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      Red fluorescent genetically encoded Ca 2+ indicators for use in mitochondria and endoplasmic reticulum

      research-article
      * , , * , , , § , * , * , § , , , , * , 1
      Biochemical Journal
      Portland Press Ltd.
      endoplasmic reticulum (ER), fluorescence Ca2+ imaging, GCaMP, mitochondrion, multicolour imaging, red fluorescent genetically encoded Ca2+ indicator for optical imaging (R-GECO), [Ca2+]i and [Ca2+]mt, free Ca2+ concentration in cytosol and mitochondrial matrix, respectively , CaM, calmodulin, cpFP, circularly permuted fluorescent protein, DMEM, Dulbecco’s modified Eagle’s medium, DRG, dorsal root ganglion, ER, endoplasmic reticulum, FP, fluorescent protein, FRET, Förster resonance energy transfer, GFP, green fluorescent protein, HBS, Hepes-buffered saline, HEK, human embryonic kidney, LAR-GECO, low-affinity red fluorescent genetically encoded Ca2+ indicator for optical imaging , LED, light-emitting diode, NA, numerical aperture, NTA, nitrilotriacetic acid, RFP, red fluorescent protein, RyR2, type 2 ryanodine receptor, SERCA, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, SOICR, store overload-induced Ca2+ release, SR, sarcoplasmic reticulum

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          Abstract

          Ca 2+ is a key intermediary in a variety of signalling pathways and undergoes dynamic changes in its cytoplasmic concentration due to release from stores within the endoplasmic reticulum (ER) and influx from the extracellular environment. In addition to regulating cytoplasmic Ca 2+ signals, these responses also affect the concentration of Ca 2+ within the ER and mitochondria. Single fluorescent protein-based Ca 2+ indicators, such as the GCaMP series based on GFP, are powerful tools for imaging changes in the concentration of Ca 2+ associated with intracellular signalling pathways. Most GCaMP-type indicators have dissociation constants ( K d) for Ca 2+ in the high nanomolar to low micromolar range and are therefore optimal for measuring cytoplasmic [Ca 2+], but poorly suited for use in mitochondria and ER where [Ca 2+] can reach concentrations of several hundred micromolar. We now report GCaMP-type low-affinity red fluorescent genetically encoded Ca 2+ indicators for optical imaging (LAR-GECO), engineered to have K d values of 24 μM (LAR-GECO1) and 12 μM (LAR-GECO1.2). We demonstrate that these indicators can be used to image mitochondrial and ER Ca 2+ dynamics in several cell types. In addition, we perform two-colour imaging of intracellular Ca 2+ dynamics in cells expressing both cytoplasmic GCaMP and ER-targeted LAR-GECO1. The development of these low-affinity intensiometric red fluorescent Ca 2+ indicators enables monitoring of ER and mitochondrial Ca 2+ in combination with GFP-based reporters.

          Abstract

          This paper describes the engineering of two red fluorescent genetically encoded Ca 2+ indicators with low affinities. These indicators enable monitoring of mitochondrial and ER Ca 2+ dynamics in combination with a green fluorescent indicator.

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          Most cited references33

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          Mitochondria as sensors and regulators of calcium signalling.

          During the past two decades calcium (Ca(2+)) accumulation in energized mitochondria has emerged as a biological process of utmost physiological relevance. Mitochondrial Ca(2+) uptake was shown to control intracellular Ca(2+) signalling, cell metabolism, cell survival and other cell-type specific functions by buffering cytosolic Ca(2+) levels and regulating mitochondrial effectors. Recently, the identity of mitochondrial Ca(2+) transporters has been revealed, opening new perspectives for investigation and molecular intervention.
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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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              A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.

              Recently, several groups have developed green fluorescent protein (GFP)-based Ca(2+) probes. When applied in cells, however, these probes are difficult to use because of a low signal-to-noise ratio. Here we report the development of a high-affinity Ca(2+) probe composed of a single GFP (named G-CaMP). G-CaMP showed an apparent K(d) for Ca(2+) of 235 nM. Association kinetics of Ca(2+) binding were faster at higher Ca(2+) concentrations, with time constants decreasing from 230 ms at 0.2 microM Ca(2+) to 2.5 ms at 1 microM Ca(2+). Dissociation kinetics (tau approximately 200 ms) are independent of Ca(2+) concentrations. In HEK-293 cells and mouse myotubes expressing G-CaMP, large fluorescent changes were observed in response to application of drugs or electrical stimulations. G-CaMP will be a useful tool for visualizing intracellular Ca2+ in living cells. Mutational analysis, together with previous structural information, suggests the residues that may alter the fluorescence of GFP.
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                Author and article information

                Journal
                Biochem J
                Biochem. J
                bic
                BJ
                Biochemical Journal
                Portland Press Ltd.
                0264-6021
                1470-8728
                28 August 2014
                23 October 2014
                15 November 2014
                : 464
                : Pt 1
                : 13-22
                Affiliations
                *Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2
                †Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, U.K.
                ‡Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA 52242, U.S.A.
                §Department of Physiology and Pharmacology, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada, T2N 4N1
                ¶Department of Biochemistry and Molecular Biology, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada, T2N 4N1
                Author notes
                1To whom correspondence should be addressed (email rc4@ 123456ualberta.ca ).
                Article
                BJ20140931
                10.1042/BJ20140931
                4214425
                25164254
                b8ac0f17-ff0a-4731-a9fc-24739122328e
                © 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC-BY)(http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 4 August 2014
                : 28 August 2014
                Page count
                Figures: 6, Tables: 1, References: 39, Pages: 10
                Categories
                Research Article

                Biochemistry
                endoplasmic reticulum (er),fluorescence ca2+ imaging,gcamp,mitochondrion,multicolour imaging,red fluorescent genetically encoded ca2+ indicator for optical imaging (r-geco),[ca2+]i and [ca2+]mt, free ca2+ concentration in cytosol and mitochondrial matrix, respectively,cam, calmodulin,cpfp, circularly permuted fluorescent protein,dmem, dulbecco’s modified eagle’s medium,drg, dorsal root ganglion,er, endoplasmic reticulum,fp, fluorescent protein,fret, förster resonance energy transfer,gfp, green fluorescent protein,hbs, hepes-buffered saline,hek, human embryonic kidney,lar-geco, low-affinity red fluorescent genetically encoded ca2+ indicator for optical imaging,led, light-emitting diode,na, numerical aperture,nta, nitrilotriacetic acid,rfp, red fluorescent protein,ryr2, type 2 ryanodine receptor,serca, sarcoplasmic/endoplasmic reticulum ca2+-atpase,soicr, store overload-induced ca2+ release,sr, sarcoplasmic reticulum

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