Optogenetic control of Ras/Erk signaling using the Phy–PIF system

Alexander G. Goglia, Maxwell Z. Wilson, Daniel B. DiGiorno, Jared E. Toettcher

Research output: Chapter in Book/Report/Conference proceedingChapter

9 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages3-20
Number of pages18
DOIs
StatePublished - Jan 1 2017

Publication series

NameMethods in Molecular Biology
Volume1636
ISSN (Print)1064-3745

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

Keywords

  • Erk
  • MAP kinase
  • Optogenetics
  • Ras
  • Signal transduction
  • Single-cell dynamics

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  • Cite this

    Goglia, A. G., Wilson, M. Z., DiGiorno, D. B., & Toettcher, J. E. (2017). Optogenetic control of Ras/Erk signaling using the Phy–PIF system. In Methods in Molecular Biology (pp. 3-20). (Methods in Molecular Biology; Vol. 1636). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7154-1_1