Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent

The mitochondrial membrane potential (ΔΨ(m)) is the main driver of oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane (IMM), consisting of cristae and inner boundary membranes (IBM), is considered to carry a uniform ΔΨ(m). However, sequestration of OXPHOS components in cristae membranes necessitates a re‐examination of the equipotential representation of the IMM. We developed an approach to monitor ΔΨ(m) at the resolution of individual cristae. We found that the IMM was divided into segments with distinct ΔΨ(m), corresponding to cristae and IBM. ΔΨ(m) was higher at cristae compared to IBM. Treatment with oligomycin increased, whereas FCCP decreased, ΔΨ(m) heterogeneity along the IMM. Impairment of cristae structure through deletion of MICOS‐complex components or Opa1 diminished this intramitochondrial heterogeneity of ΔΨ(m). Lastly, we determined that different cristae within the individual mitochondrion can have disparate membrane potentials and that interventions causing acute depolarization may affect some cristae while sparing others. Altogether, our data support a new model in which cristae within the same mitochondrion behave as independent bioenergetic units, preventing the failure of specific cristae from spreading dysfunction to the rest.