PROJECT SUMMARY Cellular function is maintained by the intricate regulation and coordination of highly dynamic organelles, which themselves contain multiple components and protein assemblies that constantly function together to promote cellular homeostasis. However, the field of cell biology has thus far largely focused on organelle dynamics at the whole-organelle level, or the crosstalk between organelles at inter-organelle membrane contact sites. In contrast, the molecular events occurring inside organelles at the intra-organelle level, such as within the mitochondria matrix, and the timing, distribution and regulation of these events has remained vastly unexplored, largely due to the previous lack of high-speed super-resolution microscopy to probe these questions at the nanoscale level. Importantly, filling in these missing pieces will be essential for the field of cell biology by dynamically mapping the cellular architecture of a living cell in real-time and advancing our knowledge of cellular homeostasis at the intra-organelle level. Mitochondria are highly critical organelles for regulating cellular function, and contain their own mtDNA which is transcribed into mtRNA within the mitochondrial matrix, as well as mitochondrial chaperones which help to promote efficient folding of the mitochondrial proteome. However, how these events are distributed inside the mitochondria and the regulation of their dynamics is still not well understood. Moreover, how the timing of these events are mechanistically regulated to maintain proper mitochondrial function and overall cellular homeostasis remains unclear. Using advanced high-speed super-resolution microscopy and single molecule tracking studies inside organelles, this proposal seeks to investigate how the dynamics of intra-organelle components and individual protein complexes are regulated within organelles such as the mitochondria. In addition, as defects in mtDNA and mitochondrial chaperones have been linked to multiple human diseases which involve neurodegeneration, these studies will also shed light on how intra-organelle events are preferentially regulated in neurons to regulate neuronal homeostasis in distinct neuronal compartments and within different neuronal populations. Finally, this work will probe the role of intra-organelle events in driving disease pathogenesis using patient- derived neurons to provide new insights into how mtDNA and mitochondrial chaperone mutations contribute to the etiology of neurological disorders, and potentially identify new therapeutic angles for targeting neurodegeneration. Together, these studies will help to answer essential questions of how key components and protein assemblies inside the mitochondria are spatially and temporally organized to dynamically regulate cellular and neuronal homeostasis. Ultimately, this work will provide a novel platform for studying the intra- organelle events occurring within other types of organelles, with the end goal of creating a dynamic cellular architecture map of the entire living cell and expanding our fundamental understanding of the real-time molecular events at nanoscale levels underlying cell biology and cellular neuroscience.

PROJECT NARRATIVE The living cell is composed of highly dynamic organelles, but the events occurring inside organelles at the intra-organelle level are still not well understood. Using advanced super-resolution microscopy, this proposal will provide new insights into intra-organelle events within organelles, such as the dynamics of mtDNA, mtRNA and mitochondrial chaperones inside the mitochondrial matrix, and shed light on how these dynamics inform mitochondrial and cellular function, neuronal homeostasis and the pathways underlying multiple mitochondrial diseases which have been linked to neurodegeneration. Elucidating the role and regulation of intra-organelle events will ultimately advance our understanding of the dynamic cellular architecture of the living cell and expand our fundamental knowledge of cellular homeostasis and neurodegenerative disease mechanisms.