title: Measuring ERK Activity Dynamics in Single Living Cells Using FRET Biosensors.
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.
authors.affiliation: Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058, Basel, Switzerland.|Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012, Bern, Switzerland.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Blum
authors.affiliation: Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058, Basel, Switzerland.
authors.forename: Rafael D
authors.initials: RD
authors.lastname: Fritz
authors.affiliation: School of Mechanical and Aerospace Engineering, Seoul National University, Seoul, 151-742, Republic of Korea.|Institute of Advanced Machinery and Design, Seoul National University, Seoul, 151-742, Republic of Korea.|Research Laboratory of Electronics, Massachusetts Institute of Technology, 77 Massachusetts avenue, Cambridge, MA, 02139, USA.
authors.forename: Hyunryul
authors.initials: H
authors.lastname: Ryu
authors.affiliation: Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058, Basel, Switzerland. olivier.pertz@izb.unibe.ch.|Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012, Bern, Switzerland. olivier.pertz@izb.unibe.ch.
authors.forename: Olivier
authors.initials: O
authors.lastname: Pertz
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods; D002460:Cell Line; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D057166:High-Throughput Screening Assays; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D015203:Reproducibility of Results; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods; D014161:Transduction, Genetic
chemical_list: D048049:Extracellular Signal-Regulated MAP Kinases
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