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Quantitative control of subcellular protein localization with a photochromic dimerizer

Abstract

Artificial control of intracellular protein dynamics with high precision provides deep insight into complicated biomolecular networks. Optogenetics and caged compound-based chemically induced dimerization (CID) systems are emerging as tools for spatiotemporally regulating intracellular protein dynamics. However, both technologies face several challenges for accurate control such as the duration of activation, deactivation rate and repetition cycles. Herein, we report a photochromic CID system that uses the photoisomerization of a ligand so that both association and dissociation are controlled by light, enabling quick, repetitive and quantitative regulation of the target protein localization upon illumination with violet and green light. We also demonstrate the usability of the photochromic CID system as a potential tool to finely manipulate intracellular protein dynamics during multicolor fluorescence imaging to study diverse cellular processes. We use this system to manipulate PTEN-induced kinase 1 (PINK1)–Parkin-mediated mitophagy, showing that PINK1 recruitment to the mitochondria can promote Parkin recruitment to proceed with mitophagy.

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Fig. 1: Design and characterization of pcDH.
Fig. 2: Photoreversible translocation of cytosolic proteins to various subcellular compartments.
Fig. 3: Quantitative translocation of cytosolic proteins by illumination light wavelength.
Fig. 4: Optical regulation of mitophagy by the photochromic CID system.
Fig. 5: Temporal control of cytPINK1 recruitment to the MOM.

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Data availability

The accession numbers in the DNA Databank of Japan, European Molecular Biology Laboratory and GenBank databases are LC739769 for eDHFR-miRFP670nano, LC739770 for Halo-mOrange2-NLS, LC739771 for Halo-mOrange2-CAAX, LC739772 for Tom20-mOrange2-Halo, LC739773 for eDHFR-miRFP670nano-NES, LC739774 for Halo-mOrange2-Cb5, LC739775 for Halo-OMP25-IRES-pSu9-mEGFP, LC739776 for Halo-OMP25-IRES-mEGFP-rLC3B, LC783899 for HA-miRFP670nano-Parkin, LC783900 for PINK1-mOrange2-eDHFR-V5, LC783901 for eDHFR-mOrange2-NES, LC783902 for eDHFR-EGFP-NES, LC783903 for Halo-miRFP670nano-NES, LC783904 for Tom20-mOrange2-eDHFR and LC783905 for Tom20-miRFP670nano-Halo. All data supporting the findings of this study are available within the paper and Supplementary Information. All other data and plasmid DNAs generated in this study are available from the corresponding author. Source data are provided with this paper.

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Acknowledgements

This work was supported by the Japan Society for the Promotion of Science and MEXT KAKENHI (JP19H05284 and JP21H05252 to S.M., JP20K05702 and JP23H02077 to T.K. and JP21H05256 to H.Y.), the Takeda Science Foundation to S.M. and T.K., the Nakatani Foundation to S.M. and T.K., the Inamori Foundation to S.M. and T.K., AMED-CREST (21gm1410006h0001) to S.M. and the ‘Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials’ Research Program in the ‘Network Joint Research Center for Materials and Devices’ to S.M. We thank the Tagen Central Analytical Facility for providing the nuclear magnetic resonance and mass spectrometry instruments. We are grateful to S. Yamaoka for the pMRXIP plasmid and T. Yasui for the pCG-gag-pol and pCG-VSV-G plasmids.

Author information

Authors and Affiliations

Authors

Contributions

T. Mashita, T.K., and S.M. conceptualized this project. T. Mashita, T.K., T. Matsui and S.M. designed the experiments. T. Mashita and S.H. performed the chemical synthesis. T. Mashita, S.H. and T.S. performed the in vitro experiments. T. Mashita, T.K. and H.Y. constructed the plasmids. T. Mashita, T.K. and T.S. performed live-cell imaging and data analysis. H.Y. generated stable cell lines. T. Mashita, T.K. and S.M. wrote the paper. All authors contributed to the revision of the paper.

Corresponding author

Correspondence to Shin Mizukami.

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The authors declare no competing interests.

