ORP1L conformation and LE positioning. (A and B, top) Mel JuSo cells expressing mRFP-ORP1L-GFP (A) or mRFP-ΔORD-GFP (B) were imaged for FRET by CLSM. The GFP image, mRFP image, calculated FRET, and donor FRET efficiency E_D images are indicated. E_D values are shown in false colors (the LUT shows the corresponding values). The position of the nucleus (N) and cell boundaries are drawn in the GFP channel. The cytosol is divided by five concentric rings, as indicated in the E_D panel. (bottom) E_D values for the constructs were collected and binned for the different rings. Values are shown for the different rings under the corresponding image. Mean ± SEM of E_D values collected for >10 cells analyzed per construct are shown. The asterisks indicate a statistically significant difference in E_D of that bin compared to bin 1 (the most internal ring; *, P = 0.024; **, P = 0.026; ***, P = 0.0063). Bars, 10 µm.