Investigating the relationship between respiratory chain organisation and mitochondrial morphology by electron cryo-tomography

Abstract

The mitochondrial respiratory chain is the primary source of cellular energy in all eukaryotic life, and therefore plays a central role in health and disease. Passage of electrons through respiratory complexes I-IV facilitates pumping of protons across the inner mitochondrial membrane; this electrochemical gradient is used to power downhill flow of protons via ATP synthase to generate energy in the form of ATP. Electron cryo-tomography has demonstrated that components of the respiratory chain can oligomerise to form higher order assemblies, the stability of which declines with age. The most abundant assembly is the ATP synthase dimer, followed by the respirasome which comprises all three proton-pumping complexes (I, III and IV) of the respiratory chain. A role for ATP synthase dimerisation in maintaining crista membrane curvature has been demonstrated, but the functional significance of the respirasome remains largely elusive. In this thesis, cryo-ET was employed to investigate the significance of both varying ATP synthase dimer architecture and respirasome formation, related to mitochondrial morphology and function. As a simple multicellular organism with a high level of homology with the human respiratory chain, C. elegans was selected as the model in which to complete these studies. Identification of a novel ATP synthase dimer architecture in C. elegans compared to other multicellular eukaryotes facilitated an investigation into the influence of dimer angle on mitochondrial morphology. Comparison with a published structure of the S. cerevisiae ATP synthase dimer, combined with an investigation of mitochondrial morphology, suggests a relationship between dimer angle and crista membrane curvature. This leads to speculation that a range of dimer angles may have evolved to suit bespoke energetic needs of different organisms. Subsequently, the role of the respirasome in C. elegans was investigated through knocking down NDUF-11 - a complex I accessory subunit located at the interface with complex III. Cryo-ET demonstrates that NDUF-11 knockdown completely abolishes respirasome formation in situ, with limited effects on complex I abundance. This validates the experimental design as an informative approach for understanding the role of the respirasome in health and disease. Quantitative analysis of mitochondrial morphology, combined with gene set enrichment analysis, reveals changes in mitochondrial morphology and protein abundance in the knockdown that mirror changes seen in ageing and age-related disease. This signifies that maintenance of respirasome stability is important for healthy ageing.