title: A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space.
abstract: Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.
authors.affiliation: Institute of Biochemistry and Genetics, Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland.
authors.forename: Rafael D
authors.initials: RD
authors.lastname: Fritz
authors.forename: Michel
authors.initials: M
authors.lastname: Letzelter
authors.forename: Andreas
authors.initials: A
authors.lastname: Reimann
authors.forename: Katrin
authors.initials: K
authors.lastname: Martin
authors.forename: Ludovico
authors.initials: L
authors.lastname: Fusco
authors.forename: Laila
authors.initials: L
authors.lastname: Ritsma
authors.forename: Bas
authors.initials: B
authors.lastname: Ponsioen
authors.forename: Erika
authors.initials: E
authors.lastname: Fluri
authors.forename: Stefan
authors.initials: S
authors.lastname: Schulte-Merker
authors.forename: Jacco
authors.initials: J
authors.lastname: van Rheenen
authors.forename: Olivier
authors.initials: O
authors.lastname: Pertz
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002454:Cell Differentiation; D002465:Cell Movement; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D015398:Signal Transduction; D015027:Zebrafish
chemical_list: C485182:Cyan Fluorescent Protein; D049452:Green Fluorescent Proteins; D048049:Extracellular Signal-Regulated MAP Kinases