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Sensors 2015, 15(6), 13052-13068; doi:10.3390/s150613052

Generation of Red-Shifted Cameleons for Imaging Ca2+ Dynamics of the Endoplasmic Reticulum

Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria
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Academic Editors: Niko Hildebrandt, Igor Medintz and Russ Algar
Received: 30 March 2015 / Revised: 21 May 2015 / Accepted: 26 May 2015 / Published: 4 June 2015
(This article belongs to the Special Issue FRET Biosensors)
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Abstract

Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store. View Full-Text
Keywords: Förster resonance energy transfer; FRET; calcium; endoplasmic reticulum; store operated calcium entry; SOCE; clover; mRuby2; dissociation constant Förster resonance energy transfer; FRET; calcium; endoplasmic reticulum; store operated calcium entry; SOCE; clover; mRuby2; dissociation constant
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Waldeck-Weiermair, M.; Bischof, H.; Blass, S.; Deak, A.T.; Klec, C.; Graier, T.; Roller, C.; Rost, R.; Eroglu, E.; Gottschalk, B.; Hofmann, N.A.; Graier, W.F.; Malli, R. Generation of Red-Shifted Cameleons for Imaging Ca2+ Dynamics of the Endoplasmic Reticulum. Sensors 2015, 15, 13052-13068.

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