|Title||Optogenetic Control of Ras/Erk Signaling Using the Phy-PIF System.|
|Publication Type||Journal Article|
|Year of Publication||2017|
|Authors||Goglia, AG, Wilson, MZ, DiGiorno, DB, Toettcher, JE|
|Journal||Methods Mol Biol|
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.
|Alternate Journal||Methods Mol. Biol.|