ABSTRACT The molecular mechanism of mitochondrial fission is not known. This process is essential and closely regulated through a variety of modifications to the major protein factor of the mitochondrial fission complex, Drp1. The goal of this proposal is to identify key molecular interactions within the mitochondrial fission complex and to understand regulatory changes that influence these interactions and ultimately affect mitochondrial fission. To begin, structures of larger, helical Drp1 oligomers on membrane templates will be studied to identify conformational changes in the Drp1 protein that promote self-assembly at the surface of mitochondria. Specifically, cryo-EM and mitochondrial isolation studies will be performed in parallel to reveal interactions within the mitochondrial fission complex adjacent to the neighboring membrane. In parallel, detailed interactions within Drp1 complexes will be identified using proteomics and mutagenesis methods. Factors that perturb Drp1 self-assembly also disrupt organelle morphology, so Drp1 interactions with the essential partner protein, Mff, will also be examined. Specifically, protein co-polymers will be characterized using structural methods to uncover the mechanism by which Drp1 is targeted to the surface of mitochondria and how Mff contributes to productive mitochondrial fission. To mimic changes associated with human disease, Drp1 sequence will be altered using site-directed mutagenesis to recapitulate natural sequence changes caused by post-translational modifications. The full effects of these changes will be assessed using structural and biochemical assays, and noticeable differences between healthy and diseased mitochondrial fission complexes will be identified. These same changes will be introduced in cell culture to correlate alterations in mitochondrial morphology to functional alterations observed in vitro. To further characterize the impact of these changes, we plan to assess the bioenergetic capacities of stable cell lines expressing phosphomimetic mutants. Moreover, the assembly properties of Drp1 and its partner proteins will be assayed in the context of post-translational modifications to reveal the underlying effects of cell signaling alterations that modify the mitochondrial network. Collectively, this project seeks to identify detailed features within the mitochondrial fission complex that contribute to specific disease states. These differences provide novel targets for the development of future therapeutics that prevent cell death in neurological disorders, which are associated with increased Drp1 activity and excessive mitochondrial fission.