Asymmetry of the spindle pole bodies and spg1p GAP segregation during mitosis in fission yeast

In the fission yeast Schizosaccharomyces pombe, the onset of septum formation is induced by a signal transduction network involving several protein kinases and a GTPase switch. One of the roles of the spg1p GTPase is to localise the cdc7p protein kinase to the poles of the mitotic spindle, from where the onset of septation is thought to be signalled at the end of mitosis. Immunofluorescence studies have shown that cdc7p is located on both spindle pole bodies early in mitosis, but only on one during the later stages of anaphase. This is mediated by inactivation of spg1p on one pole before the other. The GAP for spg1p is a complex of two proteins, cdc16p and byr4p. Localisation of cdc16p and byr4p by indirect immunofluorescence during the mitotic cell cycle showed that both proteins are present on the spindle pole body in interphase cells. During mitosis, byr4p is seen first on both poles of the spindle, then on only one. This occurs prior to cdc7p becoming asymmetric. In contrast, the signal due to cdc16p decreases to a low level during early mitosis, before being seen strongly on the same pole as byr4p. Double staining indicates that this is the opposite pole to that which retains cdc7p in late anaphase. Examination of the effect of inactivating cdc16p at various stages of the cell cycle suggests that cdc16p, together with cdc2p plays a role in restraining septum formation during interphase. The asymmetric inactivation of spg1p is mediated by recruitment of the cdc16p-byr4p GAP to one of the poles of the spindle before the other, and the asymmetry of the spindle pole bodies may be established early during mitosis. Moreover, the spindle pole bodies appear to be non-equivalent even after division has been completed.

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Evidence for cell cycle-specific, spindle pole body-mediated, nuclear positioning in the fission yeast Schizosaccharomyces pombe

Journal of Cell Science ◽

10.1242/jcs.110.16.1851 ◽

1997 ◽

Vol 110 (16) ◽

pp. 1851-1866 ◽

Cited By ~ 11

Author(s):

I. Hagan ◽

M. Yanagida

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Nuclear Positioning ◽

Central Location ◽

Pole Body ◽

Spindle Pole Bodies ◽

A Cell ◽

Multiple Spindle

Specific changes in spatial order occur during cell cycle progression in fission yeast. Growth of the rod-shaped cells is highly regulated and undergoes a cell cycle and size-regulated switch from monopolar to bipolar tip extension. During both phases of growth, the interphase nucleus is maintained in a central location. Following the separation of the genome to the cell tips in mitosis, the two nuclei migrate back towards the cell equator before stopping in two new positions that will become the middle of the two new cells. Here we use simultaneous labeling of microtubules, chromatin and spindle pole bodies in wild-type and cdc mutants, to show that nuclear positioning is achieved by regulation of spindle pole body-mediated nuclear migration. We show that the number and location of nuclear positioning signals is regulated in a cell cycle-specific manner and that spindle pole body-mediated forces are likely to be responsible for maintaining correct nuclear position once the nuclei have reached the appropriate position in the cell. Accentuating the movement of the nuclei back towards the cell equator after mitosis by artificially increasing cell length shows that the spindle pole body leads the nucleus during this migration. When multiple spindle pole bodies are associated with the same or different nuclei they all go to the same point indicating that the different spindle pole bodies are responding to the same positional cue. In a septation-defective mutant cell, which contains four nuclei, the spindle pole bodies on the four different nuclei initially group as two pairs in regions that would become the middle of the new cells, were the cell able to divide. In the subsequent interphase, the nuclei aggregate as a group of four in the centre of the cell. The presence of two or three clusters of spindle pole bodies in larger cells with eight nuclei suggests that the mechanisms specifying the normally central location for multiple nuclei may be unable to operate properly as the cells get larger. Perturbation of microtubules with the microtubule poison thiabendazole prevents the spindle pole body clustering in septation mutants, demonstrating that nuclear positioning requires a functional microtubule cytoskeleton.

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Quantifying Tubulin Concentration and Microtubule Number Throughout the Fission Yeast Cell Cycle

Biomolecules ◽

10.3390/biom9030086 ◽

2019 ◽

Vol 9 (3) ◽

pp. 86 ◽

Cited By ~ 4

Author(s):

Isabelle Loiodice ◽

Marcel Janson ◽

Penny Tavormina ◽

Sebastien Schaub ◽

Divya Bhatt ◽

...

