with a monoclonal anti-yeast-tubulin antibody on the morphogenetic mutant cdc4 (which forms multiple, abnormally elongated buds while the nuclear cycle is arrested) revealed the surprising occurrence of multiple bundles of cytoplasmic microtubules emanating from the one duplicated spindle-pole body per cell. It seems that most or all of the buds contain one or more of these bundles of microtubules, which often can be seen to extend to the very tips of the buds. These observations are consistent with the hypotheses that actin, tubulin, or both may be involved in the polarization of growth and localization of cell-wall deposition that occurs during the yeast cell cycle.

scholarly journals The Role of Actin in Spindle Orientation Changes during the Saccharomyces cerevisiae Cell Cycle

1999 ◽  
Vol 146 (5) ◽  
pp. 1019-1032 ◽  
Author(s):  
Chandra L. Theesfeld ◽  
Javier E. Irazoqui ◽  
Kerry Bloom ◽  
Daniel J. Lew
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Spindle Orientation ◽  
Cortical Actin ◽  
Yeast Saccharomyces Cerevisiae ◽  
Daughter Cells ◽  
M Phase ◽  
Actin Cables

In the budding yeast Saccharomyces cerevisiae, the mitotic spindle must align along the mother-bud axis to accurately partition the sister chromatids into daughter cells. Previous studies showed that spindle orientation required both astral microtubules and the actin cytoskeleton. We now report that maintenance of correct spindle orientation does not depend on F-actin during G2/M phase of the cell cycle. Depolymerization of F-actin using Latrunculin-A did not perturb spindle orientation after this stage. Even an early step in spindle orientation, the migration of the spindle pole body (SPB), became actin-independent if it was delayed until late in the cell cycle. Early in the cell cycle, both SPB migration and spindle orientation were very sensitive to perturbation of F-actin. Selective disruption of actin cables using a conditional tropomyosin double-mutant also led to de- fects in spindle orientation, even though cortical actin patches were still polarized. This suggests that actin cables are important for either guiding astral microtubules into the bud or anchoring them in the bud. In addition, F-actin was required early in the cell cycle for the development of the actin-independent spindle orientation capability later in the cell cycle. Finally, neither SPB migration nor the switch from actin-dependent to actin-independent spindle behavior required B-type cyclins.

scholarly journals Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae.

1995 ◽  
Vol 130 (3) ◽  
pp. 687-700 ◽  
Author(s):  
E Yeh ◽  
R V Skibbens ◽  
J W Cheng ◽  
E D Salmon ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Translocation ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Wild Type ◽  
Immunofluorescent Localization ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Dynamics ◽  
Spindle Elongation ◽  
Anaphase B

We have used time-lapse digital- and video-enhanced differential interference contrast (DE-DIC, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae. The real-time analysis reveals six stages in the spindle cycle. Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell. The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants. In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell. Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length. This stage is protracted in the dynein mutants and devoid of oscillatory motion. Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics. Genetic analysis reveals that in the absence of a functional central spindle (ndcl), dynein is essential for chromosome movement into the bud. Immunofluorescent localization of dynein-beta-galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex: with spindle pole body localization dependent on intact microtubules. A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud.

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 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 Absence of microtubule sliding and an analysis of spindle formation and elongation in isolated mitotic spindles from the yeast Saccharomyces cerevisiae.

1982 ◽  
Vol 94 (2) ◽  
pp. 341-349 ◽  
Author(s):  
S M King ◽  
J S Hyams ◽  
A Luba
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Spindles ◽  
Microtubule Organization ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Formation ◽  
Detailed Model ◽  
Thin Sections ◽  
Gold Thioglucose

Mitotic spindles were isolated from a cell division cycle mutant of the budding yeast Saccharomyces cerevisiae by the lysis of sphateroplasts on an air:buffer interface and were negatively stained with 1% gold thioglucose. Isolated spindles were incubated under conditions which promoted the sliding disintegration of parallel preparations of Tetrahymena axonemes, namely the addition of ATP to 20 microM. In no experiment was a corresponding change in microtubule organization of the spindle observed even when spindles were first pretreated with either 1-10 microgram/ml trypsin or 0.2-2% Triton X-100. During these experiments a number of spindles were isolated from cells that had passed through the imposed temperature block, and from the images obtained a detailed model of spindle formation and elongation has been constructed. Two sets of microtubules, one from each spindle pole body (SPB), completely interdigitate to form a continuous bundle, and a series of discontinuous microtubules are then nucleated by each SPB. As the spindle elongates, the number of microtubules continuous between the two SPBs decreases until, at a length of 4 micrometer, only one remains. The spindle, composed of only one microtubule, continues to elongate until it reaches the maximal nuclear dimension of 8 micrometer. The data obtained from negatively stained preparations have been verified in thin sections of wild-type cells. We suggest that, as in the later stages of mitosis only one microtubule is involved in the separation of the spindle poles, the microtubular spindle in S. cerevisiae is not a force-generating system but rather acts as a regulatory mechanism controlling the rate of separation.

scholarly journals IBD2 Encodes a Novel Component of the Bub2p-Dependent Spindle Checkpoint in the Budding Yeast Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 161 (2) ◽  
pp. 595-609
Author(s):  
Hyung-Seo Hwang ◽  
Kiwon Song
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Budding Yeast ◽  
Essential Gene ◽  
Spindle Checkpoint ◽  
Mitotic Arrest ◽  
Mitotic Exit ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Checkpoint Pathway

