The conserved Spc7 protein is required for spindle integrity and links kinetochore complexes in fission yeast - PubMed (original) (raw)

Anne Kerres et al. Mol Biol Cell. 2007 Jul.

Abstract

Spc7, a member of the conserved Spc105/KNL-1 family of kinetochore proteins, was identified as an interaction partner of the EB1 homologue Mal3. Spc7 associates with the central centromere region of the chromosome but does not affect transcriptional silencing. Here, we show that Spc7 is required for the integrity of the spindle as well as for targeting of MIND but not of Ndc80 complex components to the kinetochore. Spindle defects in spc7 mutants were severe ranging from the inability to form a bipolar spindle in early mitosis to broken spindles in midanaphase B. spc7 mutant phenotypes were partially rescued by extra alpha-tubulin or extra Mal2. Thus, Spc7 interacts genetically with the Mal2-containing Sim4 complex.

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Figures

Figure 1.

Figure 1.

Spc7 is not involved in centromere silencing. Serial dilution patch tests (104 to 101 cells) of wild-type, spc7-23, spc7-24, and spc7-30 strains grown at the indicated temperatures for 3–4 d. (B) Viability of wild-type and spc7 mutant strains incubated at 36°C for 6 or 8 h. (C) Top diagram, black bars indicate conserved Spc7 residues; bottom diagram, amino acid changes found in the Spc7 mutant proteins in comparison to wild-type Spc7. (D) Kinetochore localization of Spc7-23. Cells expressing the mutant Spc7-23-GFP protein were incubated at 25°C (top lane) or at 36°C for 6 h (bottom lane), fixed, and stained with DAPI, anti-tubulin antibody, and anti-GFP-antibody. Bar, 5 μm. (E) Endogenous Spc7-GFP or Spc7-23-GFP protein isolated from cells grown asynchronously at 32°C for 6 h. Protein extracts prepared from these strains were used for immunoprecipitations using an anti-GFP antibody, followed by Western blotting using the same antibody. Protein extracts used had a similar protein concentration. Actin was used as a loading control. Spc7-23 protein is reduced relative to the wild-type protein by 70%. (F) Serial dilution patch tests (104 to 101 cells) of wild-type, spc7-23, and sim4-193 cells that have the promoter-crippled arg3+ gene inserted at cen1 (top panels) or ura4+ inserted at otr2 (wild-type, spc7-23, and _swi6_Δ cells). Cells were incubated on selective medium with (+arg) or without (−arg) arginine or with (+ura) and without (−ura) uracil at 25°C for 6 d.

Figure 2.

Figure 2.

Temperature-sensitive spc7 mutants have severe mitotic defects. (A) Chromatin distribution in mitotic spc7 mutant cells with an elongating spindle incubated at the permissive (25°C) or restrictive temperature (6 h at 36°C). N/strain = 100. (B) Photomicrographs of spc7-23 cells incubated at 36°C. Fixed cells were stained with DAPI and anti-tubulin antibody. Shown are the three main phenotypes observed: smeared chromatin (a and c), nonseparated chromatin (b and d), and unequally/partially segregated chromatin (e and f) on an elongating spindle. Bar, 5 μm. (C) Diagrammatic representation of spc7-23 anaphase phenotypes at 6 and 10 h after the release from G1 arrest and incubation at 36°C. N/time point = 300. (D) Photomicrographs of cells expressing endogenous Spc7-GFP or Spc7-23-GFP. Synchronized G1 cells were released into the cell cycle and incubated at 36°C. Cells were fixed and stained with anti-GFP antibody. Cells shown at the 4-h time point are also representative of earlier time points. (E) Photomicrographs of cen1.gfp spc7-23 cells incubated at 36°C for 6 h. Fixed cells were stained with anti-GFP antibody, DAPI, and anti-tubulin antibody. The merged images show cen1.GFP plus spindle staining. Bar, 5 μm. Thirteen of 28 cells analyzed showed this phenotype. (F) spc7-23 interacts genetically with components of the spindle checkpoint pathway. Serial dilution patch tests of spc7-23, _mph1_Δ, _mad2_Δ, and the respective spc7-23 double mutants grown at the indicated temperatures for 3 d.

