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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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(a) Live cell imaging of SpNdc80p-GFP (b1 and b2), SpNuf2p-GFP (c1 and c2), and SpSpc24p-GFP (d1 and d2) in S. pombe. Cells about to complete anaphase A (b1, c1, and d1) show between five and six spots which within a few minutes coalesce into two spots during anaphase B (b2, c2, and d2). (e–g) Fluorescence of GFP compared with immunofluorescence with anti-Sad1 (e), antitubulin (f), and anti–HA-tagged Bub1 (g). Bars, 2.5 μm.
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Figure 8: (a) Live cell imaging of SpNdc80p-GFP (b1 and b2), SpNuf2p-GFP (c1 and c2), and SpSpc24p-GFP (d1 and d2) in S. pombe. Cells about to complete anaphase A (b1, c1, and d1) show between five and six spots which within a few minutes coalesce into two spots during anaphase B (b2, c2, and d2). (e–g) Fluorescence of GFP compared with immunofluorescence with anti-Sad1 (e), antitubulin (f), and anti–HA-tagged Bub1 (g). Bars, 2.5 μm.

Mentions: We tagged each of the three potential S. pombe homologues with GFP together with a known S. pombe SPB component Cut12 (Bridge et al. 1998) as a control. All the cells containing Cut12-GFP showed only one or two dots of staining (not shown) as was previously found (Bridge et al. 1998). Cells containing SpNdc80-GFP, SpNuf2-GFP, and SpSpc24-GFP also showed mainly one or two dots. However in larger single cells which would be expected to enter mitosis soon, the dots partly separated and moved continuously relative to each other in the center of the cell, eventually forming a procession of five to six dots (Fig. 8 a). Within a minute or slightly longer, these coalesced into two dots at either end of the procession and then moved to the ends of the cell. There were never more than six dots except in a few very large, presumably polyploid, cells. This pattern of staining suggests centromere localization (Funabiki et al. 1993; Ekwall et al. 1995; Saitoh et al. 1997). To confirm this we used immunofluorescence to compare the localization of the GFP in the three strains with an SPB marker, Sad1 (Hagan and Yanagida 1995), tubulin, and a known centromere component, Bub1 (Bernard et al. 1998). Centromeres cluster around the SPB in interphase S. pombe cells (Uzawa and Yanagida 1992) and as expected GFP staining in all three strains colocalized with Sad1 in these cells (Fig. 8 e). In mitotic cells where the centromeres are distributed along the spindle, the GFP staining was also distributed as a series of dots along the spindle (Fig. 8 f), and coincident (Fig. 8 g) with a known centromere marker, Bub1 (Bernard et al. 1998). These results suggest that SpNdc80, SpNuf2, and SpSpc24 are centromere components in S. pombe.


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) Live cell imaging of SpNdc80p-GFP (b1 and b2), SpNuf2p-GFP (c1 and c2), and SpSpc24p-GFP (d1 and d2) in S. pombe. Cells about to complete anaphase A (b1, c1, and d1) show between five and six spots which within a few minutes coalesce into two spots during anaphase B (b2, c2, and d2). (e–g) Fluorescence of GFP compared with immunofluorescence with anti-Sad1 (e), antitubulin (f), and anti–HA-tagged Bub1 (g). Bars, 2.5 μm.
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Figure 8: (a) Live cell imaging of SpNdc80p-GFP (b1 and b2), SpNuf2p-GFP (c1 and c2), and SpSpc24p-GFP (d1 and d2) in S. pombe. Cells about to complete anaphase A (b1, c1, and d1) show between five and six spots which within a few minutes coalesce into two spots during anaphase B (b2, c2, and d2). (e–g) Fluorescence of GFP compared with immunofluorescence with anti-Sad1 (e), antitubulin (f), and anti–HA-tagged Bub1 (g). Bars, 2.5 μm.
Mentions: We tagged each of the three potential S. pombe homologues with GFP together with a known S. pombe SPB component Cut12 (Bridge et al. 1998) as a control. All the cells containing Cut12-GFP showed only one or two dots of staining (not shown) as was previously found (Bridge et al. 1998). Cells containing SpNdc80-GFP, SpNuf2-GFP, and SpSpc24-GFP also showed mainly one or two dots. However in larger single cells which would be expected to enter mitosis soon, the dots partly separated and moved continuously relative to each other in the center of the cell, eventually forming a procession of five to six dots (Fig. 8 a). Within a minute or slightly longer, these coalesced into two dots at either end of the procession and then moved to the ends of the cell. There were never more than six dots except in a few very large, presumably polyploid, cells. This pattern of staining suggests centromere localization (Funabiki et al. 1993; Ekwall et al. 1995; Saitoh et al. 1997). To confirm this we used immunofluorescence to compare the localization of the GFP in the three strains with an SPB marker, Sad1 (Hagan and Yanagida 1995), tubulin, and a known centromere component, Bub1 (Bernard et al. 1998). Centromeres cluster around the SPB in interphase S. pombe cells (Uzawa and Yanagida 1992) and as expected GFP staining in all three strains colocalized with Sad1 in these cells (Fig. 8 e). In mitotic cells where the centromeres are distributed along the spindle, the GFP staining was also distributed as a series of dots along the spindle (Fig. 8 f), and coincident (Fig. 8 g) with a known centromere marker, Bub1 (Bernard et al. 1998). These results suggest that SpNdc80, SpNuf2, and SpSpc24 are centromere components in S. pombe.

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
Related in: MedlinePlus