Figure 5.

Phosphorylation of RCC1 is essential for production of the RanGTP gradient. (A) YIC FRET reports the existence of RanGTP in the interphase nucleus. tsBN2 cells expressing YIC were incubated at permissive (33.5°C or 37°C) or restrictive (39.5°C) temperatures for 2–3 h before FRET. Images of interphase cells in CFP and YFP before and after photobleaching are shown. White dashed circles outline the interphase nuclei. Histogram shows quantification of FRET: the increase in CFP fluorescence intensity in bleached (black columns) and control unbleached (white columns) cells. (B) A typical quantification curve of FRET. The cell was scanned five times before and after bleaching YFP. Fluorescence intensities (I) of CFP from the last scan before bleaching (I₅) and the first scan after bleaching (I₆) were used to quantify FRET shown in the histograms. (C) RanGTP is concentrated on mitotic chromosomes in vivo. Images of pro-metaphase and anaphase cells before and after photobleaching are shown. White dashed circles outline mitotic cells. A higher FRET signal was detected on mitotic chromosomes than in the mitotic cytosol (see the histogram below the images). (D) Both RCC1-GFP and RCC1S2,11A-GFP support RanGTP production in interphase nuclei. FRET was carried out in tsBN2 cells expressing RCC1-GFP or RCC1S2,11A-GFP grown at 39.5°C for 2–3 h. Similar FRET signals were detected. (E) Phosphorylation of RCC1 is required for the RanGTP gradient production in mitosis. tsNB2 cells expressing RCC1-GFP or RCC1S2,11A-GFP were arrested in mitosis at the permissive temperature by nocodazole, and then shifted to 39.5°C for 2–3 h before FRET. Significantly stronger FRET was detected on the mitotic chromosomes in cells expressing RCC1-GFP than cells expressing RCC1S2,11A-GFP.