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Reversible protein inactivation by optogenetic trapping in cells

Nature Methods volume 11, pages 633–636 (2014)Cite this article

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Abstract

We present a versatile platform to inactivate proteins in living cells using light, light-activated reversible inhibition by assembled trap (LARIAT), which sequesters target proteins into complexes formed by multimeric proteins and a blue light–mediated heterodimerization module. Using LARIAT, we inhibited diverse proteins that modulate cytoskeleton, lipid signaling and cell cycle with high spatiotemporal resolution. Use of single-domain antibodies extends the method to target proteins containing specific epitopes, including GFP.

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Figure 1: Blue light–induced cluster formation.
Figure 2: Inhibition of CRY2-conjugated proteins by light-inducible trapping in clusters.
Figure 3: Inhibition of target proteins by trapping via CRY2-conjugated single-domain antibodies.

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Acknowledgements

We thank C.L. Tucker (University of Colorado) for cDNAs encoding CRY2PHR-mCherry and CIBN-pmEGFP, T. Inoue (Johns Hopkins University) for cDNA encoding YFP-PHBtk and M. Matsuda (Kyoto University) for cDNA encoding Raichu-Rac1. This work was supported by the National Research Foundation of Korea Stem Cell Program (no. 2011-0019509), the Intelligent Synthetic Biology Center of Global Frontier Project (no. 2011-0031955) and the Korea Advanced Institute of Science and Technology Future Systems Healthcare Project funded by the Ministry of Science, Information and Communication Technology & Future Planning in Korea.

Author information

Author notes

  1. Sangkyu Lee and Hyerim Park: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon, Republic of Korea

    Sangkyu Lee & Won Do Heo

  2. Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

    Sangkyu Lee, Hyerim Park, Taeyoon Kyung, Na Yeon Kim, Sungsoo Kim, Jihoon Kim & Won Do Heo

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  1. Sangkyu Lee
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Contributions

W.D.H. and S.L. conceived the idea and directed the work. S.L., H.P., T.K., N.Y.K., S.K. and J.K. performed experiments. W.D.H., S.L., H.P. and T.K. wrote the manuscript.

Corresponding author

Correspondence to Won Do Heo.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–22, Supplementary Table 1 and Supplementary Note (PDF 9325 kb)

Rapid and reversible cluster formation upon blue-light illumination.

A HeLa cell co-expressing CRY2-mCherry and CIB1-mCerulean-MP was briefly stimulated by blue light (488 nm, 6.5 μW). Fluorescent images of CRY2-mCherry were captured in every 20 seconds. Numbers indicate minutes:seconds. (AVI 2742 kb)

Spatiotemporal control of cluster formation in multiple cells upon blue-light illumination.

Three HeLa cells co-expressing CRY2-mCherry and CIB1-mCerulean-MP were sequentially stimulated by blue light (488 nm, 6.5 μW for each cell) at different time points indicated by arrows. Fluorescent images of CRY2-mCherry were captured in every 20 seconds. Numbers indicate minutes:seconds. (AVI 3993 kb)

Spatiotemporal control of cluster formation at the subcellular level upon blue-light illumination.

Three different regions (12 μm in diameter) of a HeLa cell were sequentially stimulated by blue light (488 nm, 6.5 μW for each region) indicated by white-lined circles. Fluorescent images of CRY2-mCherry were captured in every 20 seconds. Numbers indicate minutes:seconds. (AVI 2960 kb)

Reversible inactivation of Vav2 visualized by cell morphological changes.

A NIH3T3 cell co-expressing mCherry-Lifeact, mCitrine-CRY2-Vav2, and CIB1-mCerulean-MP was stimulated twice by blue light (488 nm) at a 20-minute interval. Fluorescent images of mCherry-Lifeact were captured in every 20 seconds. Numbers indicate minutes:seconds. (AVI 5386 kb)

Inactivation of Vav2 in numerous cells in a wide field of view.

NIH3T3 cells co-expressing mCherry-Lifeact, mCitrine-CRY2-Vav2, and CIB1-mCerulean-MP were stimulated by blue light (488 nm). Fluorescent images of mCherry-Lifeact were captured in every 20 seconds. Numbers indicate minutes:seconds. (AVI 4714 kb)

Spatiotemporal inactivation of Vav2 at the subcellular level.

A NIH3T3 cell co-expressing mCherry-Lifeact, mCitrine-CRY2-Vav2, and CIB1-mCerulean-MP was stimulated by blue light (488 nm). Fluorescent images of mCherry-Lifeact were captured in every 20 seconds. Numbers indicate minutes:seconds. (AVI 2934 kb)

Changing cell polarity through local and sustained inactivation of Vav2.