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Genetic Model ◽

Spindle Pole Body ◽

Model Organism ◽

Spindle Pole ◽

Living Cells ◽

Fluorescent Imaging ◽

Mother And Daughter ◽

In The Wild

The fission yeast Schizosaccharomyces pombe serves as a good genetic model organism for the molecular dissection of the microtubule (MT) cytoskeleton. However, analysis of the number and distribution of individual MTs throughout the cell cycle, particularly during mitosis, in living cells is still lacking, making quantitative modelling imprecise. We use quantitative fluorescent imaging and analysis to measure the changes in tubulin concentration and MT number and distribution throughout the cell cycle at a single MT resolution in living cells. In the wild-type cell, both mother and daughter spindle pole body (SPB) nucleate a maximum of 23 ± 6 MTs at the onset of mitosis, which decreases to a minimum of 4 ± 1 MTs at spindle break down. Interphase MT bundles, astral MT bundles, and the post anaphase array (PAA) microtubules are composed primarily of 1 ± 1 individual MT along their lengths. We measure the cellular concentration of αβ-tubulin subunits to be ~5 µM throughout the cell cycle, of which one-third is in polymer form during interphase and one-quarter is in polymer form during mitosis. This analysis provides a definitive characterization of αβ-tubulin concentration and MT number and distribution in fission yeast and establishes a foundation for future quantitative comparison of mutants defective in MTs.

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Budding Yeast Bub2 Is Localized at Spindle Pole Bodies and Activates the Mitotic Checkpoint via a Different Pathway from Mad2

The Journal of Cell Biology ◽

10.1083/jcb.145.5.979 ◽

1999 ◽

Vol 145 (5) ◽

pp. 979-991 ◽

Cited By ~ 118

Author(s):

Roberta Fraschini ◽

Elisa Formenti ◽

Giovanna Lucchini ◽

Simonetta Piatti

Keyword(s):

Cell Cycle ◽

Mitotic Spindle ◽

Cell Cycle Progression ◽

Budding Yeast ◽

Spindle Pole Body ◽

Mitotic Checkpoint ◽

Spindle Pole ◽

Cycle Progression ◽

Spindle Pole Bodies

The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle. In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest. Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins. We investigated the role of Bub2 in budding yeast mitotic checkpoint. The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions. Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.

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Regulation of spindle pole body assembly and cytokinesis by the centrin-binding protein Sfi1 in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e13-11-0699 ◽

2014 ◽

Vol 25 (18) ◽

pp. 2735-2749 ◽

Cited By ~ 22

Author(s):

I-Ju Lee ◽

Ning Wang ◽

Wen Hu ◽

Kersey Schott ◽

Jürg Bähler ◽

...

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Fission Yeast ◽

Budding Yeast ◽

Binding Protein ◽

Spindle Pole Body ◽

Spindle Pole ◽

Centrosome Duplication ◽

Pole Body ◽

The Individual

Centrosomes play critical roles in the cell division cycle and ciliogenesis. Sfi1 is a centrin-binding protein conserved from yeast to humans. Budding yeast Sfi1 is essential for the initiation of spindle pole body (SPB; yeast centrosome) duplication. However, the recruitment and partitioning of Sfi1 to centrosomal structures have never been fully investigated in any organism, and the presumed importance of the conserved tryptophans in the internal repeats of Sfi1 remains untested. Here we report that in fission yeast, instead of doubling abruptly at the initiation of SPB duplication and remaining at a constant level thereafter, Sfi1 is gradually recruited to SPBs throughout the cell cycle. Like an sfi1Δ mutant, a Trp-to-Arg mutant (sfi1-M46) forms monopolar spindles and exhibits mitosis and cytokinesis defects. Sfi1-M46 protein associates preferentially with one of the two daughter SPBs during mitosis, resulting in a failure of new SPB assembly in the SPB receiving insufficient Sfi1. Although all five conserved tryptophans tested are involved in Sfi1 partitioning, the importance of the individual repeats in Sfi1 differs. In summary, our results reveal a link between the conserved tryptophans and Sfi1 partitioning and suggest a revision of the model for SPB assembly.

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Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast.

The Journal of Cell Biology ◽

10.1083/jcb.121.5.961 ◽

1993 ◽

Vol 121 (5) ◽

pp. 961-976 ◽

Cited By ~ 312

Author(s):

