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A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity

Nature Methods volume 20, pages 1553–1562 (2023)Cite this article

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Abstract

Molecular tools enabling the control and observation of the proximity of proteins are essential for studying the functional role of physical distance between two proteins. Here we present CATCHFIRE (chemically assisted tethering of chimera by fluorogenic-induced recognition), a chemically induced proximity technology with intrinsic fluorescence imaging and sensing capabilities. CATCHFIRE relies on genetic fusion to small dimerizing domains that interact upon addition of fluorogenic inducers of proximity that fluoresce upon formation of the ternary assembly, allowing real-time monitoring of the chemically induced proximity. CATCHFIRE is rapid and fully reversible and allows the control and tracking of protein localization, protein trafficking, organelle transport and cellular processes, opening new avenues for studying or controlling biological processes with high spatiotemporal resolution. Its fluorogenic nature allows the design of a new class of biosensors for the study of processes such as signal transduction and apoptosis.

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Fig. 1: CATCHFIRE.
Fig. 2: CATCHFIRE enables the control of protein dimerization with high temporal control.
Fig. 3: Control and tracking of nucleocytoplasmic shuttling.
Fig. 4: Control and tracking of secretory protein trafficking through RUCH.
Fig. 5: Control and tracking of organelle positioning.
Fig. 6: CATCHFIRE-based biosensors for studying cell signaling.

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Data availability

The data supporting the findings of this study are available within the article and supplementary information and are available from the corresponding authors upon reasonable request. The plasmids developed in this study will be available from Addgene. Source data are provided with this paper.

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Acknowledgements

We thank the imaging facility of the Institut de Biologie Paris Seine of Sorbonne Université as well as the Cell and Tissue Imaging (PICT-IBiSA), Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04). We acknowledge A.-S. Macé from the PICT-IBiSA (Institut Curie) for her help to quantify the distribution of mitochondria, A. El Marjou and B. Ouine from the Protein Facility of the Institut Curie for their help to produce and purify FIREmate. We acknowledge T. Miyamoto for the plasmid Tom20-CR (Addgene plasmid #171461), T. Inoue for the plasmid FKBP1C–CFP–Cb5 (Addgene plasmid #162438), D. Gadella for the plasmids FRB–eCFP(W66A)–Giantin (Addgene plasmid #67903) and eCFP(W66A)–FRB–MoA (Addgene plasmid #67904), W. Greene for the plasmid GFP–RelA (Addgene plasmid #23255), R. Youle for the plasmids pHAGE–mt-mKeima–P2A–FRB–Fis1 (Addgene plasmid #135295) and pEYFP–N1–Pink1 (Addgene plasmid #101874), and L. Kapitein for the the KIF17MD–flag–FRB plasmid. The work in the laboratory of A.G. has been supported by the European Research Council (ERC-2016-CoG-724705 FLUOSWITCH), the Agence Nationale de la Recherche (ANR-19-CE13-0026 ADOBE) and the Institut Universitaire de France. The work in the laboratory of F.P. has been supported by the Fondation pour la Recherche Médicale (EQU201903007925) and the Agence Nationale de la Recherche (ANR-19-CE13-0006-03; ANR-20-CE14-0017-02; ANR-19-CE13-0002-03; ANR-11-LABX-0038) and has also received support under the program Investissements d’Avenir, launched by the French Government and implemented by ANR with the references CelTisPhyBio (11-LBX-0038) and ANR10-IDEX-0001-02 PSL.

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Author notes

  1. These authors contributed equally: Octave Joliot, Zeyneb Vildan Cakil.

Authors and Affiliations

  1. Sorbonne Université, École Normale Supérieure, Université PSL, CNRS, Laboratoire des Biomolécules, Paris, France

    Sara Bottone, Zeyneb Vildan Cakil, Lina El Hajji, Louise-Marie Rakotoarison & Arnaud Gautier

  2. Institut Curie, Université PSL, Paris, France

    Octave Joliot, Gaelle Boncompain & Franck Perez

  3. Institut Universitaire de France, Paris, France

    Arnaud Gautier

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  1. Sara Bottone
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Contributions

S.B., O.J., Z.V.C., L.E.H., L.M.R., G.B., F.P. and A.G. designed the experiments. S.B., O.J., Z.V.C., L.E.H., L.M.R. and G.B. performed the experiments. S.B., O.J., Z.V.C., L.E.H., L.M.R., G.B., F.P. and A.G. analyzed the experiments. S.B., F.P. and A.G. wrote the paper with the help of all the authors.

