Mitochondria exist not just as isolated organelles, but also as highly interconnected networks, constantly shifting between these two states via a balance between opposing fusion and fission forces. This ability to transition between fission/fusion states is essential for the functions of mitochondria in maintaining optimal cellular bioenergetics, in regulating intracellular Ca2+, and in cellular stress responses. Disruption of this homeostasis can have major consequences for mitochondrial health and function. In humans, mitofusin 1 and 2 orchestrate mitocondrial fusion together with optic atrophy-1 (OPA1). Mitochondrial fission is mediated by another member of the dynamin family, dynamin-related protein 1 (DRP1). In C. elegans, FZO-1 is the sole mitofusin orthologue, EAT-3 is the orthologue of OPA1, and the orthologue of DRP1 is DRP-1.
Methods
Large populations of age-synchronised worms were prepared. Briefly, gravid hermaphrodites were bleached to release eggs, which were allowed to hatch overnight before plating onto OP50-seeded 60 mm NGM plates and incubation at 20 °C until the day of experiment. Synchronised L4 or 3DOA worms were initially washed off the plate with M9 buffer into micro-centrifuge tubes, and subsequent washing of the worms in the tubes was performed at least twice to remove residual OP50 and offspring. For analysis at L4stage, 100 μL of 60–70 L4 worms was then plated into five wells per genotype of a 96-well plate across three replicate experiments. For analysis of adults, 30–50 3DOA worms were plated into six wells of a 96-well plate per genotype, across two replicate experiments. The plates containing the worms were placed in a WMicrotracker instrument and run for 3 h.
Results
As shown in Fig. 3a, all three mitochondrial fission and fusion mutants displayed significant deficits in movement over a 3 h period compared to wild type animals at the L4 stage. Loss of either of the fusion genes led to a twofold reduction in animal movement, whereas loss of fission induced an intermediate phenotype. In adult animals (3-day-old adult, 3DOA), both fusion mutants again displayed severely reduced activity compared to the wild type, but eat-3 mutants were now much reduced in movement compared to the fzo-1 mutants. Interestingly, adult drp-1mutants displayed no significant difference to wild type animals (Fig. 3b). These data demonstrate that the overall activity of C. elegans is strongly impaired in the absence of the mitochondrial fusion and fission proteins.
Cell Mol Life Sci. 2019 May;76(10):1967-1985. doi: 10.1007/s00018-019-03024-5. Epub 2019 Mar 6.
Byrne JJ , Soh MS , Chandhok G , Vijayaraghavan T, Teoh JS, Crawford S, Cobham AE, Yapa NMB, Mirth CK, Neumann B.