PROJECT SUMMARY Lipid droplets (LDs) are found in virtually all eukaryotic cells where they serve to store neutral lipids, such as triglycerides (TGs), as reservoirs of energy and membrane lipids. Owing to their roles in cellular metabolism, metabolic diseases, and industrial oil production, there has been a huge increase in investigation of LD biology and progress is being made. However, the central question of LD biology – how these monolayer-bound organelles are formed from the ER bilayer – remains unanswered. In recent years, investigations by our laboratory and others have revealed that LD formation occurs in distinct steps that are regulated by specific proteins. We propose these steps are: a) TG synthesis in the ER membrane; b) collection of TG lenses within the ER membrane; c) budding/separation of TG lenses from the ER to form “nascent” LDs (less than 200 nm diameter); and d) growth and maturation of nascent LDs to mature iLDs (400-600 nm diameter). The overarching goal of our proposal is to reveal the mechanistic bases for the individual steps of LD formation. Proteins governing these steps appear to be involved in determining the number of LD formation sites, whereas other proteins are involved in promoting the budding/separation of LDs from the ER. Moreover, we recently showed that the ER protein seipin promotes the maturation of budded nascent LDs to mature initial LDs, although the precise mechanism is unclear. In this proposal, we will utilize cutting edge cell biology tools (CRISPR engineering of cells; protein markers of LD formation; confocal, lattice light-sheet and FIB-SEM microscopy) and in vitro assay systems to unravel specific steps in LD formation. In Aim 1, we will study how LD formation sites are determined and test the hypothesis that specific ER proteins that control ER shape, such as atlastin, regulate LD formation in the ER. Aim 2 focuses on budding (i.e., complete separation) of LDs from the ER. Here we will utilize an in vitro assay to identify cytoplasmic factors that promote LD formation and budding/separation from the ER in an in vitro system. We will identify these factors and determine how they promote LD formation in vitro and in cells. Aim 3 focuses on maturation of budded nascent LDs to mature initial LDs, a process we showed recently that involves seipin. We will determine the mechanism by which seipin causes growth of nascent LDs during LD formation, and we will determine which proteins, in addition to seipin, maintain LD-ER contact during LD formation. Successful completion of our aims will have a major impact on the field of LD biology and reveal the basic biology that underlies numerous metabolic diseases of excess lipid droplet accumulation, such as obesity and related diseases.