H Funabiki ◽

I Hagan ◽

S Uzawa ◽

M Yanagida

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Topoisomerase Ii ◽

Nuclear Periphery ◽

Spindle Pole Body ◽

Critical Length ◽

Spindle Pole ◽

Dna Topoisomerase ◽

Motor Cells ◽

Cycle Dependent

Fluorescence in situ hybridization (FISH) shows that fission yeast centromeres and telomeres make up specific spatial arrangements in the nucleus. Their positioning and clustering are cell cycle regulated. In G2, centromeres cluster adjacent to the spindle pole body (SPB), while in mitosis, their association with each other and with the SPB is disrupted. Similarly, telomeres cluster at the nuclear periphery in G2 and their associations are disrupted in mitosis. Mitotic centromeres interact with the spindle. They remain undivided until the spindle reaches a critical length, then separate and move towards the poles. This demonstrated, for the first time, that anaphase A occurs in fission yeast. The mode of anaphase A and B is similar to that of higher eukaryotes. In nda3 and cut7 mutants defective in tubulin of a kinesin-related motor, cells are blocked in early stages of mitosis due to the absence of the spindle, and centromeres dissociate but remain close to the SPB, whereas in a metaphase-arrested nuc2 mutant, they reside at the middle of the spindle. FISH is therefore a powerful tool for analyzing mitotic chromosome movement and disjunction using various mutants. Surprisingly, in top2 defective in DNA topoisomerase II, while most chromatid DNAs remain undivided, sister centromeres are separated. Significance of this finding is discussed. In contrast, most chromatid DNAs are separated but telomeric DNAs are not in cut1 mutant. In cut1, the dependence of SPB duplication on the completion of mitosis is abolished. In crm1 mutant cells defective in higher-order chromosome organization, the interphase arrangements of centromeres and telomeres are disrupted.

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Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast

Wellcome Open Research ◽

10.12688/wellcomeopenres.10507.1 ◽

2017 ◽

Vol 2 ◽

pp. 2 ◽

Cited By ~ 10

Author(s):

Colette Fox ◽

Juan Zou ◽

Juri Rappsilber ◽

Adele L. Marston

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Chromosome Segregation ◽

Budding Yeast ◽

Spindle Pole Body ◽

Initial Step ◽

Mitotic Cell ◽

Spindle Pole ◽

Fluorescence And Electron Microscopy ◽

Meiosis I

Background Gametes are generated through a specialized cell division called meiosis, in which ploidy is reduced by half because two consecutive rounds of chromosome segregation, meiosis I and meiosis II, occur without intervening DNA replication. This contrasts with the mitotic cell cycle where DNA replication and chromosome segregation alternate to maintain the same ploidy. At the end of mitosis, CDKs are inactivated. This low CDK state in late mitosis/G1 allows for critical preparatory events for DNA replication and centrosome/spindle pole body (SPB) duplication. However, their execution is inhibited until S phase, where further preparatory events are also prevented. This “licensing” ensures that both the chromosomes and the centrosomes/SPBs replicate exactly once per cell cycle, thereby maintaining constant ploidy. Crucially, between meiosis I and meiosis II, centrosomes/SPBs must be re-licensed, but DNA re-replication must be avoided. In budding yeast, the Cdc14 protein phosphatase triggers CDK down regulation to promote exit from mitosis. Cdc14 also regulates the meiosis I to meiosis II transition, though its mode of action has remained unclear. Methods Fluorescence and electron microscopy was combined with proteomics to probe SPB duplication in cells with inactive or hyperactive Cdc14. Results We demonstrate that Cdc14 ensures two successive nuclear divisions by re-licensing SPBs at the meiosis I to meiosis II transition. We show that Cdc14 is asymmetrically enriched on a single SPB during anaphase I and provide evidence that this enrichment promotes SPB re-duplication. Cells with impaired Cdc14 activity fail to promote extension of the SPB half-bridge, the initial step in morphogenesis of a new SPB. Conversely, cells with hyper-active Cdc14 duplicate SPBs, but fail to induce their separation. Conclusion Our findings implicate reversal of key CDK-dependent phosphorylations in the differential licensing of cyclical events at the meiosis I to meiosis I transition.

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Components of the yeast spindle and spindle pole body.

The Journal of Cell Biology ◽

10.1083/jcb.111.5.1913 ◽

1990 ◽

Vol 111 (5) ◽

pp. 1913-1927 ◽

Cited By ~ 162

Author(s):

M P Rout ◽

J V Kilmartin

Keyword(s):

