title: Generation of Red-Shifted Cameleons for Imaging Ca²⁺ Dynamics of the Endoplasmic Reticulum.
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.
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. markus.weiermair@medunigraz.at.
authors.forename: Markus
authors.initials: M
authors.lastname: Waldeck-Weiermair
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. bischof.helmut@gmail.com.
authors.forename: Helmut
authors.initials: H
authors.lastname: Bischof
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. sandra.blass@medunigraz.at.
authors.forename: Sandra
authors.initials: S
authors.lastname: Blass
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. andras.deak@medunigraz.at.
authors.forename: Andras T
authors.initials: AT
authors.lastname: Deak
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. christiane.klec@medunigraz.at.
authors.forename: Christiane
authors.initials: C
authors.lastname: Klec
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. thomas.graier@medunigraz.at.
authors.forename: Thomas
authors.initials: T
authors.lastname: Graier
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. clara.roller@gmx.at.
authors.forename: Clara
authors.initials: C
authors.lastname: Roller
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. rene.rost@medunigraz.at.
authors.forename: Rene
authors.initials: R
authors.lastname: Rost
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. emrah.eroglu@medunigraz.at.
authors.forename: Emrah
authors.initials: E
authors.lastname: Eroglu
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. benjamin.gottschalk@medunigraz.at.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Gottschalk
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. nicole.hofmann@medunigraz.at.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Hofmann
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. wolfgang.graier@medunigraz.at.
authors.forename: Wolfgang F
authors.initials: WF
authors.lastname: Graier
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. roland.malli@medunigraz.at.
authors.forename: Roland
authors.initials: R
authors.lastname: Malli
mesh_terms: D002118:Calcium / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D002118:Calcium
keywords: FRET; Förster resonance energy transfer; SOCE; calcium; clover; dissociation constant; endoplasmic reticulum; mRuby2; store operated calcium entry