NDC10: a gene involved in chromosome segregation in Saccharomyces cerevisiae.

A mutant, ndc10-1, was isolated by anti-tubulin staining of temperature-sensitive mutant banks of budding yeast. ndc10-1 has a defect chromosome segregation since chromosomes remains at one pole of the anaphase spindle. This produces one polyploid cell and one aploid cell, each containing a spindle pole body (SPD. NDC10 was cloned and sequenced and is identical to CBF2 (Jiang, W., J. Lechnermn and J. Carbon. 1993. J. Cell Biol. 121:513) which is the 110-kD component of a centromere DNA binding complex (Lechner, J., and J. Carbon. 1991. Cell. 61:717-725). NDC10 is an essential gene. Antibodies to Ndc10p labeled the SPB region in nearly all the cells examined including nonmitotic cells. In some cells with short spindles which may be in metaphase, staining was also observed along the spindle. The staining pattern and the phenotype of ndc10-1 are consistent with Cbf2p/Ndc10p being a kinetochore protein, and provide in vivo evidence for its role in the attachment of chromosomes to the spindle.

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The Yeast Spindle Pole Body Component Spc72p Interacts with Stu2p and Is Required for Proper Microtubule Assembly

The Journal of Cell Biology ◽

10.1083/jcb.141.5.1169 ◽

1998 ◽

Vol 141 (5) ◽

pp. 1169-1179 ◽

Cited By ~ 70

Author(s):

Xiaoyue Peter Chen ◽

Hongwei Yin ◽

Tim C. Huffaker

Keyword(s):

Chromosome Segregation ◽

Spindle Pole Body ◽

Spindle Pole ◽

Microtubule Assembly ◽

Temperature Sensitive ◽

Pole Body ◽

Cytoplasmic Microtubules ◽

Spindle Elongation

We have previously shown that Stu2p is a microtubule-binding protein and a component of the Saccharomyces cerevisiae spindle pole body (SPB). Here we report the identification of Spc72p, a protein that interacts with Stu2p. Stu2p and Spc72p associate in the two-hybrid system and can be coimmunoprecipitated from yeast extracts. Stu2p and Spc72p also interact with themselves, suggesting the possibility of a multimeric Stu2p-Spc72p complex. Spc72p is an essential component of the SPB and is able to associate with a preexisting SPB, indicating that there is a dynamic exchange between soluble and SPB forms of Spc72p. Unlike Stu2p, Spc72p does not bind microtubules in vitro, and was not observed to localize along microtubules in vivo. A temperature-sensitive spc72 mutation causes defects in SPB morphology. In addition, most spc72 mutant cells lack cytoplasmic microtubules; the few cytoplasmic microtubules that are observed are excessively long, and some of these are unattached to the SPB. spc72 cells are able to duplicate and separate their SPBs to form a bipolar spindle, but spindle elongation and chromosome segregation rarely occur. The chromosome segregation block does not arrest the cell cycle; instead, spc72 cells undergo cytokinesis, producing aploid cells and polyploid cells that contain multiple SPBs.

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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

Molecular Biology of the Cell ◽

10.1091/mbc.e17-08-0497 ◽

2017 ◽

Vol 28 (25) ◽

pp. 3647-3659 ◽

Cited By ~ 23

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.

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Novel Phosphorylation States of the Yeast Spindle Pole Body

10.1101/245340 ◽

2018 ◽

Author(s):

Kimberly K. Fong ◽

Alex Zelter ◽

Beth Graczyk ◽

Jill M. Hoyt ◽

Michael Riffle ◽

...

Keyword(s):

Cell Cycle ◽

Spindle Pole Body ◽

Mitotic Checkpoint ◽

Spindle Pole ◽

Temperature Sensitive ◽

Phosphorylation Sites ◽

Microtubule Nucleation ◽

Data Set ◽

Pole Body

ABSTRACTPhosphorylation regulates yeast spindle pole body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of purifiedSaccharomyces cerevisiaeSPBs for two cell cycle arrests, G1/S and the mitotic checkpoint, expanding on previously reported phosphoproteomic data sets. We present a novel phosphoproteomic state of SPBs arrested in G1/S by acdc4-1temperature sensitive mutation, with particular interest in phosphorylation events on the γ-tubulin small complex (γ-TuSC). Thecdc4-1arrest is the earliest arrest at which microtubule nucleation has occurred at the newly duplicated SPB. Several novel phosphorylation sites were identified in G1/S and during mitosis on the microtubule nucleating γ-TuSC. These sites were analyzedin vivoby fluorescence microscopy and were shown to be required for proper regulation of spindle length. Additionally,in vivoanalysis of two mitotic sites in Spc97 found that phosphorylation of at least one of these sites is required for progression through the cell cycle. This phosphoproteomic data set not only broadens the scope of the phosphoproteome of SPBs, it also identifies several γ-TuSC phosphorylation sites influencing microtubule regulation.

