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

Sensors - Tập 15 Số 6 - Trang 13052-13068
Markus Waldeck‐Weiermair1, Helmut Bischof1, Sandra Blaß1, András T. Deak1, Christiane Klec1, Thomas Graier1, Clara A. Roller1, René Rost1, Emrah Eroğlu1, Benjamin Gottschalk1, Nicole Hofmann1, Wolfgang F. Graier1, Roland Malli1
1Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.

Tóm tắt

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.

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Sensors

Tập 15 Số 6

13052-13068

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