FIGURE 1:

RCC1-dependent increase in Ran⋅GTP accelerates the cell cycle. (A and B) Changes in cellular Ran⋅GTP concentration analyzed by computational models of the minimal Ran system in a fibroblast-like cell (A) and a HeLa-like cell (B). (C) Immunoblots of RCC1 in the hTERT-RPE1^WT and hTERT-RPE1^RCC1-V5 cell lysates. (D) Schematic of the RBP-4 FRET sensor for Ran⋅GTP detection with FLIM. The binding of Ran⋅GTP to RBP-4 increases the donor–acceptor distance, resulting in a longer donor fluorescence lifetime, τ_donor. (E) Detection of mitotic Ran⋅GTP gradients in hTERT-RPE1^WT and hTERT-ΔRPE1^RCC1-V5 cells, using FLIM with RBP-4. The top row shows the donor intensity images and the bottom row shows the pseudocolor FLIM images. The range of the displayed values (corresponding to τ_donor values) is indicated beneath the FLIM panels. Scale bar: 10 μm. (F) Scatter plot of the mitotic Ran⋅GTP gradients quantified as the difference between the cytoplasmic and chromatin E in each cell (ΔE; single-cell data; means ± SD; t test). (G) Scatter plot of the inverse of the average cellular RBP-4 E, which is proportional to Ran⋅GTP concentration (E⁻¹; single-cell data; means ± SD; t test). (H) Cell number in cultures of hTERT-RPE1^WT and hTERT-RPE1^RCC1-V5 cells grown in parallel. Means ± SD from two experiments performed in triplicate were fitted with exponential growth equations after 2 d from the start of culture (dashed lines) to calculate the PDT. The null hypothesis was tested: one curve for both data sets.