Supplemental Materials

This article contains the following supporting material:

• Supplemental Figure S1. - Acceptor Photobleaching of YIC in fixed Hela cells. (A) Graph of acceptor photobleaching and donor dequenching of YIC. Bleaching occurred between time points 5 (t = 12 sec) and 6 (t = 14 sec). Three unstressed cells are shown. (B) Pseudo-colored image of CFP and YFP pre and post bleach.
• Supplemental Figure S2. - Depletion of energy by NaN₃ and deoxyglucose does not reduce RCC1 mobility. (A) FRAP of RCC1-GFP in HeLa cells. Shown is T _1/2 of RCC1 recovery as a function of binned time in NaN₃ and deoxyglucose. Error bars represent standard deviation. (B) Immobile fraction of RCC1 plotted as a function of binned time in NaN₃ and deoxyglucose, derived from the same experiment as A. Error bars represent standard deviation. (C) NaN₃ and deoxyglucose effect on Ran and Mog1 distributions using immunofluorescence microscopy of endogenous protein, at 0 and 5 min of treatment.
• Supplemental Figure S3. - Behavior of the FRET sensor YFP-Importin β Binding domain-CFP (YIC) in fixed cells. To show the FRET readout is responsive to conditions that disrupt the Ran gradient and that this can be measured in fixed cells, we did the following experiment. HeLa cells were transfected with YIC for 24 hours. In order to induce a change in FRET of the YIC sensor to analyze its response under live and fixed conditions, NaN₃ and deoxyglucose were used to deplete energy levels in the cell. Cells were treated with NaN₃ and deoxyglucose for 10 minutes, and then were either measured directly, or fixed in 3.75% formaldehyde. FRET efficiency was lower in live cells than in fixed cells, however, the response to energy depletion gave an increase in FRET efficiency in both cases, showing that, while the absolute value of FRET efficiency may be affected by fixation, the relative changes in FRET efficiency are not.