Spindle Pole Body ◽

Spindle Pole ◽

The Other ◽

Yeast Cells ◽

Immunofluorescent Staining ◽

Cell Extracts ◽

Pole Body ◽

Spindle Pole Bodies ◽

Cytoplasmic Face ◽

Spindle Microtubules

Yeast spindle pole bodies (SPBs) with attached nuclear microtubles were enriched approximately 600-fold from yeast cell extracts. 14 mAbs prepared against this enriched SPB fraction define at least three components of the SPB and spindle. Immunofluorescent staining of yeast cells showed that throughout the cell cycle two of the components (110 and 90 kD) were localized exclusively to the SPB region, and the other (80 kD) was localized both to the SPB region and to particulate dots in short spindles. Immunoelectron microscopy confirmed and extended most of these findings. Thus the 110-kD component was localized to a layer in the SPB just to the nuclear side of the plane of the inner nuclear membrane. The 90-kD component was localized in a layer across the cytoplasmic face of intact SPBs, and, in SPBs where nuclear microtubules were removed by extraction with DEAE-dextran, the 90-kD component was also found in an inner nuclear layer close to where spindle microtubules emerge. In intact SPBs with attached nuclear microtubules the anit-80-kD mAb labels microtubules, particularly those close to the SPB. These results begin to provide a preliminary molecular map of the SPB and should also enable the corresponding genes to be isolated.

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In vivo localisation of fission yeast cyclin-dependent kinase cdc2p and cyclin B cdc13p during mitosis and meiosis

Journal of Cell Science ◽

10.1242/jcs.114.14.2627 ◽

2001 ◽

Vol 114 (14) ◽

pp. 2627-2640 ◽

Cited By ~ 1

Author(s):

Anabelle Decottignies ◽

Patrick Zarzov ◽

Paul Nurse

Keyword(s):

Fission Yeast ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Nuclear Periphery ◽

Spindle Pole Body ◽

Spindle Pole ◽

Cyclin Dependent Kinase ◽

Spindle Pole Bodies ◽

Mitosis And Meiosis

We investigated the in vivo localisation of fission yeast cyclin-dependent kinase cdc2p during mitosis and meiosis. Fusion to yellow fluorescent protein (YFP) revealed that cdc2-YFP is present in the cytoplasm at all stages of the cell cycle. Nuclear cdc2-YFP fluorescence oscillates with that of cdc13-YFP cyclin. At G1/S, at least one of cdc13p, cig1p or cig2p B-type cyclins is required for the accumulation of cdc2-YFP into the nucleus. Cdc2-YFP and cdc13-YFP are highly enriched on the spindle pole body of cells in late G2 or arrested at S phase. Both accumulate on the spindle pole bodies and the spindle in prophase and metaphase independently of the microtubule-associated protein dis1p. In anaphase, the cdc2p/cdc13p complex leaves the spindle prior to sister chromatid separation, and cdc13-YFP is enriched at the nuclear periphery before fluorescence disappears. If cdc13p cannot be recognized by the anaphase-promoting complex, cdc2-YFP and cdc13-YFP remain associated with the spindle. In mating cells, cdc2-YFP enters the nucleus as soon as the cells undergo fusion. During karyogamy and meiotic prophase, cdc2-YFP is highly enriched on the centromeres. In meiosis I, association of cdc2-YFP with the spindle and the spindle pole bodies shows differences to mitotic cells, suggesting different mechanisms of spindle formation. This study suggests that changes in cdc2p localisation are important for both mitosis and meiosis regulation.

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Spindle pole body components are reorganized during fission yeast meiosis

Molecular Biology of the Cell ◽

10.1091/mbc.e11-11-0951 ◽

2012 ◽

Vol 23 (10) ◽

pp. 1799-1811 ◽

Cited By ~ 24

Author(s):

Midori Ohta ◽

Masamitsu Sato ◽

Masayuki Yamamoto

Keyword(s):

Fission Yeast ◽

Meiotic Prophase ◽

Spindle Pole Body ◽

Mitotic Cell ◽

Spindle Pole ◽

Nuclear Movement ◽

Pole Body ◽

Proper Number ◽

Body Components ◽

Yeast Meiosis

During meiosis, the centrosome/spindle pole body (SPB) must be regulated in a manner distinct from that of mitosis to achieve a specialized cell division that will produce gametes. In this paper, we demonstrate that several SPB components are localized to SPBs in a meiosis-specific manner in the fission yeast Schizosaccharomyces pombe. SPB components, such as Cut12, Pcp1, and Spo15, which stay on the SPB during the mitotic cell cycle, disassociate from the SPB during meiotic prophase and then return to the SPB immediately before the onset of meiosis I. Interestingly, the polo kinase Plo1, which normally localizes to the SPB during mitosis, is excluded from them in meiotic prophase, when meiosis-specific, horse-tail nuclear movement occurs. We found that exclusion of Plo1 during this period was essential to properly remodel SPBs, because artificial targeting of Plo1 to SPBs resulted in an overduplication of SPBs. We also found that the centrin Cdc31 was required for meiotic SPB remodeling. Thus Plo1 and a centrin play central roles in the meiotic SPB remodeling, which is essential for generating the proper number of meiotic SPBs and, thereby provide unique characteristics to meiotic divisions.

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