In mammary epithelial cells cytoplasmic lipid droplets that are formed and secreted into milk provide essential fatty acids and cholesterol for membrane synthesis and a large percentage of neonatal calories. Since the mammary gland is one of the most active lipogenic organs of the body it provides an ideal model system to understand the cellular and molecular processes involved in lipid droplet formation. The overall goal of this project is to define how lipids, triglycerides and cholesterol esters, synthesized in the endoplasmic reticulum, are packaged into cytoplasmic lipid droplets. The PAT family (Perilipin, Adipophilin, TIP47) of lipid droplet associated proteins and caveolins have been shown to play an essential role in the formation, stabilization and metabolism of lipid droplets in other tissues. In the mammary gland this process is developmentally coordinated and regulated by the hormones of pregnancy. We hypothesize that the endoplasmic reticulum of mammary alveolar epithelial cells undergoes structural alterations during pregnancy resulting in the formation of specific lipid droplet assembly domains that contain the enzymes of lipid synthesis as well as the proteins necessary for lipid droplet assembly. In Specific Aim 1 we use morphological techniques to define the temporal alterations in the structure of the endoplasmic reticulum during lipid droplet formation. These changes will be correlated with expression of lipid synthetic enzymes and lipid synthesis in Specific Aim 2. The expression levels of candidate genes will be altered in Specific Aim 3 to develop a precise molecular model for the interactions of proteins involved in formation of lipid droplets. These experiments are designed to identify the structural and biochemical processes involved in formation of lipid droplets and to provide a framework for understanding the role of hormones in this process. The unique aspect of this proposal is the hypothesis that CLD form within specialized domains in the ER containing all the synthetic machinery. If this hypothesis is correct it will substantially alter our current understanding of the cell biological mechanisms involved in the initiation of CLD formation.