Project Summary Mitochondria regulate a number of critical cellular pathways including energy homeostasis, calcium handling and lipid production. In a number of cell types, distinct populations of mitochondria are created and maintained within subcellular compartments driving unique responses to physiological challenges in different regions of the cell. While many of the molecular players that modulate mitochondrial shape, and therefore function, have been identified, complete understanding of their functions and interactions in establishing these subpopulations of mitochondria within cells remain difficult to define. The deficit in understanding subcellular mitochondrial shape and function is largely due to a limited ability to visualize, and manipulate, these dynamic organelles in a truly physiological environment at high spatial and temporal resolution. Our approaches are designed to address these gaps in knowledge by leveraging newly developed technologies enabling genetic labelling and manipulation, across multiple cell types, with high spatial and temporal imaging of mitochondrial morphology, dynamics and function in vivo. In project one, members of the laboratory will target the four known mammalian receptors (MFF, FIS1, MIEF1/2) of the dynamin-like protein one (DRP1), the main effector of mitochondrial fission, to test their roles in the creation and maintenance of different mitochondrial subpopulations in cortical neurons and skeletal myocytes in vivo. Through the use of loss of function experiments, CRISPR/Cas labeling and targeting-motif analysis coupled with high resolution imaging we will map the molecular mechanisms regulating subcellular mitochondrial fission dynamics across multiple mitochondrial subpopulations. In project two, members of the laboratory will implement methods for sparse, bright labeling of cortical neuron and skeletal myocyte mitochondria with fluorescent reporters for adenosine triphosphate, calcium, pH and reactive oxygen species, and couple it with 2-photon imaging in living mice to reveal how these mitochondrial subpopulations inform mitochondrial and cellular function in vivo. By manipulating different subpopulations and visualizing the effects on mitochondrial and cellular function in multiple cell types in vivo, we will provide a uniquely integrated approach to understanding the universal and cell-specific roles of mitochondrial subpopulations found within cells.