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Mitochondrial Ca²⁺ Dynamics Reveals Limited Intramitochondrial Ca²⁺ Diffusion

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary; and Neurobiochemical Group, Hungarian Academy of Sciences, Budapest, Hungary

Correspondence: Address reprint requests to Prof. Vera Adam-Vizi, MD, PhD, Dept. of Medical Biochemistry, Semmelweis University, PO Box 262, H-1444 Budapest, Hungary. Tel.: 361-266-2773; Fax: 361-267-0031; E-mail: av{at}puskin.sote.hu.

To reveal heterogeneity of mitochondrial function on the single-mitochondrion level we have studied the spatiotemporal dynamics of the mitochondrial Ca²⁺ signaling and the mitochondrial membrane potential using wide-field fluorescence imaging and digital image processing techniques. Here we demonstrate first-time discrete sites—intramitochondrial hotspots—of Ca²⁺ uptake after Ca²⁺ release from intracellular stores, and spreading of Ca²⁺ rise within the mitochondria. The phenomenon was characterized by comparison of observations in intact cells stimulated by ATP and in plasma membrane permeabilized or in ionophore-treated cells exposed to elevated buffer [Ca²⁺]. The findings indicate that Ca²⁺ diffuses laterally within the mitochondria, and that the diffusion is limited for shorter segments of the mitochondrial network. These observations were supported by mathematical simulation of buffered diffusion. The mitochondrial membrane potential was investigated using the potentiometric dye TMRM. Irradiation-induced fluctuations (flickering) of TMRM fluorescence showed synchronicity over large regions of the mitochondrial network, indicating that certain parts of this network form electrical syncytia. The spatial extension of these syncytia was decreased by 2-aminoethoxydiphenyl borate (2-APB) or by propranolol (blockers of nonclassical mitochondrial permeabilities). Our data suggest that mitochondria form syncytia of electrical conductance whereas the passage of Ca²⁺ is restricted to the individual organelle.

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