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.

Altered Bioenergetics in Primary Dermal Fibroblasts from Adult Carriers of the FMR1 Premutation Before the Onset of the Neurodegenerative Disease Fragile X-Associated Tremor/Ataxia Syndrome

“…Images were analyzed with the ImageJ software discarding the color information and adjusting the threshold to minimize the background level. Pattern of mitochondrial network was analyzed using the Fiji (ImageJ) plugin MitoMap and the percentage of cells exhibiting disrupted mitochondrial network were calculated as described in [19]. …”

“…At least 10 cells per line were imaged and analyzed with the Fiji’s MitoMap plugin. Relative volumes (V s ) were calculated as described in [19]. Objects with V s ≤ 20% of total cellular V s were considered fragmented.…”

Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins

“…In multi-scale representation systems, mitochondrial morphology is depicted on a linear scale ranging from “fragmented” to “filamentous”, before adding two or more categories (e.g. “compact” versus “branched”, “non-tubular” versus “tubular”) (Vowinckel et al, 2015). When consensus clustering was applied to automatically define morphological subtypes in mitochondrial images, a total of 19 distinct morphological clusters were identified, suggesting that mitochondrial shape complexity is under-appreciated even when a single cell type is considered (Peng et al, 2011).…”

Monitoring the Effects of Pharmacological Reagents on Mitochondrial Morphology

“…Therefore, they depolarize mitochondrial membrane potential resulting in mitochondrial dysfunction. Consequently, mitochondria undergo fission and become morphologically fragmented (Li et al., ; Vowinckel, Hartl, Butler, & Ralser, ). Oligomycin A is another uncoupler of oxidative phosphorylation and an inhibitor of F 1 F 0 ATP synthase (Chappell & Greville, ; Lardy, Connelly, & Johnson, ), that causes mitochondrial dysfunction and fragmentation (Fu et al., ; Wang et al., ).…”