Corresponding authors

Correspondence to Franck Perez or Arnaud Gautier.

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Competing interests

A.G., F.P., S.B., Z.V.C. and O.J. are listed as inventors on a patent application related to the present work and filed by Sorbonne Université, the École Normale Supérieure – PSL University, the CNRS and the Institut Curie. A.G. and F.P. are also co-founders of The Twinkle Factory, a start-up that will commercially distribute the match molecules for research use. All other authors declare no competing interests.

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Peer review information

Nature Methods thanks Alexander Lohman, Akihiko Nakano, and the other, anonymous, reviewer for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 Match-induced complementation of FIREmate (a.k.a. pFAST1–114) and FIREtag (a.k.a. pFAST115–125).

Normalized average fluorescence of about 20,000 HEK293T cells coexpressing the FK506-binding protein (FKBP) fused to FIREtag and the FKBP-rapamycin-binding domain of mammalian target of rapamycin (FRB) fused to FIREmate treated without or with 500 nM of rapamycin, and with a 1, 5, 10, 25 or 50 μM of match550 (a.k.a. HBR-2,5DM), b 0.1, 0.25, 1 or 2.5 μM of match550 (a.k.a. HBR-2,5DM), and c 0.1, 0.25, 1, 2.5, 5, 10 or 25 μM of match540 (a.k.a. HMBR). Individual cell fluorescence was analyzed by flow cytometry. Data represent the mean values ± SD of three independent experiments. A figure presenting the gating strategy in presented on Supplementary Fig. 4.

Extended Data Fig. 2 Fluorescence activation of match550 and match540 upon complementation of FIREmate and FIREtag.

a Fluorescence spectra of match550 when free (black dotted line) or in presence of recombinant FIREmate and mCherry-FIREtag (red solid line). [match550] = 6 μM, [FIREmate] = 12 μM, [mCherry-FIREmate] = 12 μM, λexc = 470 nm. Note the observed shoulder at 615 nm corresponds to the signal resulting from the FRET between bound match550 and mCherry. b Fluorescence spectra of match550 when free (black dotted line) or in presence of recombinant pFAST (blue solid line). [match550] = 6 μM, [pFAST] = 6 μM, λexc = 470 nm. c Fluorescence spectra of match550 within CATCHFIRE vs pFAST. Spectra were normalized by the absorbance of bound match550 at 470 nm for allowing direct comparison. d Fluorescence spectra of match540 when free (black dotted line) or in presence of recombinant FIREmate and mCherry-FIREtag (red solid line). [match540] = 6 μM, [FIREmate] = 12 μM, [mCherry-FIREmate] = 12 μM, λexc = 470 nm. Note the observed shoulder at 615 nm corresponds to the signal resulting from the FRET between bound match540 and mCherry. e Fluorescence spectra of match540 when free (black dotted line) or in presence of recombinant pFAST (blue solid line). [match540] = 6 μM, [pFAST] = 6 μM, λexc = 470 nm. f Fluorescence spectra of match540 within CATCHFIRE vs pFAST. Spectra were normalized by the absorbance of bound match540 at 470 nm for allowing direct comparison.

Extended Data Fig. 3 Fluorogenic induced recruitment of cytoplasmic proteins to mitochondria.

a, b HeLa cells coexpressing mCherry-FIREtag and Tom20-ECFP-FIREmate were treated with 10 μM match550 and imaged by timelapse confocal microscopy at 1 image per 5 seconds. a Representative timelapse of CATCHFIRE (ex/em 488/508-570 nm) (see also Supplementary Video 2). Experiment was repeated three times with similar results. Scale bars are 20 μm. b Temporal evolution of the CATCHFIRE signal. Data represents the mean value ± SD of three independent experiments (n = 17 cells). c, d U2OS cells coexpressing mCherry-FIREtag and Tom20-ECFP-FIREmate were treated with match550 and image by timelapse confocal microscopy at 1 image every 2 minutes. c Representative timelapse (mCherry: ex/em 561/606-675 nm; CATCHFIRE: ex/em 488/508-570 nm; ECFP: ex/em 445/455-499 nm) (see also Supplementary Video 3). Experiment was repeated three times with similar results. Scale bars are 20 μm. d Temporal evolution of the CATCHFIRE signal. Data represents the mean value ± SD of three independent experiments (n = 13 cells).

