title: 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.
authors.affiliation: Dept. of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, 80 Tennis Court Road, Cambridge, UK.
authors.forename: Jakob
authors.initials: J
authors.lastname: Vowinckel
authors.affiliation: Dept. of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, 80 Tennis Court Road, Cambridge, UK.
authors.forename: Johannes
authors.initials: J
authors.lastname: Hartl
authors.affiliation: The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Butler
authors.affiliation: Dept. of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, 80 Tennis Court Road, Cambridge, UK; The Francis Crick Institute, Mill Hill Laboratory, London NW7 1AA, UK.
authors.forename: Markus
authors.initials: M
authors.lastname: Ralser
mesh_terms: D001699:Biometry; D017930:Genes, Reporter; D021621:Imaging, Three-Dimensional; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000502:physiology* / Q000648:ultrastructure*; D008962:Models, Theoretical; D059010:Single-Cell Analysis; D013194:Staining and Labeling
keywords: Membrane potential; Mitochondrial morphology; Quantitative microscopy; Super resolution microscopy