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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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ImmunoEM of Nuf2p-GFP in short spindles (a and b) and a longer spindle in three consecutive serial sections (c1–c3). Bars, 0.1 μm.
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Figure 2: ImmunoEM of Nuf2p-GFP in short spindles (a and b) and a longer spindle in three consecutive serial sections (c1–c3). Bars, 0.1 μm.

Mentions: Ndc80p, Spc25p, and Spc24p are all spindle-associated proteins, localizing to a subset of nuclear microtubules (Rout and Kilmartin 1990; Wigge et al. 1998), whereas Nuf2p has been described as associated with the nuclear face of the SPB (Osborne et al. 1994; Kahana et al. 1995). Some potential spindle localization of Nuf2p was observed by immunofluorescence (Osborne et al. 1994), but because of antibody accessibility problems necessitating the use of spheroplasts, the precise relationship between Nuf2p, the SPB, and the nuclear microtubules was difficult to establish by light microscopy. We reinvestigated this relationship by immunoEM (Adams and Kilmartin 1999), and avoided accessibility problems by using GFP as a tag. Two types of staining patterns were observed. In short spindles (0.5–0.8 μm, n = 8), staining was observed all along the spindle (Fig. 2, a and b), whereas in longer spindles (1–2.4 μm, n = 4), staining was observed close to the SPB (Fig. 2, c1–c3). The number of spindles examined was small because the cells tend to break up during processing (Adams and Kilmartin 1999), and in order to measure spindle length (estimated as the distance between the nuclear edges of the two central plaques), it was necessary for the spindle to be in, or very close to, the plane of the section. In addition, all single SPBs (n = 30), some of which may have been produced by breakup of longer spindles, had staining close to the nuclear face of the SPB (as in Fig. 2, c1 and c3). Very similar results were found for GFP-tagged Ndc80p (data not shown), confirming the earlier immunoEM results (Rout and Kilmartin 1990). These results show that all four components of the Ndc80p complex are localized to a subset of spindle microtubules.


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)

ImmunoEM of Nuf2p-GFP in short spindles (a and b) and a longer spindle in three consecutive serial sections (c1–c3). Bars, 0.1 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199619&req=5

Figure 2: ImmunoEM of Nuf2p-GFP in short spindles (a and b) and a longer spindle in three consecutive serial sections (c1–c3). Bars, 0.1 μm.
Mentions: Ndc80p, Spc25p, and Spc24p are all spindle-associated proteins, localizing to a subset of nuclear microtubules (Rout and Kilmartin 1990; Wigge et al. 1998), whereas Nuf2p has been described as associated with the nuclear face of the SPB (Osborne et al. 1994; Kahana et al. 1995). Some potential spindle localization of Nuf2p was observed by immunofluorescence (Osborne et al. 1994), but because of antibody accessibility problems necessitating the use of spheroplasts, the precise relationship between Nuf2p, the SPB, and the nuclear microtubules was difficult to establish by light microscopy. We reinvestigated this relationship by immunoEM (Adams and Kilmartin 1999), and avoided accessibility problems by using GFP as a tag. Two types of staining patterns were observed. In short spindles (0.5–0.8 μm, n = 8), staining was observed all along the spindle (Fig. 2, a and b), whereas in longer spindles (1–2.4 μm, n = 4), staining was observed close to the SPB (Fig. 2, c1–c3). The number of spindles examined was small because the cells tend to break up during processing (Adams and Kilmartin 1999), and in order to measure spindle length (estimated as the distance between the nuclear edges of the two central plaques), it was necessary for the spindle to be in, or very close to, the plane of the section. In addition, all single SPBs (n = 30), some of which may have been produced by breakup of longer spindles, had staining close to the nuclear face of the SPB (as in Fig. 2, c1 and c3). Very similar results were found for GFP-tagged Ndc80p (data not shown), confirming the earlier immunoEM results (Rout and Kilmartin 1990). These results show that all four components of the Ndc80p complex are localized to a subset of spindle microtubules.

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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