scholarly journals Characterization of a Fission Yeast SUMO-1 Homologue, Pmt3p, Required for Multiple Nuclear Events, Including the Control of Telomere Length and Chromosome Segregation

1999 ◽  
Vol 19 (12) ◽  
pp. 8660-8672 ◽  
Author(s):  
Katsunori Tanaka ◽  
Junko Nishide ◽  
Koei Okazaki ◽  
Hiroaki Kato ◽  
Osami Niwa ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Telomere Length ◽  
Chromosome Segregation ◽  
Protein Modification ◽  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Nuclear Events ◽  
Posttranslational Protein Modification ◽  
Green Fluorescent

ABSTRACT Unlike ubiquitin, the ubiquitin-like protein modifier SUMO-1 and its budding yeast homologue Smt3p have been shown to be more important for posttranslational protein modification than for protein degradation. Here we describe the identification of the SUMO-1 homologue of fission yeast, which we show to be required for a number of nuclear events including the control of telomere length and chromosome segregation. A disruption of thepmt3 + gene, the Schizosaccharomyces pombe homologue of SMT3, was not lethal, but mutant cells carrying the disrupted gene grew more slowly. Thepmt3Δ cells showed various phenotypes such as aberrant mitosis, sensitivity to various reagents, and high-frequency loss of minichromosomes. Interestingly, we found thatpmt3 + is required for telomere length maintenance. Loss of Pmt3p function caused a striking increase in telomere length. When Pmt3p synthesis was restored, the telomeres became gradually shorter. This is the first demonstration of involvement of one of the Smt3p/SUMO-1 family proteins in telomere length maintenance. Fusion of Pmt3p to green fluorescent protein (GFP) showed that Pmt3p was predominantly localized as intense spots in the nucleus. One of the spots was shown to correspond to the spindle pole body (SPB). During prometaphase- and metaphase, the bright GFP signals at the SPB disappeared. These observations suggest that Pmt3p is required for kinetochore and/or SPB functions involved in chromosome segregation. The multiple functions of Pmt3p described here suggest that several nuclear proteins are regulated by Pmt3p conjugation.

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

1997 ◽  
Vol 110 (15) ◽  
pp. 1805-1812 ◽  
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.

scholarly journals A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast

2017 ◽  
Vol 28 (25) ◽  
pp. 3647-3659 ◽  
Author(s):  
Masashi Yukawa ◽  
Tomoki Kawakami ◽  
Masaki Okazaki ◽  
Kazunori Kume ◽  
Ngang Heok Tang ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Bipolar Spindle ◽  
Pole Body ◽  
Cross Linker ◽  
Spindle Elongation

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.

UNC-84 localizes to the nuclear envelope and is required for nuclear migration and anchoring during C. elegans development

Development ◽  
1999 ◽  
Vol 126 (14) ◽  
pp. 3171-3181 ◽  
Author(s):  
C.J. Malone ◽  
W.D. Fixsen ◽  
H.R. Horvitz ◽  
M. Han
Keyword(s):  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Spindle Pole Body ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Dna Lesions ◽  
Body Protein ◽  
C Elegans ◽  
Green Fluorescent

Nuclear migrations are essential for metazoan development. Two nuclear migrations that occur during C. elegans development require the function of the unc-84 gene. unc-84 mutants are also defective in the anchoring of nuclei within the hypodermal syncytium and in the migrations of the two distal tip cells of the gonad. Complementation analyses of 17 unc-84 alleles defined two genetically separable functions. Both functions are required for nuclear and distal tip cell migrations, but only one is required for nuclear anchorage. The DNA lesions associated with these 17 mutations indicate that the two genetically defined functions correspond to two distinct regions of the UNC-84 protein. The UNC-84 protein has a predicted transmembrane domain and a C-terminal region with similarity to the S. pombe spindle pole body protein Sad1 and to two predicted mammalian proteins. Analysis of a green fluorescent protein reporter indicated that UNC-84 is widely expressed and localized to the nuclear envelope. We propose that UNC-84 functions to facilitate a nuclear-centrosomal interaction required for nuclear migration and anchorage.

scholarly journals FH3, A Domain Found in Formins, Targets the Fission Yeast Formin Fus1 to the Projection Tip During Conjugation

1998 ◽  
Vol 141 (5) ◽  
pp. 1217-1228 ◽  
Author(s):  
Janni Petersen ◽  
Olaf Nielsen ◽  
Richard Egel ◽  
Iain M. Hagan
Keyword(s):  
Fluorescent Protein ◽  
Protein Localization ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Amino Terminal ◽  
Gfp Fusion Protein ◽  
Pole Body ◽  
Body Component ◽  
Green Fluorescent ◽  
Amino Terminal Domain

Formins are involved in diverse aspects of morphogenesis, and share two regions of homology: FH1 and FH2. We describe a new formin homology region, FH3. FH3 is an amino-terminal domain that differs from the Rho binding site identified in Bni1p and p140mDia. The Schizosaccharomyces pombe formin Fus1 is required for conjugation, and is localized to the projection tip in cells of mating pairs. We replaced genomic fus1+ with green fluorescent protein (GFP)- tagged versions that lacked either the FH1, FH2, or FH3 domain. Deletion of any FH domain essentially abolished mating. FH3, but neither FH1 nor FH2, was required for Fus1 localization. An FH3 domain–GFP fusion protein localized to the projection tips of mating pairs. Thus, the FH3 domain alone can direct protein localization. The FH3 domains of both Fus1 and the S. pombe cytokinesis formin Cdc12 were able to localize GFP to the spindle pole body in half of the late G2 cells in a vegetatively growing population. Expression of both FH3-GFP fusions also affected cytokinesis. Overexpression of the spindle pole body component Sad1 altered the distribution of both Sad1 and the FH3-GFP domain. Together these data suggest that proteins at multiple sites can interact with FH3 domains.

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

2001 ◽  
Vol 12 (12) ◽  
pp. 3933-3946 ◽  
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.

scholarly journals 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

2002 ◽  
Vol 13 (3) ◽  
pp. 930-946 ◽  
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.

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

2001 ◽  
Vol 114 (14) ◽  
pp. 2627-2640 ◽  
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.

scholarly journals Time-Lapse Video Microscopy Analysis Reveals Astral Microtubule Detachment in the Yeast Spindle Pole Mutantcnm67

2000 ◽  
Vol 11 (4) ◽  
pp. 1197-1211 ◽  
Author(s):  
Dominic Hoepfner ◽  
Arndt Brachat ◽  
Peter Philippsen
Keyword(s):  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Attachment Site ◽  
Time Lapse ◽  
Spindle Pole ◽  
Cell Cycles ◽  
Astral Microtubule ◽  
Normal Behavior ◽  
Green Fluorescent ◽  
Diploid Cells

Saccharomyces cerevisiae cnm67Δ cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein–labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the γ-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Δ cells Spc72–γ-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Δ cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.

scholarly journals Coordinated Spindle Assembly and Orientation Requires Clb5p-Dependent Kinase in Budding Yeast

2000 ◽  
Vol 148 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Marisa Segal ◽  
Duncan J. Clarke ◽  
Paul Maddox ◽  
E.D. Salmon ◽  
Kerry Bloom ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Time Lapse ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Dynamic Interactions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Green Fluorescent

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Δ cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Δ cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.

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

2005 ◽  
Vol 16 (5) ◽  
pp. 2325-2338 ◽  
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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