Deciphering the lipid composition of primary cilia in human metabolic disease

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PROJECT SUMMARY The primary cilium is a cell surface organelle that plays critical roles in human health. Multiple G protein-coupled receptors (GPCRs) are trafficked to the primary cilium where they carry extracellular signals across the membrane to initiate intracellular signaling events. Genetic studies have linked GPCR signaling at primary cilia to human metabolic disease. For example, ciliary GPCRs regulate diverse metabolic processes such as the perception of satiety, the secretion of insulin, and the formation of adipocytes. With 42% of U.S. adults classified as obese, understanding how the primary cilium functions as a GPCR signaling center is critical to addressing this major human health concern. In this proposal I will test how the primary cilium membrane composition controls GPCR signaling. Lipids have profound effects on GPCR signaling through (1) direct binding and (2) indirect modulation by changing membrane bilayer properties. Through these interactions, GPCRs play fundamental roles in cellular lipid signaling and organismal lipid homeostasis. A key barrier to understanding how GPCRs regulate human metabolism is the lack of tools to study lipids in cells. I have developed a unique skillset and a set of tools to visualize, quantify and manipulate lipids at primary cilia to address these challenges. This proposal tests three fundamental questions related to cilia lipid signaling. First, despite its importance in regulating metabolism, it is unknown whether the primary cilia membrane is altered by dietary lipids. External stimuli robustly change the plasma membrane (PM) composition to drive cell signaling events required for cellular homeostasis. To test this at cilia, I will treat cells with fatty acids or cholesterol to mimic the human diet and ask whether the membrane composition is altered. Cilia lipids will be further examined in mice fed different chow diets and in mice with metabolic disease (Aim 1). Second, despite having a membrane that is structurally continuous with the PM, the primary cilium maintains a distinct membrane composition that is critical for GPCR signaling through unknown mechanisms. I will test whether lipids exchange between the PM and cilia membrane, and then determine how lipids are delivered to cilia by disrupting membrane trafficking pathways (Aim 2). Finally, I will determine what lipids are required for FFAR4 activity at cilia. FFAR4 is a GPCR that intimately relies on the integrity of the cilia membrane environment for signaling. By cross comparing this screen with a screen I previously performed to find regulators of the ciliary GPCR SMO, I will identify generalizable pathways controlling ciliary lipid homeostasis. Importantly, this screen will also determine the specific lipid requirements of FFAR4, which is a promising target for treating metabolic disease due to its role in insulin secretion and adipogenesis. Through this research I will develop robust methodologies that can be utilized to study cilia in diverse contexts, and I will advance our understanding of cilia biology, membrane biology, and GPCR signaling. The long-term vision of this project is to identify new strategies for correcting lipid or GPCR signaling defects found in human metabolic disease.