Fig. 2: FLIM–FRET measurements of the NUP98 FG domain inside the NPC.
From: Visualizing the disordered nuclear transport machinery in situ
a, Schematic of the FLIM–FRET analysis pipeline. Different chain segments of the NUP98 FG domain were labelled with a FRET dye pair, and the donor fluorescence intensity was measured on a cell-by-cell basis. Each nuclear rim was selected as a region of interest, and the measured donor fluorescence intensity profiles before and after acceptor photobleaching were extracted and analysed. b,c, Acceptor photobleaching assays were performed for a single-amber-mutated sample (NUP98A221TAG) in permeabilized cells labelled with a AZDye594–LD655 mixture (b) and living cells labelled with a JF549–JF646 mixture (c). In b, the average fluorescence lifetime of the donor dye did not change before and after acceptor photobleaching, indicating the absence of intermolecular FRET. In c, the average fluorescence lifetime of the donor dye changed before and after acceptor photobleaching changed, indicating that intermolecular FRET was detected in highly overexpressing living cells with a bright nuclear rim. d, Acceptor photobleaching assay was performed for a double-amber-mutated sample (NUP98A221TAG–A312TAG) labelled with the AZDye594–LD655 mixture. The average fluorescence lifetime of the donor dye changed before and after acceptor photobleaching, validating the presence of intramolecular FRET. For b–d, n = 5 experiments were repeated independently with the same conclusion; scale bars, 5 μm. e, Fluorescence decay profile before photobleaching was subtracted from the one after photobleaching for the 18 different chain segments of the NUP98 FG domain. Each profile represents an averaged result of approximately 100 cells. The higher peak shows a greater difference in the intensity profiles, indicating higher FRET efficiency and smaller inter-residue distance. f, Phasor plot showing donor lifetimes of the measured 18 chain segments on a single-cell basis (here approximately 2,000 cells in total), in which each point represents the fluorescence decay of one nuclear rim. The left-shifted points represent longer lifetimes.
