Project Summary / Abstract The long-term goal of my lab is to better understand cellular mechanisms that regulate secretory granule maturation, efficient packaging of hormone, and secretion. In doing so, we will identify drug targets that improve secretory efficiency during disease. Defects in storage, maturation, or secretion of peptide hormones can lead to several mental and metabolic disorders, including depression, bipolar disorder, and diabetes. Despite the importance of this widespread secretion mechanism, the cellular signals that regulate secretory granule maturation remain unclear. Dense core secretory granules (DCSG) are organelles for the intracellular storage and stimulus-dependent exocytosis in regulated secretory cells. The hallmarks of granule maturation include: losing the clathrin coat, acidifying lumen, processing of hormone peptide, and formation of a dense core. This is consistent for many peptide processing cells. In this proposal, we will focus proinsulin processing in β cells and proopiomelanocortin (POMC) processing in neurons. In both cell types, distinct subpopulations of DCSG exist. These subpopulations have different protein and lipid content which affects their secretory capacity. For example, specific signals lead to preferential secretion of subsets of DCSG granules. We have a limited understanding of how many subpopulations exist and even less is understood regarding their spatial localization. A key gap in knowledge is understanding how different signaling networks affect maturation and the formation of distinct DCSG subpopulations. Additionally, DCSG maturation and the locations it occurs within the cell are still considered a black box. Thus, use of single-cell unbiased imaging approaches, or those with no requirement for stains or probes, are ideal for capturing DCSG maturation in the context of the entire cell. Over the next 5 years, we will establish a high-throughput, and generalizable quantitative structural cell biology platform. To do this, we will leverage soft X-ray tomography (SXT) and fluorescence lifetime imaging microscopy (FLIM). SXT will be used to map cell organization and quantify the molecular packing of secretory granules (or maturation), which has not been possible with other approaches. We will complement these studies with functional live-cell imaging, using a FLIM-based pH probe developed in my lab to monitor granule pH (maturation). We will explore how different signaling pathways influence DCSG maturation and the spatial localization of specific DCSG subpopulations. For a holistic understanding of how the rest of the cell contributes to maturation, we will quantify cellular reorganization by analyzing organelle volumes and inter-organelle contacts. We hypothesize that cAMP signaling and DCSG interactions with the mitochondria enhance secretory efficiency. The proposed work will validate our pipeline as a generalizable approach for studying DCSG maturation and uncover new mechanistic insi...