Background: Vigorous chromosome movements driven by cytoskeletal assemblies are a widely conserve... more Background: Vigorous chromosome movements driven by cytoskeletal assemblies are a widely conserved feature of sexual differentiation to facilitate meiotic recombination. In fission yeast, this process involves the dramatic conversion of arrays of cytoplasmic microtubules (MTs), generated from multiple MT organizing centers (MTOCs), into a single radial MT (rMT) array associated with the spindle pole body (SPB), the major MTOC during meiotic prophase. The rMT is then dissolved upon the onset of meiosis I when a bipolar spindle emerges to conduct chromosome segregation. Structural features and molecular mechanisms that govern these dynamic MT rearrangements are poorly understood. Results: Electron tomography of the SPBs showed that the rMT emanates from a newly recognized amorphous structure, which we term the rMTOC. The rMTOC, which resides at the cytoplasmic side of the SPB, is highly enriched in g-tubulin reminiscent of the pericentriolar material of higher eukaryotic centrosomes. Formation of the rMTOC depends on Hrs1/ Mcp6, a meiosis-specific SPB component that is located at the rMTOC. At the onset of meiosis I, Hrs1/Mcp6 is subject to strict downregulation by both proteasome-dependent degradation and phosphorylation leading to complete inactivation of the rMTOC. This ensures rMT dissolution and bipolar spindle formation. Conclusions: Our study reveals the molecular basis for the transient generation of a novel MTOC, which triggers a program of MT rearrangement that is required for meiotic differentiation.
Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. ... more Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.
Much remains unknown about the molecular regulation of meiosis. Here we show that meiosis-specifi... more Much remains unknown about the molecular regulation of meiosis. Here we show that meiosis-specific transcripts are selectively removed if expressed during vegetative growth in fission yeast. These messenger RNAs contain a cis-acting region-which we call the DSR-that confers this removal via binding to a YTH-family protein Mmi1. Loss of Mmi1 function severely impairs cell growth owing to the untimely expression of meiotic transcripts. Microarray analysis reveals that at least a dozen such meiosis-specific transcripts are eliminated by the DSR-Mmi1 system. Mmi1 remains in the form of multiple nuclear foci during vegetative growth. At meiotic prophase these foci precipitate to a single focus, which coincides with the dot formed by the master meiosis-regulator Mei2. A meiotic arrest due to the loss of the Mei2 dot is released by a reduction in Mmi1 activity. We propose that Mei2 turns off the DSR-Mmi1 system by sequestering Mmi1 to the dot and thereby secures stable expression of meiosis-specific transcripts.
Background: Yeast mating-pheromone receptors facilitate the study of G protein-coupled signal tra... more Background: Yeast mating-pheromone receptors facilitate the study of G protein-coupled signal transduction. To date, molecular dissection of the budding yeast a-factor receptor has been done extensively, but little analysis has been performed with pheromone receptors of fission yeast, another genetically tractable yeast species.
Autophagy is a conserved bulk protein degradation process that is proposed to play a role in even... more Autophagy is a conserved bulk protein degradation process that is proposed to play a role in events that arise when organisms are forced to radically change their fate, including nutritional starvation, differentiation and development. In our present study, we have identified fission yeast autophagy as a bulk protein degradation process induced by the deprivation of environmental nitrogen, the effects of which are known to trigger sexual differentiation as an adaptive response. Autophagy-defective mutants were found to be sterile in the absence of environmental nitrogen, but could complete sexual differentiation when nitrogen was supplied, suggesting that the major function of autophagy is to provide a nitrogen source. In addition, the environmental nitrogen levels act as an autophagy "on/off" switch, whereas components essential for sexual differentiation were dispensable for this regulation. We propose that fission yeast autophagy functions to supply nitrogen and is activated when cells cannot access exogenous nitrogen, thus ensuring that they can adapt and subsequently propagate.
