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

Sensors

Rene Rost¹  Nicole A. Hofmann¹  Markus Waldeck-Weiermair¹  Emrah Eroglu¹  Andras T. Deak¹  Christiane Klec¹  Thomas Graier¹  Helmut Bischof¹  Roland Malli¹  Sandra Blass¹  Wolfgang F. Graier¹  Clara Roller¹  Benjamin Gottschalk¹ 

[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: DOAJ

【 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. WhileGFP/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】

Unknown