Journal: Journal of Cell Science

Article Title: OptoLoop – an optogenetic tool to probe the functional role of genome organization

doi: 10.1242/jcs.264574

Figure Lengend Snippet: Exploration of optogenetic clustering properties of CRY2. (A) Top panels, time-lapse images of a NIH3T3 cell expressing CRY2high–mCherry activated with a 488-nm microscope laser starting at time t =0 (blue vertical arrow, 1.5 s pulses every 10 s). Scale bars: 5 µm. Bottom panel, coefficient of variation (CV) of fluorescence intensity calculated as the ratio between the nuclear intensity standard deviation and the nuclear intensity mean, presented as relative to the CV at time t =0. Data points corresponding to the images are marked in red. (B) Top panel, protein sequence of the C-terminus of the CRY2 PHR domain and part of the artificial linker used for C-terminal fusions for wild-type CRY2 (CRY2wt) and for CRY2 mutants. The newly generated variant CRY2hiclu is marked in bold. Mutations relative to the CRY2wt sequence are highlighted in gray. Bottom panel, images of NIH3T3 cells expressing CRY2 mutants fused to mCherry, illuminated with 1 s blue light pulses every 10 s for 15 min, and then fixed. The nucleus is delimited with a yellow line. Scale bars: 5 µm. (C) CV calculated from images obtained from NIH3T3 cells expressing CRY2 variants fused to mCherry, illuminated with pulsed blue light for 15 min, and then fixed, plotted as a function of mCherry nuclear intensity. ∼25 cells were analyzed per sample (each dot represents one cell). Continuous lines represent simple logistic fits. (D) Time-lapse images of a NIH3T3 cell expressing CRY2hiclu–mCherry activated once with the 488-nm microscope laser for 15 s at time t =0 (marked with a blue arrow). Scale bars: 5 µm. (E) Mean ( n =25) CV calculated from time-lapse images obtained from NIH3T3 cells expressing CRY2olig-mCherry, illuminated with blue light at time t =0, and then kept without blue light. The clustering ( t c ) and declustering times ( t d ) were determined from individual kinetic curves. (F) t c (top panel) and t d (bottom panel) represented as a function of mCherry nuclear intensity. ∼25–40 cells were analyzed per sample (each dot represents one cell). Continuous lines represent simple exponential (clustering) and linear (declustering) fits.

Article Snippet: NIH3T3 (mouse fibroblasts, ATCC #CRL-1658), U2OS (from human osteosarcoma, ATCC #HTB-96), HeLa (from human cervical adenocarcinoma, ATCC #CRM-CCL-2) and Lenti-X HEK-293T (from human embryonic kidney, cat. #632180 from Takara Bio, Japan) cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco, Waltham, MA, USA) supplemented with 10% (15% for NIH3T3) fetal bovine serum (Gibco, Waltham, MA, USA) plus 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco, Waltham, MA, USA) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Microscopy, Fluorescence, Standard Deviation, Sequencing, Generated, Variant Assay