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
Mitochondria are central metabolic organelles for eukaryotic cells
The method exploits mitochondrial localisation signals that are dependent on the membrane potential for protein import (Bevis and Glick, 2002; Veatch et al, 2009)
To generate the second marker, an mCherry protein was fused to the N-terminal localisation sequence of cytochrome C oxidase 4 ((COX4), preCOX4, [1:28]), which is imported into mitochondria proportional to the membrane potential (MMP) (Bevis and Glick, 2002; Garipler et al, 2014; Veatch et al, 2009)
Summary
Mitochondria are central metabolic organelles for eukaryotic cells. They are required in energetic and biosynthetic metabolism and as such are crucial for cellular growth and survival. Descending from Archean symbiotic proteobacteria, most eukaryotic mitochondria carry their own genetic material, host the tricarboxylic acid (TCA) cycle, are required for the assembly of iron–sulphur clusters, the respiratory chain, and are integral part of the apoptotic machinery (Frazzon et al, 2002; Madeo et al, 2009; Schatz, 1995; Veatch et al, 2009; Wallace, 1999). This central role requires mitochondria to constantly adapt to changes in cellular physiology. The MMP is required for mitochondrial ATP production by the F1F0 ATPase (Complex V), for import of mitochondrial proteins, and is essential for the maintenance of mitochondrial ion homeostasis (O'Rourke et al, 2005)
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