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

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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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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A Cytoplasmic Dynein Heavy Chain Is Required for Oscillatory Nuclear Movement of Meiotic Prophase and Efficient Meiotic Recombination in Fission Yeast

The Journal of Cell Biology ◽

10.1083/jcb.145.6.1233 ◽

1999 ◽

Vol 145 (6) ◽

pp. 1233-1250 ◽

Cited By ~ 175

Author(s):

Ayumu Yamamoto ◽

Robert R. West ◽

J. Richard McIntosh ◽

Yasushi Hiraoka

Keyword(s):

Fission Yeast ◽

Heavy Chain ◽

Meiotic Recombination ◽

Meiotic Prophase ◽

Fluorescent Protein ◽

Cytoplasmic Dynein ◽

Spindle Pole ◽

Nuclear Movement ◽

Homologous Chromosomes ◽

Dynein Heavy Chain

Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination.

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Dynamic Behavior of Microtubules during Dynein-dependent Nuclear Migrations of Meiotic Prophase in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.12.12.3933 ◽

2001 ◽

Vol 12 (12) ◽

pp. 3933-3946 ◽

Cited By ~ 51

Author(s):

Ayumu Yamamoto ◽

Chihiro Tsutsumi ◽

Hiroaki Kojima ◽

Kazuhiro Oiwa ◽

Yasushi Hiraoka

Keyword(s):

Fission Yeast ◽

Dynamic Behavior ◽

Meiotic Prophase ◽

Fluorescent Protein ◽

Cortical Microtubule ◽

Spindle Pole Body ◽

Cytoplasmic Dynein ◽

Spindle Pole ◽

Microtubule Assembly ◽

Cell Cortex

During meiotic prophase in fission yeast, the nucleus migrates back and forth between the two ends of the cell, led by the spindle pole body (SPB). This nuclear oscillation is dependent on astral microtubules radiating from the SPB and a microtubule motor, cytoplasmic dynein. Here we have examined the dynamic behavior of astral microtubules labeled with the green fluorescent protein during meiotic prophase with the use of optical sectioning microscopy. During nuclear migrations, the SPB mostly follows the microtubules that extend toward the cell cortex. SPB migrations start when these microtubules interact with the cortex and stop when they disappear, suggesting that these microtubules drive nuclear migrations. The microtubules that are followed by the SPB often slide along the cortex and are shortened by disassembly at their ends proximal to the cortex. In dynein-mutant cells, where nuclear oscillations are absent, the SPB never migrates by following microtubules, and microtubule assembly/disassembly dynamics is significantly altered. Based on these observations, together with the frequent accumulation of dynein at a cortical site where the directing microtubules interact, we propose a model in which dynein drives nuclear oscillation by mediating cortical microtubule interactions and regulating the dynamics of microtubule disassembly at the cortex.

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The 14-kDa Dynein Light Chain-Family Protein Dlc1 Is Required for Regular Oscillatory Nuclear Movement and Efficient Recombination during Meiotic Prophase in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.01-11-0543 ◽

2002 ◽

Vol 13 (3) ◽

pp. 930-946 ◽

Cited By ~ 65

Author(s):

Futaba Miki ◽

Koei Okazaki ◽

Mizuki Shimanuki ◽

Ayumu Yamamoto ◽

Yasushi Hiraoka ◽

...

Keyword(s):

