scholarly journals Mutations in SPC110, encoding the yeast spindle pole body calmodulin-binding protein, cause defects in cell integrity as well as spindle formation

2000 ◽  
Vol 1499 (1-2) ◽  
pp. 85-100 ◽  
Author(s):  
Douglas A Stirling ◽  
Michael J.R Stark
Keyword(s):  
Binding Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cell Integrity ◽  
Spindle Formation ◽  
Calmodulin Binding ◽  
Pole Body

The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (12) ◽  
pp. 7913-7924
Author(s):  
J R Geiser ◽  
H A Sundberg ◽  
B H Chang ◽  
E G Muller ◽  
T N Davis
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Binding Site ◽  
Hybrid System ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Amino Acid Residues ◽  
Calmodulin Binding ◽  
Pole Body ◽  
Two Hybrid

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.

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

1996 ◽  
Vol 133 (1) ◽  
pp. 111-124 ◽  
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.

scholarly journals Bim1p/Yeb1p Mediates the Kar9p-dependent Cortical Attachment of Cytoplasmic Microtubules

2000 ◽  
Vol 11 (9) ◽  
pp. 2949-2959 ◽  
Author(s):  
Rita K. Miller ◽  
Soo-Chen Cheng ◽  
Mark D. Rose
Keyword(s):  
Fluorescent Protein ◽  
Binding Protein ◽  
Spindle Pole Body ◽  
Attachment Site ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Microtubule Binding ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Two Hybrid

In Saccharomyces cerevisiae, positioning of the mitotic spindle depends on the interaction of cytoplasmic microtubules with the cell cortex. In this process, cortical Kar9p in the bud acts as a link between the actin and microtubule cytoskeletons. To identify Kar9p-interacting proteins, a two-hybrid screen was conducted with the use of full-length Kar9p as bait, and three genes were identified: BIM1, STU2, andKAR9 itself. STU2 encodes a component of the spindle pole body. Bim1p is the yeast homologue of the human microtubule-binding protein EB1, which is a binding partner to the adenomatous polyposis coli protein involved in colon cancer. Eighty-nine amino acids within the third quarter of Bim1p was sufficient to confer interaction with Kar9p. The two-hybrid interactions were confirmed with the use of coimmunoprecipitation experiments. Genetic analysis placed Bim1p in the Kar9p pathway for nuclear migration. Bim1p was not required for Kar9p's cortical or spindle pole body localization. However, deletion ofBIM1 eliminated Kar9p localization along cytoplasmic microtubules. Furthermore, in the bim1 mutants, the cytoplasmic microtubules no longer intersected the cortical dot of Green Fluorescent Protein–Kar9p. These experiments demonstrate that the interaction of cytoplasmic microtubules with the Kar9p cortical attachment site requires the microtubule-binding protein Bim1p.

scholarly journals Regulation of spindle pole body assembly and cytokinesis by the centrin-binding protein Sfi1 in fission yeast

2014 ◽  
Vol 25 (18) ◽  
pp. 2735-2749 ◽  
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.

scholarly journals The 110-kD spindle pole body component of Saccharomyces cerevisiae is a phosphoprotein that is modified in a cell cycle-dependent manner.

1996 ◽  
Vol 132 (5) ◽  
pp. 903-914 ◽  
Author(s):  
D B Friedman ◽  
H A Sundberg ◽  
E Y Huang ◽  
T N Davis
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Wild Type ◽  
Spindle Formation ◽  
Pole Body ◽  
A Cell ◽  
In Cells

Spc110p (Nuf1p) is an essential component of the yeast microtubule organizing center, or spindle pole body (SPB). Asynchronous wild-type cultures contain two electrophoretically distinct isoforms of Spc110p as detected by Western blot analysis, suggesting that Spc110p is modified in vivo. Both isoforms incorporate 32Pi in vivo, suggesting that Spc110p is post-translationally modified by phosphorylation. The slower-migrating 120-kD Spc110p isoform after incubation is converted to the faster-migrating 112-kD isoform after incubation with protein phosphatase PP2A, and specific PP2A inhibitors block this conversion. Thus, additional phosphorylation of Spc110p at serine and/or threonine residues gives rise to the slower-migrating 120-kD isoform. The 120-kD isoform predominates in cells arrested in mitosis by the addition of nocodazole. However, the 120-kD isoform is not detectable in cells grown to stationary phase (G0) or in cells arrested in G1 by the addition of alpha-factor. Temperature-sensitive cell division cycle (cdc) mutations demonstrate that the presence of the 120-kD isoform correlates with mitotic spindle formation but not with SPB duplication. In a synchronous wild-type population, the additional serine/threonine phosphorylation that gives rise to the 120-kD isoform appears as cells are forming the mitotic spindle and diminishes as cells enter anaphase. None of several sequences similar to the consensus for phosphorylation by the Cdc28p (cdc2p34) kinase is important for these mitosis-specific phosphorylations or for function. Carboxy-terminal Spc110p truncations lacking the calmodulin binding site can support growth and are also phosphorylated in a cell cycle-specific manner. Further truncation of the Spc110p carboxy terminus results in mutant proteins that are unable to support growth and now migrate as single species. Collectively, these results provide the first evidence of a structural component of the SPB that is phosphorylated during spindle formation and dephosphorylated as cells enter anaphase.

