Journal: Nature methods

Article Title: A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity.

doi: 10.1038/s41592-023-01988-8

Figure Lengend Snippet: Fig. 2 | CATCHFIRE enables the control of protein dimerization with high temporal control. a–f, HeLa cells coexpressing mCherry–FIREtag and FIREmate– eCFP–Giantin (a,b), FIREmate–eCFP–Cb5 (c,d), Lyn11–FIREmate–eCFP (e,f) were treated with 10 μM match550 and imaged by timelapse confocal microscopy. Representative confocal micrographs of cells before (0 min) and after (5 min) addition of match550 (Supplementary Videos 9–11) (a,c,e). Experiments were repeated three times with similar results. Temporal evolution of the CATCHFIRE signal (b,d,f). Data represent the mean ± s.d. of three independent experiments (n = 11 cells (b), 15 cells (d) and 15 cells (f)). g,h, 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 match540 for 212 s and washed

Article Snippet: The plasmid pAG1159 allowing the expression of TOM20– eCFP–FIREmate was constructed by replacing the sequence coding for FRB by the FIREmate sequence in the Addgene vector #171461 Tom20–CR coding for TOM20–eCFP–FRB30.

Techniques: Control, Confocal Microscopy

Journal: Nature methods

Article Title: A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity.

doi: 10.1038/s41592-023-01988-8

Figure Lengend Snippet: Fig. 3 | Control and tracking of nucleocytoplasmic shuttling. a–d, HeLa cells coexpressing NLS–mCherry–FIREtag and NES–eCFP–FIREmate were treated with 10 μM match550 and imaged by timelapse confocal microscopy. Representative confocal micrographs before and after treatment with match550 (see also Supplementary Video 17) (b). Experiments were repeated three times with similar results. Temporal evolution of the CATCHFIRE signal (c). Data represent the mean ± s.d. of n = 5 cells from two independent experiments. Temporal evolution of the ratio nucleus-to-cytoplasm mCherry fluorescence of n = 5 cells from two independent experiments (d). e–h, HeLa cells coexpressing NLS–mCherry–

Article Snippet: The plasmid pAG1159 allowing the expression of TOM20– eCFP–FIREmate was constructed by replacing the sequence coding for FRB by the FIREmate sequence in the Addgene vector #171461 Tom20–CR coding for TOM20–eCFP–FRB30.

Techniques: Control, Confocal Microscopy, Fluorescence

Journal: Nature methods

Article Title: A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity.

doi: 10.1038/s41592-023-01988-8

Figure Lengend Snippet: Fig. 4 | Control and tracking of secretory protein trafficking through RUCH. a, Schematic illustrating how FIREmate–KDEL acts as a hook to retain FIREtagged reporter in the ER 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 coexpressing FIREmate–KDEL and TNF–mCherry–FIREtag were treated with match550 for 24 h and imaged by spinning disk microscopy after washout of match550. Representative micrographs before washout, 15 min and 60 min after washout (see also Supplementary Video 19) (b). Experiments were repeated three times with similar results. Temporal evolution of the global CATCHFIRE signal (observed in the ER, green) and the mCherry signal quantified in the Golgi apparatus (red) (c). Data represent the mean ± s.d. of eight cells. d, Schematic illustrating how the RUCH approach can be used to study the

Article Snippet: The plasmid pAG1159 allowing the expression of TOM20– eCFP–FIREmate was constructed by replacing the sequence coding for FRB by the FIREmate sequence in the Addgene vector #171461 Tom20–CR coding for TOM20–eCFP–FRB30.

Techniques: Control, Microscopy

Journal: Nature methods

Article Title: A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity.

doi: 10.1038/s41592-023-01988-8

Figure Lengend Snippet: Fig. 5 | Control and tracking of organelle positioning. a, Schematic illustrating how match-induced interaction between LAMP1–mCherry–FIREtag and FIREmate– KIF17 allows the chemically induced anterograde transport of lysosomes. b, 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.

Article Snippet: The plasmid pAG1159 allowing the expression of TOM20– eCFP–FIREmate was constructed by replacing the sequence coding for FRB by the FIREmate sequence in the Addgene vector #171461 Tom20–CR coding for TOM20–eCFP–FRB30.

Techniques: Control, Microscopy