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Nature Chemical Biology thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Suppression of recruitment to the PM upon addition of TMP.

a, Photochromism-induced recruitment of cytosolic proteins to the PM followed by diffusion by the addition of TMP. Confocal fluorescence images of HeLa cells expressing Halo-mOrange2-CAAX and eDHFR-miRFP670nano-NES before and after illumination at 405 nm (violet) and 555 nm (green). Violet light (405 nm) was illuminated at −15 and −12.5 min and every 2.5 min starting at −5 min. Green light (555 nm) was illuminated at −10 min. Representative images in three independent experiments are shown. Scale bar, 20 µm. Line profiles indicate the fluorescence intensity of mOrange2 (orange) and miRFP670nano (magenta), measured along the yellow lines in the images. B, Time–course changes in the PCC (eDHFR-miRFP670nano-NES vs. Halo-mOrange2-CAAX). Arrows indicate light irradiation at 405 nm (violet) and 555 nm (green). The addition of TMP (final concentration: 50 µM, red triangle) at 0 min induced eDHFR diffusion from the PM to the cytosol, the rate of which was slower than the rate of diffusion induced by the green light (green arrow at −10 min). The solid lines and shaded area indicate mean and s.d., respectively. c, Evaluation of suppression of eDHFR recruitment to the PM upon TMP addition. Red dots represent the mean value. A circular dot indicates each data point. n = 13 cells (50 µM TMP) or 14 cells (TMP (−)) in three independent experiments (b,c). Friedman repeated-measures ANOVA followed by the two-sided Durbin-Conover pairwise comparisons. **P < 0.01. d, Correlation between fluorescence intensity of Halo-mOrange2-CAAX and the background recruitment of eDHFR-fused protein to the PM (ΔPCCBG = PCCDark – PCCTMP). r represents Pearson’s correlation coefficient.

Source data

Extended Data Fig. 2 Photochromism-induced recruitment of cytosolic proteins to the MOM.

a, Schematic illustration of photoreversible translocation of pcDH-labeled Halo-miRFP670nano between the cytosol and MOM. b,c, Confocal fluorescence images of HeLa cells expressing Tom20-mOrange2-eDHFR and Halo-miRFP670nano-NES before and after light illumination at 405 nm (violet) and 555 nm (green). The cells were treated with 3 µM pcDH (b) or DMSO (c) prior to imaging. Representative images in three independent experiments are shown. Scale bar: 20 µm. d, Time–course changes of PCC (Tom20-mOrange2-eDHFR vs. Halo-miRFP670nano-NES). Violet light (405 nm) was illuminated at 5, 15, and 25 min, and green light (555 nm) was illuminated at 10, 20, and 30 min. Solid lines and shaded area indicate mean and s.d., respectively (n = 19 cells (3 µM pcDH) or 17 cells (pcDH (−), three independent experiments).

Source data

Extended Data Fig. 3 Quantitative recruitment of cytosolic proteins induced by different concentrations of TMP-HTL.

a, Recruitment of cytosolic proteins to the PM upon addition of a conventional chemical dimerizer TMP-HTL. Confocal fluorescence images of HeLa cells expressing Halo-mOrange2-CAAX and eDHFR-miRFP670nano-NES treated with 0, 0.5, or 5 µM TMP-HTL. Representative images in three independent experiments are shown. Scale bars, 20 µm. b, Quantification of eDHFR recruitment using TMP-HTL. Blue and red circular dots represent data points and mean values, respectively (n = 26 cells (0 µM), 25 cells (0.5 µM) or 34 cells (5 µM), three independent experiments). Welch’s ANOVA followed by two-sided Games-Howell post-hoc test. ****P < 0.0001.

Source data

Extended Data Fig. 4 Evaluation of excitation light on protein translocation.

a, HeLa cells expressing eDHFR-EGFP-NES and Tom20-miRFP670nano-Halo were incubated with 3 µM pcDH, and 470-nm light (12 µW for 150 ms) was used to visualize eDHFR-EGFP-NES. Scale bar, 20 µm. Fluorescence signal of eDHFR-EGFP-NES in the mitochondria was monitored. Violet light (405 nm) was illuminated just before the excitation number of 40, and green light (555 nm) was illuminated just before the excitation number of 60. Solid lines and shaded area indicate mean and s.d., respectively (n = 21 cells, three independent experiments). b, HeLa cells expressing eDHFR-mOrange2-NES and Tom20-miRFP670nano-Halo were incubated with 3 µM pcDH, and 555-nm light (14 µW for 150 ms) was used to visualize eDHFR-mOrange2-NES. Scale bar, 20 µm. Fluorescence signal of eDHFR-mOrange2-NES in the mitochondria was monitored. Violet light (405 nm) was illuminated just before the excitation number of 20, and green light (555 nm) was illuminated just before the excitation number of 60. Solid lines and shaded area indicate mean and s.d., respectively (n = 25 cells, three independent experiments).

