Limits...
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.

Show MeSH
(a) Immunoblot of HeLa cell extract with anti-hNuf2R. Double label immunofluorescent staining by confocal microscopy of mitotic HeLa cells with anti-hNuf2R (b and d), anti-HEC (e), and anti–CENP-B (c, f, and g). Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199619&req=5

Figure 9: (a) Immunoblot of HeLa cell extract with anti-hNuf2R. Double label immunofluorescent staining by confocal microscopy of mitotic HeLa cells with anti-hNuf2R (b and d), anti-HEC (e), and anti–CENP-B (c, f, and g). Bars, 5 μm.

Mentions: We prepared antibodies against hNuf2R which in immunoblots of whole HeLa cells (Fig. 9 a) reacted with a band (∼52 kD) close to the expected size for hNuf2R (54.3 kD). No clear immunofluorescent staining pattern was seen in formaldehyde-fixed cells, even for fixation times as short as 1 min. In methanol-acetone–fixed cells, again no clear staining pattern was seen in interphase cells; however, in prophase and metaphase cells dots appeared (Fig. 9b and Fig. d) which had a very similar (Fig. 9c and Fig. f) but not completely identical staining pattern to anti–CENP-B (Earnshaw et al. 1987). Two sets of double-labeled images are shown, one where the CENP-B staining is predominantly dots (Fig. 9 c) and another (Fig. 9 f) where the staining is elongated probably by stretching of the centromere (Shelby et al. 1996). We also stained these cells with antibodies against HEC, the mammalian homologue of Ndc80p (Zheng et al. 1999), and found a very similar staining pattern to anti-hNuf2R (Fig. 9 e). This also coincided in part with anti–CENP-B (Fig. 9 g) and was susceptible to formaldehyde fixation. This susceptibility probably explains why earlier results with anti-HEC (Chen et al. 1997) did not show centromere staining in formaldehyde-fixed cells but did with isolated chromosomes. The extensive cross-linking of cells by formaldehyde can reduce accessibility to buried antigens (Kilmartin et al. 1993), and isolated chromosomes probably have a more open structure. The overall pattern of dots found with both anti-hNuf2R and anti-HEC is similar to the pattern with anti–CENP-B, but when the CENP-B staining was extended, the anti-hNuf2R and HEC staining was associated only with the ends of the extensions (Fig. 9h and Fig. i) and appeared to extend beyond them. These results suggest that the centromeres were stained. By anaphase, the staining in HeLa cells appeared much weaker (data not shown), but we do not know whether this reflects occupancy of the centromere by the antigens or an increase in the accessibility problem. In Indian muntjac cells, which have large centromeres (Brinkley et al. 1984) and thus brighter staining, we saw centromere staining at all stages of mitosis including anaphase (data not shown). In conclusion we have shown that two mammalian homologues of components of the Ndc80p complex, HEC and hNuf2R, are also located at the centromere.


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)

(a) Immunoblot of HeLa cell extract with anti-hNuf2R. Double label immunofluorescent staining by confocal microscopy of mitotic HeLa cells with anti-hNuf2R (b and d), anti-HEC (e), and anti–CENP-B (c, f, and g). Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: (a) Immunoblot of HeLa cell extract with anti-hNuf2R. Double label immunofluorescent staining by confocal microscopy of mitotic HeLa cells with anti-hNuf2R (b and d), anti-HEC (e), and anti–CENP-B (c, f, and g). Bars, 5 μm.
Mentions: We prepared antibodies against hNuf2R which in immunoblots of whole HeLa cells (Fig. 9 a) reacted with a band (∼52 kD) close to the expected size for hNuf2R (54.3 kD). No clear immunofluorescent staining pattern was seen in formaldehyde-fixed cells, even for fixation times as short as 1 min. In methanol-acetone–fixed cells, again no clear staining pattern was seen in interphase cells; however, in prophase and metaphase cells dots appeared (Fig. 9b and Fig. d) which had a very similar (Fig. 9c and Fig. f) but not completely identical staining pattern to anti–CENP-B (Earnshaw et al. 1987). Two sets of double-labeled images are shown, one where the CENP-B staining is predominantly dots (Fig. 9 c) and another (Fig. 9 f) where the staining is elongated probably by stretching of the centromere (Shelby et al. 1996). We also stained these cells with antibodies against HEC, the mammalian homologue of Ndc80p (Zheng et al. 1999), and found a very similar staining pattern to anti-hNuf2R (Fig. 9 e). This also coincided in part with anti–CENP-B (Fig. 9 g) and was susceptible to formaldehyde fixation. This susceptibility probably explains why earlier results with anti-HEC (Chen et al. 1997) did not show centromere staining in formaldehyde-fixed cells but did with isolated chromosomes. The extensive cross-linking of cells by formaldehyde can reduce accessibility to buried antigens (Kilmartin et al. 1993), and isolated chromosomes probably have a more open structure. The overall pattern of dots found with both anti-hNuf2R and anti-HEC is similar to the pattern with anti–CENP-B, but when the CENP-B staining was extended, the anti-hNuf2R and HEC staining was associated only with the ends of the extensions (Fig. 9h and Fig. i) and appeared to extend beyond them. These results suggest that the centromeres were stained. By anaphase, the staining in HeLa cells appeared much weaker (data not shown), but we do not know whether this reflects occupancy of the centromere by the antigens or an increase in the accessibility problem. In Indian muntjac cells, which have large centromeres (Brinkley et al. 1984) and thus brighter staining, we saw centromere staining at all stages of mitosis including anaphase (data not shown). In conclusion we have shown that two mammalian homologues of components of the Ndc80p complex, HEC and hNuf2R, are also located at the centromere.

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