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.
Optogenetics Signal transduction Single-cell dynamics MAP kinase Ras Erk
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We thank Mohammad Seyedsayamdost for assistance and advice with HPLC purification. This work was supported by the NIH National Institute of Biomedical Imaging and Bioengineering (grant DP2EB024247 to J.E.T.), the NIH National Cancer Institute (fellowship F30CA206408 to A.G.G.), and a Princeton University Dean of Research Innovation Award to J.E.T.
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