Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells

Abstract

The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.

Figure 1. Development of light-inducible nuclear localization signals (LINuSs) for use in yeast and mammalian cells.

(a) Schematic of LINuS function. In the dark state, the hybrid Jα helix is folded and interacts with the AsLOV2 core domain. Upon blue light exposure, it unfolds rendering the NLS accessible to endogenous importins. (b) Schematic of the mCherry-LINuS expression construct based on the SV40 NLS for use in S. cerevisiae. TEF, constitutive promoter. White space, glycine–serine linker. LINuS, AsLOV2 domain-NLS chimera (colour code as in a). Numbers show the position of the corresponding residues in the full-length AsLOV2 domain. (c) Representative fluorescence microscopy images of S. cerevisiae cells expressing mCherry fused to the wild-type AsLOV2 domain (truncated at residue 540) or to the LINuS tag, before and after continuous illumination with blue light for 10 min. Scale bar, 5 μm. (d) Quantification of the mean nuclear localization score for the mCherry-LINuS construct in c, normalized to the mean nuclear localization score for the mCherry-AsLOV2 construct in c. Mean nuclear localization scores were calculated for a population (n≥36) of cells before and after blue light illumination. Error bars, s.d. of three independent experiments. (e) Schematic of the expression construct for mCherry-LINuS based on the mutated c-Myc^P1A NLS for use in mammalian cells. CMV, strong constitutive promoter. White spaces, glycine-serine linkers. NES, PKIt nuclear export sequence. Grey boxes highlight residues identical to those in the AsLOV2 domain. (f) Representative fluorescence microscopy images of the indicated cell lines transiently transfected with the mCherry-LINuS construct shown in e. Illumination was performed with 1 s blue light pulses every 30 s, for 20 min followed by a 20-min recovery phase in the dark. Scale bars, 15 μm. Quantification of the import and export dynamics in HEK 293T cells (g) and HepG2 and HeLa cells (h). NES only control, construct shown in e without the NLS. As this construct remains cytoplasmic (Supplementary Fig. 6), only the initial value is shown for simplicity. Data represent mean±s.e.m. (n=21, two independent experiments (g) and n=20 cells, two independent experiments (h)).

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

(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-Myc^P1AK4A) 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.

Figure 3. Light-induced entry into mitosis mediated by LINuS.

(a) Schematic of the expression construct CDK1AF-mCherry-LINuS-IRES-Cyclin B1^S147E-mCherry-LINuS. The white spaces represent glycine–serine linkers. (b) Top, representative average fluorescence intensity projections showing HeLa TetON cells transiently transfected with the construct in a before (left image) and after (right image) light activation. Yellow arrows depict cells that were counted as mitotic, based on their regular round shape, absence of a nucleus and, in some instances, presence of visible metaphase plates (shown in the zoomed image below, corresponding to one z-plane of the area in the yellow box above). Red box highlights cells that were not counted as mitotic despite being round, due to either the presence of a nucleus (red star, zoomed image) or blebs (red arrow, zoomed image). Light induction was performed by scanning the field of view for 8 s with 458 nm laser light every 30 s for 80 min. Scale bars, 20 μm. (c) Bar plot representing the percentage of mitotic cells in the dark and light for the indicated constructs. Error bars indicate s.d. (data from at least two independent experiments were pooled; n≥126 cells). *P<0.05 (Students t-test).

Figure 4. LINuS design based on bipartite NLSs.

(a) Schematic representation of a bipartite NLS. (b) Multiple sequence alignment of the wild type AsLOV2 C-terminal helix and corresponding biLINuS variants used in this study. Blue colouring of amino-acid residues indicates the degree of identity among the sequences shown (darker blue indicates higher identity). Colour code of the boxes as in a.

Figure 5. Qualitative assessment of biLINuS variants.

Representative fluorescence microscopy images of HEK 293T cells transiently transfected with the indicated mCherry-biLINuS variants, before and after 15 min of illumination. All biLINuS variants carry a constitutive PKIt NES. Illumination was performed with 1 s blue light pulses every 30 s. Red boxes indicate mCherry-biLINuS variants quantified in Fig. 6a. Scale bar, 15 μm.

Figure 6. Characterization and optimization of biLINuS variants.

(a) Quantification of the relative nuclear localization of the indicated mCherry-biLINuS variants in HEK 293T cells before activation (Before), after 20 min of illumination (After) and after recovery phase in the dark (Recovery). Data represent the mean ± s.d. (n=20 cells, two independent experiments). (b) Quantification of import and export dynamics of the indicated mCherry-biLINuS variants in HEK 293T cells. Data represent the mean± s.e.m. (n=20 cells, two independent experiments). (a,b) All biLINuS variants carry a constitutive PKIt NES. (c) Representative fluorescence microscopy images of HEK 293T cells transiently transfected with mCherry-biLINuS2 variant bearing the indicated NES before and after 20 min of illumination. 2xPKIt indicates two repeats of the PKIt NES. Scale bar, 15 μm. (d) Quantification of the relative nuclear localization of biLINuS2 variants with the indicated NESs in HEK 293T cells before activation (Before), after 20 min of illumination (After) and after recovery phase in the dark (Recovery). Data represent the mean±s.d. (n=20 cells, two independent experiments). (e) Quantification of import and export dynamics of the mCherry-biNLS2 variant carrying the IkBα NES in HEK 293T cells. Data represent the mean±s.e.m. (n=20 cells, two independent experiments). (a–e) Illumination was performed with 1 s blue light pulses every 30 s for 20 min, followed by 20 min recovery phase in the dark.

Figure 7. Light-induced gene expression mediated by biLINuS.

(a) Schematic of the expression construct for biLINuS-based synthetic mammalian TFs. Besides bearing different biLINuS variants, they differ by the presence or absence of an NES and by the nature of the NES, when present. DBD, DNA-binding domain. TA, transactivation domain. LINuS indicates the AsLOV2 domain-NLS chimera (colour code as in Fig. 1a). The white spaces represent glycine–serine linkers. (b) Representative fluorescence microscopy images of HEK 293T cells transiently transfected with biLINuS10 variant of the construct in (a) lacking an NES (103 kDa) before and after illumination with blue light. Illumination was performed with 1 s blue light pulses every 30 s, for 15 min. Scale bar, 15 μm. (c) Quantification of luciferase activity in HEK 293T cells transiently co-transfected with the indicated TF, a reporter construct consisting of the firefly luciferase gene driven from a minimal promoter coupled to four LexA-binding sites, and a constitutive renilla expression construct. Illumination started 15 h post transfection and was performed for 24 h with constant blue light (λ_max =460 nm, ~10 μmol m⁻² s light intensity). Firefly luciferase activity was normalized to renilla luciferase in each sample. Data represent mean±s.d. (three independent experiments).