Extended Data Fig. 4 FIREtag position does not influence CATCHFIRE efficiency.

HeLa cells coexpressing FIREtag-mCherry (a,b) or mCherry-FIREtag-mCherry (c,d) and Tom20-ECFP-FIREmate were treated with 10 μM match550 and imaged by timelapse confocal microscopy. a, c Representative confocal micrographs of cells before (0 min) and after (5 min) addition of match550 (mCherry: ex/em 561/606-675 nm; CATCHFIRE: ex/em 488/508-570 nm; ECFP: ex/em 445/455-499 nm) (see also Supplementary Videos 4, 5). Experiments were repeated three times with similar results. Scale bars are 20 μm. b, d Temporal evolution of the CATCHFIRE signal. Data represents the mean values ± SD of three independent experiments (n = 19 cells (b) and 12 cells (d)).

Extended Data Fig. 5 CATCHFIRE to various organelles.

HeLa cells co-expressing mCherry-FIREtag and FIREmate-ECFP-Giantin (a, b), FIREmate-ECFP-Cb5 (c, d), Lyn11-FIREmate-ECFP (e, f) were treated with 10 μM match540, and imaged by time-lapse confocal microscopy. a,c,e Representative confocal micrographs of cells before (0 min) and after (5 min) addition of match540 (see Supplementary Videos 12–14). Experiments were repeated three times with similar results. b,d,f Temporal evolution of the CATCHFIRE signal. Data represents the mean values ± SD of three independent experiments (n = 12 cells (b), 16 cells (d), 13 cells (f)).

Extended Data Fig. 6 Dual control with CATCHFIRE and FRB-rapamycin-FKBP.

HeLa cells co-expressing mCherry-FKBP-FIREtag, TOM20-FIREmate and FRB-ECFP-Giantin were treated with 10 μM match550 and then, after 2 min, with 500 nM rapamycin, and imaged by time-lapse confocal microscopy. Representative confocal micrographs of cells before, after addition of match550, and after addition of rapamycin. Red: ex/em 561/606-675 nm; green: ex/em 488/508-570 nm; cyan: ex/em 445/455-499 nm. Scale bars are 20 μm.

Extended Data Fig. 7 Design of cagedFIREtag.

Fusion of FIREtag at the N-terminal domain of the apo photoactive yellow protein (PYP2-114) resulted in a protein (cagedFIREtag) that folds like a circular permutation of PYP because of the intramolecular interaction between FIREtag and PYP1–114, masking thus FIREmate. The structure of apo PYP was generated from the crystal structure (PDB: 1NWZ). The model of fluorogen-bound pFAST was generated by homology modeling and molecular dynamics in ref. 14. The model of cagedFIREtag was predicted using Alphafold (ref. 36).

Extended Data Fig. 8 Caging FIREtag avoids residual self-association.

a Design of cagedFIREtag. b Normalized average fluorescence of about 50,000 HEK293T cells co-expressing the FK506-binding protein (FKBP) fused to cagedFIREtag and the FKBP-rapamycin-binding domain of mammalian target of rapamycin (FRB) fused to FIREmate treated without or with 500 nM of rapamycin, and with 1, 5, 10, 25 or 50 μM of match550. Individual cell fluorescence was analyzed by flow cytometry. Data represent the mean values ± SD of three independent experiments. c, d HeLa cells co-expressing mCherry-cagedFIREtag and FIREmate-ECFP-Giantin were treated with 10 μM match550, and imaged by time-lapse confocal microscopy. c Representative confocal micrographs of cells before (0 min) and after (9 min) addition of match550 (see also Supplementary Video 16). Experiments were repeated three times with similar results. Red: ex/em 561/606-675 nm; green: ex/em 488/508-570 nm; cyan: ex/em 445/455-499 nm. Scale bars are 20 μm. d Temporal evolution of the CATCHFIRE signal. Data represents the mean values ± SD of three independent experiments (n = 7 cells).

