Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, before the onset of anaphase, the spindle apparatus is always positioned with one spindle pole at, or through, the neck between the mother cell and the growing bud. This spindle orientation enables proper chromosome segregation to occur during anaphase, allowing one replicated genome to be segregated into the bud and the other to remain in the mother cell. In this study, we synchronized a population of cells before the onset of anaphase such that > 90% of the cells in the population had spindles with the correct orientation, and then disrupted specific cytoskeletal elements using temperature-sensitive mutations. Disruption of either the astral microtubules or actin function resulted in improper spindle orientation in approximately 40-50% of the cells. When cells with disrupted astral microtubules or actin function entered into anaphase, there was a 100-200-fold increase in the frequency of binucleated cell bodies. Thus, the maintenance of proper spindle orientation by these cytoskeletal elements was essential for proper chromosome segregation. These data are consistent with the model that proper spindle orientation is maintained by directly or indirectly tethering the astral microtubules to the actin cytoskeleton. After nuclear migration, but before anaphase, bulk chromosome movement occurs within the nucleus apparently because the chromosomes are attached to a mobile spindle. The frequency and magnitude of bulk chromosome movement is greatly diminished by disruption of the astral microtubules but not by disruption of the nonkinetochore spindle microtubules. These results suggest that astral microtubules are not only important for spindle orientation before anaphase, but they also mediate force on the spindle, generating spindle displacement and in turn chromosome movement. Potential roles for this force in spindle assembly and orientation are discussed.

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Nuclear behaviour and cell wall composition in relation to budding in mutants of the yeast Saccharomyces cerevisiae

Canadian Journal of Botany ◽

10.1139/b86-027 ◽

1986 ◽

Vol 64 (1) ◽

pp. 193-200 ◽

Cited By ~ 4

Author(s):

Mario Lachapelle ◽

E. Roger Boothroyd

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Spindle Pole ◽

Minor Role ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Bud Emergence ◽

Spindle Elongation ◽

10 Nm Filaments ◽

A Minor

A temperature-sensitive, cell division cycle mutant (cdc24–1) and karyogamy-deficient (kar1) mutant of Saccharomyces cerevisiae, both of which can produce binucleate or multinucleate cells, were used to study certain aspects of budding, after fluorescent staining for mannan, chitin, and nuclei (DNA). In most binucleate cells the two nuclei lay close together and divided into the same bud. In a few, however, the nuclei were far apart and one or two buds were formed, each proximal to a nucleus. The proximity of daughter nuclei in most blocked cdc24–1 cells suggests a role for the CDC24 gene product in spindle elongation. The relationship between the nuclei and the number and location of buds supports the theory of a preponderant role for the nucleus in budding. Although buds develop preferentially in regions of low chitin content in kar1 heterokaryons, the ability of cdc24–1 cells to bud even with a uniformly high content of chitin and mannan suggests a minor role for these cell wall constituents in determining the sites of bud emergence. The chitin ring is not needed for bud emergence but seems to play a role in normal bud development and in septum formation. Electron microscopy of cdc24–1 cells blocked (37 °C) for 8 h and released (23 °C) for 30 min showed morphologically normal spindle pole bodies, cytoplasmic microtubules, and intranuclear spindles. Although the chitin ring was absent, the ring of 10-nm filaments was present, consistent with its proposed role in bud emergence.

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

The Journal of Cell Biology ◽

10.1083/jcb.146.5.1019 ◽

1999 ◽

Vol 146 (5) ◽

pp. 1019-1032 ◽

Cited By ~ 59

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.

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A mutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4351 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4351-4364 ◽

Cited By ~ 65

Author(s):

W E Payne ◽

M Fitzgerald-Hayes

Keyword(s):

Saccharomyces Cerevisiae ◽

Phospholipase C ◽

Chromosome Segregation ◽

Mitotic Chromosome ◽

Fold Increase ◽

Temperature Sensitive ◽

Aberrant Chromosome ◽

Yeast Strains ◽

Ef Hand ◽

Exogenous Calcium

We identified a putative Saccharomyces cerevisiae homolog of a phosphoinositide-specific phospholipase C (PI-PLC) gene, PLC1, which encodes a protein most similar to the delta class of PI-PLC enzymes. The PLC1 gene was isolated during a study of yeast strains that exhibit defects in chromosome segregation. plc1-1 cells showed a 10-fold increase in aberrant chromosome segregation compared with the wild type. Molecular analysis revealed that PLC1 encodes a predicted protein of 101 kDa with approximately 50 and 26% identity to the highly conserved X and Y domains of PI-PLC isozymes from humans, bovines, rats, and Drosophila melanogaster. The putative yeast protein also contains a consensus EF-hand domain that is predicted to bind calcium. Interestingly, the temperature-sensitive and chromosome missegregation phenotypes exhibited by plc1-1 cells were partially suppressed by exogenous calcium.