Source data

Extended Data Fig. 5 Intermittent illumination protocol.

a,b, Schematic illustration of intermittent illumination with green light (555 nm) for deactivation (a) and violet light (405 nm) for activation (b). c,d, Three successive confocal fluorescence images from the time-lapse imaging (Fig. 4c) of HeLa cells expressing cytPINK1-mOrange2-eDHFR, miRFP670nano-Parkin, pSu9-mEGFP, and Halo-OMP25. Intermittent green (555 nm, 1.1 mW, 500 ms) (c) or violet (405 nm, 130 µW, 100 ms) (d) was used. Scale bar, 10 µm.

Extended Data Fig. 6 Colocalization of an autophagy marker mEGFP-rLC3B and Parkin after violet light illumination.

a, Confocal fluorescence images of HeLa cells expressing cytPINK1-mOrange2-eDHFR, miRFP670nano-Parkin, mEGFP-rLC3B, and Halo-OMP25, treated with 7 µM pcDH. Violet (405 nm) light was illuminated before the 0-min image acquisition and after every image acquisition (0–60 min). Scale bar, 20 µm. Two independent experiments were performed. b, Enlarged images of the area indicated by the yellow squares in a. Scale bar, 5 µm.

Extended Data Fig. 7 Evaluation of mitophagy progress after violet light illumination by immunostaining of WIPI2 and LAMP1.

Confocal fluorescence images of live and fixed HeLa cells expressing cytPINK1-mOrange2-eDHFR, miRFP670nano-Parkin, pSu9-mEGFP, and Halo-OMP25. To the cells treated with 7 µM pcDH (a, c) or DMSO (b, d), green light (555 nm) was illuminated after the image acquisition in the first 9 min, and violet light (405 nm) was illuminated before the 0-min image acquisition and after every image acquisition (0–60 min). After the time-lapse imaging, the cells were immunostained (magenta) for WIPI2 (a, b) and LAMP1 (c, d). Representative images in three independent experiments are shown. Scale bars, 5 µm. Arrowheads indicate that WIPI2 puncta (a) or lysosomes (LAMP1) (c) were colocalized with the mitochondria (pSu9-mEGFP, green). Yellow rectangles indicate magnified area.

Extended Data Fig. 8 Temporal recruitment of cytPINK1 to the MOM under alternating violet and green light illumination.

Confocal fluorescence images of HeLa cells expressing cytPINK1-mOrange2-eDHFR, miRFP670nano-Parkin, pSu9-mEGFP, and Halo-OMP25. Violet (405 nm) light was irradiated before the 0-min image acquisition and after every image acquisition during 0–29 min (a), 0–9 min (b), 0–2 min (c), or 0 min (d). Green (555 nm) light was irradiated before the 30-min (a), 10-min (b), 3-min (d), or 1-min (d) image acquisition and after every image acquisition (30–60 min (a), 10–60 min (b), 3–60 min (c), 1–60 min (d)). Representative images in three independent experiments are shown. Scale bars, 20 µm.

Supplementary information

Supplementary Information

Supplementary Figs. 1–16, Tables 1 and 2, Notes 1 (plasmid DNA construction) and 2 (synthesis of pcDH, TMP-Fl, TMP-HTL and pcDH0) and NMR spectra (15, pcDH, TMP-Fl, TMP-HTL, 2831 and pcDH0).

Reporting Summary

Supplementary Video 1

Photoreversible translocation of eDHFR-miRFP670nano-NES to the PM. Confocal fluorescence images of HeLa cells expressing Halo-mOrange2-CAAX and eDHFR-miRFP670nano-NES before and after light illumination at 405 and 555 nm. Scale bar, 20 µm.

Supplementary Video 2

Suppression of eDHFR-miRFP670nano-NES recruitment to the PM upon TMP addition. Confocal fluorescence images of HeLa cells expressing Halo-mOrange2-CAAX and eDHFR-miRFP670nano-NES before and after light illumination at 405 and 555 nm. TMP (final concentration, 50 µM) was added at 0 min. Scale bar, 20 µm.

