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

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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Metazoan-like kinetochore arrangement masked by the interphase RabI configuration

10.1101/2020.09.09.289066 ◽

2020 ◽

Author(s):

Alberto Jiménez-Martín ◽

Alberto Pineda-Santaella ◽

Daniel León-Periñán ◽

David Delgado-Gestoso ◽

Laura Marín-Toral ◽

...

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Cell Cycle Progression ◽

Spindle Pole Body ◽

Model Organism ◽

Spindle Pole ◽

Chromosome Configuration ◽

Kinetochore Assembly ◽

Entire Cell ◽

Crucial Part

AbstractDuring cell cycle progression in metazoan, the kinetochore, the protein complex attached to centromeres which directly interacts with the spindle microtubules, the vehicle of chromosome segregation, is assembled at mitotic onset and disassembled during mitotic exit. This program is assumed to be absent in budding and fission yeast because kinetochore proteins are stably maintained at the centromeres throughout the entire cell cycle. In this work, we show that the assembly program at the mitotic onset of the Ndc80 complex, a crucial part of the outer kinetochore, is unexpectedly conserved in Schizosaccharomyces pombe. We have identified this behavior by removing the Rabl chromosome configuration during interphase, in which centromeres are permanently associated with the nuclear envelope beneath the spindle pole body. Hence, the Rabl configuration masks the presence of a program to recruit Ndc80 at mitotic onset in fission yeast, similar to that taking place in metazoan. Besides the evolutionary implications of our observations, we think that our work will help understand the molecular processes behind the kinetochore assembly program during mitotic entry using fission yeast as the model organism.

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

Journal of Cell Science ◽

10.1242/jcs.112.14.2313 ◽

1999 ◽

Vol 112 (14) ◽

pp. 2313-2321 ◽

Cited By ~ 22

Author(s):

L. Cerutti ◽

V. Simanis

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Spindle Pole Body ◽

Mitotic Cell ◽

Spindle Pole ◽

The Other ◽

Septum Formation ◽

Spindle Pole Bodies ◽

Interphase Cells ◽

Early Mitosis

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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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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Cell cycle control of spindle pole body duplication and splitting by Sfi1 and Cdc31 in fission yeast

Journal of Cell Science ◽

10.1242/jcs.159657 ◽

2015 ◽

Vol 128 (8) ◽

pp. 1481-1493 ◽

Cited By ~ 21

Author(s):

I. B. Bouhlel ◽

M. Ohta ◽

A. Mayeux ◽

N. Bordes ◽

F. Dingli ◽

...

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Cell Cycle Control ◽

Spindle Pole Body ◽

Spindle Pole ◽

Pole Body

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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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Measuring Affinities of Fission Yeast Spindle Pole Body Proteins in Live Cells across the Cell Cycle

Biophysical Journal ◽

10.1016/j.bpj.2013.08.017 ◽

2013 ◽

Vol 105 (6) ◽

pp. 1324-1335 ◽

Cited By ~ 11

Author(s):

Chad D. McCormick ◽

Matthew S. Akamatsu ◽

Shih-Chieh Ti ◽

Thomas D. Pollard

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Live Cells ◽

Pole Body

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Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules, as revealed by continuous observation of chromosomes and microtubules in living cells

Journal of Cell Science ◽

10.1242/jcs.111.6.701 ◽

1998 ◽

Vol 111 (6) ◽

pp. 701-712 ◽

Cited By ~ 17

Author(s):

D.Q. Ding ◽

Y. Chikashige ◽

T. Haraguchi ◽

Y. Hiraoka

Keyword(s):

Fission Yeast ◽

Meiotic Prophase ◽

Fluorescent Protein ◽

Dynamic Instability ◽

Spindle Pole Body ◽

Spindle Pole ◽

Living Cells ◽

Continuous Observation ◽

Nuclear Movement ◽

Pole Body

Using a computerized fluorescence microscope system to observe fluorescently stained cellular structures in vivo, we have examined the dynamics of chromosomes and microtubules during the process of meiosis in the fission yeast Schizosaccharomyces pombe. Fission yeast meiotic prophase is characterized by a distinctive type of nuclear movement that is led by telomeres clustered at the spindle-pole body (the centrosome-equivalent structure in fungi): the nucleus oscillates back and forth along the cell axis, moving continuously between the two ends of the cell for some hours prior to the meiotic divisions. To obtain a dynamic view of this oscillatory nuclear movement in meiotic prophase, we visualized microtubules and chromosomes in living cells using jellyfish green fluorescent protein fused with alpha-tubulin and a DNA-specific fluorescent dye, Hoechst 33342, respectively. Continuous observation of chromosomes and microtubules in these cells demonstrated that the oscillatory nuclear movement is mediated by dynamic reorganization of astral microtubules originating from the spindle-pole body. During each half-oscillatory period, the microtubules extending rearward from the leading edge of the nucleus elongate to drive the nucleus to one end of the cell. When the nucleus reversed direction, its motion during the second half of the oscillation was not driven by the same microtubules that drove its motion during the first half, but rather by newly assembled microtubules. Reversible inhibition of nuclear movement by an inhibitor of microtubule polymerization, thiabendazole, confirmed the involvement of astral microtubules in oscillatory nuclear movement. The speed of the movement fluctuated within a range 0 to 15 micron/minute, with an average of about 5 microm/minute. We propose a model in which the oscillatory nuclear movement is mediated by dynamic instability and selective stabilization of astral microtubules.

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