MiPsummer Programme pdf - Mitochondrial Physiology Society

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Abstract # 36
Hyperactivation of UNC-105 causes loss of mitochondrial membrane potential and impaired
mitochondrial ATP production in C. elegans
C. J. Gaffney, D. Constantin-Teodosiu, P. L. Greenhaff, and N. J. Szewczyk.
MRC/ARUK Centre for Musculoskeletal Ageing Research, University of Nottingham, UK.
Background C. elegans is a free-living nematode known for its utility in genomics research. A
dominant gain-of-function mutation in unc-105, a putative mechano-sensitive ion channel of the
ENaC/Degenerin family, causes mitochondrial fragmentation in body-wall muscles (Szewczyk et
al. unpublished data) and a movement defect 1 . These defects are ameliorated in unc-105; let-2
suppressed mutants (let-2 encodes a collagen).
Objectives Mitochondrial membrane potential and maximal rates of ATP production (MRAP) were
determined in wild-type (WT) and unc-105 mutants to quantify disruption, if any, of mitochondrial
function. The unc-105; let-2 mutants were also assessed to quantify any potential rescue of
mitochondrial function.
Methods Any disruption of mitochondrial membrane potential was quantified using JC-10 and
Mitotracker® CMXRos in vivo staining, which exhibit potential-dependent accumulation in
mitochondria. JC-10 fluorescence was quantified using ImageJ. MRAP and maximal citrate
synthase (CS) activity were determined in mitochondria isolated from mixed-age worms (n300 per
assay). MRAP was determined through incubation with a bioluminescent luciferase-based
monitoring reagent, and a combination of respiratory substrates and ADP.
Results Mitochondrial accumulation of JC-10 was reduced in unc-105 versus WT (P