ABSTRACT The human protein kinase Nek2 is related to the NIMA cell cycle regulatory kinase of Asp... more ABSTRACT The human protein kinase Nek2 is related to the NIMA cell cycle regulatory kinase of Aspergillus nidulans. Whereas NIMA has been shown to be essential for cell cycle progression into mitosis in this fungus, the function of mammalian Nek2 remains to be elucidated. In this study, we isolated a cDNA coding for a mouse ortholog of human Nek2 and analyzed the expression of this kinase in different organs. RNase protection assays performed on RNAs from mouse adult organs showed very high expression of Nek2 in testis. Lower levels of transcripts were detected in intestine, thymus, and skin, three mitotically active organs, and whole-mount in situ hybridization performed on 10.5-day embryos allowed the detection of Nek2 transcripts in the brain. In situ hybridization analysis of testis sections revealed that the transcription of Nek2 occurred in a stage-specific manner during spermatogenesis. The strongest signals were seen in cells undergoing meiosis, but Nek2 transcripts could also be detected in haploid cells (stage I and II spermatids). Extending these results, in situ hybridization performed on ovary sections revealed strong signals in meiotically active oocytes. In addition, some Nek2 transcription was observed in actively dividing follicle cells surrounding the oocytes and in the oviduct. Finally, indirect immunofluorescence staining of testis sections with Nek2-specific antibodies confirmed that this kinase is highly expressed in spermatocytes and, to a lesser extent, in early spermatids. Taken together, these results indicate that Nek2 may play an important role not only during mitosis but also during meiosis.
Microtubules and the motor protein dynein play pivotal roles in the movement and positioning of t... more Microtubules and the motor protein dynein play pivotal roles in the movement and positioning of the nucleus and cytoplasmic organelles in a cell. In fission yeast, oscillatory movement of the nucleus termed horsetail nuclear movement (HNM) has been observed during meiotic prophase. HNM is led by an astral microtubule array emanating from the spindle pole body (SPB), a centrosome-equivalent organelle in yeasts, aided by the dynein-dynactin complex, and is proposed to facilitate the alignment of homologous chromosomes necessary for efficient meiotic recombination. Here we show that a meiosis-specific SPB component Hrs1p (also known as Mcp6p) is a key molecule to remodel microtubules into the horsetail-astral array (HAA). Deletion of Hrs1p impaired HAA formation, leading to compromised HNM. Ectopic expression of Hrs1p during the mitotic cell cycle resulted in the formation of a HAA-like astral microtubule array, which drove an oscillatory nuclear movement in interphase cells. Hrs1p interacted with components of the gamma-tubulin ring complex (gamma-TuRC) as well as with a meiotic SPB component. We propose that Hrs1p facilitates formation of the HAA, responsible for the vigorous HNM, by stabilizing connection between the SPB and minus ends of microtubules.
In eukaryotic cells, the nuclear envelope partitions the nucleus from the cytoplasm. The fission ... more In eukaryotic cells, the nuclear envelope partitions the nucleus from the cytoplasm. The fission yeast Schizosaccharomyces pombe undergoes closed mitosis in which the nuclear envelope persists rather than being broken down, as in higher eukaryotic cells . It is therefore assumed that nucleocytoplasmic transport continues during the cell cycle . Here we show that nuclear transport is, in fact, abolished specifically during anaphase of the second meiotic nuclear division. During that time, both nucleoplasmic and cytoplasmic proteins disperse throughout the cell, reminiscent of the open mitosis of higher eukaryotes, but the architecture of the S. pombe nuclear envelope itself persists. This functional alteration of the nucleocytoplasmic barrier is likely induced by spore wall formation, because ectopic induction of sporulation signaling leads to premature dispersion of nucleoplasmic proteins. A photobleaching assay demonstrated that nuclear envelope permeability increases abruptly at the onset of anaphase of the second meiotic division. The permeability was not altered when sporulation was inhibited by blocking the trafficking of forespore-membrane vesicles from the endoplasmic reticulum to the Golgi. The evidence indicates that yeast gametogenesis produces vesicle transport-mediated forespore membranes by inducing nuclear envelope permeabilization.
Plo1-associated casein kinase activity peaked during mitosis before septation. Phosphatase treatm... more Plo1-associated casein kinase activity peaked during mitosis before septation. Phosphatase treatment abolished this activity. Mitotic Plo1 activation had a requirement for prior activation of M-phase promoting factor (MPF), suggesting that Plo1 does not act as a mitotic trigger kinase to initiate MPF activation during mitotic commitment. A link between Plo1 and the septum initiating network (SIN) has been suggested by the inability of plo1D cells to septate and the proli®c septation following plo1 + overexpression. Interphase activation of Spg1, the G protein that modulates SIN activity, induced septation but did not stimulate Plo1-associated kinase activity. Conversely, SIN inactivation did not affect the mitotic stimulation of Plo1-associated kinase activity. plo1.ts4 cells formed a misshapen actin ring, but rarely septated at 36°C. Forced activation of Spg1 enabled plo1.ts4 mutant cells, but not cells with defects in the SIN component Sid2, to convert the actin ring to a septum. The ability of plo1 + overexpression to induce septation was severely compromised by SIN inactivation. We propose that Plo1 acts before the SIN to control septation.
