title: Mag-Fluo4 in T cells: Imaging of intra-organelle free Ca2+concentrations.
abstract: Ca2+signaling is a major signal transduction pathway involved in T cell activation, but also in apoptosis of T cells. Since T cells make use of several Ca2+-mobilizing second messengers, such as nicotinic acid adenine dinucleotide phosphate, d-myo-inositol 1,4,5-trisphosphate, and cyclic ADP-ribose, we intended to analyze luminal Ca2+concentration upon cell activation. Mag-Fluo4/AM, a low-affinity Ca2+dye known to localize to the endoplasmic reticular lumen in many cell types, showed superior brightness and bleaching stability, but, surprisingly, co-localized with mito-tracker, but not with ER-tracker in Jurkat T cells. Thus, we used Mag-Fluo4/AM to monitor the free luminal mitochondrial Ca2+concentration ([Ca2+]mito) in these cells. Simultaneous analysis of the free cytosolic Ca2+concentration ([Ca2+]i) and [Ca2+]mitoupon cell stimulation revealed that Ca2+signals in the majority of mitochondria were initiated at [Ca2+]i≥approx. 400 to 550nM. In primary murine CD4+T cells, Mag-Fluo4 showed two different localization patterns: either co-localization with mito-tracker, as in Jurkat T cells, or with ER-tracker. Thus, in single primary murine CD4+T cells, either decreases of [Ca2+]ERor increases of [Ca2+]mitowere observed upon cell stimulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
authors.affiliation: The Calcium Signalling Group, Department of Biochemistry and Molecular Cell Biology, University Medical Centre Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
authors.forename: Björn-Philipp
authors.initials: BP
authors.lastname: Diercks
authors.affiliation: The Calcium Signalling Group, Department of Biochemistry and Molecular Cell Biology, University Medical Centre Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Fliegert
authors.affiliation: The Calcium Signalling Group, Department of Biochemistry and Molecular Cell Biology, University Medical Centre Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. Electronic address: guse@uke.de.
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Guse
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008928:Mitochondria / Q000378:metabolism; D015388:Organelles / Q000378:metabolism*; D014966:Xanthenes / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D014966:Xanthenes; D002118:Calcium
keywords: Ca(2+) signaling; Endoplasmic reticular Ca(2+) concentration; Mag-Fluo4; Mitochondrial Ca(2+) concentration; Signal transduction; T cell