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© The Rockefeller University Press,
0021-9525/1998//1091 $5.00
The Journal of Cell Biology, Volume 140, Number 5,
, 1998 1091-1099
Article |
Functional Imaging of Mitochondria in Saponin-permeabilized Mice Muscle Fibers
Arbeitsgruppe Molekulare Mustererkennung des Instituts für Medizinische Neurobiologie; and Institut für Medizinische Mikrobiologie, Universitätsklinikum der Otto-von-Guericke-Universität, D-39120 Magdeburg, Germany
Confocal laser-scanning and digital fluorescence imaging microscopy were used to quantify the mitochondrial autofluorescence changes of NAD(P)H and flavoproteins in unfixed saponin-permeabilized myofibers from mice quadriceps muscle tissue. Addition of mitochondrial substrates, ADP, or cyanide led to redox state changes of the mitochondrial NAD system. These changes were detected by ratio imaging of the autofluorescence intensities of fluorescent flavoproteins and NAD(P)H, showing inverse fluorescence behavior. The flavoprotein signal was colocalized with the potentiometric mitochondria-specific dye dimethylaminostyryl pyridyl methyl iodide (DASPMI), or with MitoTrackerTM Green FM, a constitutive marker for mitochondria. Within individual myofibers we detected topological mitochondrial subsets with distinct flavoprotein autofluorescence levels, equally responding to induced rate changes of the oxidative phosphorylation. The flavoprotein autofluorescence levels of these subsets differed by a factor of four. This heterogeneity was substantiated by flow-cytometric analysis of flavoprotein and DASPMI fluorescence changes of individual mitochondria isolated from mice skeletal muscle. Our data provide direct evidence that mitochondria in single myofibers are distinct subsets at the level of an intrinsic fluorescent marker of the mitochondrial NAD–redox system. Under the present experimental conditions these subsets show similar functional responses.
Abbreviations used in this paper: DASPMI, 2-(p-dimethylaminostyryl) pyridyl methyl iodide; FAD, flavine adenine dinucleotide; IMM, intermyofibrillar mitochondria; SSM, subsarcolemmal mitochondria; TTFB, 4, 5, 6, 7-tetrachloro-2-trifluoromethyl-benzimidazole.
The excellent technical assistance of K. Kaiser (Neurobiochemisches Labor der Klinik für Neurologie, Magdeburg, Germany) is gratefully acknowledged.
A. Kuznetsov and O. Mayboroda contributed equally to this work.
Address all correspondence to Wolfram S. Kunz, Neurobiochemisches Labor der Klinik für Neurologie, Universitätsklinikum der Otto-von- Guericke Universität Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany. Tel.: (49) 391-671-5219. Fax: (49) 391-671-5228. E-mail: wolfram. kunz{at}medizin.uni-magdeburg.de
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