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. 2011 Nov 14;195(4):563-72.
doi: 10.1083/jcb.201106078.

CENP-A exceeds microtubule attachment sites in centromere clusters of both budding and fission yeast

Valerie C Coffman  1 , Pengcheng Wu, Mark R Parthun, Jian-Qiu Wu
Affiliations

CENP-A exceeds microtubule attachment sites in centromere clusters of both budding and fission yeast

Valerie C Coffman et al. J Cell Biol. .

Abstract

The stoichiometries of kinetochores and their constituent proteins in yeast and vertebrate cells were determined using the histone H3 variant CENP-A, known as Cse4 in budding yeast, as a counting standard. One Cse4-containing nucleosome exists in the centromere (CEN) of each chromosome, so it has been assumed that each anaphase CEN/kinetochore cluster contains 32 Cse4 molecules. We report that anaphase CEN clusters instead contained approximately fourfold more Cse4 in Saccharomyces cerevisiae and ~40-fold more CENP-A (Cnp1) in Schizosaccharomyces pombe than predicted. These results suggest that the number of CENP-A molecules exceeds the number of kinetochore-microtubule (MT) attachment sites on each chromosome and that CENP-A is not the sole determinant of kinetochore assembly sites in either yeast. In addition, we show that fission yeast has enough Dam1-DASH complex for ring formation around attached MTs. The results of this study suggest the need for significant revision of existing CEN/kinetochore architectural models.

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Figures

Figure 1.
Figure 1.
Relative quantification of Cnp1 and S. pombe cytokinesis proteins. (A) Differential interference contrast (DIC) and fluorescence image of S. pombe cells showing Cdc12-3YFP nodes (strain KV346; open arrowhead) or Cnp1-mYFP anaphase clusters (strain JW1469; closed arrowheads). (B) Box plots (see Materials and methods) of the number of molecules in individual 3YFP- or mYFP-tagged cytokinesis nodes using Cnp1 as a standard (set at 15 Cnp1 molecules/anaphase cluster, n = 60 clusters; strain JW1469). From left to right, strains KV346, JW1110, and JW949 are shown. (C) Relative intensity of individual Cdc12-3YFP nodes in a heterozygous diploid strain and a haploid strain (JW1404-1). au, arbitrary unit. (D) DIC and fluorescence images of cells quantified in C. Arrowheads mark cells with nodes. Asterisks on the DIC images mark interphase cells with speckles (haploid) or without speckles (diploid). (E) Quantification of Cdc12-3YFP in nodes and speckles in haploid cells. (F) Micrographs and quantification of spindle pole body protein Sad1 (JW1141) and Cnp1 (JW1469) during anaphase. Dashed lines on micrographs show cell boundaries. Bars, 5 µm.
Figure 2.
Figure 2.
Quantification of node proteins and CENP-As using MotB as a reference. (A) Micrographs of Myo2 nodes (JW1109; open arrowhead), Cnp1 anaphase clusters (JW1470; closed arrowheads), and MotB motor of E. coli (JPA750; arrows) with the same imaging settings and contrast adjustment. (B) Comparison of EGFP (MLP198) and mEGFP (JW948) intensity (mean ± SD). au, arbitrary unit. (C) Quantification of the intensity of a single EGFP molecule and MotB molecules in each motor using stepwise bleaching experiments. (top) A representative bleaching trace of EGFP-MotB. Each plateau ranges from 10 to 50 frames. The yellow line shows raw data after background subtraction, whereas the blue line shows Chung-Kennedy filtered data. (bottom left) The histogram shows the step sizes from 20 bleaching traces. (bottom right) The number of EGFP-MotB molecules per motor calculated from the measured step size. The arrows indicate the peak (9.52 units [left] and 22.5 ± 3.4 molecules [right]). (D) Molecules measured in individual cytokinesis nodes and Cnp1 anaphase clusters (JW1470) compared with the MotB motor. Red diamonds indicate published mean values (Wu and Pollard, 2005; Leake et al., 2006; Joglekar et al., 2008). (E) Quantification of Mid1-mECitrine (JW1790) in interphase nodes using stepwise bleaching as in C from eight bleaching traces. The arrows indicate the peak. (F) Number of Mid1 molecules in interphase nodes using the bleaching method (left) or intensity ratio to the MotB motor (right). (G, left) Comparison of Cnp1 (JW1469) and Cse4 (JW2686-2) in anaphase clusters using MotB as a standard. (right) Cells expressing mYFP-tagged CENP-As in the same field for comparison. Dashed lines on micrographs show cell boundaries. Bars, 5 µm.
Figure 3.
Figure 3.
Cnp1 displays similar dynamics to Cse4 at anaphase clusters. (A) Percentage of total cellular CENP-A intensity (mean ± SD) contained in anaphase clusters or the nucleus of S. pombe (JW1469) and S. cerevisiae (JW2687) cells. (B) Quantification of Cnp1 at early anaphase B, late anaphase, and G1/S phase (JW1469) with cellular features diagrammed below the graph (see Materials and methods). (C) FRAP is plotted as the percentage of recovery (left axis), whereas the unbleached cluster’s intensity is plotted as the percentage of prebleach intensity (right axis; mean ± SEM). One CEN cluster in each cell was bleached at early anaphase B, and the recovery was monitored with a 30-s delay for Cnp1 (JW1470) and 1 min for Cse4 (KBY7006). The change in intensity of the unbleached cluster was also monitored. (D) A kymograph of a representative bleached cell expressing Cnp1-mEGFP. The arrowhead marks the bleach point. Bar, 5 µm.
Figure 4.
Figure 4.
Some Cse4 in S. cerevisiae binds to 2-µm, and most is lost from the CEN cluster during G1 phase. (A) Molecules in each anaphase cluster in Cse4-linker-mYFP strains with and without 2-µm. From left to right, strains JW2687, JW2679, and JW2683 are shown. (B) Maximum and sum intensity projections of representative anaphase clusters of the strains in A. Dashed lines on micrographs show cell boundaries. (C) Cells expressing Cse4-linker-mYFP (JW2687) imaged with the same settings and contrast adjustments. (D) Molecules in each CEN cluster for cells, color coded as in C. Prolonged G1 arrest is defined as cells with a long shmoo. Bars, 5 µm.
Figure 5.
Figure 5.
Fission yeast kinetochores have sufficient Dam1–DASH complex to form a ring. (A) Molecules of Dam1 (JW3667), Ask1 (JW3666), Ndc80 (JW3668), and Cnp1 (JW1469) in anaphase clusters using Cse4-mYFP in the same field as a standard. (B) Quantification of Dam1 foci along the spindle. (C) Representative images of budding and fission yeast anaphase cells used for quantification in A and B. The arrowhead marks a Dam1 structure as quantified in B. Dashed lines on micrographs show cell boundaries. Bar, 5 µm.
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