Abstract During mitosis, genomic integrity is maintained by the proper coordination of mitotic events through the spindle checkpoint. The bifurcated spindle checkpoint blocks cell cycle progression at metaphase by monitoring unattached kinetochores and inhibits mitotic exit in response to the incorrect orientation of the mitotic spindle. Bfa1p is a spindle checkpoint regulator of budding yeast in the Bub2p checkpoint pathway for proper mitotic exit. We have isolated a novel Bfa1p interacting protein named Ibd2p in the budding yeast Saccharomyces cerevisiae. We found that IBD2 (Inhibition of Bud Division 2) is not an essential gene but its deletion mutant proceeded through the cell cycle in the presence of microtubule-destabilizing drugs, thereby inducing a sharp decrease in viability. In addition, overexpression of Mps1p caused partial mitotic arrest in ibd2Δ as well as in bub2Δ, suggesting that IBD2 encodes a novel component of the spindle checkpoint downstream of MPS1. Overexpression of Ibd2p induced mitotic arrest with increased levels of Clb2p in wild type and mad2Δ, but not in deletion mutants of BUB2 and BFA1. Pds1p was also stabilized by the overexpression of Ibd2p in wild-type cells. The mitotic arrest defects observed in ibd2Δ in the presence of nocodazole were restored by additional copies of BUB2, BFA1, and CDC5, whereas an extra copy of IBD2 could not rescue the mitotic arrest defects of bub2Δ and bfa1Δ. The mitotic arrest defects of ibd2Δ were not recovered by MAD2, or vice versa. Analysis of the double mutant combinations ibd2Δmad2Δ, ibd2Δbub2Δ, and ibd2Δdyn1Δ showed that IBD2 belongs to the BUB2 epistasis group. Taken together, these data demonstrate that IBD2 encodes a novel component of the BUB2-dependent spindle checkpoint pathway that functions upstream of BUB2 and BFA1.

Centromere clustering is a major determinant of yeast interphase nuclear organization

2000 ◽  
Vol 113 (11) ◽  
pp. 1903-1912 ◽  
Author(s):  
Q.W. Jin ◽  
J. Fuchs ◽  
J. Loidl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Organization ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genome Separation ◽  
Pole Body ◽  
Opposite Pole ◽  
Somatic Pairing ◽  
Higher Eukaryotes

During interphase in the budding yeast, Saccharomyces cerevisiae, centromeres are clustered near one pole of the nucleus as a rosette with the spindle pole body at its hub. Opposite to the centromeric pole is the nucleolus. Chromosome arms extend outwards from the centromeric pole and are preferentially directed towards the opposite pole. Centromere clustering is reduced by the ndc10 mutation, which affects a kinetochore protein, and by the microtubule poison nocodazole. This suggests that clustering is actively maintained or enforced by the association of centromeres with microtubules throughout interphase. Unlike the Rabl-orientation known from many higher eukaryotes, centromere clustering in yeast is not only a relic of anaphase chromosome polarization, because it can be reconstituted without the passage of cells through anaphase. Within the rosette, homologous centromeres are not arranged in a particular order that would suggest somatic pairing or genome separation.

scholarly journals The 110-kD spindle pole body component of Saccharomyces cerevisiae is a phosphoprotein that is modified in a cell cycle-dependent manner.

1996 ◽  
Vol 132 (5) ◽  
pp. 903-914 ◽  
Author(s):  
D B Friedman ◽  
H A Sundberg ◽  
E Y Huang ◽  
T N Davis
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Wild Type ◽  
Spindle Formation ◽  
Pole Body ◽  
A Cell ◽  
In Cells

Spc110p (Nuf1p) is an essential component of the yeast microtubule organizing center, or spindle pole body (SPB). Asynchronous wild-type cultures contain two electrophoretically distinct isoforms of Spc110p as detected by Western blot analysis, suggesting that Spc110p is modified in vivo. Both isoforms incorporate 32Pi in vivo, suggesting that Spc110p is post-translationally modified by phosphorylation. The slower-migrating 120-kD Spc110p isoform after incubation is converted to the faster-migrating 112-kD isoform after incubation with protein phosphatase PP2A, and specific PP2A inhibitors block this conversion. Thus, additional phosphorylation of Spc110p at serine and/or threonine residues gives rise to the slower-migrating 120-kD isoform. The 120-kD isoform predominates in cells arrested in mitosis by the addition of nocodazole. However, the 120-kD isoform is not detectable in cells grown to stationary phase (G0) or in cells arrested in G1 by the addition of alpha-factor. Temperature-sensitive cell division cycle (cdc) mutations demonstrate that the presence of the 120-kD isoform correlates with mitotic spindle formation but not with SPB duplication. In a synchronous wild-type population, the additional serine/threonine phosphorylation that gives rise to the 120-kD isoform appears as cells are forming the mitotic spindle and diminishes as cells enter anaphase. None of several sequences similar to the consensus for phosphorylation by the Cdc28p (cdc2p34) kinase is important for these mitosis-specific phosphorylations or for function. Carboxy-terminal Spc110p truncations lacking the calmodulin binding site can support growth and are also phosphorylated in a cell cycle-specific manner. Further truncation of the Spc110p carboxy terminus results in mutant proteins that are unable to support growth and now migrate as single species. Collectively, these results provide the first evidence of a structural component of the SPB that is phosphorylated during spindle formation and dephosphorylated as cells enter anaphase.

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