Figure 4. Centromeres becomes uncoupled from the MAS in ima1Δ cells.

A. GFPIma1 associates with centromeres. Schematic representation of the Chromosome I centromere. otr=outer repeat, imr=innermost repeat, cnt1=center region, dg=dg repeats, dh=dh repeats. Arrows indicate positions of primer sets used in real-time PCR analysis. Graph of fold-enrichment of GFP-Ima1 by chromatin immunoprecipitation at the given region over the control actin gene location, act1. The average of three independent immunoprecipitations are presented with their standard deviations. B. Centromeres are separated from Sad1-DsRed in ima1Δ cells. For each cell, the degree of colocalization of LacI-GFP associated with Lac operators integrated into centromere II (cen2:lacOp, green, labeled GFP-Cen) with Sad1-DsRed (red) was categorized as illustrated in the cartoons with yellow representing colocalization. The percentage of cells displaying each localization pattern was plotted. The graphs represent the average of three separate experiments consisting of greater than 100 cells each. C. In WT cells, GFP-Cen remains closely associated with the MAS during SPB oscillation. Time-lapse images of GFP-LacI and Sad1-DsRed in WT cells with integrated cen2:lacOp (MKSP71) taken every 30 seconds for 5 minutes. D. In ima1Δ cells the centromeres of sister chromatids often separate (asterisk) and no longer colocalize with Sad1-DsRed. Time-lapse images of GFP-LacI and Sad1-DsRed in an ima1Δ cell with integrated cen2:lacOp (MKSP158) were taken every 30 seconds for 5 minutes. The arrow indicates the distended nucleus (green channel) led by a weak focus of Sad1-DsRed (red channel), which is not associated with the centromeres.