Supplementary Video 3

Photoreversible translocation of eDHFR-miRFP670nano to the nucleus. Confocal fluorescence images of HeLa cells expressing Halo-mOrange2-NLS and eDHFR-miRFP670nano before and after light illumination at 405 and 555 nm. Scale bar, 20 µm.

Supplementary Video 4

Photoreversible translocation of eDHFR-miRFP670nano-NES to the MOM. Confocal fluorescence images of HeLa cells expressing Tom20-mOrange2-Halo and eDHFR-miRFP670nano-NES before and after light illumination at 405 and 555 nm. Scale bar, 20 µm.

Supplementary Video 5

Photoreversible translocation of eDHFR-miRFP670nano-NES to the ER. Confocal fluorescence images of HeLa cells expressing Halo-mOrange2-Cb5 and eDHFR-miRFP670nano-NES before and after light illumination at 405 and 555 nm. Scale bar, 20 µm.

Supplementary Video 6

Translocation rate of eDHFR-miRFP670nano-NES to the MOM. Confocal fluorescence images of HeLa cells expressing Tom20-mOrange2-Halo and eDHFR-miRFP670nano-NES before and after light illumination at 405 and 555 nm. Scale bar, 20 µm.

Supplementary Video 7

Wavelength-dependent recruitment of eDHFR-miRFP670nano-NES to the PM. Confocal fluorescence images of HeLa cells expressing Halo-mOrange2-CAAX and eDHFR-miRFP670nano-NES before and after light illumination at 405, 445 and 555 nm. Scale bar, 20 µm.

Supplementary Video 8

Violet-light-dependent recruitment of cytPINK1-mOrange2-eDHFR to the MOM followed by miRFP670nano-Parkin accumulation. Confocal fluorescence images of HeLa cells expressing cytPINK1-mOrange2-eDHFR (green), miRFP670nano-Parkin (magenta), pSu9-mEGFP and Halo-OMP25. Scale bar, 20 µm. Violet light (405 nm) was illuminated before image acquisition at 0 min and after every image acquisition from 0 to 60 min.

Supplementary Video 9

Temporal recruitment of cytPINK1 to mitochondria. Confocal fluorescence images of HeLa cells expressing cytPINK1-mOrange2-eDHFR (green), miRFP670nano-Parkin (magenta), pSu9-mEGFP and Halo-OMP25. Violet light (405 nm) was irradiated before the 0-min image acquisition and after every image acquisition from 0 to 29 min (a), from 0 to 9 min (b), from 0 to 2 min (c) or at 0 min (d). Green light (555 nm) was irradiated before the 30-min (a), 10-min (b), 3-min (c) and 1-min (d) image acquisition and after every image acquisition (30–60 min, a; 10–60 min, b; 3–60 min, c; 1–60 min, d). Scale bars, 20 µm.

Supplementary Dataset 1

Source data for absorption spectra in Supplementary Fig. 2.

Supplementary Dataset 2

Source data for HPLC chromatograms in Supplementary Fig. 3.

Supplementary Dataset 3

Source data for unprocessed gels and band intensity plots in Supplementary Fig. 4.

Supplementary Dataset 4

Source data for fluorescence polarization plots in Supplementary Fig. 5.

Supplementary Dataset 5

Source data for fluorescence polarization plots in Supplementary Fig. 6.

Supplementary Dataset 6

Source data for line profiles in Supplementary Fig. 8.

Supplementary Dataset 7

Statistical source data and source data for labeling efficiency in Supplementary Fig. 9.

Supplementary Dataset 8

Source data for plots in Supplementary Fig. 10.

Supplementary Dataset 9

Source data for time–course plots in Supplementary Fig. 14.

Supplementary Dataset 10

Source data for time–course plots and slopes in Supplementary Fig. 16.

Source data

Source Data Fig. 1

Source data for plots.

Source Data Fig. 2

Source data for time–course plots.

Source Data Fig. 3

Source data for time–course plots and statistical source data.

Source Data Fig. 4

Source data for time–course plots and statistical source data.

Source Data Fig. 5

Source data for time–course plots and statistical source data.

Extended Data Fig. 1

Source data for time–course plots and statistical source data.

Extended Data Fig. 2

Source data for time–course plots.

Extended Data Fig. 3

Statistical source data.

Extended Data Fig. 4

Source data for time–course plots.

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Mashita, T., Kowada, T., Yamamoto, H. et al. Quantitative control of subcellular protein localization with a photochromic dimerizer. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01654-w

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