Light Chain ◽

Meiotic Prophase ◽

Fluorescent Protein ◽

Spindle Pole Body ◽

Cytoplasmic Dynein ◽

Spindle Pole ◽

Cell Cortex ◽

Dynein Light Chain ◽

Nuclear Movement ◽

Green Fluorescent

A Schizosaccharomyces pombe spindle pole body (SPB) protein interacts in a two-hybrid system with Dlc1, which belongs to the 14-kDa Tctex-1 dynein light chain family. Green fluorescent protein-tagged Dlc1 accumulated at the SPB throughout the life cycle. During meiotic prophase, Dlc1 was present along astral microtubules and microtubule-anchoring sites on the cell cortex, reminiscent of the cytoplasmic dynein heavy chain Dhc1. In a dlc1-null mutant, Dhc1-dependent nuclear movement in meiotic prophase became irregular in its duration and direction. Dhc1 protein was displaced from the cortex anchors and the formation of microtubule bundle(s) that guide nuclear movement was impaired in the mutant. Meiotic recombination in the dlc1 mutant was reduced to levels similar to that in the dhc1 mutant. Dlc1 and Dhc1 also have roles in karyogamy and rDNA relocation during the sexual phase. Strains mutated in both the dlc1 and dhc1loci displayed more severe defects in recombination, karyogamy, and sporulation than in either single mutant alone, suggesting that Dlc1 is involved in nuclear events that are independent of Dhc1. S. pombe contains a homolog of the 8-kDa dynein light chain, Dlc2. This class of dynein light chain, however, is not essential in either the vegetative or sexual phases.

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Dissociation of the Nuf2-Ndc80 Complex Releases Centromeres from the Spindle-Pole Body during Meiotic Prophase in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e04-11-0996 ◽

2005 ◽

Vol 16 (5) ◽

pp. 2325-2338 ◽

Cited By ~ 48

Author(s):

Haruhiko Asakawa ◽

Aki Hayashi ◽

Tokuko Haraguchi ◽

Yasushi Hiraoka

Keyword(s):

Fission Yeast ◽

Chromosome Segregation ◽

Meiotic Prophase ◽

Spindle Pole Body ◽

Spindle Pole ◽

Mating Pheromone ◽

Pole Body ◽

Pheromone Signaling ◽

Ndc80 Complex ◽

Mutant Cells

In the fission yeast Schizosaccharomyces pombe, centromeres remain clustered at the spindle-pole body (SPB) during mitotic interphase. In contrast, during meiotic prophase centromeres dissociate from the SPB. Here we examined the behavior of centromere proteins in living meiotic cells of S. pombe. We show that the Nuf2-Ndc80 complex proteins (Nuf2, Ndc80, Spc24, and Spc25) disappear from the centromere in meiotic prophase when the centromeres are separated from the SPB. The centromere protein Mis12 also dissociates during meiotic prophase; however, Mis6 remains throughout meiosis. When cells are induced to meiosis by inactivation of Pat1 kinase (a key negative regulator of meiosis), centromeres remain associated with the SPB during meiotic prophase. However, inactivation of Nuf2 by a mutation causes the release of centromeres from the SPB in pat1 mutant cells, suggesting that the Nuf2-Ndc80 complex connects centromeres to the SPB. We further found that removal of the Nuf2-Ndc80 complex from the centromere and centromere-SPB dissociation are caused by mating pheromone signaling. Because pat1 mutant cells also show aberrant chromosome segregation in the first meiotic division and this aberration is compensated by mating pheromone signaling, dissociation of the Nuf2-Ndc80 complex may be associated with remodeling of the kinetochore for meiotic chromosome segregation.

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Membrane proteins Bqt3 and -4 anchor telomeres to the nuclear envelope to ensure chromosomal bouquet formation

The Journal of Cell Biology ◽

10.1083/jcb.200902122 ◽

2009 ◽

Vol 187 (3) ◽

pp. 413-427 ◽

Cited By ~ 76

Author(s):

Yuji Chikashige ◽

Miho Yamane ◽

Kasumi Okamasa ◽

Chihiro Tsutsumi ◽

Tomoko Kojidani ◽

...

Keyword(s):

Nuclear Envelope ◽

Fission Yeast ◽

Nuclear Membrane ◽

Meiotic Prophase ◽

Spindle Pole Body ◽

Spindle Pole ◽

Null Mutant ◽

Vegetative Cells ◽

Nuclear Membranes ◽

Pole Body

In many organisms, telomeres cluster to form a bouquet arrangement of chromosomes during meiotic prophase. Previously, we reported that two meiotic proteins, Bqt1 and -2, are required for tethering telomeres to the spindle pole body (SPB) during meiotic prophase in fission yeast. This study has further identified two novel, ubiquitously expressed inner nuclear membrane (INM) proteins, Bqt3 and -4, which are required for bouquet formation. We found that in the absence of Bqt4, telomeres failed to associate with the nuclear membranes in vegetative cells and consequently failed to cluster to the SPB in meiotic prophase. In the absence of Bqt3, Bqt4 protein was degraded during meiosis, leading to a phenotype similar to that of the bqt4-null mutant. Collectively, these results show that Bqt4 anchors telomeres to the INM and that Bqt3 protects Bqt4 from protein degradation. Interestingly, the functional integrity of telomeres is maintained even when they are separated from the nuclear envelope in vegetative cells.

