The interactions between lysosomes and lipid droplets represent the processes of mobilization of lipids and energy required for cell metabolism and lipid homeostasis. Lipophagy, for lipid degradation, is a known type of lysosome-lipid droplet interaction. Our preliminary studies and results from others suggest there is a second type of lysosome-lipid droplet interaction, which mediates lipid transport from lysosomes to lipid droplets for lipid synthesis and storage. This process is probably mediated by the organelle membrane contact sites (MCS), which are recently defined as tethered organelles, organized by protein-protein and protein-lipid interactions. MCS provide vectorial transport of lipids between heterologous organelles and are increasingly appreciated for their role in lipid homeostasis. Our group will study lysosome-lipid droplet interactions with a focus of lysosome-lipid droplet MCS in the context of fatty acid metabolism. Regulation of free fatty acids within the cell is critical for normal cell functions, as excess cytosolic free fatty acids cause toxic effects to cells and tissues (named lipotoxicity) and thus have been recognized as a causative factor in many metabolic disorders, including non-alcoholic fatty liver disease (NAFLD). One source of cytosolic free fatty acids is the lysosome, which releases free fatty acids after digesting endocytosed extracellular lipids and autophagic intracellular membranes and lipids. Increasing the transport and storage of free fatty acids into lipid droplets protects cells, including liver cells, from lipotoxicity. Therefore, lysosome-lipid droplet MCS may play an important role in prevention of lysosomal free fatty acid-induced lipotoxicity. However, it is not clear whether MCS exist between lysosomes and lipid droplets and whether lysosome-lipid droplet MCS transport free fatty acids. Moreover, it is not known whether lysosome-lipid droplet MCS play a role in preventing lysosome- triggered lipotoxicity and regulating fatty acid metabolism. Our recent studies support the existence of lysosome-lipid droplet MCS in human liver cells, and we identified the protein-protein interactions at lysosome-lipid droplet MCS through a genetic screening in yeast. In the next five years, we will define the principal components and function of lysosome-lipid droplet MCS, and test if promoting MCS formation will prevent or reduce lipotoxicity and thus NAFLD progression in a mouse model. By investigating the features, mechanisms, and physiological functions of lysosome-lipid droplet MCS, our work will reveal a previously undefined interaction between lysosomes and lipid droplets and uncover a novel mechanism of free fatty acid transport. This mechanism will deepen the understandings to fatty acid metabolism, lipotoxicity, and the pathology of NAFLD.