Abstract Regulated Mitochondrial Morphology The mitochondrial reticulum performs an astonishing number of essential cellular functions, including respiratory energy production, anabolic production of critical metabolites, and regulated cell death. Mutations, injuries, and infections degrade mitochondrial activity; and damaged or dysfunctional mitochondria are increasingly recognized as contributing if not causative factors for a long and still growing list of diseases. The most commonly observed defect seen in aging, injured, or diseased cells is a breakdown of the inter-connected reticulum into hyper-fragmented organelle units that lose their chemical potential and the integrity of their genomes. The observation of hyper-fission in disease settings generated clinical interest in specific inhibitors of the mitochondrial fission machinery to ameliorate a range of illness: from chronic neurodegeneration and certain cancers to more acute injuries like heart attack and stroke—with promising proof-of-concept studies in animal models. Progress has been slow, however, in part because we do not understand the molecular mechanisms that govern mitochondrial fission. Recent biochemical breakthroughs in our lab—in combination with the resolution revolution in electron cryo-microscopy or cryoEM—have finally prepared us to resolve the mechanisms that drive these fission machines in unprecedented detail. We propose to determine the structural mechanisms that govern recruitment and assembly of the fission machine on the surface of mitochondria through the activity of specialized receptors (Aim1). We further propose to determine the allosteric protein motions that harness the chemical energy present in guanine nucleotides to perform mechanical, constricting work on mitochondrial tubules (Aim 2). Finally, we propose to determine how post-translational modifications—including phosphorylation and SUMOylation—tune or turn off the activity of the fission machinery (Aim 3). Together, accomplishing these objectives will provide new and unique insights into how these fundamental cellular machines work and will enable a new generation of structure-guided studies to identify and characterize novel therapeutic opportunities.