Defining Molecular Interactions that Drive Mitochondrial Fission

项目总结（摘要）

PROJECT SUMMARY (ABSTRACT) Mitochondria are double-membrane organelles that change shape, size and abundance in response to specific stimuli. Protein interactions that control mitochondrial division are tightly regulated and directly impact ATP production, Ca2+ homeostasis, and regulation of programmed cell death. Therefore, mitochondrial dynamics has recently come to the forefront as a therapeutic target in several degenerative diseases, including neurodegeneration, cancer, and cardiovascular disease. But the lack of insight into the regulation of this process is a major limitation. The major driver of mitochondrial division is a cytosolic GTPase, dynamin-related protein 1 (Drp1). To mediate membrane scission, Drp1 recruitment and self-assembly is coordinated through combinatorial interactions with lipids, proteins and nucleotides at the surface of mitochondria. This proposal seeks to identify key attributes of the mitochondrial division machinery and how dysregulation of Drp1 leads to organelle damage and cellular degeneration. This will be accomplished using a multifaceted approach that combines molecular studies with functional cell experiments to provide a comprehensive evaluation of Drp1 interactions that govern membrane remodeling. Under Specific Aim 1 of the renewal, cryo-EM studies will examine auto-inhibitory interactions that limit Drp1 oligomerization in a cytosolic state. Distinct conformations will be studied to identify and characterize intermediate structures during recruitment and assembly of Drp1 into a functional fission complex. We propose that regulated rearrangements “open” the molecule for functional assembly at defined sites of mitochondrial division. For Specific Aim 2, reconstitution experiments provide a means to evaluate macromolecular interactions that drive mitochondrial membrane remodeling. Specific mitochondrial cues, including lipids and partner proteins, will be studied to evaluate the contribution of each component to membrane remodeling. Constriction of protein-lipid tubules will be encouraged to evaluate the magnitude of constriction using advanced structural methods. Liquid-EM will visualize dynamic narrowing of Drp1-lipid tubules in real time, and cryo-ET will be used to resolve 3D structures of assorted Drp1 constriction events in parallel. In Specific Aim 3, defects in mitochondrial fission will be examined at the cellular level to establish how deleterious changes in Drp1 can directly influence mitochondrial bioenergetics. The integrity of ETC complexes will be studied to reveal how altered organelle morphology informs metabolic stress. Concurrently, the impact of this stress on ROS signaling and mitophagy will be monitored. In summary, the structural and functional insight gained from this proposal will catalyze directed therapeutic strategies that counteract mitochondrial damage in various disease states.