Rapid blue-light-mediated induction of protein interactions in living cells - PubMed (original) (raw)
Rapid blue-light-mediated induction of protein interactions in living cells
Matthew J Kennedy et al. Nat Methods. 2010 Dec.
Abstract
Dimerizers allowing inducible control of protein-protein interactions are powerful tools for manipulating biological processes. Here we describe genetically encoded light-inducible protein-interaction modules based on Arabidopsis thaliana cryptochrome 2 and CIB1 that require no exogenous ligands and dimerize on blue-light exposure with subsecond time resolution and subcellular spatial resolution. We demonstrate the utility of this system by inducing protein translocation, transcription and Cre recombinase-mediated DNA recombination using light.
Figures
Figure 1
Mapping of interacting domains of CRY2 and CIB1. (a) Schematic showing full-length CRY2 and CIB1 constructs used in experiments. The numbers below the proteins indicate amino acid residue position. (b)β-galactosidase activity of CRY2 and CIB1 constructs tested for interaction in the dark or in blue light (461 nm, 1.9 mW, 4 hr). The Gal4 binding domain (Gal4BD-X) and Gal4 activation domain (Gal4AD-Y) fusions used are indicated. The control vector was pGBKT7rec containing no insert. Error bars represent standard deviation (n = 3 samples). The inset panel shows immunoblot analysis of Gal4BD fusion proteins in yeast.
Figure 2
Light-triggered translocation of CRY2 in mammalian cells. (a) Schematic showing fusion proteins. CIBN-pmGFP contains a CaaX box prenylation motif for targeting to the plasma membrane. (b) Images of CIBN-pmGFP (left panel) and CRY2-mCh (right two panels) coexpressed in HEK293T cells. The localization of CRY2-mCh is shown prior to light excitation (middle panel) and 20 s following a 100 ms pulse of blue light (488 nm, 25μW) (right panel). Scale bar 5 μm. (c) Time course of CRY2-mCh recruitment to the plasma membrane following a single 100 ms pulse of 488 nm light (25 μW). CIBN-pmGFP localization is shown in the left panel. Scale bar 2 μm. (d) CRY2-mCh translocation kinetics following a 100 ms pulse of 488 nm light (arrow). The distribution of CIBN-pmGFP and the line used to generate the CRY2-mCh kymograph is shown in the upper left panel. Scale bar 1 μm. The bottom panel shows quantification of CRY2-mCh in the cytoplasm and at the plasma membrane, using the regions shown in (c) by the dotted and solid lines respectively. Each fraction was normalized between 0 and 1. (e) Activation and reversal time course of the CRY2PHR-CIBN interaction. The top panel shows cells coexpressing the indicated constructs before and after delivery of two 100 ms pulses of blue light (25 μW) spaced 12.5 min apart. The bottom panel shows quantification of cytoplasmic CRY2PHR-mCh, with light pulses delivered at t = 0 and t = 12.5 min (arrows).
Figure 3
Light induced activation of transcription and DNA recombination. (a) Schematic showing split Gal4 modules (Gal4BD-CRY2 and Gal4AD-CIB1) expressed in yeast cells containing an HA-tagged reporter protein under control of a galactose-inducible promoter. (b) Immunoblot analysis of the HA-tagged reporter (top panel) in response to blue light pulses (10 s pulses, 1.7 mW, 8 min apart). The control lane contains lysates from cells expressing only the reporter. The graph at bottom shows the quantification of western blot bands. (c) Schematic showing the two split Cre constructs (CIBN-CreC and CRY2-CreN) and the Cre reporter. (d) The plot shows % Cre reporter recombination (# GFP expressing cells / # mCherry expressing cells) measured 48 hours after transfection of HEK293T cells with the Cre reporter and indicated constructs. Cells were exposed to blue light pulses (450 nm, 4.5 mW) for indicated times (15 min, 1 hr, or 24 hrs), or kept in the dark for the duration (−). The error bars represent standard deviation for 3 samples from 3 independent experiments. (e) GFP fluorescence images from samples containing both CRY2-CreN and CIBN-CreC that were exposed to 24 hours of blue light or dark. Scale bar 20 μm.
Similar articles
- Optogenetic control of transcription in zebrafish.
Liu H, Gomez G, Lin S, Lin S, Lin C. Liu H, et al. PLoS One. 2012;7(11):e50738. doi: 10.1371/journal.pone.0050738. Epub 2012 Nov 30. PLoS One. 2012. PMID: 23226369 Free PMC article. - Arabidopsis CRY2 and ZTL mediate blue-light regulation of the transcription factor CIB1 by distinct mechanisms.
