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A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space

Fritz, R. D. and Letzelter, M. and Reimann, A. and Martin, K. and Fusco, L. and Ritsma, L. and Ponsioen, B. and Fluri, E. and Schulte-Merker, S. and van Rheenen, J. and Pertz, O.. (2013) A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space. Science signaling, Vol. 6, H. 285 , rs12.

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Official URL: http://edoc.unibas.ch/dok/A6338216

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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.
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Former Units at DBM > Cell Migration and Neuritogenesis (Pertz)
UniBasel Contributors:Pertz, Olivier
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:American Association for the Advancement of Science
ISSN:1937-9145
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:10 Apr 2015 09:12
Deposited On:10 Apr 2015 09:12

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