Extended Data Fig. 9 Control and tracking of secretory protein trafficking.

a Schematic illustrating how FIREmate-KDEL acts as a hook to retain FIREtag-ged reporter in the endoplasmic reticulum in presence of the match. Release is induced by washing of the match, allowing trafficking of the reporter to its acceptor compartment. b,c HeLa cells co-expressing FIREmate-KDEL and mApple-FIREtag-GPI were treated with match550 for 24 h and image by spinning-disk microscopy after washout of match550. b Representative micrographs before washout, 20 min and 70 min after washout (see also Supplementary Video 20). Experiments were repeated five times with similar results. c Temporal evolution of the CATCHFIRE signal in the ER (green) and the mCherry signal in the Golgi apparatus (red). Data represents the mean ± SD of 14 cells. Red: ex/em 561/604-664 nm; green: ex/em 488/545-575 nm. Scale bars are 10 μm. All images were enhanced by 2D-deconvolution on NIS-Elements Ver.5.42.

Extended Data Fig. 10 Fluorogenic induced recruitment of PINK1 to mitochondria induces mitophagy.

a-d HeLa cells co-expressing ECFP-FIREmate-Fis1 and either a, b mCherry-FIREtag (negative control) or c, d PINK1-mCherry-FIREtag were treated without or with 10 μM match550 for 2 h. a, c Representative micrographs (see also slementary Fig. 3 for additional examples). Experiments were repeated three times with similar results. Red: ex/em 561/606-675 nm; green: ex/em 488/508-570 nm; cyan: ex/em 445/455-499 nm. Scale bars are 20 μm. b, d Quantification of the mitochondria area in individual cells. The mean ± SD is reported (b n = 29 (– match550), n = 34 (+ match550); d n = 44 (– match550), n = 19 (+ match550)). A two-tailed Welch’s t-test was used to compare the two distributions. ** p value = 0.0022, ns: non significant p value = 0.9859.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4 and Supplementary Tables 1 and 2.

Reporting Summary

Supplementary Video 1

Fluorogenic rerouting of cytosolic proteins to mitochondria with match550. HeLa cells coexpressing mCherry–FIREtag together with Tom20–eCFP–FIREmate were treated with 10 μM match550 and imaged by timelapse confocal microscopy (Fig. 1c,d). Experiments were repeated three times with similar results. Red (mCherry), ex/em 561/606–675 nm; green (CATCHFIRE), ex/em 488/508–570 nm; cyan (eCFP), ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 2

Kinetics of CATCHFIRE. HeLa cells coexpressing mCherry–FIREtag together with Tom20–eCFP–FIREmate were treated with 10 μM match550 and imaged by fast timelapse confocal microscopy (see also Extended Data Fig. 3a,b). Experiments were repeated three times with similar results. Green (CATCHFIRE), ex/em 488/508–570 nm. Scale bar, 20 μm.

Supplementary Video 3

Stability of CATCHFIRE. U2OS cells coexpressing mCherry–FIREtag and Tom20–eCFP–FIREmate were treated with match550 and image by timelapse confocal microscopy at one image every 2 min (see also Extended Data Fig. 3c,d). Experiment was repeated three times with similar results. Red (mCherry), ex/em 561/606–675 nm; green (CATCHFIRE), ex/em 488/508–570 nm; cyan (eCFP), ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 4

Fluorogenic rerouting of cytosolic proteins to mitochondria with match550. HeLa cells coexpressing FIREtag–mCherry together with Tom20–eCFP–FIREmate were treated with 10 μM match550 and imaged by timelapse confocal microscopy (see also Extended Data Fig. 4a,b). Experiments were repeated three times with similar results. Red (mCherry), ex/em 561/606–675 nm; green (CATCHFIRE), ex/em 488/508–570 nm; cyan (eCFP), ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 5

Fluorogenic rerouting of cytosolic proteins to mitochondria with match550. HeLa cells coexpressing mCherry–FIREtag–mCherry together with Tom20–eCFP–FIREmate were treated with 10 μM match550 and imaged by timelapse confocal microscopy (see also Extended Data Fig. 4c,d). Experiments were repeated three times with similar results. Red (mCherry), ex/em 561/606–675 nm; green (CATCHFIRE), ex/em 488/508–570 nm; cyan (eCFP), ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 6

Fluorogenic rerouting of cytosolic proteins to mitochondria with match540. HeLa cells coexpressing mCherry–FIREtag together with Tom20–eCFP–FIREmate were treated with 10 μM match540 and imaged by timelapse confocal microscopy (see also Fig. 1e,f). Experiments were repeated three times with similar results. Red (mCherry), ex/em 561/606–675 nm; green (CATCHFIRE), ex/em 488/508–570 nm; cyan (eCFP), ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 7

