A genetically encoded fluorescent sensor of ERK activity.
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.
Harvey
Christopher D
CD
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.
Ehrhardt
Anka G
AG
Cellurale
Cristina
C
Zhong
Haining
H
Yasuda
Ryohei
R
Davis
Roger J
RJ
Svoboda
Karel
K
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D016477:Artifacts; D001426:Bacterial Proteins / Q000235:genetics; D002460:Cell Line; D003712:Dendrites / Q000201:enzymology; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics; D020935:MAP Kinase Signaling System; D009924:Organ Culture Techniques; D018408:Patch-Clamp Techniques; D017966:Pyramidal Cells / Q000201:enzymology*; D051381:Rats
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D048049:Extracellular Signal-Regulated MAP Kinases