Figure 1: Engineering and characterization of LEXY, a light-inducible nuclear export system. | Nature Communications

Figure 1: Engineering and characterization of LEXY, a light-inducible nuclear export system.

From: Optogenetic control of nuclear protein export

Figure 1

(a) Schematic of LEXY function. Blue light irradiation releases the photocaged NES from the AsLOV2 core, thereby inducing nuclear export. (b) Schematic of a construct encoding NLS-mCherry-LEXY and a H2B-GFP nuclear marker. NLS, cMycP1A NLS; 2A, T2A peptide. Green boxes indicate amino-acid residues altered as compared with the wild-type AsLOV2 Jα helix (Wt Jα). Numbers show the position of the corresponding residues in the full-length AsLOV2 domain. (c) Fluorescence images of HEK 293T cells transiently transfected with the construct in b before and after 15 min of pulsatile blue light irradiation. Scale bar, 20μm. (d) Relative nuclear fluorescence of HEK 293T expressing NLS-mCherry-LEXY (LEXY) or NLS-mCherry-AsLOV2 (control) over time. Cells were incubated in the dark for 3 min before blue light irradiation for 15 min followed by a 20-min dark-recovery phase (mean±s.e.m., n=22 cells from 3 independent experiments). (e) Fluorescence images of NLS-mCherry-LEXY-expressing cells undergoing repeated illumination and recovery cycles. Scale bar, 20μm. (f) Fluorescence images of cells expressing NLS-mCherry-LEXY individually irradiated. Green circles depict regions scanned with a blue laser beam for 10 min following 20 min of dark recovery. Scale bar, 20μm. (g) Quantification of the relative nuclear fluorescence of individually irradiated cells (i) and uninduced control cells (u) at the indicated time points. Data represent box plots and individual data points, n=8 induced cells and 13 uninduced cells from 3 independent experiments; ***P=1.69 × 10−17 by Welch’s t-test. NS, not significant.

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