A small region (indicated by yellow circles) of a NIH3T3 cell co-expressing mCherry-Lifeact, mCitrine-CRY2-Vav2, and CIB1-mCerulean-MP was repeatedly illuminated by blue light (488 nm) at 2-minute intervals for 46 minutes. Fluorescent images of mCherry-Lifeact were captured in every 20 seconds. Numbers indicate minutes:seconds. (AVI 8119 kb)

Inactivation of GFP-Vav2 by cluster-trapping with a CRY2-conjugated anti-GFP nanobody.

NIH3T3 cells co-expressing mCherry-Lifeact, CLIP-CRY2-VHH(GFP), CIB1-SNAP-MP, and EGFP-Vav2 were exposed to blue light (488 nm). Fluorescent images of mCherry-Lifeact were captured in every 20 seconds. Numbers indicate minutes:seconds. (AVI 2968 kb)

Inactivation of GFP-Tiam1 by cluster-trapping with a CRY2-conjugated anti-GFP nanobody.

NIH3T3 cells co-expressing mCherry-Lifeact, CLIP-CRY2-VHH(GFP), CIB1-SNAP-MP, and EGFP-Tiam1 were exposed to blue light (488 nm). Fluorescent images of mCherry-Lifeact were captured in every 10 seconds. Numbers indicate minutes:seconds. (AVI 3713 kb)

Inactivation of GFP-Rac1 by cluster-trapping with a CRY2-conjugated anti-GFP nanobody.

NIH3T3 cells co-expressing mCherry-Lifeact, CLIP-CRY2-VHH(GFP), CIB1-SNAP-MP, and EGFP-Rac1 were exposed to blue light (488 nm). Fluorescent images of mCherry-Lifeact were captured in every 10 seconds. Numbers indicate minutes:seconds. (AVI 3371 kb)

Inactivation of GFP-RhoG by cluster-trapping with a CRY2-conjugated anti-GFP nanobody.

NIH3T3 cells co-expressing mCherry-Lifeact, CLIP-CRY2-VHH(GFP), CIB1-SNAP-MP, and EGFP-RhoG were exposed to blue light (488 nm). Fluorescent images of mCherry-Lifeact were captured in every 10 seconds. Numbers indicate minutes:seconds. (AVI 2376 kb)

Inactivation of GFP-Cdc42 by cluster-trapping with a CRY2-conjugated anti-GFP nanobody.

NIH3T3 cells co-expressing mCherry-Lifeact, CLIP-CRY2-VHH(GFP), CIB1-SNAP-MP, and EGFP-Cdc42 were exposed to blue light (488 nm). Fluorescent images of mCherry-Lifeact were captured in every 10 seconds. Numbers indicate minutes:seconds. (AVI 4269 kb)

Inactivation of CFP-PI3KCAAX visualized by dissociation of PIP3 biosensor from the plasma membrane.

NIH3T3 cells co-expressing mCherry-PHBtk, CLIP-CRY2-VHH(GFP), CIB1-SNAP-MP, and ECFP-p110CAAX were exposed to blue light (488 nm). Fluorescent images of mCherry-PHBtk were captured in every 10 seconds. Numbers indicate minutes:seconds. (AVI 1210 kb)

Inactivation of CFP-PI3KCAAX visualized by cell morphological changes upon cluster-trapping with a CRY2-conjugated anti-GFP nanobody.

NIH3T3 cells co-expressing mCherry-Lifeact, CLIP-CRY2-VHH(GFP), CIB1-SNAP-MP, and ECFP-p110CAAX were exposed to blue light (488 nm). Fluorescent images of mCherry-Lifeact were captured in every 10 seconds. Numbers indicate minutes:seconds. (AVI 6245 kb)

Inhibition of microtubule function in the course of mitosis.

HeLa cells co-expressing mCherry-H2B and GFP-tubulin with CIB1-MP (the left video) or CRY2-VHH(GFP) (the center video) or both CRY2-VHH(GFP) and CIB1-MP (the right video) were exposed to blue light (488 nm) at 5-minute intervals for 24 hours. Fluorescent images of mCherry-H2B and GFP-tubulin were simultaneously captured in every 5 minutes. Numbers indicate hours:minutes. (AVI 23919 kb)

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Lee, S., Park, H., Kyung, T. et al. Reversible protein inactivation by optogenetic trapping in cells. Nat Methods 11, 633–636 (2014). https://doi.org/10.1038/nmeth.2940

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