Generation of Red-Shifted Cameleons for Imaging Ca2+ Dynamics of the Endoplasmic Reticulum
AbstractCameleons 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
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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.
Waldeck-Weiermair M, Bischof H, Blass S, Deak AT, Klec C, Graier T, Roller C, Rost R, Eroglu E, Gottschalk B, Hofmann NA, Graier WF, Malli R. Generation of Red-Shifted Cameleons for Imaging Ca2+ Dynamics of the Endoplasmic Reticulum. Sensors. 2015; 15(6):13052-13068.Chicago/Turabian Style
Waldeck-Weiermair, Markus; Bischof, Helmut; Blass, Sandra; Deak, Andras T.; Klec, Christiane; Graier, Thomas; Roller, Clara; Rost, Rene; Eroglu, Emrah; Gottschalk, Benjamin; Hofmann, Nicole A.; Graier, Wolfgang F.; Malli, Roland. 2015. "Generation of Red-Shifted Cameleons for Imaging Ca2+ Dynamics of the Endoplasmic Reticulum." Sensors 15, no. 6: 13052-13068.