Abstract
The mitochondrial inner membrane is a protein-rich environment containing large multimeric complexes, including complexes of the mitochondrial electron transport chain, mitochondrial translocases and quality control machineries. Although the inner membrane is highly proteinaceous, with 40-60% of all mitochondrial proteins localised to this compartment, little is known about the spatial distribution and organisation of complexes in this environment. We set out to survey the arrangement of inner membrane complexes using stochastic optical reconstruction microscopy (STORM). We reveal that subunits of the TIM23 complex, TIM23 and TIM44 (also known as TIMM23 and TIMM44, respectively), and the complex IV subunit COXIV, form organised clusters and show properties distinct from the outer membrane protein TOM20 (also known as TOMM20). Density based cluster analysis indicated a bimodal distribution of TIM44 that is distinct from TIM23, suggesting distinct TIM23 subcomplexes. COXIV is arranged in larger clusters that are disrupted upon disruption of complex IV assembly. Thus, STORM super-resolution microscopy is a powerful tool for examining the nanoscale distribution of mitochondrial inner membrane complexes, providing a 'visual' approach for obtaining pivotal information on how mitochondrial complexes exist in a cellular context.