Figure 5: Optogenetic changes in cell contractility regulate mechanosensitive signalling pathways.

(a) iRFP images of one representative cell expressing optoGEF-RhoA, CIBN-GFP-CAAX and iRFP-YAP before and during blue illumination (7.5 min of imaging followed by 52.5 min of imaging and activation, one activation pulse every 45 s). (b) Nuclear YAP fluorescence intensity over time for the example shown in a. (c) Quantification of relative nuclear YAP over time for control cells expressing CRY2-mcherry, CIBN-GFP-CAAX, and iRFP-YAP (black, n=330 cells) and for cells expressing optoGEF-RhoA, CIBN-GFP-CAAX and iRFP-YAP (green, n=270 cells) subjected to the same activating routine as in a. (c, right) Distribution of relative nuclear YAP in the experimental population after 50 min of activation. (d) Quantification of relative nuclear YAP over time for cells subjected to two activation periods (same period as in a) separated by 1 h of no illumination (n=222 cells). (e) iRFP images of 2 representative cells expressing optoGEF-RhoA, mito-CIBN-GFP and iRFP-YAP during blue light illumination (same illumination protocol as in a). (f) Nuclear YAP fluorescence intensity over time for the example represented in e. (g) Quantification of relative nuclear YAP over time for control cells expressing CRY2-mCherry, mito-CIBN-GFP and iRFP-YAP (black, n=177 cells) and for cells expressing optoGEF-RhoA, mito-CIBN-GFP, and iRFP-YAP (red, n=250 cells) subjected to the same activating routine as in a. (g, right) Distribution of relative nuclear YAP in the experimental population after 20 min of activation. (h) Quantification of relative nuclear YAP over time for cells subjected to two activation routines (60 min of illumination, one activation pulse every 60 s) each of them followed by 1 h of no illumination (n=166 cells). Error bars are the s.d. between the mean curves of each independent experiment (three independent experiments for each graph). Scale bars, 20 μm.