Development and validation of optogenetics-based live-cell nuclear transport assay. (A and B) Measurement of nuclear import and export rates using light-inducible nuclear transport systems, LINuS (import), and LEXY (export). Upon 447-nm laser illumination, NES-mCherry-LINuS and NLS-mCherry-LEXY probes translocate from the cytoplasm to the nucleus and vice versa, respectively. Nuclear transport rates are determined by fitting the light-induced change of the nuclear mCherry intensity to a monoexponential decay model. When the activation radiation ceases, the probes return to their preillumination locations, allowing repeated measurements. Scale bar, 10 µm. Norm., normalized. (C) Automated acquisition and analysis of nuclear transport assay. U2OS cells stably expressing the transport probes and histone marker are imaged in the absence and presence of the 447-nm activation laser illumination. The cycle of activation and recovery is repeated at multiple locations or time points, if necessary. Nuclei are segmented and tracked based on the histone or DNA images. Nuclear import or export rate of each nucleus is determined by monoexponential decay model fitting. Measured transport rates are aggregated for correlation analysis. (D–F) Validation of the nuclear transport assay by measuring the effects of nuclear transport perturbations: NUP RNAi, n > 180 nuclei for each condition (D). Neg Ctrl, negative control. (E) GFP-bimax2 transfection for importin-α sequestration. n = 491 (GFP) and 289 (GFP-bimax2) nuclei. Locally weighted scatterplot smoothing lines (GFP, solid line; GFP-bimax2, dashed line) drawn to show trends. (F) 30-min, 1-µM KPT-330 treatment for export inhibition. n > 90 nuclei. P values were calculated by two-sided Welch’s t test for comparison with negative control. n.s., P > 0.01; ***, P < 1 × 10⁻⁴.