FRET imaging reveals conformational changes in RCC1 bound to chromatin upon binding Ran(T24N) and during mitosis in MDCK cells. (A) Ran(T24N) binding reduces FRET efficiency of the CFP-RCC1-YFP biosensor bound to chromatin. Cotransfection of CFP and YFP was used as a negative control for FRET (top), and CFP-YFP fusion was used as a positive control (middle). After permeabilization with 0.1% Triton X-100, cells were imaged ± recombinant Ran(T24N) (middle) or Ran(Q69L) (bottom). After subtracting background, raw FRET images were corrected for donor and acceptor bleedthrough and for expression levels of donor and acceptor to obtain N_FRET images. (B) FRET efficiency is slightly but consistently reduced in mitotic cells. FRET imaging was performed on live cells expressing the RCC1 biosensor. Representative N_FRET images of interphase cells and anaphase cells. (C) Quantification of N_FRET efficiencies in interphase, prophase, metaphase, anaphase, and telophase cells. N_FRET intensities of 61 cells at different phases were measured, and the mean N_FRET values are compared. Error bars represent ±SD. *, P < 0.05; **, P < 0.001.