Engineering Improved Photoswitches for the Control of Nucleocytoplasmic Distribution

Abstract

Optogenetic techniques use light-responsive proteins to study dynamic processes in living cells and organisms. These techniques typically rely on repurposed naturally occurring light-sensitive proteins to control subcellular localization and activity. We previously engineered two optogenetic systems, the light activated nuclear shuttle (LANS) and the light-inducible nuclear exporter (LINX), by embedding nuclear import or export sequence motifs into the C-terminal helix of the light-responsive LOV2 domain of Avena sativa phototropin 1, thus enabling light-dependent trafficking of a target protein into and out of the nucleus. While LANS and LINX are effective tools, we posited that mutations within the LOV2 hinge-loop, which connects the core PAS domain and the C-terminal helix, would further improve the functionality of these switches. Here, we identify hinge-loop mutations that favorably shift the dynamic range (the ratio of the on- to off-target subcellular accumulation) of the LANS and LINX photoswitches. We demonstrate the utility of these new optogenetic tools to control gene transcription and epigenetic modifications, thereby expanding the optogenetic "tool kit" for the research community.

Keywords: LANS; LINX; LOV2; dynamic range; nucleocytoplasmic shuttle; optogenetics.

Figure 1.

Designs and amino acid sequences of the nuclear import and export photoswitches and the mutations tested herein. (a) Schematic showing light-induced unfolding of the Jα helix and subsequent nuclear import of the Light Activated Nuclear Shuttle (LANS) in purple and nuclear export of the Light Induced Nuclear Exporter (LINX) in green (blue arrows) as well as dark-induced refolding of the Jα helix and subsequent export of LANS and import of LINX (black arrows). Mutations in the hinge-loop region (red). (b) Sequence alignment of wild type LANS depicting the NLS (highlighted gray), the hinge-loop region (bold), and mutations to the hinge-loop (red). (c) Sequence alignment of wild type LINX depicting the NES (highlighted pink) as well as the hinge-loop region and mutations as in (b).

Figure 2.

Binding of LANS proteins in the light and dark. Fluorescence polarization competitive binding assay of importin α7 bound to TAMRA-labelled NLS against titrated LANS_RLR (a) or LANS_RVH (b) at 25°C. For comparison, the dotted lines show the titration against wild type LANS.

Figure 3.

Control of protein localization in the dark and with a lit mimetic using LANS4 and LINXa3. Quantification of nuclear/cytoplasmic distribution from still images of HeLa cells expressing wild type, RLR, or RVH variants of LANS4 (a) and LINXa3 (b) either in the dark (black circles) or in the dark but expressing a lit mimetic (I539E) variant of the photoswitch (blue triangles). Mean ± s.d. were calculated from images of multiple cells (n ≥ 11). **P < 0.01, ***P < 0.001, and ****P < 0.0001 by unpaired two-tailed t-test relative to the wild type dark or lit mimetic; n.s. – not significant.

Figure 4.

Control of protein localization with LANS4. (a) Photoactivation of wild type LANS4, LANS4_RLR, or LANS4_RVH fused to fluorescent proteins in HeLa cells. Snapshots from before blue light exposure (pre-activation), after 10 minutes of blue light exposure (activation), and after 10 minutes of dark (post-activation) from activation of a field of cells over time (Supplementary Videos 1-3; scale bars are 15 μm). Asterisks indicate an adjacent cell out of plane and not used for quantification. (b) Quantification of nuclear/cytoplasmic fluorescence intensity change upon blue light activation (activations, n = 3). The dotted gray line is the LANS4 WT mean for comparison. Mean ± s.e.m. was calculated from images of multiple cells (wild type LANS4, n = 3; LANS4_RLR, n = 4; LANS4_RVH, n = 4).

Figure 5.

Control of protein localization with LINXa3. (a) Photoactivation of wild type LINXa, LINXa3_RLR, or LINXa3_RVH fused to fluorescent proteins in HeLa cells. Snapshots from before blue light exposure (pre-activation), after 10 minutes of blue light exposure (activation), and after 10 minutes of dark (post-activation) from activation of a field of cells over time (Supplementary Videos 4-6; scale bars are 15 μm). Asterisks indicate an adjacent cell out of plane and not used for quantification. (b) Quantification of nuclear/cytoplasmic fluorescence intensity change upon blue light activation (activations, n = 3). The dotted gray line is the LINXa3 WT mean for comparison. Mean ± s.e.m. was calculated from images of multiple cells (wild type LINXa3, n = 3; LINXa3_RLR, n = 4; LINXa3_RVH, n = 6).

Figure 6.

Quantification of protein localization using still images from LANS4 and LINXa3 activations. Comparison of nuclear/cytoplasmic distribution of wild type, RLR, and RVH variants in LANS4 (a) and LINXa3 (b) from pre-activation, activation, and post-activation frames quantified in Figures 4b and 5b. Mean ± s.e.m. was calculated from images of multiple activations and multiple cells as in Figures 4b and 5b. *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired two-tailed t-test.

Figure 7.

Control of gene transcription with LANS4 and LINXa3. β-galactosidase activity in the light (blue bars) or dark (black bars) induced with a GAL4 activation domain and LexA DNA binding domain fused to wild type, RLR, or RVH variants for LANS4 (a) or LINXa3 (b). (n = 3, mean ± s.e.m). **P < 0.01, ***P < 0.001, and ****P < 0.0001 by unpaired two-tailed t-test relative to wild type.

Figure 8.

Control of histone H2B monoubiquitylation with LINXa4. (a) Immunoblots demonstrating light-mediated control of Bre1, an E3 ligase, in a BRE1 deletion strain (bre1∆) by fusing it to wild type LINXa4, LINXa4_RLR, or LINXa4_RVH. Asterisks indicate nonspecific bands. (b) Quantifications of dark state band intensities from replicate western blots as in (a); (n = 3, mean ± s.e.m.; see Supplementary Figure 4 for controls and replicate blots). *P < 0.05 by unpaired two-tailed t-test.