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      Live-Cell Imaging of Physiologically Relevant Metal Ions Using Genetically Encoded FRET-Based Probes

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          Abstract

          Essential biochemical reactions and processes within living organisms are coupled to subcellular fluctuations of metal ions. Disturbances in cellular metal ion homeostasis are frequently associated with pathological alterations, including neurotoxicity causing neurodegeneration, as well as metabolic disorders or cancer. Considering these important aspects of the cellular metal ion homeostasis in health and disease, measurements of subcellular ion signals are of broad scientific interest. The investigation of the cellular ion homeostasis using classical biochemical methods is quite difficult, often even not feasible or requires large cell numbers. Here, we report of genetically encoded fluorescent probes that enable the visualization of metal ion dynamics within individual living cells and their organelles with high temporal and spatial resolution. Generally, these probes consist of specific ion binding domains fused to fluorescent protein(s), altering their fluorescent properties upon ion binding. This review focuses on the functionality and potential of these genetically encoded fluorescent tools which enable monitoring (sub)cellular concentrations of alkali metals such as K +, alkaline earth metals including Mg 2+ and Ca 2+, and transition metals including Cu +/Cu 2+ and Zn 2+. Moreover, we discuss possible approaches for the development and application of novel metal ion biosensors for Fe 2+/Fe 3+, Mn 2+ and Na +.

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          Most cited references203

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          Calcium signaling.

          Calcium ions (Ca(2+)) impact nearly every aspect of cellular life. This review examines the principles of Ca(2+) signaling, from changes in protein conformations driven by Ca(2+) to the mechanisms that control Ca(2+) levels in the cytoplasm and organelles. Also discussed is the highly localized nature of Ca(2+)-mediated signal transduction and its specific roles in excitability, exocytosis, motility, apoptosis, and transcription.
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            Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.

            Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.
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              Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.

              Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca(2+)-saturated form, thereby increasing the Ca(2+)-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca(2+) dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET.
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                Author and article information

                Journal
                Cells
                Cells
                cells
                Cells
                MDPI
                2073-4409
                22 May 2019
                May 2019
                : 8
                : 5
                : 492
                Affiliations
                [1 ]Gottfried Schatz Research Center, Chair of Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria; sandra.burgstaller@ 123456medunigraz.at (S.B.); markus.weiermair@ 123456medunigraz.at (M.W.-W.); thomas.rauter@ 123456medunigraz.at (T.R.); maximilian.schinagl@ 123456medunigraz.at (M.S.); jeta.ramadani@ 123456medunigraz.at (J.R.-M.); wolfgang.graier@ 123456medunigraz.at (W.F.G.); roland.malli@ 123456medunigraz.at (R.M.)
                [2 ]BioTechMed Graz, Mozartgasse 12/II, 8010 Graz, Austria
                Author notes
                [* ]Correspondence: helmut.bischof@ 123456medunigraz.at ; Tel.: +43-(0)-316-385-71958
                Author information
                https://orcid.org/0000-0003-2380-600X
                https://orcid.org/0000-0003-1871-3298
                https://orcid.org/0000-0001-6327-8729
                Article
                cells-08-00492
                10.3390/cells8050492
                6562680
                31121936
                16f99af1-c75a-4137-9092-d0d614818a8a
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 30 April 2019
                : 21 May 2019
                Categories
                Review

                fret,fluorescent protein,fp,fluorescence microscopy,genetically encoded probes,imaging,ca2+,cu+,cu2+,fe2+,fe3+,k+,mg2+,mn2+,zn2+

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