The interplay of mtDNA quality, physiological heterogeneity, and cristae structure within the mitochondrial network and its effect on purifying selection

• Applicants: Dr. David Hörl; Professor Dr. Christof Osman
• Subject Area: Biochemistry ; Cell Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 513597632

Mitochondria fulfil crucial roles in energy supply of eukaryotic cells through a process known as oxidative phosphorylation (OXPHOS). The dynamic mitochondrial network consists of interconnected tubules and possesses a unique ultrastructure characterized by invaginations of the inner membrane called cristae. Many factors influencing cristae structure have been described, including the ATP synthase subunits e and g that facilitate dimerization of the ATP synthase to stabilize cristae edges. Regulatory mechanisms that determine ATP synthase dimerization and thereby cristae shape and abundance according to cellular needs, however, remain largely unknown. Mitochondria contain multiple copies of the mitochondrial genome (mtDNA), coding mainly for OXPHOS components. Mutations of mtDNA are implicated in a multitude of human diseases and ageing processes. How cells maintain a healthy pool of mtDNA copies over generations remains largely unknown. We previously could show that purifying selection against mutant mtDNA occurs in yeast cells in a continuous mitochondrial network. Our findings support a sphere-of-influence model in which individual mtDNA copies locally affect physiological parameters in a cristae-dependent manner. In our proposed studies we will use S. cerevisiae to expand on our previous work and address open questions regarding the principles that govern the interplay between mitochondrial physiology, cristae structure and mtDNA quality control. In the first aim, we will test our hypothesis that local mitochondrial fitness depends on the integrity of nearby mtDNA copies. We will use fluorescent biosensors and live-cell microscopy to assess the local relationships between physiological parameters (e.g., NADH/ATP concentrations, pH, redox potential) and mtDNA genotype in heteroplasmic cells containing WT and mutant mtDNA. To understand the link between fitness and mtDNA quality control, we will use established genetic assays and novel image-based lineage tracing, to monitor purifying selection of mtDNA upon targeted perturbations to mitochondrial physiology. In the second aim, we will test our hypothesis that phosphorylation of the yeast ATP synthase subunit g homolog Atp20 regulates cristae structure and abundance. First, we will characterize the phosphorylation status of Atp20 under different growth conditions and use super-resolution and electron microscopy to determine the effect of phosphomimetic and -ablative mutants of Atp20 on cristae (ultra)structure. We will further assess the role of Atp20 phosphorylation in mtDNA genotype-physiology relationships and mtDNA quality control. Finally, we will identify kinases and phosphatases that set the Atp20 phosphorylation status and might link mitochondrial ultrastructure to other cellular processes.

• DFG Programme: Research Grants