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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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The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability.

The Journal of Cell Biology ◽

10.1083/jcb.129.4.1033 ◽

1995 ◽

Vol 129 (4) ◽

pp. 1033-1047 ◽

Cited By ~ 265

Author(s):

I Hagan ◽

M Yanagida

Keyword(s):

Fission Yeast ◽

Nuclear Membrane ◽

Essential Gene ◽

Nuclear Periphery ◽

Spindle Pole Body ◽

Spindle Pole ◽

Temperature Sensitive ◽

Spindle Formation ◽

Isolation And Characterization ◽

Pole Body

Spindle formation in fission yeast occurs by the interdigitation of two microtubule arrays extending from duplicated spindle pole bodies which span the nuclear membrane. By screening a bank of temperature-sensitive mutants by anti-tubulin immunofluorescence microscopy, we previously identified the sad1.1 mutation (Hagan, I., and M. Yanagida. 1990. Nature (Lond.). 347:563-566). Here we describe the isolation and characterization of the sad1+ gene. We show that the sad1.1 mutation affected both spindle formation and function. The sad1+ gene is a novel essential gene that encodes a protein with a predicted molecular mass of 58 kD. Deletion of the gene was lethal resulting in identical phenotypes to the sad1.1 mutation. Sequence analysis predicted a potential membrane-spanning domain and an acidic amino terminus. Sad1 protein migrated as two bands of 82 and 84 kD on SDS-PAGE, considerably slower than its predicted mobility, and was exclusively associated with the spindle pole body (SPB) throughout the mitotic and meiotic cycles. Microtubule integrity was not required for Sad1 association with the SPB. Upon the differentiation of the SPB in metaphase of meiosis II, Sad1-staining patterns similarly changed from a dot to a crescent supporting an integral role in SPB function. Moderate overexpression of Sad1 led to association with the nuclear periphery. As Sad1 was not detected in the cytoplasmic microtubule-organizing centers activated at the end of anaphase or kinetochores, we suggest that Sad1 is not a general component of microtubule-interacting structures per se, but is an essential mitotic component that associates with the SPB but is not required for microtubule nucleation. Sad1 may play a role in SPB structure, such as maintaining a functional interface with the nuclear membrane or in providing an anchor for the attachment of microtubule motor proteins.

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Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication

The Journal of Cell Biology ◽

10.1083/jcb.200307064 ◽

2003 ◽

Vol 162 (7) ◽

pp. 1211-1221 ◽

Cited By ~ 135

Author(s):

John V. Kilmartin

Keyword(s):

Single Molecule ◽

Protein A ◽

Spindle Pole Body ◽

Spindle Pole ◽

Binding Motif ◽

Temperature Sensitive ◽

Pole Body ◽

Human Proteins ◽

Z Domain ◽

Essential Function

Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds centrin in the repeat region, showing that this centrin-binding motif is conserved.

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Stu2p: A Microtubule-Binding Protein that Is an Essential Component of the Yeast Spindle Pole Body

The Journal of Cell Biology ◽

10.1083/jcb.139.5.1271 ◽

1997 ◽

Vol 139 (5) ◽

pp. 1271-1280 ◽

Cited By ~ 117

Author(s):

Peijing Jeremy Wang ◽

Tim C. Huffaker

Keyword(s):

Essential Gene ◽

Spindle Pole Body ◽

Spindle Pole ◽

Cold Sensitivity ◽

Gene Encoding ◽

Microtubule Binding ◽

Amino Acid Region ◽

Pole Body ◽

Spindle Function

Previously we isolated tub2-423, a cold-sensitive allele of the Saccharomyces cerevisiae gene encoding β-tubulin that confers a defect in mitotic spindle function. In an attempt to identify additional proteins that are important for spindle function, we screened for suppressors of the cold sensitivity of tub2-423 and obtained two alleles of a novel gene, STU2. STU2 is an essential gene and encodes a protein whose sequence is similar to proteins identified in a variety of organisms. Stu2p localizes primarily to the spindle pole body (SPB) and to a lesser extent along spindle microtubules. Localization to the SPB is not dependent on the presence of microtubules, indicating that Stu2p is an integral component of the SPB. Stu2p also binds microtubules in vitro. We have localized the microtubule-binding domain of Stu2p to a highly basic 100-amino acid region. This region contains two imperfect repeats; both repeats appear to contribute to microtubule binding to similar extents. These results suggest that Stu2p may play a role in the attachment, organization, and/or dynamics of microtubule ends at the SPB.