Figure 3.

Figure 3.

Interaction between spc7+ and other components of the Ndc80-MIND-Spc7 kinetochore complex. (A) Diagrammatic representation of the kinetochore localization of Spc7-GFP in nuf2-1 and mis12-537 ts mutants and that of the Spc24-GFP, Nuf2-GFP, Mis12-GFP, and Mis14-GFP fusion proteins in the spc7-23 ts mutant. (B and C) Photomicrographs of spc7-23 cells expressing Spc24-GFP or Mis12-GFP. The strains were incubated at 25°C or for 6 h at 36°C, fixed, and stained with DAPI, anti-tubulin antibody, and anti-GFP antibody. Bar, 5 μm. (D) Mis12-GFP and Spc24-GFP in spc7-23 cells. Protein extracts prepared from wild-type or spc7-23 strains that expressed Mis12-GFP or Spc24-GFP endogenously were used for immunoprecipitations using an anti-GFP-antibody, followed by Western blotting using the same antibody. Protein extracts used had a similar protein concentration. Actin was used as a loading control. The strains were incubated at 36°C for 6 h before protein extraction. (E) Photomicrographs of mis12-537 expressing endogenous Spc7-GFP. The strain was treated as described in B.

Figure 4.

Figure 4.

spc7 interacts with components of the Sim4 complex. (A) Diagrammatic representation of the kinetochore localization of Fta2-GFP, Mal2-GFP, Mis6-GFP, Dad1-GFP, and Sim4-GFP in the _spc7-23_ts mutant and Spc7-GFP in a mal2-1 and fta2-291 mutants incubated at the nonpermissive temperature. *Data taken from Kerres et al. (2006). (B) Representation of the genetic interactions between spc7-23 and mutant components of the Sim4 complex. + to +++; weak to strong genetic interaction. (C) Serial dilution patch test of spc7-23 transformants grown on selective medium at the indicated temperatures for 3 (32°C) or 4 (25°C) days. Vector control (v) indicates plasmid without insert, mal2+ and spc7+ denote the presence of wild-type mal2+ or spc7+ expressed from the wild-type promoter or from the thiamine-repressible nmt41+ promoter in the absence of thiamine, respectively. (D) Diagrammatic representation of anaphases observed in spc7-23 cells transformed with a vector control or a plasmid overexpressing mal2+. Cells were incubated for 6 h at 32°C before fixation. N/strain = 100. (E) Immunoprecipitations of GFP-tagged Spc7 proteins. Protein extracts prepared from a wild-type (wt) strain expressing Spc7-GFP from a plasmid and a spc7-23-gfp strain transformed with a vector control (v) or a plasmid expressing mal2+ were analyzed by Western blot analysis using an anti-GFP antibody. Strains were grown for 6 h at 32°C. Actin was used as a loading control.

Figure 5.

Figure 5.

Spc7 is required for the integrity of the spindle. (A) Photomicrographs of spindle defects observed in spc7-23 cells incubated at 36°C for 6 h: thin spindle midzones (a and b), two half-spindles per cell (c and d), bent spindle (e), and unequally stained spindle (f). Fixed cells were stained with anti-tubulin antibody. (B) Serial dilution patch tests (104 to 101 cells) of wild-type, spc7-30, and spc7-24 cells carrying the integrated nmt81-GFP-atb2. Strains were grown at the indicated temperatures for 5 d under derepressed conditions. (C) Chromosome segregation and spindle phenotypes observed in spc7-23 and spc7-23 nmt81-GFP-atb2 (spc7-23 gfp-atb2) strains grown at 34°C. Right diagram (from left to right), wild-type anaphase spindle, anaphase spindles with thinly staining midzone, disintegrating/broken anaphase spindles, bent spindles and unequally stained spindles. N/strain = 100. (D) Immunoprecipitations of Spc7-HA and GFP-Atb2 proteins. Protein extracts prepared from strains expressing Spc7-HA, GFP-Atb2, or both were used for immunoprecipitations (IP) with an anti-GFP or anti-HA antibody, followed by Western blot analysis using anti-GFP and anti-HA antibodies.