Liu H, Wang Q, Liu Y, Zhao X, Imaizumi T, Somers DE, Tobin EM, Lin C. Liu H, et al. Proc Natl Acad Sci U S A. 2013 Oct 22;110(43):17582-7. doi: 10.1073/pnas.1308987110. Epub 2013 Oct 7. Proc Natl Acad Sci U S A. 2013. PMID: 24101505 Free PMC article. - Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase.
Taslimi A, Zoltowski B, Miranda JG, Pathak GP, Hughes RM, Tucker CL. Taslimi A, et al. Nat Chem Biol. 2016 Jun;12(6):425-30. doi: 10.1038/nchembio.2063. Epub 2016 Apr 11. Nat Chem Biol. 2016. PMID: 27065233 Free PMC article. - Signaling mechanisms of plant cryptochromes in Arabidopsis thaliana.
Liu B, Yang Z, Gomez A, Liu B, Lin C, Oka Y. Liu B, et al. J Plant Res. 2016 Mar;129(2):137-48. doi: 10.1007/s10265-015-0782-z. Epub 2016 Jan 25. J Plant Res. 2016. PMID: 26810763 Free PMC article. Review. - Cryptochromes Orchestrate Transcription Regulation of Diverse Blue Light Responses in Plants.
Yang Z, Liu B, Su J, Liao J, Lin C, Oka Y. Yang Z, et al. Photochem Photobiol. 2017 Jan;93(1):112-127. doi: 10.1111/php.12663. Epub 2017 Jan 27. Photochem Photobiol. 2017. PMID: 27861972 Free PMC article. Review.
Cited by
- Optogenetic Control of the Mitochondrial Protein Import in Mammalian Cells.
Althoff LFJ, Kramer MM, Bührer B, Gaspar D, Radziwill G. Althoff LFJ, et al. Cells. 2024 Oct 9;13(19):1671. doi: 10.3390/cells13191671. Cells. 2024. PMID: 39404433 Free PMC article. - C9orf72 poly-PR forms anisotropic condensates causative of nuclear TDP-43 pathology.
Hodgson RE, Rayment JA, Huang WP, Sanchez Avila A, Ellis BCS, Lin YH, Soni N, Hautbergue GM, Shelkovnikova TA. Hodgson RE, et al. iScience. 2024 Sep 14;27(10):110937. doi: 10.1016/j.isci.2024.110937. eCollection 2024 Oct 18. iScience. 2024. PMID: 39391721 Free PMC article. - Perspectives on Synthetic Protein Circuits in Mammalian Cells.
Aldrete CA, An C, Call CC, Gao XJ, Vlahos AE. Aldrete CA, et al. Curr Opin Biomed Eng. 2024 Dec;32:100555. doi: 10.1016/j.cobme.2024.100555. Epub 2024 Aug 14. Curr Opin Biomed Eng. 2024. PMID: 39372446 - Notch1 Phase Separation Coupled Percolation facilitates target gene expression and enhancer looping.
Foran G, Hallam RD, Megaly M, Turgambayeva A, Antfolk D, Li Y, Luca VC, Necakov A. Foran G, et al. Sci Rep. 2024 Sep 19;14(1):21912. doi: 10.1038/s41598-024-71634-6. Sci Rep. 2024. PMID: 39300145 Free PMC article. - Ubiquitin-driven protein condensation stabilizes clathrin-mediated endocytosis.
Yuan F, Gollapudi S, Day KJ, Ashby G, Sangani A, Malady BT, Wang L, Lafer EM, Huibregtse JM, Stachowiak JC. Yuan F, et al. PNAS Nexus. 2024 Aug 21;3(9):pgae342. doi: 10.1093/pnasnexus/pgae342. eCollection 2024 Sep. PNAS Nexus. 2024. PMID: 39253396 Free PMC article.
References
- Spencer DM, Wandless TJ, Schreiber SL, Crabtree GR. Controlling signal transduction with synthetic ligands. Science. 1993;262:1019–1024. - PubMed
- Bishop A, et al. Unnatural ligands for engineered proteins: new tools for chemical genetics. Annu Rev Biophys Biomol Struct. 2000;29:577–606. - PubMed
- Shimizu-Sato S, Huq E, Tepperman JM, Quail PH. A light-switchable gene promoter system. Nat Biotechnol. 2002;20:1041–1044. - PubMed
- Yazawa M, Sadaghiani AM, Hsueh B, Dolmetsch RE. Induction of protein-protein interactions in live cells using light. Nat Biotechnol. 2009;27:941–945. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
- HHMI/Howard Hughes Medical Institute/United States
- R01 MH064748/MH/NIMH NIH HHS/United States
- R01 DK081584/DK/NIDDK NIH HHS/United States
- R01 DK81584/DK/NIDDK NIH HHS/United States
- R01 NS039402/NS/NINDS NIH HHS/United States
- R01 DK081584-02/DK/NIDDK NIH HHS/United States
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases
Research Materials