Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins

Abstract

Intracellular antibodies have become powerful tools for imaging, modulating and neutralizing endogenous target proteins. Here, we describe an optogenetically activated intracellular antibody (optobody) consisting of split antibody fragments and blue-light inducible heterodimerization domains. We expanded this optobody platform by generating several optobodies from previously developed intracellular antibodies, and demonstrated that photoactivation of gelsolin and β2-adrenergic receptor (β2AR) optobodies suppressed endogenous gelsolin activity and β2AR signaling, respectively.

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Fig. 1: Optobody design and development.
Fig. 2: Applications of optobodies for inducible protein targeting and manipulation.
Fig. 3: Optogenetic inhibition of endogenous GSN activity and β2AR signaling with a GSN optobody and β2AR optobody, respectively.

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information files. Extra data are available from the corresponding author upon reasonable request.

Code availability

The code for high-throughput analysis of two-channel time-lapse colocalization in individual cells are available in Supplementary code files.

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Acknowledgements

We thank J. Gettemans for kindly providing the GSN nanobody and S.-C. Kim for helpful discussions about intrabodies. We are grateful to N. Kim for statistical analysis and discussion, to J.-H. Kim and Y. Lee for experimental support and to S. Lee for discussions. This work was supported by a grant from the Institute for Basic Science (no. IBS-R001-G1), the National Research Fund (NRF-2018R1A2B3004764) and the KAIST Institute for the BioCentury, Republic of Korea.

Author information

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Authors

Contributions

D.Y., B.O.P. and W.D.H. conceived the idea and directed the work. D.Y., H.L., B.-H.O., B.O.P. and W.D.H. designed the experiments. D.Y., B.O.P., H.L., H.J. and J.H. performed the experiments. Y.J. created the custom MATLAB scripts. D.Y., H.L., B.O.P. and W.D.H. wrote the manuscript.

Corresponding authors

Correspondence to Byung Ouk Park or Won Do Heo.

Ethics declarations

Competing interests

South Korean patent no. 10-2016-0013121 has been awarded to Institute for Basic Science (to D.Y., B.O.P. and W.D.H., being the inventors) for the optobody and chemobody technology described in this paper. The technology has been sold to Hulux and W.D.H. is a shareholder.

Additional information

Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–19 and Note 1.

Reporting Summary

Supplementary Video 1

GFP Optobody activation on light illumination HeLa cells co-expressing GFP optobody and Mito-GFP were imaged for 20 min without blue-light stimulation and then illuminated with 488 nm light every 2 min for 2 h. Numbers indicate hours:minutes:seconds.

Supplementary Video 2

Spatiotemporal control of GFP Optobody two HeLa cells co-expressing GFP optobody and Mito-GFP were sequentially stimulated by five pulses of 488-nm light with 2 min intervals at different time points indicated by arrows. Fluorescent images of pMagHigh1-VHHGFP(C66)-FuRed were captured in every 2 min. Numbers indicate hours:minutes:seconds.

Supplementary Video 3

Irreversible activation of GFP Optobody HeLa cells co-expressing Mito-GFP and GFP optobody conjugated with iLID and SspB R73Q were imaged for 10 min without blue-light stimulation, and then illuminated with 488-nm light every 10 s for 2 h. The stimulated cells incubated for 4 h in darkness, capturing images every 10 min. Numbers indicate hours:minutes:seconds.

Supplementary Video 4

Actin Optobody activation on light illumination HeLa cells co-expressing Actin optobody and iRFP-LifeAct were imaged for 20 min without blue-light stimulation, and then illuminated with 488 nm light every 2 min for 1 h. Numbers indicate hours:minutes:seconds

Supplementary Video 5

β2AR Optobody activation on light illumination HEK293 cells co-expressing β2AR Optobody and β2AR-iRFP were incubated in 20 min, captured every 2 min. The cells stimulated with 488-nm light every 2 min for 2 h. Numbers indicate hours:minutes:seconds.

Supplementary Video 6

GSN Optobody activation on light illumination Mitochondria-conjugated GSN optobody of HeLa cells, cotransfected with GSN optobody-Mito and EGFP-GSN, were imaged for 20 min. The cells were stimulated with 488 nm light every 2 min for 90 min by capturing EGFP-GSN. Numbers indicate hours:minutes:seconds

Supplementary Video 7

HeLa cells co-expressing GCN4 optobody and mitochondria-conjugated GCN4 peptides were imaged for 20 min without blue light stimulation and then illuminated with 488 nm light every 2 min for 100 min. Numbers indicate hours:minutes:seconds.

Supplementary Code

A semi-automated computational pipeline for high-throughput analysis of two-channel time-lapse colocalization in individual cells. The pipeline consists of the modules for cell segmentation, cell tracking, fluorescent signal quantification and morphological profiling.

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Yu, D., Lee, H., Hong, J. et al. Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins. Nat Methods 16, 1095–1100 (2019). https://doi.org/10.1038/s41592-019-0592-7

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