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Role of calmodulin and Spc110p interaction in the proper assembly of spindle pole body compenents.

The Journal of Cell Biology ◽

10.1083/jcb.133.1.111 ◽

1996 ◽

Vol 133 (1) ◽

pp. 111-124 ◽

Cited By ~ 70

Author(s):

H A Sundberg ◽

L Goetsch ◽

B Byers ◽

T N Davis

Keyword(s):

Spindle Pole Body ◽

Spindle Pole ◽

Immunofluorescent Staining ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Nonpermissive Temperature ◽

Carboxy Terminus ◽

Calmodulin Binding ◽

Pole Body ◽

Mutant Cells

Previously we demonstrated that calmodulin binds to the carboxy terminus of Spc110p, an essential component of the Saccharomyces cerevisiae spindle pole body (SPB), and that this interaction is required for chromosome segregation. Immunoelectron microscopy presented here shows that calmodulin and thus the carboxy terminus of Spc110p localize to the central plaque. We created temperature-sensitive SPC110 mutations by combining PCR mutagenesis with a plasmid shuffle strategy. The temperature-sensitive allele spc110-220 differs from wild type at two sites. The cysteine 911 to arginine mutation resides in the calmodulin-binding site and alone confers a temperature-sensitive phenotype. Calmodulin overproduction suppresses the temperature sensitivity of spc110-220. Furthermore, calmodulin levels at the SPB decrease in the mutant cells at the restrictive temperature. Thus, calmodulin binding to Spc110-220p is defective at the nonpermissive temperature. Synchronized mutant cells incubated at the nonpermissive temperature arrest as large budded cells with a G2 content of DNA and suffer considerable lethality. Immunofluorescent staining demonstrates failure of nuclear DNA segregation and breakage of many spindles. Electron microscopy reveals an aberrant nuclear structure, the intranuclear microtubule organizer (IMO), that differs from a SPB but serves as a center of microtubule organization. The IMO appears during nascent SPB formation and disappears after SPB separation. The IMO contains both the 90-kD and the mutant 110-kD SPB components. Our results suggest that disruption of the calmodulin Spc110p interaction leads to the aberrant assembly of SPB components into the IMO, which in turn perturbs spindle formation.

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Spc24 interacts with Mps2 and is required for chromosome segregation, but is not implicated in spindle pole body duplication

Molecular Microbiology ◽

10.1046/j.1365-2958.2002.02844.x ◽

2002 ◽

Vol 43 (6) ◽

pp. 1431-1443 ◽

Cited By ~ 14

Author(s):

Ivan Le Masson ◽

Cosmin Saveanu ◽

Anne Chevalier ◽

Abdelkader Namane ◽

Renee Gobin ◽

...

Keyword(s):

Chromosome Segregation ◽

Spindle Pole Body ◽

Spindle Pole ◽

Pole Body

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Nuf2, a spindle pole body-associated protein required for nuclear division in yeast.

The Journal of Cell Biology ◽

10.1083/jcb.125.4.853 ◽

1994 ◽

Vol 125 (4) ◽

pp. 853-866 ◽

Cited By ~ 43

Author(s):

M A Osborne ◽

G Schlenstedt ◽

T Jinks ◽

P A Silver

Keyword(s):

Nuclear Division ◽

Spindle Pole Body ◽

Coiled Coil ◽

Temperature Shift ◽

Spindle Pole ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Culture Cells ◽

Pole Body ◽

Associated Proteins

The NUF2 gene of the yeast Saccharomyces cerevisiae encodes an essential 53-kd protein with a high content of potential coiled-coil structure similar to myosin. Nuf2 is associated with the spindle pole body (SPB) as determined by coimmunofluorescence with known SPB proteins. Nuf2 appears to be localized to the intranuclear region and is a candidate for a protein involved in SPB separation. The nuclear association of Nuf2 can be disrupted, in part, by 1 M salt but not by the detergent Triton X-100. All Nuf2 can be removed from nuclei by 8 M urea extraction. In this regard, Nuf2 is similar to other SPB-associated proteins including Nufl/SPC110, also a coiled-coil protein. Temperature-sensitive alleles of NUF2 were generated within the coiled-coil region of Nuf2 and such NUF2 mutant cells rapidly arrest after temperature shift with a single undivided or partially divided nucleus in the bud neck, a shortened mitotic spindle and their DNA fully replicated. In sum, Nuf2 is a protein associated with the SPB that is critical for nuclear division. Anti-Nuf2 antibodies also recognize a mammalian 73-kd protein and display centrosome staining of mammalian tissue culture cells suggesting the presence of a protein with similar function.

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