Mitochondrial DNA (mtDNA) encodes RNAs and proteins critical for cell function. In human cells, 100–1000s mtDNA copies are replicated asynchronously, packaged into protein-DNA nucleoids, and distributed within a dynamic mitochondrial network. The mechanisms that govern how nucleoids are chosen for replication and distribution are not understood. Mitochondrial distribution depends on division, which occurs at endoplasmic reticulum (ER)-mitochondria contact sites. These sites were spatially linked to a subset of nucleoids selectively marked by mtDNA polymerase and engaged in mtDNA synthesis, events that occurred upstream of mitochondrial constriction and division machine assembly. Our data suggest that ER tubules proximal to nucleoids were necessary but not sufficient for mtDNA synthesis. Thus, ER-mitochondria contacts coordinate licensing of mtDNA synthesis with division to distribute newly replicated nucleoids to daughter mitochondria.

Balanced rates of mitochondrial division and fusion are required to maintain mitochondrial function, as well as cellular and organismal homeostasis. In mammals, the cellular machines that mediate these processes are dynamin-related GTPases; the cytosolic DRP1 mediates division, while the outer membrane MFN1/2 and inner membrane OPA1 mediate fusion. Unbalanced mitochondrial dynamics are linked to varied pathologies, including cell death and neurodegeneration, raising the possibility that small molecules that target the division and fusion machines to restore balance may have therapeutic potential. Here we describe the discovery of novel small molecules that directly and selectively inhibit assembly-stimulated GTPase activity of the division dynamin, DRP1. In addition, these small molecules restore wild type mtDNA copy number in MFN1 knockout mouse embryonic fibroblast cells, a phenotype linked to deficient mitochondrial fusion activity. Thus, these compounds are unique tools to explore the roles of mitochondrial division in cells, and to assess the potential therapeutic efficacy of rebalancing mitochondrial dynamics in pathologies associated with excessive mitochondrial division.

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