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

Fritz, Rafael D; Letzelter, Michel; Reimann, Andreas; Martin, Katrin; Fusco, Ludovico; Ritsma, Laila; Ponsioen, Bas; Fluri, Erika; Schulte-Merker, Stefan; van Rheenen, Jacco; Pertz, Olivier (2013). A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space. Science signaling, 6(285), rs12. American Association for the Advancement of Science 10.1126/scisignal.2004135

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

08 Faculty of Science > Department of Biology > Institute of Cell Biology

UniBE Contributor:

Pertz, Olivier

Subjects:

500 Science > 570 Life sciences; biology

ISSN:

1937-9145

Publisher:

American Association for the Advancement of Science

Language:

English

Submitter:

Olivier Pertz

Date Deposited:

03 May 2016 10:22

Last Modified:

03 May 2016 10:22

Publisher DOI:

10.1126/scisignal.2004135

PubMed ID:

23882122

URI:

https://boris.unibe.ch/id/eprint/81857

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