PROJECT SUMMARY AND RELEVANCE The long-term goals of this project are to define the intracellular functions of the bioactive sphingolipid metabolites, sphingosine-1-phosphate (S1P) and sphingosine. Most of the known actions of S1P, produced intracellularly from sphingosine by two closely related sphingosine kinases, SphK1 and SphK2, are initiated by signaling through S1P receptors. However, lower organisms have similar evolutionary conserved sphingolipid metabolic enzymes and although their sphingolipid metabolites have important functions, they do not have S1P receptors. Based on published and exciting preliminary results, our overarching hypothesis is that recruitment of SphK1 to sphingosine-containing vesicles and conversion of sphingosine to S1P plays a critical role in membrane trafficking, formation and functions of membrane contact sites between the endoplasmic reticulum and late endosomes and lysosomes. These membrane contact sites are highly regulated regions of close membrane apposition between organelles that serve as platforms for transfer and metabolism of lipids, including cholesterol and sphingolipids, calcium homeostasis, and to ensure inter-organellar communication. To test this concept, we propose the following specific aims: Aim 1. Identify the key steps in endocytic trafficking and autophagic fluxes that rely on sphingosine to S1P conversion by SphK1; Aim 2. Evaluate the role of the sphingosine/SphK1/S1P axis in regulation of ER-membrane contact sites and consequently on cholesterol and sphingolipid metabolism; Aim 3. Determine the molecular mechanisms by which sphingolipid metabolites regulate membrane trafficking and membrane contact sites and identify their intracellular targets. This proposal will utilize several innovative technologies, including cutting edge electron microscopy and systems-level live cell multispectral fluorescence-based spectroscopy imaging, as well as trifunctional sphingosine click chemistry and state of the art sphingolipidomic mass spectrometry. The new conceptual groundwork in this proposal will alter the view of the enigmatic nature of sphingolipid metabolite signaling and will reveal their ancient but understudied intracellular roles in regulation of membrane dynamics and contact sites with the endoplasmic reticulum as sensors that integrate cell growth signals and coordinate cholesterol and sphingolipid metabolism. This should also provide deeper understanding of how perturbations of these fundamental biological processes contribute to human diseases. !