Mag-Fluo4 in T cells: Imaging of intra-organelle free Ca2+ concentrations

Publication date: June 2017
Source:Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Volume 1864, Issue 6
Author(s): Björn-Philipp Diercks, Ralf Fliegert, Andreas H. Guse
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⁺]mito upon 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⁺ ]ER or increases of [Ca²⁺ ]mito were observed upon cell stimulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

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