Figure 6.

Figure 6.

Overexpression of spc7-c does not affect localization of members of the Ndc80-MIND-Spc7 complex. (A) Immunoprecipitations of Spc7-HA and Spc7-C-GFP proteins. Protein extracts prepared from a wild-type and a Spc7-HA strain expressing plasmid-borne Spc7-C-GFP under the control of the nmt41 promoter were used for immunoprecipitations using anti-GFP or anti-HA antibodies. The immunoprecipitates were halved and analyzed by Western blot analysis using anti-GFP and anti-HA antibodies. (B) Photomicrographs of Spc24-GFP, Spc7-GFP, and Mis14-GFP strains overexpressing spc7-c from the nmt1+ promoter for 24 h at 30°C. Fixed cells were stained with DAPI and anti-GFP antibody. Bar, 5 μm.

Figure 7.

Figure 7.

Mitosis in living _spc7-c_–expressing cells. Time-lapse images of mitosis in wild-type nmt81-GFP-atb2 cells transformed with a vector control (A) or overexpressing spc7-c (B–G) from the nmt1+ promoter for 36–48 h at 25°C. Time interval between images was 100 s (A–F) or 150 s (G). Numbers beside photomicrographs indicate the number of spindles with this phenotype. (A) Normal spindle elongation. The appearance of the PAA is indicated by an arrow. (B) Delay in spindle elongation possibly caused by an active spindle checkpoint. (C) Inability to form a bipolar spindle. The separated spindle pole bodies (second panel) collapse into a single fluorescent signal. Bar, 1.5 μm. (D) Failure of bipolar spindle elongation. In the time frame measured the spindle shows cycles of spindle elongation (up to 4.7 μm) followed by shrinkage. (E) Elongating spindle that collapses in midanaphase B, followed by fusion of the elongating spindle fragments and further elongation of the bipolar spindle. (F) Normal elongation of the spindle and delay in PAA appearance. The cell shown did not have a PAA in the time measured as shown for the wild-type mitosis in A. (G) Aberrant interphase microtubule cytoskeleton in _spc7-c_–overexpressing cells. Cells showed fewer microtubule bundles, which often curved around the cell. (H) Quantitation of spindle length for nmt81-GFP-atb2 cells transformed with a vector control (black graphs, 3 spindles) or overexpressing spc7-c (gray graphs, 4 spindles). The graphs marked with A, B, and D represent the spindles shown in A, B, and D, respectively.

Figure 8.

Figure 8.

Interaction of spc7 with components of the mitotic spindle. (A) Subcellular localization of the Spc7 variants fused to GFP and expressed on a plasmid under the control of the nmt1+ promoter. Cells were grown under promoter-derepressing conditions for 24 h at 30°C, fixed, and stained with anti-tubulin and anti-GFP antibodies. Bar, 5 μm. (B) Overexpression of Spc7-N or Spc7-C in a wild-type strain leads to TBZ hypersensitivity. Panels show serial dilution patch tests of wild-type cells expressing low (left panels) and high (middle and right panels) amounts of the indicated Spc7 variants. Cells shown in the right panels were grown on medium containing TBZ. (C) Serial dilution patch tests (104 to 101 cells) of spc7-23, _mal3_Δ, peg1-1, _alp14_Δ, and the respective double mutants grown on YE5S at the indicated temperatures for 3–6 d.

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