Background: Vigorous chromosome movements driven by cytoskeletal assemblies are a widely conserve... more Background: Vigorous chromosome movements driven by cytoskeletal assemblies are a widely conserved feature of sexual differentiation to facilitate meiotic recombination. In fission yeast, this process involves the dramatic conversion of arrays of cytoplasmic microtubules (MTs), generated from multiple MT organizing centers (MTOCs), into a single radial MT (rMT) array associated with the spindle pole body (SPB), the major MTOC during meiotic prophase. The rMT is then dissolved upon the onset of meiosis I when a bipolar spindle emerges to conduct chromosome segregation. Structural features and molecular mechanisms that govern these dynamic MT rearrangements are poorly understood. Results: Electron tomography of the SPBs showed that the rMT emanates from a newly recognized amorphous structure, which we term the rMTOC. The rMTOC, which resides at the cytoplasmic side of the SPB, is highly enriched in g-tubulin reminiscent of the pericentriolar material of higher eukaryotic centrosomes. Formation of the rMTOC depends on Hrs1/ Mcp6, a meiosis-specific SPB component that is located at the rMTOC. At the onset of meiosis I, Hrs1/Mcp6 is subject to strict downregulation by both proteasome-dependent degradation and phosphorylation leading to complete inactivation of the rMTOC. This ensures rMT dissolution and bipolar spindle formation. Conclusions: Our study reveals the molecular basis for the transient generation of a novel MTOC, which triggers a program of MT rearrangement that is required for meiotic differentiation.
Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. ... more Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.
Much remains unknown about the molecular regulation of meiosis. Here we show that meiosis-specifi... more Much remains unknown about the molecular regulation of meiosis. Here we show that meiosis-specific transcripts are selectively removed if expressed during vegetative growth in fission yeast. These messenger RNAs contain a cis-acting region-which we call the DSR-that confers this removal via binding to a YTH-family protein Mmi1. Loss of Mmi1 function severely impairs cell growth owing to the untimely expression of meiotic transcripts. Microarray analysis reveals that at least a dozen such meiosis-specific transcripts are eliminated by the DSR-Mmi1 system. Mmi1 remains in the form of multiple nuclear foci during vegetative growth. At meiotic prophase these foci precipitate to a single focus, which coincides with the dot formed by the master meiosis-regulator Mei2. A meiotic arrest due to the loss of the Mei2 dot is released by a reduction in Mmi1 activity. We propose that Mei2 turns off the DSR-Mmi1 system by sequestering Mmi1 to the dot and thereby secures stable expression of meiosis-specific transcripts.
Background: Yeast mating-pheromone receptors facilitate the study of G protein-coupled signal tra... more Background: Yeast mating-pheromone receptors facilitate the study of G protein-coupled signal transduction. To date, molecular dissection of the budding yeast a-factor receptor has been done extensively, but little analysis has been performed with pheromone receptors of fission yeast, another genetically tractable yeast species.
Autophagy is a conserved bulk protein degradation process that is proposed to play a role in even... more Autophagy is a conserved bulk protein degradation process that is proposed to play a role in events that arise when organisms are forced to radically change their fate, including nutritional starvation, differentiation and development. In our present study, we have identified fission yeast autophagy as a bulk protein degradation process induced by the deprivation of environmental nitrogen, the effects of which are known to trigger sexual differentiation as an adaptive response. Autophagy-defective mutants were found to be sterile in the absence of environmental nitrogen, but could complete sexual differentiation when nitrogen was supplied, suggesting that the major function of autophagy is to provide a nitrogen source. In addition, the environmental nitrogen levels act as an autophagy "on/off" switch, whereas components essential for sexual differentiation were dispensable for this regulation. We propose that fission yeast autophagy functions to supply nitrogen and is activated when cells cannot access exogenous nitrogen, thus ensuring that they can adapt and subsequently propagate.
