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. 2012 Dec 27;10(12):e1001434. doi: 10.1371/journal.pbio.1001434

Figure 6. Chromosome-specific differences in centromere and kinetochore protein levels.

Figure 6

(a, b) Double labeling of X/Y; cid-EGFP spermatocytes with anti-ModC (a), which marks the X-Y chromosome territory, and analysis of X/0; cid-EGFP spermatocytes (b) indicated that the Y centromere contains ∼2-fold higher levels of Cid-EGFP compared to the other centromeres. Dots in the diagrams below the images indicate relative intensity of individual Cid-EGFP dots in S5 stage spermatocytes representing either a chromosome 2 or 3 centromere (2/3), the paired chromosome 4 centromeres (4p), the X centromere (X), or the Y centromere (Y). The sum of all the individually measured centromeric signals within each analyzed spermatocyte was set to 100%. Averages (long horizontal line) are given with s.d. (short horizontal lines). n>22. (c) Analogous analysis of Cenp-C-EGFP spermatocytes during stage S5 indicated that the Y centromere contains ∼2-fold higher levels of Cenp-C-EGFP compared to the other centromeres. (d) In case of Spc25-EGFP, meiotic cells were analyzed because this kinetochore protein is only present at centromeres during the meiotic M phases. The diagrams display data from cells during prometaphase of meiosis I from either X/Y (XY prometa I) or X/0 (X/0 prometa I) males but only if eight or seven distinct EGFP signals, respectively, could be resolved. In the case of the diagram of prometaphase II in X/Y males (XY prometa II) exclusively, cells with four distinct signals are displayed. Dots in the diagrams below the images indicate relative intensity of individual Spc25-EGFP spots after setting the sum of all the individually measured kinetochore signals within each analyzed cell to 100%. Each column of dots represents one of the analyzed cells. Red dots indicate the values proposed to correspond to the Y centromere. (e) Spreads of mitotic chromosomes were prepared from syncytial blastoderm embryos expressing Cid-EGFP, Cenp-C-EGFP, or Spc25-EGFP and stained for DNA. As illustrated by the image panels, individual chromosomes could be identified based on chromosome size, pattern of intensely staining heterochromatin blocks, and centromere position. Dots in the diagram indicate total centromeric EGFP intensity per chromosome in arbitrary units (a.u.) chosen to result in an average intensity on the Y chromosome of 100 a.u. Averages (long horizontal line) are given with s.d. (short horizontal lines). n>15 chromosomes.