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Calmodulin localizes to the spindle pole body of Schizosaccharomyces pombe and performs an essential function in chromosome segregation

Journal of Cell Science ◽

10.1242/jcs.110.15.1805 ◽

1997 ◽

Vol 110 (15) ◽

pp. 1805-1812 ◽

Cited By ~ 8

Author(s):

M.J. Moser ◽

M.R. Flory ◽

T.N. Davis

Keyword(s):

Cell Growth ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Chromosome Segregation ◽

Fluorescent Protein ◽

Budding Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Temperature Sensitive ◽

Pole Body

The essential calmodulin genes in both Saccharomyces cerevisiae and Schizosaccharomyces pombe were precisely replaced with genes encoding fusions between calmodulin and the green fluorescent protein (GFP). In living budding yeast the GFP-calmodulin fusion protein (GFP-Cmd1p) localized simultaneously to sites of cell growth and to the spindle pole body (SPB), the yeast analog of the centrosome. Having demonstrated proper localization of GFP-calmodulin in budding yeast, we examined the localization of a fusion between GFP and calmodulin (GFP-Camlp) in fission yeast, where calmodulin had not been localized by any method. We find GFP-Camlp also localizes both to sites of polarized cell growth and to the fission yeast SPB. The localization of calmodulin to the SPB by GFP fusion was confirmed by indirect immunofluorescence. Antiserum to S. pombe calmodulin labeled the ends of the mitotic spindle stained with anti-tubulin antiserum. This pattern was identical to that seen using antiserum to Sad1p, a known SPB component. We then characterized the defects in a temperature-sensitive S. pombe calmodulin mutant. Mutant cam1-E14 cells synchronized in S phase completed DNA synthesis, but lost viability during transit of mitosis. Severe defects in chromosome segregation, including hypercondensation, fragmentation, and unequal allocation of chromosomal material were observed. Immunofluorescence analysis of tubulin revealed a population of cells containing either broken or mislocalized mitotic spindles, which were never observed in wild-type cells. Taken together with the subcellular localization of calmodulin, the observed spindle and chromosome segregation defects suggest that calmodulin performs an essential role during mitosis at the fission yeast SPB.

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Bqt2p is essential for initiating telomere clustering upon pheromone sensing in fission yeast

The Journal of Cell Biology ◽

10.1083/jcb.200602152 ◽

2006 ◽

Vol 173 (6) ◽

pp. 845-851 ◽

Cited By ~ 23

Author(s):

Xie Tang ◽

Ye Jin ◽

W. Zacheus Cande

Keyword(s):

Fission Yeast ◽

Chromosome Pairing ◽

Meiotic Prophase ◽

Homologous Chromosome ◽

Spindle Pole Body ◽

Spindle Pole ◽

Meiotic Spindle ◽

Meiotic Progression ◽

Pole Body ◽

Homologous Chromosome Pairing

The telomere bouquet, i.e., telomere clustering on the nuclear envelope (NE) during meiotic prophase, is thought to promote homologous chromosome pairing. Using a visual screen, we identified bqt2/im295, a mutant that disrupts telomere clustering in fission yeast. Bqt2p is required for linking telomeres to the meiotic spindle pole body (SPB) but not for attachment of telomeres or the SPB to the NE. Bqt2p is expressed upon pheromone sensing and colocalizes thereafter to Sad1p, an SPB protein. This localization only depends on Bqt1p, not on other identified proteins required for telomere clustering. Upon pheromone sensing, generation of Sad1p foci next to telomeres depends on Bqt2p. However, depletion of Bqt2p from the SPB is dispensable for dissolving the telomere bouquet at the end of meiotic prophase. Therefore, telomere bouquet formation requires Bqt2p as a linking component and is finely regulated during meiotic progression.

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