Figure 1.

Construction of an optogenetic tool to control RhoA activity.A, Schematic of opto-RhoA. Spatiotemporal control of RhoA activity is achieved using an optogenetic probe to recruit RhoA-specific GEF LARG to the plasma membrane. An iLID molecule is anchored to the plasma membrane via the CAAX motif, while a protein consisting of SspB fused to the DH domain of LARG is distributed throughout the cytosol. When irradiated with blue light, iLID undergoes a conformational change exposing a binding site for SspB, and driving LARG-DH to the plasma membrane, where it activates RhoA. B, Representative images of HeLa cells expressing opto-RhoA before (458-nm light OFF) and after (458-nm light ON) light irradiation with a 458-nm laser. Scale bar = 20 μm. C, Representative images of HeLa cells expressing opto-RhoA and mCherry-RBD_rhotekin. Opto-RhoA was locally activated by a 458-nm laser within 300–600 s every 20 s. Blue boxes in images indicate the activated area, and lower panels showed high magnification images for this area. D, Quantification of local intensity increases of mVenus-SspB-LARG-DH (top) and mCherry-RBD_rhotekin (bottom) from the images shown in (C). The entire area of the cell excluding the region of activation is indicated as the control area. Activation period is indicated by a blue background. Experiments were repeated seven times.