MitoLoc: A method for the simultaneous quantification of mitochondrial network morphology and membrane potential in single cells

Abstract

Mitochondria assemble into flexible networks. Here we present a simple method for the simultaneous quantification of mitochondrial membrane potential and network morphology that is based on computational co-localisation analysis of differentially imported fluorescent marker proteins. Established in, but not restricted to, Saccharomyces cerevisiae, MitoLoc reproducibly measures changes in membrane potential induced by the uncoupling agent CCCP, by oxidative stress, in respiratory deficient cells, and in ∆ fzo1, ∆ ref2, and ∆ dnm1 mutants that possess fission and fusion defects. In combination with super-resolution images, MitoLoc uses 3D reconstruction to calculate six geometrical classifiers which differentiate network morphologies in ∆ fzo1, ∆ ref2, and ∆ dnm1 mutants, under oxidative stress and in cells lacking mtDNA, even when the network is fragmented to a similar extent. We find that mitochondrial fission and a decline in membrane potential do regularly, but not necessarily, co-occur. MitoLoc hence simplifies the measurement of mitochondrial membrane potential in parallel to detect morphological changes in mitochondrial networks. Marker plasmid open-source software as well as the mathematical procedures are made openly available.

Highlights

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  MitoLoc is a workflow for measuring changes mitochondrial membrane potential and morphology in parallel.

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  Pixel-by pixel co-localisation of two fluorescent reporters is used to detect changes in mitochondrial membrane potential.

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  Automated 3D reconstruction of super resolution images to calculate mitochondrial morphological classifiers

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  We make fluorescent markers, mathematical procedures and software openly available.

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  We demonstrate the application of MitoLoc on several examples.