Optogenetic Control of Ras/Erk Signaling Using the Phy-PIF System.

<p>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.</p>