Fluorogenic rerouting of cytosolic proteins to mitochondria with match600. HeLa cells coexpressing EGFP–FIREtag together with Tom20–eCFP–FIREmate were treated with 10 μM match600 and imaged by timelapse confocal microscopy (see also Fig. 1g,h). Experiments were repeated three times with similar results. Red (mCherry): ex/em 561/606–675 nm; green (CATCHFIRE): ex/em 488/508–570 nm; cyan (eCFP), ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 8

Fluorogenic rerouting of cytosolic proteins to mitochondria with matchdark. HeLa cells coexpressing mCherry–FIREtag together with Tom20–eCFP–FIREmate were treated with 10 μM matchdark and imaged by timelapse confocal microscopy (see also Fig. 1i,j). Experiments were repeated three times with similar results. Red (mCherry), ex/em 561/606–675 nm; green (CATCHFIRE), ex/em 488/508–570 nm; cyan (eCFP), ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 9

Fluorogenic rerouting of cytosolic proteins to the Golgi apparatus with match550. HeLa cells coexpressing mCherry–FIREtag together with FIREmate–eCFP–Giantin were treated with 10 μM match550 and imaged by timelapse confocal microscopy (see also Fig. 2a,b). Experiments were repeated three times with similar results. Red (mCherry), ex/em 561/606–675 nm; green (CATCHFIRE), ex/em 488/508–570 nm; cyan (eCFP), ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 10

Fluorogenic rerouting of cytosolic proteins to the ER with match550. HeLa cells coexpressing mCherry–FIREtag together with FIREmate–eCFP–Cb5 were treated with 10 μM match550 and imaged by timelapse confocal microscopy (see also Fig. 2c,d). Experiments were repeated three times with similar results. Red (mCherry), ex/em 561/606–675 nm; green (CATCHFIRE), ex/em 488/508–570 nm; cyan (eCFP), ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 11

Fluorogenic rerouting of cytosolic proteins to the plasma membrane with match550. HeLa cells coexpressing mCherry–FIREtag together with Lyn11–FIREmate–eCFP were treated with 10 μM match550 and imaged by timelapse confocal microscopy (see also Fig. 2e,f). Experiments were repeated three times with similar results. Red (mCherry), ex/em 561/606–675 nm; green (CATCHFIRE), ex/em 488/508–570 nm; cyan (eCFP), ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 12

Fluorogenic rerouting of cytosolic proteins to the Golgi apparatus with match540. HeLa cells coexpressing mCherry–FIREtag together with FIREmate–eCFP–Giantin were treated with 10 μM match540 and imaged by timelapse confocal microscopy (see also Extended Data Fig. 5a,b). Experiments were repeated three times with similar results. Red (mCherry), ex/em 561/606–675 nm; green (CATCHFIRE), ex/em 488/508–570 nm; cyan (eCFP), ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 13

Fluorogenic rerouting of cytosolic proteins to the ER with match540. HeLa cells coexpressing mCherry–FIREtag together with FIREmate–eCFP–Cb5 were treated with 10 μM match540 and imaged by timelapse confocal microscopy (see also Extended Data Fig. 5c,d). Experiments were repeated three times with similar results. Red (mCherry), ex/em 561/606–675 nm; green (CATCHFIRE), ex/em 488/508–570 nm; cyan (eCFP), ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 14

Fluorogenic rerouting of cytosolic proteins to the plasma membrane with match540. HeLa cells coexpressing mCherry–FIREtag together with Lyn11–FIREmate–eCFP were treated with 10 μM match540 and imaged by timelapse confocal microscopy (see also Extended Data Fig. 5e,f). Experiments were repeated three times with similar results. Red (mCherry), ex/em 561/606–675 nm; green (CATCHFIRE), ex/em 488/508–570 nm; cyan (eCFP), ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 15

Fluorogenic recruitment of proteins is reversible through washout. HeLa cells coexpressing mCherry–FIREtag and Giantin–IRFP–FIREmate were treated with 10 μM match550 for 240 s, washed for 212 s, and then treated with 10 μM match550 for 212 s, and then washed again. Cells were imaged by timelapse spinning-disk microscopy (see also Fig. 2g). Experiments were repeated five times with similar results. Red (mCherry), ex/em 561/604–664 nm; green (CATCHFIRE), ex/em 488/545–575 nm; cyan (IRFP), ex/em 633/700–770 nm. Scale bar, 10 μm.