ABSTRACT The human protein kinase Nek2 is related to the NIMA cell cycle regulatory kinase of Asp... more ABSTRACT The human protein kinase Nek2 is related to the NIMA cell cycle regulatory kinase of Aspergillus nidulans. Whereas NIMA has been shown to be essential for cell cycle progression into mitosis in this fungus, the function of mammalian Nek2 remains to be elucidated. In this study, we isolated a cDNA coding for a mouse ortholog of human Nek2 and analyzed the expression of this kinase in different organs. RNase protection assays performed on RNAs from mouse adult organs showed very high expression of Nek2 in testis. Lower levels of transcripts were detected in intestine, thymus, and skin, three mitotically active organs, and whole-mount in situ hybridization performed on 10.5-day embryos allowed the detection of Nek2 transcripts in the brain. In situ hybridization analysis of testis sections revealed that the transcription of Nek2 occurred in a stage-specific manner during spermatogenesis. The strongest signals were seen in cells undergoing meiosis, but Nek2 transcripts could also be detected in haploid cells (stage I and II spermatids). Extending these results, in situ hybridization performed on ovary sections revealed strong signals in meiotically active oocytes. In addition, some Nek2 transcription was observed in actively dividing follicle cells surrounding the oocytes and in the oviduct. Finally, indirect immunofluorescence staining of testis sections with Nek2-specific antibodies confirmed that this kinase is highly expressed in spermatocytes and, to a lesser extent, in early spermatids. Taken together, these results indicate that Nek2 may play an important role not only during mitosis but also during meiosis.
Microtubules and the motor protein dynein play pivotal roles in the movement and positioning of t... more Microtubules and the motor protein dynein play pivotal roles in the movement and positioning of the nucleus and cytoplasmic organelles in a cell. In fission yeast, oscillatory movement of the nucleus termed horsetail nuclear movement (HNM) has been observed during meiotic prophase. HNM is led by an astral microtubule array emanating from the spindle pole body (SPB), a centrosome-equivalent organelle in yeasts, aided by the dynein-dynactin complex, and is proposed to facilitate the alignment of homologous chromosomes necessary for efficient meiotic recombination. Here we show that a meiosis-specific SPB component Hrs1p (also known as Mcp6p) is a key molecule to remodel microtubules into the horsetail-astral array (HAA). Deletion of Hrs1p impaired HAA formation, leading to compromised HNM. Ectopic expression of Hrs1p during the mitotic cell cycle resulted in the formation of a HAA-like astral microtubule array, which drove an oscillatory nuclear movement in interphase cells. Hrs1p interacted with components of the gamma-tubulin ring complex (gamma-TuRC) as well as with a meiotic SPB component. We propose that Hrs1p facilitates formation of the HAA, responsible for the vigorous HNM, by stabilizing connection between the SPB and minus ends of microtubules.
In eukaryotic cells, the nuclear envelope partitions the nucleus from the cytoplasm. The fission ... more In eukaryotic cells, the nuclear envelope partitions the nucleus from the cytoplasm. The fission yeast Schizosaccharomyces pombe undergoes closed mitosis in which the nuclear envelope persists rather than being broken down, as in higher eukaryotic cells . It is therefore assumed that nucleocytoplasmic transport continues during the cell cycle . Here we show that nuclear transport is, in fact, abolished specifically during anaphase of the second meiotic nuclear division. During that time, both nucleoplasmic and cytoplasmic proteins disperse throughout the cell, reminiscent of the open mitosis of higher eukaryotes, but the architecture of the S. pombe nuclear envelope itself persists. This functional alteration of the nucleocytoplasmic barrier is likely induced by spore wall formation, because ectopic induction of sporulation signaling leads to premature dispersion of nucleoplasmic proteins. A photobleaching assay demonstrated that nuclear envelope permeability increases abruptly at the onset of anaphase of the second meiotic division. The permeability was not altered when sporulation was inhibited by blocking the trafficking of forespore-membrane vesicles from the endoplasmic reticulum to the Golgi. The evidence indicates that yeast gametogenesis produces vesicle transport-mediated forespore membranes by inducing nuclear envelope permeabilization.
Plo1-associated casein kinase activity peaked during mitosis before septation. Phosphatase treatm... more Plo1-associated casein kinase activity peaked during mitosis before septation. Phosphatase treatment abolished this activity. Mitotic Plo1 activation had a requirement for prior activation of M-phase promoting factor (MPF), suggesting that Plo1 does not act as a mitotic trigger kinase to initiate MPF activation during mitotic commitment. A link between Plo1 and the septum initiating network (SIN) has been suggested by the inability of plo1D cells to septate and the proli®c septation following plo1 + overexpression. Interphase activation of Spg1, the G protein that modulates SIN activity, induced septation but did not stimulate Plo1-associated kinase activity. Conversely, SIN inactivation did not affect the mitotic stimulation of Plo1-associated kinase activity. plo1.ts4 cells formed a misshapen actin ring, but rarely septated at 36°C. Forced activation of Spg1 enabled plo1.ts4 mutant cells, but not cells with defects in the SIN component Sid2, to convert the actin ring to a septum. The ability of plo1 + overexpression to induce septation was severely compromised by SIN inactivation. We propose that Plo1 acts before the SIN to control septation.
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Papers by Kayoko Tanaka