Two-hybrid search for proteins that interact with Sad1 and Kms1, two membrane-bound components of the spindle pole body in fission yeast

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7913-7924.1993 ◽

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.

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Cut1/separase C-terminus affects spindle pole body positioning in interphase of fission yeast: pointed nuclear formation

Genes to Cells ◽

10.1046/j.1365-2443.2002.00586.x ◽

2002 ◽

Vol 7 (11) ◽

pp. 1113-1124 ◽

Cited By ~ 9

Author(s):

Takahiro Nakamura ◽

Koji Nagao ◽

Yukinobu Nakaseko ◽

Mitsuhiro Yanagida

Keyword(s):

Fission Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Pole Body ◽

C Terminus ◽

Body Positioning

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Spindle Pole Body Duplication in Fission Yeast Occurs at the G1/S Boundary but Maturation Is Blocked until Exit from S by an Event Downstream of Cdc10+

Molecular Biology of the Cell ◽

10.1091/mbc.e04-03-0255 ◽

2004 ◽

Vol 15 (12) ◽

pp. 5219-5230 ◽

Cited By ~ 31

Author(s):

Satoru Uzawa ◽

Fei Li ◽

Ye Jin ◽

Kent L. McDonald ◽

Michael B. Braunfeld ◽

...

Keyword(s):

Fission Yeast ◽

Spindle Pole Body ◽

Specific Inhibitor ◽

Spindle Pole ◽

Feedback Mechanism ◽

G1 Arrest ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Pole Body ◽

In Cells

The regulation and timing of spindle pole body (SPB) duplication and maturation in fission yeast was examined by transmission electron microscopy. When cells are arrested at G1 by nitrogen starvation, the SPB is unduplicated. On release from G1, the SPBs were duplicated after 1–2 h. In cells arrested at S by hydroxyurea, SPBs are duplicated but not mature. In G1 arrest/release experiments with cdc2.33 cells at the restrictive temperature, SPBs remained single, whereas in cells at the permissive temperature, SPBs were duplicated. In cdc10 mutant cells, the SPBs seem not only to be duplicated but also to undergo partial maturation, including invagination of the nuclear envelope underneath the SPB. There may be an S-phase–specific inhibitor of SPB maturation whose expression is under control of cdc10+. This model was examined by induction of overreplication of the genome by overexpression of rum1p or cdc18p. In cdc18p-overexpressing cells, the SPBs are duplicated but not mature, suggesting that cdc18p is one component of this feedback mechanism. In contrast, cells overexpressing rum1p have large, deformed SPBs accompanied by other features of maturation and duplication. We propose a feedback mechanism for maturation of the SPB that is coupled with exit from S to trigger morphological changes.

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

Molecular Biology of the Cell ◽

10.1091/mbc.11.9.2949 ◽

2000 ◽

Vol 11 (9) ◽

pp. 2949-2959 ◽

Cited By ~ 102

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.

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