In situ structural cell biology - studying bioenergetics at the nanoscope.

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Mitochondria are the powerhouse of eukaryotic cells and the principal site for the regeneration of the cellular energy unit adenosine triphosphate (ATP). A cell's survival and function is highly dependent on matching energy production with energy consumption and thus the production of ATP in mitochondria is highly regulated. Perturbance in energy production regulation is a strong feature of many human diseases including cancer and neuropathies. ATP production is carried out via oxidative phosphorylation (OXPHOS) by five large membrane proteins (CI-CV) embedded in the highly folded internal membranes of mitochondria (cristae). CI-CIV harness the energy from the oxidation of electron rich substrates to build up a transmembrane gradient of protons which is used by CV to power ATP regeneration.It is hypothesised that as energy demand of cells increase, the infolding of the inner membrane increases to increase the surface area available to house more OXPHOS proteins. Furthermore, CI-CIV form supercomplexes (SCs) that are more prevalent when cellular energy demand is high suggesting a role for SCs in regulating OXPHOS. In addition, it is known that the composition of SCs varies specifically in relation to CIV number. This is particularly interesting as CIV (cytochrome c oxidase) is the terminal electron acceptor of these protein chains and is often presented as the overall regulator of OXPHOS. Recent proteomics analysis suggested that the multiple isoforms of a CIV subunit Cox7A could control whether CIV exists as a monomer, dimer or part of a supercomplex and that the expression level of the isoforms varies between cell type, developmental stage and health versus disease.In this proposal we aim to link the physiological energy demand of cells with changes in the molecular structure of mitochondria - namely the amount of infolding and surface area of mitochondrial cristae, and the number, variability, and organisation of supercomplex in cristae under different growth conditions and in different Cox7A isoform mutants. To achieve our aim, we will be using existing wildtype and mutant human cell lines from our collaborator Dr. Cristina Ugalde which express only one Cox7A isoform. These cells will be grown under different growth conditions including high/low glucose and ATP in culture media and under synchronized cell cycles. Mitochondria will be harvested at different time points and analysed biochemically for mitochondrial function, SC formation and bioenergetic capabilities. Growth conditions which show greatest variation will be selected for nanoscale imaging at Diamond light source (DLS). To analyse changes in cristae structure and surface area, cells will be grown on EM-grids and imaged at Diamond's B24 soft x-ray beamline. To analyse changes in supercomplex composition and organisation in cristae membranes, cryo-electron tomograms of isolated cristae membranes will be collected and analysed at Diamond's cryo-electron microscopy facility (eBIC).Through the combination of DLS's bioimaging technologies with lab-based biochemical, and bioenergetic profiling we will be able to determine the nanoscopic changes in mitochondrial membrane and protein organisation which accompany changes in energy production - a necessary step for understanding energy regulation and disease.