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

Sensors

Markus Waldeck-Weiermair¹  Helmut Bischof²  Sandra Blass²  Andras T. Deak²  Christiane Klec²  Thomas Graier²  Clara Roller²  Rene Rost²  Emrah Eroglu²  Benjamin Gottschalk²  Nicole A. Hofmann²  Wolfgang F. Graier²  Roland Malli²  Niko Hildebrandt²  Igor Medintz² 

[1] Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria;

关键词: Förster resonance energy transfer;    FRET;    calcium;    endoplasmic reticulum;    store operated calcium entry;    SOCE;    clover;    mRuby2;    dissociation constant;   

DOI  :  10.3390/s150613052

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.