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The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation.

Wigge PA, Kilmartin JV - J. Cell Biol. (2001)

Bottom Line: Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere.Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells.Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.

ABSTRACT
We have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

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Phenotype of spc24-1 and spc25-1. Immunofluorescent staining of unsynchronized spc24-1 (a–c) and spc25-1 cells (d–f) after 4 h at 36°C with antitubulin (a and d), anti-Tub4p (b and e), and DAPI (c and f). Arrowheads show aploid cells with SPBs, arrows postanaphase cells. spc24-1 (g–i) and spc25-1 (j–l) cells synchronized in G1 with α-factor and released at 36°C for 1.5 h were stained similarly. (m) EM of the same synchronized spc24-1 cells at 1.5 h. Four consecutive serial sections are shown (m1–m4), and overlapping microtubules are present between the two SPBs. The same SPB is shown in m1 and m2. Arrowheads show some of the nuclear pore complexes in m3, and the arrow shows an apparent discontinuity in the spindle in m3 caused by the microtubules being slightly out of the plane of the section. The microtubules can be seen in the equivalent position in the next section (arrow in m4) and even followed if viewed end on at twice the magnification of m3 (m3a). (n) Flow cytometry of K699 (wt), spc24-1, and spc25-1 cells synchronized in G1 and released at 36°C. Bars: (a–l) 2 μm; (m 1–4) 0.2 μm.
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Figure 3: Phenotype of spc24-1 and spc25-1. Immunofluorescent staining of unsynchronized spc24-1 (a–c) and spc25-1 cells (d–f) after 4 h at 36°C with antitubulin (a and d), anti-Tub4p (b and e), and DAPI (c and f). Arrowheads show aploid cells with SPBs, arrows postanaphase cells. spc24-1 (g–i) and spc25-1 (j–l) cells synchronized in G1 with α-factor and released at 36°C for 1.5 h were stained similarly. (m) EM of the same synchronized spc24-1 cells at 1.5 h. Four consecutive serial sections are shown (m1–m4), and overlapping microtubules are present between the two SPBs. The same SPB is shown in m1 and m2. Arrowheads show some of the nuclear pore complexes in m3, and the arrow shows an apparent discontinuity in the spindle in m3 caused by the microtubules being slightly out of the plane of the section. The microtubules can be seen in the equivalent position in the next section (arrow in m4) and even followed if viewed end on at twice the magnification of m3 (m3a). (n) Flow cytometry of K699 (wt), spc24-1, and spc25-1 cells synchronized in G1 and released at 36°C. Bars: (a–l) 2 μm; (m 1–4) 0.2 μm.

Mentions: Ts mutants in both NUF2 and NDC80 show defects in mitosis. Nuf2-61 cells arrest in mitosis (Osborne et al. 1994) and ndc80-1 cells segregate SPBs but not chromosomes (Wigge et al. 1998), suggesting a specific defect in chromosome segregation. We examined ts mutants in both SPC25 and SPC24 and found that they had a similar phenotype to ndc80-1. We looked at both asynchronous and synchronized cells because some aspects of the phenotype are easier to interpret in synchronized cells. After an asynchronous block for 4 h at 36°C, about half the cells in both spc24-1 (45%) and spc25-1 (50%) are apparently aploid containing SPBs but little DNA (arrowheads in Fig. 3b and Fig. e). All of the postanaphase spindles examined in both mutants (19% of the cells in spc24-1, 3% in spc25-1) showed segregation of SPBs but not DNA (arrows in Fig. 3, a and d). However, we could not be sure that these were spindles because most had a break in the microtubules in the neck region. The rest of the cells in both mutants were cells containing SPBs with DNA (36% in spc24-1 and 40% in spc25-1) and cells containing short spindles with DNA (1% in spc24-1 and 7% in spc25-1).


The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation.

Wigge PA, Kilmartin JV - J. Cell Biol. (2001)

Phenotype of spc24-1 and spc25-1. Immunofluorescent staining of unsynchronized spc24-1 (a–c) and spc25-1 cells (d–f) after 4 h at 36°C with antitubulin (a and d), anti-Tub4p (b and e), and DAPI (c and f). Arrowheads show aploid cells with SPBs, arrows postanaphase cells. spc24-1 (g–i) and spc25-1 (j–l) cells synchronized in G1 with α-factor and released at 36°C for 1.5 h were stained similarly. (m) EM of the same synchronized spc24-1 cells at 1.5 h. Four consecutive serial sections are shown (m1–m4), and overlapping microtubules are present between the two SPBs. The same SPB is shown in m1 and m2. Arrowheads show some of the nuclear pore complexes in m3, and the arrow shows an apparent discontinuity in the spindle in m3 caused by the microtubules being slightly out of the plane of the section. The microtubules can be seen in the equivalent position in the next section (arrow in m4) and even followed if viewed end on at twice the magnification of m3 (m3a). (n) Flow cytometry of K699 (wt), spc24-1, and spc25-1 cells synchronized in G1 and released at 36°C. Bars: (a–l) 2 μm; (m 1–4) 0.2 μm.
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Related In: Results  -  Collection

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Figure 3: Phenotype of spc24-1 and spc25-1. Immunofluorescent staining of unsynchronized spc24-1 (a–c) and spc25-1 cells (d–f) after 4 h at 36°C with antitubulin (a and d), anti-Tub4p (b and e), and DAPI (c and f). Arrowheads show aploid cells with SPBs, arrows postanaphase cells. spc24-1 (g–i) and spc25-1 (j–l) cells synchronized in G1 with α-factor and released at 36°C for 1.5 h were stained similarly. (m) EM of the same synchronized spc24-1 cells at 1.5 h. Four consecutive serial sections are shown (m1–m4), and overlapping microtubules are present between the two SPBs. The same SPB is shown in m1 and m2. Arrowheads show some of the nuclear pore complexes in m3, and the arrow shows an apparent discontinuity in the spindle in m3 caused by the microtubules being slightly out of the plane of the section. The microtubules can be seen in the equivalent position in the next section (arrow in m4) and even followed if viewed end on at twice the magnification of m3 (m3a). (n) Flow cytometry of K699 (wt), spc24-1, and spc25-1 cells synchronized in G1 and released at 36°C. Bars: (a–l) 2 μm; (m 1–4) 0.2 μm.
Mentions: Ts mutants in both NUF2 and NDC80 show defects in mitosis. Nuf2-61 cells arrest in mitosis (Osborne et al. 1994) and ndc80-1 cells segregate SPBs but not chromosomes (Wigge et al. 1998), suggesting a specific defect in chromosome segregation. We examined ts mutants in both SPC25 and SPC24 and found that they had a similar phenotype to ndc80-1. We looked at both asynchronous and synchronized cells because some aspects of the phenotype are easier to interpret in synchronized cells. After an asynchronous block for 4 h at 36°C, about half the cells in both spc24-1 (45%) and spc25-1 (50%) are apparently aploid containing SPBs but little DNA (arrowheads in Fig. 3b and Fig. e). All of the postanaphase spindles examined in both mutants (19% of the cells in spc24-1, 3% in spc25-1) showed segregation of SPBs but not DNA (arrows in Fig. 3, a and d). However, we could not be sure that these were spindles because most had a break in the microtubules in the neck region. The rest of the cells in both mutants were cells containing SPBs with DNA (36% in spc24-1 and 40% in spc25-1) and cells containing short spindles with DNA (1% in spc24-1 and 7% in spc25-1).

Bottom Line: Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere.Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells.Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.

ABSTRACT
We have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

Show MeSH