Fig. 1. Fluorescent protein tags strongly influence the sensitivity of LOV2-based optogenetic nuclear export.

a Blue-light induces export of mTurquoise2-tagged optogenetic nuclear export construct, mTq2-optoNES (Supplementary Fig. 4) in hippocampal neurons (n = 8 wells at 40× magnification, scalebar 10 µm; other examples in Supplementary Fig. 5). b Fluorescent protein tags (XFP) fused to optogenetic constructs might influence their sensitivity to light via RET. c Illumination scheme used to quantify light-dependency of nuclear export rates. d Example data from a single 10× field treated as shown in c (four cycles, at least 10 min apart, mean ± SEM, for 23 and 31 cells, respectively), showing faster export at higher photon flux. e Nuclear export rate dependence on photon flux is shown for optoNES constructs fused to fluorescent proteins mTurquoise2, Ypet and mScarlet (in turquoise, yellow and red, respectively) expressed in different cultures. The mTq2 construct is more sensitive (significant by F-test, F(2,66) = 41.34, P < 0.0001). Data are normalised to fitted maximal rates to facilitate sensitivity comparison (non-normalised versions in Supplementary Fig. 9). f Corresponding experiments with a spacer between fluorescent protein and LOV2 domain to reduce resonance energy transfer (RET, Supplementary Fig. 8). All constructs are equally sensitive to light, thus in e, mTurquoise had increased sensitivity whereas Ypet/mScarlet decreased sensitivity. This suggests RET can either increase or decrease optogenetic switch sensitivity. In e-f, all channels were imaged regardless of which XFP was used, to ensure identical illumination conditions. Data are means ± SEM, n = 6 wells. Best-fit ED₅₀s in µmol 438 nm photons.m⁻².flash⁻¹ indicated on the graphs for clarity and in Supplementary Table 1 with standard errors. g Introduction of RET-limiting spacer reduces sensitivity (ED₅₀) of the mTq2 optoNES >twofold; F-test, F(1,44) = 10.33. h Corresponding sensitivities for the Ypet and mScarlet optoNES (from e to f) showing introduction of spacer to limit RET increases sensitivity >threefold; F(1,44) = 23.19 and 49.86, for Ypet and mScarlet respectively; i Dronpa-optoNES shows significant difference in sensitivity in Dronpa-off and Dronpa-on states; F(1, 45) = 49.51. g–i show best-fit values ± S.E. (**P = 0.0025, ***P < 0.0001) from curve-fitting of datasets in e, f and Supplementary Fig. 9F. Source data are provided as a Source Data file.