Journal: bioRxiv

Article Title: STIM1 and Endoplasmic Reticulum-Plasma Membrane Contact Sites Oscillate Independently of Calcium-Induced Calcium Release

doi: 10.1101/2025.03.16.643575

Figure Lengend Snippet: (a) Top : Schematic of light-induced plasma membrane translocation of STIM1-Cry2-miRFP670 by Lyn11-CIBN-GFP. Bottom : Representative TIRF images of a cell before and after blue light exposure without antigen stimulation (n=20 cells from 4 experiments). (b) Kymographs and intensity profile of STIM1-Cry2-miRFP670 and GECO1 during exposure to blue light pulses. Movies were acquired 10-30min after antigen stimulation. n=15 cells from 4 experiments. For TIRF images, scale bar =10 µm; for kymographs, scale bar =1min (horizontal bar), 5 µm (vertical bar).

Article Snippet: GCaMP3 (addgene #22692), R-GECO1 (addgene #32444), mCherry-Sec61b (addgene #90994), GFP-MAPPER (addgene #117721), G-CEPIA1er (addgene #58215), CIBN (deltaNLS)-pmGFP (shown as CIBN-GFP-CAAX in text)(addgene #26867), Lyn11-CIBN-GFP (addgene #79572), iRFP (addgene #31857), pmiRFP670-N1 (#79987) from Addgene.

Techniques: Membrane, Translocation Assay

Journal: bioRxiv

Article Title: A tripartite chemogenetic fluorescent reporter for imaging ternary protein interactions

doi: 10.1101/2023.10.19.563144

Figure Lengend Snippet: a,b Normalized average fluorescence of about 20,000 HEK293T cells co-expressing the triplet pFAST 99-114 – N FRB, C FRB–pFAST 1-98 and FKBP– pFAST 115-125 treated without or with 500 nM of rapamycin, and with 0, 1, 5, 10, 25 or 50 μM of HBR– 2,5DM. Data represent the mean ± SD of two independent experiments. The concentrations of fluorogen for half maximal complementation in absence (EC 50,–interaction ) or presence (EC 50,+interaction ) of rapamycin are given. c-e HEK293T cells expressing the triplet pFAST 99-114 – N FRB, C FRB–pFAST 1-98 and FKBP– pFAST 115-125 were treated with 10 μM HBR-2,5DM. Cells were imaged by time-lapse confocal microscopy after addition of 500 nM of rapamycin. Experiments were repeated three times with similar results. c Representative micrographs before and after addition of rapamycin (see also Supplementary Movie 3 ). Scale bars 20 μm. d Temporal evolution of the fluorescence signal after addition of rapamycin n = 45 cells from three independent experiments. e Fluorescence fold increase upon addition of rapamycin. f-h HEK293T cells expressing the triplet pFAST 99-114 – N FRB–ECFP–CAAX, Lyn11-mCherry– C FRB–pFAST 1-98 and FKBP–pFAST 115-125 were treated with 10 μM HBR-2,5DM. Cells were imaged by time-lapse confocal microscopy after addition of 500 nM of rapamycin. Experiments were repeated three times with similar results. g Representative micrographs before and after addition of rapamycin (see also Supplementary Movie 4 ). Scale bars 20 μm. h Temporal evolution of the fluorescence signal after addition of rapamycin n = 20 cells from three independent experiments. d,e,h Each cell is color-coded according to the biological replicate it came from. d,h The dot lines represent the mean value of each biological replicate, while the black line represents the mean of the three biological replicates. e The solid circles correspond to the mean of each biological replicate. The black line represents the mean ± SD of the three biological replicates.

Article Snippet: The plasmid pAG1240 allowing the mammalian expression of TOM20-mCherry- C FRB-pFAST 1-98 was constructed by replacing the Lyn11 sequence by TOM20 sequence, amplified from the Addgene vector #171461 Tom20-CR coding for TOM20-ECFP-FRB , in the vector pAG1223.

Techniques: Fluorescence, Expressing, Confocal Microscopy

Journal: bioRxiv

Article Title: A tripartite chemogenetic fluorescent reporter for imaging ternary protein interactions

doi: 10.1101/2023.10.19.563144

Figure Lengend Snippet: a-c HeLa cells co-expressing pFAST 99-114 – N FRB–ECFP–CAAX, Tom20-mCherry– C FRB–pFAST 1-98 and FKBP– pFAST 115-125 were treated with 10 μM HBR-2,5DM. Cells were imaged by time-lapse confocal microscopy after addition of 500 nM of rapamycin. Experiments were repeated three times with similar results. b Representative micrographs before and after addition of rapamycin (see also Supplementary Movie 5 ). c Correlation of the EGFP and mCherry signals before and after rapamycin addition. d-f HeLa cells co-expressing pFAST 99-114 – N FRB–ECFP–Cb5, Lyn11-mCherry– C FRB–pFAST 1-98 and FKBP– pFAST 115-125 were treated with 10 μM HBR-2,5DM. Cells were imaged by time-lapse confocal microscopy after addition of 500 nM of rapamycin. Experiments were repeated three times with similar results. e Representative micrographs before and after addition of rapamycin (see also Supplementary Movie 6 ). f Correlation of the ECFP and mCherry signals before and after rapamycin addition. c Correlation of the EGFP and mCherry signals before and after rapamycin addition. g-i HeLa cells co-expressing pFAST 99-114 – N FRB–ECFP–Cb5, Tom20-mCherry– C FRB–pFAST 1-98 and FKBP–pFAST 115-125 were treated with 10 μM HBR-2,5DM. Cells were imaged by time-lapse confocal microscopy after addition of 500 nM of rapamycin. Experiments were repeated three times with similar results. h Representative micrographs before and after addition of rapamycin (see also Supplementary Movie 7 ). i Correlation of the EGFP and mCherry signals before and after rapamycin addition. j-l HeLa cells co-expressing pFAST 99-114 – N FRB–ECFP–Cb5, Lyn11-mCherry– C FRB–pFAST 1-98 , and Tom20–FKBP– pFAST 115-125 were treated with 10 μM HBR-2,5DM. Cells were imaged by time-lapse confocal microscopy after addition of 500 nM of rapamycin. Experiments were repeated three times with similar results. k Representative micrographs before and after addition of rapamycin (see also Supplementary Movie 8 ). l Correlation of the EGFP and mCherry signals before and after rapamycin addition. c,f,i,l Data represent the mean of n = 10 (c), 9 (f), 10 (i), 11 (l) from three independent experiments. Two-tailed Student’s t-test assuming equal variance was used to compare correlations before and after addition rapamycin. Scale bars 20 μm.

Article Snippet: The plasmid pAG1240 allowing the mammalian expression of TOM20-mCherry- C FRB-pFAST 1-98 was constructed by replacing the Lyn11 sequence by TOM20 sequence, amplified from the Addgene vector #171461 Tom20-CR coding for TOM20-ECFP-FRB , in the vector pAG1223.

Techniques: Expressing, Confocal Microscopy, Two Tailed Test