Project Summary/Abstract The long-term objective of this research program is to understand the molecular mechanisms of Ca2+ signaling in neurons intimately linked to neural health and function. In this project, our particular emphasis is on uncovering the structural and molecular foundations underlying Ca2+ signaling at specialized membrane contact sites between the endoplasmic reticulum (ER) and mitochondria. These ER-mitochondria contacts (ERMCs) are hotspots for Ca2+-transport from the ER, the cell’s major Ca2+ storage organelle, to mitochondria. Inositol 1,4 5- trisphosphate receptors (IP3Rs) are the most widely expressed Ca2+ release channels residing in the ER membranes and are essential components of ERMCs, participating in both membrane tethering and as a Ca2+ transport system that is activated by IP3 to liberate Ca2+ sequestered in ER stores. IP3Rs serve as scaffolds at these ERMCs, where they interact with other proteins and form multiprotein complexes, responsible for the precise regulation of Ca2+ signaling in response to various extracellular and intracellular signals. This regulation is crucial for many cellular processes, including cell signaling, metabolism, and apoptosis. Emerging evidence suggests that functional IP3Rs cluster within ER membranes, and this spatial organization ensures that Ca2+ release is appropriately coordinated and tailored to specific cellular needs. Dysregulation of IP3Rs or loss of IP3Rs by knockout have been reported to reduce the ERMCs that leads to pathological conditions by either an exaggerated cell death, as in neurodegenerative diseases, or escape from cell death as in some types of cancer. Despite the rapid progress carried out in recent years, the structural basis and molecular mechanisms underlying protein-protein interactions at ER-mitochondrial membrane contacts remain largely obscure. The lack of any satisfactory explanation for how IP3Rs are assembled into clusters and communicate with other organelles highlights the urgent need to structurally characterize the subcellular geography of IP3Rs and their interactions with mitochondria. In this project to address this long-standing conundrum, we will utilize cutting-edge cryogenic electron tomography (cryoET) to study frozen-hydrated IP3R complexes within ER-mitochondrial membrane junctions isolated from mouse brain (aim 1) and in situ within mammalian cells expressing recombinant IP3Rs as well as in native neurons cultured on EM grids (aim 2). To accomplish these studies, we endeavor to develop an experimental workflow for in situ cryoET analysis that will have broad applicability to studies of other subcellular macromolecular complexes. The proposed work is highly important as it will shed light on the complex network of IP3R molecular interactions within neurons, with implications for a wide range of biological processes and diseases. Anticipated structural and molecular insights will serve as a platform for the development of potential therap...