Figure 1: Control of optoGEF-RhoA localization.

(a) Scheme of the optogenetic system to control cell contractility. The system is based on overexpressing a RhoA activator (DHPH domain of ARHGEF11) fused to the light-sensitive protein CRY2-mcherry. The resulting protein is called optoGEF-RhoA. In the absence of blue light, a fraction of the RhoA pool is active because of endogenous activity and overexpression of the RhoA activator (left scheme). Upon illumination, CRY2 changes conformation and binds to its optogenetic partner CIBN. To increase contractility, we forced translocation of optoGEF-RhoA to the cell surface, where RhoA is located, by targeting CIBN-GFP to the plasma membrane (top right panel). To decrease contractility, we sequestered optoGEF-RhoA at mitochondria by targeting CIBN-GFP to the mitochondrial membrane (bottom right panel). (b,c) Confluent MDCK cells stably expressing CIBN-GFP-CAAX (b) and optoGEF-RhoA before and after blue light illumination (c). Illumination was restricted to the central area of the field of view represented by a blue square. The temporal pattern of illumination is indicated by the upper blue line. (d,e) Subconfluent MDCK cells co-transfected with mito-CIBN-GFP (d) and optoGEF-RhoA (e). In b–e, bottom panels show zoomed areas marked by the white rectangles. (f,g) Quantification of optoGEF-RhoA both at the cell membrane (f) and at the cell mitochondria (g) over the 20 min of experiment (n=8 and n=14 fields of view, respectively). Data are shown as mean±s.e.m. Cells were illuminated with 2 pulses of blue light separated by 10 s at time t=0. Scale bars, 20 μm.