title: Spatiotemporal correlations between cytosolic and mitochondrial Ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon.
abstract: The transfer of Ca(2+) from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca(2+) ([Ca(2+)](cyto)) can be excellently quantified with the ratiometric Ca(2+) probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca(2+) sensors, the cameleons, are efficiently used to specifically measure Ca(2+) within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca(2+)](cyto) and mitochondrial Ca(2+) ([Ca(2+)](mito)) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca(2+) probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca(2+)](cyto) and [Ca(2+)](mito) within same individual cells. Our data indicate that depending on the kinetics of [Ca(2+)](cyto) rises there is a significant lag between onset of [Ca(2+)](cyto) and [Ca(2+)](mito) signals, pointing to a certain threshold of [Ca(2+)](cyto) necessary to activate mitochondrial Ca(2+) uptake. The temporal correlation between [Ca(2+)](mito) and [Ca(2+)](cyto) as well as the efficiency of the transfer of Ca(2+) from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca(2+) extrusion and a desensitization of mitochondrial Ca(2+) uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca(2+) oscillations of pancreatic beta-cells in response to D-glucose.
authors.affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Graz, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Waldeck-Weiermair
authors.forename: Muhammad Rizwan
authors.initials: MR
authors.lastname: Alam
authors.forename: Muhammad Jadoon
authors.initials: MJ
authors.lastname: Khan
authors.forename: Andras T
authors.initials: AT
authors.lastname: Deak
authors.forename: Neelanjan
authors.initials: N
authors.lastname: Vishnu
authors.forename: Felix
authors.initials: F
authors.lastname: Karsten
authors.forename: Hiromi
authors.initials: H
authors.lastname: Imamura
authors.forename: Wolfgang F
authors.initials: WF
authors.lastname: Graier
authors.forename: Roland
authors.initials: R
authors.lastname: Malli
mesh_terms: D020746:Calcium Channels, L-Type / Q000378:metabolism; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D016257:Fura-2 / Q000737:chemistry / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D020746:Calcium Channels, L-Type; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005947:Glucose; D016257:Fura-2