Differential optogenetic stimulation of Rac and Cdc42 induces lamellipodia and directed migration on a FN substrate. Differential optogenetic stimulation.

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Differential optogenetic stimulation of Rac and Cdc42 induces lamellipodia and directed migration on a FN substrate. Differential optogenetic stimulation of Rac and Cdc42 induces lamellipodia and directed migration on a FN substrate. (A) Schematic diagram of optogenetic switch to regulate GTPases by localizing specific GEF DH/PH domains to the cell membrane. T represents GTP-bound GTPase, while D represents GDP-bound GTPase. (B) Representative fluorescence micrographs of live IA32 fibroblasts being optogenetically activated. Top panels show optogenetic recruitment of RFP control, while the middle and bottom show recruitment of the Tiam1 and ITSN1 DH/PH domains over time. Right panels are kymographs along the yellow line. Blue arrows denote activated areas. Scale bars: 50 µm (fluorescence images); 5 µm and 250 s (kymographs). (C) Schematic diagram of the optotaxis chamber. (D) Image of the light gradient produced by the optotaxis chamber. (E) FMI graph for fibroblasts expressing Venus-iLID-CAAX and Tiam1-RFP to determine haptotaxis in response to light intensities at the positions demonstrated in D. Results are mean±95% c.i. (F) Rose plots, FMI graphs and velocity plots representing migration vectors for control fibroblasts, and fibroblasts expressing Tiam1- and ITSN1-Micro (with iLID-CAAX). The blue triangle represents the light gradient. Error bars are the 95% c.i. *P<0.05. In B,E and F, cells were plated on 10 µg/ml FN. Refer to Table S1 for experimental details. Seth P. Zimmerman et al. J Cell Sci 2017;130:2971-2983 © 2017. Published by The Company of Biologists Ltd