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PMID: 19033456 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

A genetically encoded fluorescent sensor of ERK activity.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 105 ·No. 49 ·2008-12-09 ·Pages 19264-9

Harvey CD, Ehrhardt AG, Cellurale C, Zhong H, Yasuda R, Davis RJ, Svoboda K

Abstract

The activity of the ERK has complex spatial and temporal dynamics that are important for the specificity of downstream effects. However, current biochemical techniques do not allow for the measurement of ERK signaling with fine spatiotemporal resolution. We developed a genetically encoded, FRET-based sensor of ERK activity (the extracellular signal-regulated kinase activity reporter, EKAR), optimized for signal-to-noise ratio and fluorescence lifetime imaging. EKAR selectively and reversibly reported ERK activation in HEK293 cells after epidermal growth factor stimulation. EKAR signals were correlated with ERK phosphorylation, required ERK activity, and did not report the activities of JNK or p38. EKAR reported ERK activation in the dendrites and nucleus of hippocampal pyramidal neurons in brain slices after theta-burst stimuli or trains of back-propagating action potentials. EKAR therefore permits the measurement of spatiotemporal ERK signaling dynamics in living cells, including in neuronal compartments in intact tissues.

MeSH Terms
Action Potentials/physiology Animals Artifacts Bacterial Proteins/genetics Cell Line Dendrites/enzymology Endothelial Cells/cytology,enzymology Enzyme Activation Extracellular Signal-Regulated MAP Kinases/genetics,metabolism Fluorescence Resonance Energy Transfer/methods Fluorescent Dyes Genes, Reporter Green Fluorescent Proteins/genetics Hippocampus/cytology Humans Kidney/cytology Luminescent Proteins/genetics MAP Kinase Signaling System Organ Culture Techniques Patch-Clamp Techniques Pyramidal Cells/enzymology Rats
Chemicals
Bacterial Proteins Fluorescent Dyes Luminescent Proteins yellow fluorescent protein, Bacteria Green Fluorescent Proteins Extracellular Signal-Regulated MAP Kinases
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Harvey Christopher D
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.
Ehrhardt Anka G
Cellurale Cristina
Zhong Haining
Yasuda Ryohei
Davis Roger J
Svoboda Karel
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
1091-6490
DOI
10.1073/pnas.0804598105
Published
2008-12-09
Epub
2008-00-25
Pages
19264-9
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC2614750
Subset
IM
Grants
NIMH NIH HHS · R01 MH080047 · United States
NIMH NIH HHS · R01 MH080047-01 · United States
Howard Hughes Medical Institute · United States
External Links
PubMed source DOI: 10.1073/pnas.0804598105 PMC Full Text View journal details
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