PROJECT SUMMARY Sophisticated compartmentalization into membrane enclosed organelles of dedicated function is a hallmark of eukaryotic cells. Organelles come in close apposition to each other forming membrane contact sites that are nodes for communication, critical for many cellular functions. Despite their ubiquitous presence, little remains known about how the molecular components of these contact organize into functional signaling conduits between disparate organelles. This is largely because the dynamic nature and low cellular copy number of these contact sites make it impossible to use conventional biochemical approaches to purify them and embark on classical structure-function studies using X-crystallography, NMR, and single particle cryo-electron microscopy. Here, we propose integration of novel cutting-edge microscopy modalities to visualize organellar interactions directly within unperturbed cellular context. We will establish our workflow on one contact site formed by proteins on endoplasmic reticulum, mitochondria, and lysosomes, respectively. Proteins at this contact site will be tagged within iPSCs, which will be differentiated into neurons on microscopy grids. Confocal microscopy at cryogenic temperatures will enable detection of these contacts with high subcellular precision. Guided by the fluorescence localization, thin electron-transparent windows will be micromachined inside these cells. Subsequent cryo- electron tomography, subtomogram averaging and deep-classification will enable three-dimensional structure determination of these contact sites. Our work will help answer a vital biological question: how ER-mitochondria- lysosome contact site marks mitochondria for fission, a process essential for maintaining their healthy supply in the cell. The pipelines for automated sample preparation, data collection, and processing established during this project will serve as a transformative blueprint for future structural studies in-situ. This project will lay a foundation towards high-resolution structural characterization of the emerging, underexplored and complex field of membrane contact sites and a mechanistic basis for understanding their function in health and disease. This project will provide much needed directional framework to researchers elsewhere who are interested in studying protein structures directly in cells, isolated tissues and organoids.

PROJECT NARRATIVE Organelles come in close proximity to one another to form membrane contact sites. These sites are hubs for metabolite, protein, and lipid exchange and regulate multitude of important cellular functions. Molecular architecture of these sites will not only give insights into the inner working of fundamental cellular processes but also the role of inter-organelle miscommunication in human diseases.