Fig. 2: EEA1 endosomes exist in two spatially distinct populations characterised by opposite membrane binding.

a Schematic diagram of EEA1 FLIM experiment logic. A shorter fluorescence lifetime (right) indicates a FRET interaction between EEA1-EGFP and Rab5-RFP and therefore indicates EEA1 is bound via its N-terminal binding domain. Correspondingly, a longer fluorescence lifetime (left) indicates no FRET interaction and thus that EEA1 is bound via its C-terminal binding domains to the membrane. b Normalised frequency histograms of the detected fluorescence lifetimes of EEA1-EGFP photons measured in peripheral endosomes (blue) and perinuclear endosomes (green); bars represent standard errors of the mean; dashed curves show Gaussian fits for reference. Mean lifetimes calculated from these data are 1.87 and 2.10 ns for the peripheral and perinuclear curves, respectively. Endosomes were measured across n = 6 cells. c Representative FLIM-FRET experiments of RPE1 cells transfected with EEA1-EGFP and Rab5-RFP. Coloured scale bar represents the donor lifetime ranging from 1.8 ns (blue) to 2.3 ns (red). Left panel shows the FLIM image of EEA1 (donor) lifetime, the middle panel shows EEA1 fluorescence intensity and the right panel shows Rab5 fluorescence intensity; boxes indicate the regions of the zoomed inset, with dotted lines corresponding to perinuclear endosomes and solid lines to peripheral endosomes. Scale bar = 10 µm. Zoomed insert scale bar = 1 µm.