scholarly journals The N-terminus of Sfi1 and yeast centrin Cdc31 provide the assembly site for a new spindle pole body

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Diana Rüthnick ◽  
Jlenia Vitale ◽  
Annett Neuner ◽  
Elmar Schiebel
Keyword(s):  
Cell Cycle ◽  
Binding Sites ◽  
Binding Protein ◽  
Spindle Pole Body ◽  
Inhibitory Effect ◽  
Spindle Pole ◽  
Satellite Formation ◽  
Pole Body ◽  
N Terminus ◽  
Assembly Site

The spindle pole body (SPB) provides microtubule-organizing functions in yeast and duplicates exactly once per cell cycle. The first step in SPB duplication is the half-bridge to bridge conversion via the antiparallel dimerization of the centrin (Cdc31)-binding protein Sfi1 in anaphase. The bridge, which is anchored to the old SPB on the proximal end, exposes free Sfi1 N-termini (N-Sfi1) at its distal end. These free N-Sfi1 promote in G1 the assembly of the daughter SPB (dSPB) in a yet unclear manner. This study shows that N-Sfi1 including the first three Cdc31 binding sites interacts with the SPB components Spc29 and Spc42, triggering the assembly of the dSPB. Cdc31 binding to N-Sfi1 promotes Spc29 recruitment and is essential for satellite formation. Furthermore, phosphorylation of N-Sfi1 has an inhibitory effect and delays dSPB biogenesis until G1. Taking these data together, we provide an understanding of the initial steps in SPB assembly and describe a new function of Cdc31 in the recruitment of dSPB components.

scholarly journals The Yeast Centrin, Cdc31p, and the Interacting Protein Kinase, Kic1p, Are Required for Cell Integrity

1998 ◽  
Vol 143 (3) ◽  
pp. 751-765 ◽  
Author(s):  
Donald S. Sullivan ◽  
Sue Biggins ◽  
Mark D. Rose
Keyword(s):  
Protein Kinase ◽  
Calcium Binding ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Permissive Temperature ◽  
Cell Integrity ◽  
Interacting Protein ◽  
Pole Body

Cdc31p is the yeast homologue of centrin, a highly conserved calcium-binding protein of the calmodulin superfamily. Previously centrins have been implicated only in microtubule-based processes. To elucidate the functions of yeast centrin, we carried out a two-hybrid screen for Cdc31p-interacting proteins and identified a novel essential protein kinase of 1,080 residues, Kic1p (kinase that interacts with Cdc31p). Kic1p is closely related to S. cerevisiae Ste20p and the p-21– activated kinases (PAKs) found in a wide variety of eukaryotic organisms. Cdc31p physically interacts with Kic1p by two criteria; Cdc31p coprecipitated with GST–Kic1p and it bound to GST–Kic1p in gel overlay assays. Furthermore, GST–Kic1p exhibited in vitro kinase activity that was CDC31-dependent. Although kic1 mutants were not defective for spindle pole body duplication, they exhibited a variety of mutant phenotypes demonstrating that Kic1p is required for cell integrity. We also found that cdc31 mutants, previously identified as defective for spindle pole body duplication, exhibited lysis and morphological defects. The cdc31 kic1 double mutants exhibited a drastic reduction in the range of permissive temperature, resulting in a severe lysis defect. We conclude that Kic1p function is dependent upon Cdc31p both in vivo and in vitro. We postulate that Cdc31p is required both for SPB duplication and for cell integrity/morphogenesis, and that the integrity/morphogenesis function is mediated through the Kic1p protein kinase.

scholarly journals gamma-Tubulin-like Tub4p of Saccharomyces cerevisiae is associated with the spindle pole body substructures that organize microtubules and is required for mitotic spindle formation.

1996 ◽  
Vol 134 (2) ◽  
pp. 429-441 ◽  
Author(s):  
A Spang ◽  
S Geissler ◽  
K Grein ◽  
E Schiebel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Checkpoint Control ◽  
Microtubule Organizing Center ◽  
Microtubule Organization ◽  
Spindle Formation ◽  
Pole Body ◽  
Gamma Tubulin

Tub4p is a novel tubulin in Saccharomyces cerevisiae that most closely resembles gamma-tubulin. We report in this manuscript that the essential Tub4p is associated with the inner and outer plaques of the yeast microtubule organizing center, the spindle pole body (SPB). These SPB substructures are involved in the attachment of the nuclear and cytoplasmic microtubules, respectively (Byers, B., and L. Goetsch. 1975. J. Bacteriol. 124:511-523). Study of a temperature sensitive tub4-1 allele revealed that TUB4 has essential functions in microtubule organization. Remarkably, SPB duplication and separation are not impaired in tub4-1 cells incubated at the nonpermissive temperature. However, SPBs from such cells contain less or misdirected nuclear microtubules. Further analysis revealed that tub4-1 cells are able to assemble a short bipolar spindle, suggesting that the defect in microtubule organization occurs after spindle formation. A role of Tub4p in microtubule organization is further suggested by an increase in chromosome loss in tub4-1 cells. In addition, cell cycle arrest and survival of tub4-1 cells is dependent on the mitotic checkpoint control gene BUB2 (Hoyt, M.A., L. Totis, B.T. Roberts. 1991. Cell. 66:507-517), one of the cell's monitors of spindle integrity.

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

1995 ◽  
Vol 129 (4) ◽  
pp. 1033-1047 ◽  
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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