Measuring ERK Activity Dynamics in Single Living Cells Using FRET Biosensors

Yannick Blum; Rafael D Fritz; Hyunryul Ryu; Olivier Pertz

doi:10.1007/978-1-4939-6424-6_15

Measuring ERK Activity Dynamics in Single Living Cells Using FRET Biosensors

Methods Mol Biol. 2017:1487:203-221. doi: 10.1007/978-1-4939-6424-6_15.

Authors

Yannick Blum^{1

2}, Rafael D Fritz¹, Hyunryul Ryu^{3

4

5}, Olivier Pertz^{6

7}

Affiliations

¹ Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058, Basel, Switzerland.
² Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012, Bern, Switzerland.
³ School of Mechanical and Aerospace Engineering, Seoul National University, Seoul, 151-742, Republic of Korea.
⁴ Institute of Advanced Machinery and Design, Seoul National University, Seoul, 151-742, Republic of Korea.
⁵ Research Laboratory of Electronics, Massachusetts Institute of Technology, 77 Massachusetts avenue, Cambridge, MA, 02139, USA.
⁶ Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058, Basel, Switzerland. olivier.pertz@izb.unibe.ch.
⁷ Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012, Bern, Switzerland. olivier.pertz@izb.unibe.ch.

PMID: 27924569
DOI: 10.1007/978-1-4939-6424-6_15

Abstract

Fluorescence resonance energy transfer (FRET)-based biosensors are powerful tools for measuring spatio-temporal signaling dynamics in single living cells with subcellular resolution. There are quite a number of already existing sensors and this technology is increasingly used to obtain quantitative dynamic datasets. In this chapter, we describe the analysis of endogenous extracellular signal-regulated kinase (ERK) activity in living cells using the EKAR2G (ERK activity reporter second generation) probe. We focus on the generation of stable cell lines expressing the EKAR2G sensor as well as data acquisition and analysis.

Keywords: Biosensors; Cell fate; Cell-cell variability; EKAR2G; ERK dynamics; Fluorescence resonance energy transfer (FRET); High throughput; Live-cell microscopy; PC12 cells; Ratiometric FRET imaging; Signaling heterogeneity.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Biosensing Techniques* / methods
Cell Line
Enzyme Activation
Extracellular Signal-Regulated MAP Kinases / metabolism*
Fluorescence Resonance Energy Transfer* / methods
Gene Expression
Genes, Reporter
Genetic Vectors / genetics
High-Throughput Screening Assays
Humans
Lentivirus / genetics
Mice
Microscopy, Fluorescence
Reproducibility of Results
Signal Transduction
Single-Cell Analysis* / methods
Transduction, Genetic

Substances

Extracellular Signal-Regulated MAP Kinases