@inbook{81ed1198e1094cdb9411b2ef0bfd152d,
title = "Optogenetic control of Ras/Erk signaling using the Phy–PIF system",
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 = "Erk, MAP kinase, Optogenetics, Ras, Signal transduction, Single-cell dynamics",
author = "Goglia, {Alexander G.} and Wilson, {Maxwell Z.} and DiGiorno, {Daniel B.} and Toettcher, {Jared E.}",
note = "Publisher Copyright: {\textcopyright} 2017, Springer Science+Business Media LLC.",
year = "2017",
doi = "10.1007/978-1-4939-7154-1_1",
language = "English (US)",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "3--20",
booktitle = "Methods in Molecular Biology",
}