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. 2014 Jul 14;5:4404. doi: 10.1038/ncomms5404

Figure 2. Tunability and reversibility of LINuS in mammalian cells.

Figure 2

(a) Graph showing the relative nuclear localization of mCherry-LINuS over time for activation performed using increasing light intensities (1, 10, 32 and 100%). Transiently transfected HEK 293T cells were illuminated with 1 s blue light pulses of the indicated intensities every 30 s for 20 min. Data represent mean±s.e.m. (n=26 cells, two independent experiments). (b) As in a, but illumination was performed with 1 s blue light pulses every 30 s using either 100% light for 30 min (dashed line) or a sequence of pulses of the indicated increasing intensities (continuous line). Data represent mean±s.e.m. (n=20 cells, two independent experiments). (c) Box plot of the relative nuclear localization of mCherry-LINuS calculated for a population of HEK 293T cells over three cycles of 20 min illumination and 20 min recovery in the dark. Illumination was performed with 1 s blue light every 30 s for 20 min. Error bars indicate s.d. (n=13 cells, two independent experiments). Inset, relative nuclear localization of mCherry-LINuS over time for a representative cell. (d) Representative fluorescence time-lapse images of HEK 293T cells transfected with mCherry-LINuS and selectively illuminated to trigger nuclear import. Indicated cells (cyan asterisks) were illuminated with a blue (458 nm multiline argon) laser beam directed to a confined area in the cytoplasm. Light induction was performed by scanning the ROI for ~30 ms every 30 s for 20 min followed by 20 min dark recovery. Scale bar, 15 μm. (e) Quantification of the relative nuclear localization for the two indicated cells in d. Cell 2 was not illuminated. (f) Fold increase in nuclear localization of mCherry-LINuS in several individual cells activated for 20 min or non-activated. A LINuS construct bearing a mutated NLS (mut NLS, c-MycP1AK4A) impaired in importin binding was used as control. Illumination was performed as described in d. Data from at least two independent experiments are shown. (a–f) mCherry-LINuS is the construct shown in Fig. 1e. wt, wild type.