Optogenetic Control of Ras/Erk Signaling Using the Phy-PIF System. Author Alexander Goglia, Maxwell Wilson, Daniel DiGiorno, Jared Toettcher Publication Year 2017 Type Journal Article Abstract The Ras/Erk signaling pathway plays a central role in diverse cellular processes ranging from development to immune cell activation to neural plasticity to cancer. In recent years, this pathway has been widely studied using live-cell fluorescent biosensors, revealing complex Erk dynamics that arise in many cellular contexts. Yet despite these high-resolution tools for measurement, the field has lacked analogous tools for control over Ras/Erk signaling in live cells. Here, we provide detailed methods for one such tool based on the optical control of Ras activity, which we call "Opto-SOS." Expression of the Opto-SOS constructs can be coupled with a live-cell reporter of Erk activity to reveal highly quantitative input-to-output maps of the pathway. Detailed herein are protocols for expressing the Opto-SOS system in cultured cells, purifying the small molecule cofactor necessary for optical stimulation, imaging Erk responses using live-cell microscopy, and processing the imaging data to quantify Ras/Erk signaling dynamics. Keywords Animals, Mice, Humans, Signal Transduction, Microscopy, Fluorescence, Biosensing Techniques, Molecular Imaging, ras Proteins, Cell Line, Tumor, Optogenetics, Phytochrome B, Extracellular Signal-Regulated MAP Kinases, Statistics as Topic, Basic Helix-Loop-Helix Transcription Factors, Transduction, Genetic Journal Methods Mol Biol Volume 1636 Pages 3-20 Date Published 2017 ISSN Number 1940-6029 DOI 10.1007/978-1-4939-7154-1_1 Alternate Journal Methods Mol Biol PMID 28730469 PubMedGoogle ScholarBibTeXEndNote X3 XML