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. 2004 Jul;15(7):3015–3030. doi: 10.1091/mbc.E04-03-0183

Figure 5.

Figure 5.

Rnt1p release from the nucleolus is cell division dependent. (A) Cells carrying a temperature sensitive allele of separin (esp1-1) were grown to log phase and then synchronized in G1 with α-factor. The synchronized cells were released either at the permissive (26°C) or the restrictive (37°C) temperatures and fixed once they reached the G2/M phase of the cell cycle. Antibodies against Rnt1p (red) were used to determine its localization pattern. The position of the nucleolus was highlighted with antibodies against the nucleolar protein Nop1p (green). DNA stained with DAPI is indicated in blue. Yellow bar, 2 μm. The graph shown on the right indicates the percentage of cells in which Rnt1p is nucleolar (No) or nucleoplasmic (Nu). The data shown represent the results of three independent experiments. (B) Cells carrying a temperature sensitive allele of cohesin (mcd1-1) were arrested in G2/M with nocodazole and then shifted to either the permissive (26°C) or the restrictive (37°C) temperature. Rnt1p was visualized and the number of cells with different localization pattern was quantified as described in A. (C) Wild-type cells (RPA190), or cells expressing a temperature-sensitive component of RNA polymerase I (rpa190-3), were either grown at the permissive temperature (23°C) or shifted to the restrictive temperature (37°C). Samples were taken at different intervals and the localization pattern of Rnt1p and two other nucleolar proteins (Nop1p and Nhp2) were visualized using immunofluorescence. The graphs shown on the left indicate the percentages of budded cells (black box) and of cells where Rnt1p is either nucleolar (open circle) or nucleoplasmic (black circle) as a function of time. The graph shown represents the average of three experiments with average error rates of ± 2.4%. (D) Photos of rpa190-3 cells grown at 37°C for either 1 or 4 h are shown on the right. The cells shown in the left panel were stained with antibodies against Nop1p (green) and Rnt1p (red), whereas those in the right panel were stained with antibodies against Nop1p (green) and Nhp2p (red). In both cases the DNA was colored by DAPI (blue). Yellow bar, 2 μm.