Supplementary Video 16

Fluorogenic recruitment of proteins with cagedFIREtag. HeLa cells coexpressing mCherry–cagedFIREtag and FIREmate–eCFP–Giantin were treated with 10 μM match550 and imaged by timelapse confocal microscopy (see also Extended Data Fig. 8c,d). Experiments were repeated three times with similar results. Red, ex/em 561/606–675 nm; green, ex/em 488/508–570 nm; cyan, ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 17

Fluorogenic induction of nuclear export. HeLa cells coexpressing NLS–mCherry–FIREtag and NES–eCFP– FIREmate were treated with 10 μM match550 and imaged by timelapse confocal microscopy (see also Fig. 3a–d). Experiments were repeated three times with similar results. Red, ex/em 561/606–675 nm; green, ex/em 488/508–570 nm; cyan, ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 18

Fluorogenic induction of nuclear import. HeLa cells coexpressing NLS–mCherry–FIREtag–NES and H2B–eCFP– FIREmate were treated with 10 μM match550 and imaged by timelapse confocal microscopy (see also Fig. 3e–h). Experiments were repeated three times with similar results. Red, ex/em 561/606–675 nm; green, ex/em 488/508–570 nm; cyan, ex/em 445/455–499 nm. Scale bar, 20 μm.

Supplementary Video 19

Control of secretory protein trafficking. HeLa cells coexpressing FIREmate–KDEL and TNF–mCherry–FIREtag were treated with match550 for 24 h and imaged by spinning-disk microscopy after washout of match550 (see also Fig. 4a–c). Experiments were repeated three times with similar results. Red (mCherry), ex/em 561/604–664 nm; green (CATCHFIRE), ex/em 488/545–575 nm. Scale bar, 10 μm. Images were enhanced by 2D deconvolution on NIS-Elements v.5.42.

Supplementary Video 20

Control of secretory protein trafficking. HeLa cells coexpressing FIREmate–KDEL and mApple–FIREtag–GPI were treated with match550 for 24 h and imaged by spinning-disk microscopy after washout of match550 (see also Extended Data Fig. 9). Experiments were repeated five times with similar results. Red (mApple), ex/em 561/604–664 nm; green (CATCHFIRE), ex/em 488/545–575 nm. Scale bar, 10 μm. Images were enhanced by 2D deconvolution on NIS-Elements v.5.42.

Supplementary Video 21

Control of secretory protein trafficking. HeLa cells coexpressing FIREmate–KDEL and ManII–mApple–FIREtag were treated with match550 for 24 h and imaged by spinning-disk microscopy after washout of match550 (see also Fig. 4d–f). Match550 was re-added after 50 min. Experiments were conducted in 25 μg ml−1 cycloheximide. Experiments were repeated five times with similar results. Red (mApple), ex/em 561/604–664 nm; green (CATCHFIRE), ex/em 488/545–575 nm. Scale bar, 10 μm. Images were enhanced by 2D deconvolution on NIS-Elements v.5.42.

Supplementary Video 22

Control and tracking of organelle positioning. HeLa cells coexpressing LAMP1–mCherry–FIREtag and FIREmate–KIF17 were imaged by spinning-disk microscopy for 35 min, then match550 was added. After 50 min of treatment, match550 was washed out and cells were imaged for 75 min (see also Fig. 5). Experiments were repeated five times with similar results. Red (mCherry), ex/em 561/604–664 nm; green (CATCHFIRE), ex/em 488/545–575 nm. Scale bar, 10 μm. Images were enhanced by 2D deconvolution on NIS-Elements v.5.42.

Supplementary Video 23

Fluorogenic rerouting of PINK1 to the outer mitochondrial membrane. HeLa cells coexpressing eCFP–FIREmate–Fis1 and PINK1–mCherry–FIREtag were treated without or with 10 μM match550 and imaged by timelapse confocal microscopy (see also Supplementary Fig. 2). Experiments were repeated three times with similar results. Red, ex/em 561/606–675 nm; green, ex/em 488/508–570 nm; cyan, ex/em 445/455–499 nm. Scale bar, 20 μm.

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Bottone, S., Joliot, O., Cakil, Z.V. et al. A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity. Nat Methods 20, 1553–1562 (2023). https://doi.org/10.1038/s41592-023-01988-8

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