Figure 6.

Rapamycin treatment in the absence of condensin causes the fragmented nucleolus phenotype as a result of ERC formation. (A) Rapamycin treatment of condensin mutant causes fragmented nucleolus phenotype. Early log-phase WT and ycs4-2 cells in YPD were shifted from 23 to 37°C for 2 h, followed by incubation with 200 nM rapamycin (+Rap) or a drug carrier (−Rap) for 0, 1, 3 and 6 h. Nucleolar structure was visualized by IF with a Nop1 antibody (bright white). Merged images of representative cells with DAPI staining (light gray) are shown. A color version of this figure is available at The EMBO Journal Online. (B) Quantification of results from (A). N=100. (C) top1Δtop2-4 cells show similar fragmented nucleolus phenotype and distribution of rDNA throughout the entire nucleus. top1Δtop2-4 cells cultured at 23°C were analyzed for nucleolar and rDNA structures by IF and FISH, respectively. Merged images with DAPI staining are shown. A color version of this figure is available at The EMBO Journal Online. (D) Rapamycin treatment in the absence of condensin causes rDNA to distribute throughout the entire nucleus. Early log-phase ycs4-2 cells in YPD were shifted from 23 to 37°C for 2 h, followed by incubation with 200 nM rapamycin (+Rap) or drug carrier (−Rap) for 6 h. rDNA structure was visualized by FISH with a 25S rDNA probe (bright white). Merged images with DAPI staining are shown. The arrowhead points to a cell containing rDNA distributed throughout the entire nucleus. A color version of this figure is available at The EMBO Journal Online. (E) Loss of condensin causes accumulation of extrachromosomal rDNA circles (ERCs). Early log-phase WT and ycs4-2 (y-4) cells in YPD were shifted from 23 to 37°C for 0 and 6 h. Total DNA was isolated, separated by agarose DNA gel electrophoresis and analyzed by Southern blot with a ³²P-labeled 25S rDNA probe. top1Δtop2-4 cells cultured at 37°C for 2 h were used as a control. Gen rDNA, genomic rDNA.