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Sli15 Associates with the Ipl1 Protein Kinase to Promote Proper Chromosome Segregation in Saccharomyces cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.145.7.1381 ◽

1999 ◽

Vol 145 (7) ◽

pp. 1381-1394 ◽

Cited By ~ 104

Author(s):

Jae-hyun Kim ◽

Jung-seog Kang ◽

Clarence S.M. Chan

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Chromosome Segregation ◽

Genetic Interaction ◽

Spindle Pole ◽

Mutant Phenotype ◽

Yeast Saccharomyces Cerevisiae ◽

Synthetic Lethal ◽

Mutant Cells

The conserved Ipl1 protein kinase is essential for proper chromosome segregation and thus cell viability in the budding yeast Saccharomyces cerevisiae. Its human homologue has been implicated in the tumorigenesis of diverse forms of cancer. We show here that sister chromatids that have separated from each other are not properly segregated to opposite poles of ipl1-2 cells. Failures in chromosome segregation are often associated with abnormal distribution of the spindle pole–associated Nuf2-GFP protein, thus suggesting a link between potential spindle pole defects and chromosome missegregation in ipl1 mutant cells. A small fraction of ipl1-2 cells also appears to be defective in nuclear migration or bipolar spindle formation. Ipl1 associates, probably directly, with the novel and essential Sli15 protein in vivo, and both proteins are localized to the mitotic spindle. Conditional sli15 mutant cells have cytological phenotypes very similar to those of ipl1 cells, and the ipl1-2 mutation exhibits synthetic lethal genetic interaction with sli15 mutations. sli15 mutant phenotype, like ipl1 mutant phenotype, is partially suppressed by perturbations that reduce protein phosphatase 1 function. These genetic and biochemical studies indicate that Sli15 associates with Ipl1 to promote its function in chromosome segregation.

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A mutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4351-4364.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4351-4364

Author(s):

W E Payne ◽

M Fitzgerald-Hayes

Keyword(s):

Saccharomyces Cerevisiae ◽

Phospholipase C ◽

Chromosome Segregation ◽

Mitotic Chromosome ◽

Fold Increase ◽

Temperature Sensitive ◽

Aberrant Chromosome ◽

Yeast Strains ◽

Ef Hand ◽

Exogenous Calcium

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A Mutation in the ATP2 Gene Abrogates the Age Asymmetry Between Mother and Daughter Cells of the Yeast Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/162.1.73 ◽

2002 ◽

Vol 162 (1) ◽

pp. 73-87 ◽

Cited By ~ 1

Author(s):

Chi-Yung Lai ◽

Ewa Jaruga ◽

Corina Borghouts ◽

S Michal Jazwinski

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Division ◽

Life Span ◽

Mother Cell ◽

Yeast Cells ◽

Mitochondrial Morphology ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Daughter Cells ◽

Mother And Daughter

Abstract The yeast Saccharomyces cerevisiae reproduces by asymmetric cell division, or budding. In each cell division, the daughter cell is usually smaller and younger than the mother cell, as defined by the number of divisions it can potentially complete before it dies. Although individual yeast cells have a limited life span, this age asymmetry between mother and daughter ensures that the yeast strain remains immortal. To understand the mechanisms underlying age asymmetry, we have isolated temperature-sensitive mutants that have limited growth capacity. One of these clonal-senescence mutants was in ATP2, the gene encoding the β-subunit of mitochondrial F1, F0-ATPase. A point mutation in this gene caused a valine-to-isoleucine substitution at the ninetieth amino acid of the mature polypeptide. This mutation did not affect the growth rate on a nonfermentable carbon source. Life-span determinations following temperature shift-down showed that the clonal-senescence phenotype results from a loss of age asymmetry at 36°, such that daughters are born old. It was characterized by a loss of mitochondrial membrane potential followed by the lack of proper segregation of active mitochondria to daughter cells. This was associated with a change in mitochondrial morphology and distribution in the mother cell and ultimately resulted in the generation of cells totally lacking mitochondria. The results indicate that segregation of active mitochondria to daughter cells is important for maintenance of age asymmetry and raise the possibility that mitochondrial dysfunction may be a normal cause of aging. The finding that dysfunctional mitochondria accumulated in yeasts as they aged and the propensity for old mother cells to produce daughters depleted of active mitochondria lend support to this notion. We propose, more generally, that age asymmetry depends on partition of active and undamaged cellular components to the progeny and that this “filter” breaks down with age.

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Nonrecombinant meiosis I nondisjunction in Saccharomyces cerevisiae induced by tRNA ochre suppressors.

Genetics ◽

10.1093/genetics/123.1.81 ◽

1989 ◽

Vol 123 (1) ◽

pp. 81-95 ◽

Cited By ~ 1

Author(s):

E J Louis ◽

J E Haber

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitotic Chromosome ◽

Stop Codon ◽

Fold Increase ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Nonsense Mutations ◽

Chromosome Iii ◽

Meiosis I ◽

Chromosome Nondisjunction

Abstract The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.

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Genes Involved in Sister Chromatid Separation and Segregation in the Budding Yeast Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/159.2.453 ◽

2001 ◽

Vol 159 (2) ◽

pp. 453-470

Author(s):

Sue Biggins ◽

Needhi Bhalla ◽

Amy Chang ◽

Dana L Smith ◽

Andrew W Murray

Keyword(s):

Chromosome Segregation ◽

Sister Chromatid ◽

Mitotic Spindle ◽

Budding Yeast ◽

Temperature Sensitive ◽

Chromosome Behavior ◽

Yeast Saccharomyces Cerevisiae ◽

Sister Chromatids ◽

Marked Chromosome ◽

Sister Chromatid Separation

Abstract Accurate chromosome segregation requires the precise coordination of events during the cell cycle. Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase. At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles. To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome. Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified. We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior. We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation. We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair α-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication. We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor.

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SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2154 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2154-2164 ◽

Cited By ~ 50

Author(s):

D J DeMarini ◽

M Winey ◽

D Ursic ◽

F Webb ◽

M R Culbertson

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Gene Product ◽

Signal Sequence ◽

Null Alleles ◽

Nucleoside Triphosphate ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

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

The Journal of Cell Biology ◽

10.1083/jcb.130.3.687 ◽

1995 ◽

Vol 130 (3) ◽

pp. 687-700 ◽

Cited By ~ 272

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.

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