Deciphering the lipid composition of primary cilia in human metabolic disease

项目总结 初生纤毛是细胞表面的细胞器，在人类健康中起着至关重要的作用。多个G蛋白偶联 受体(GPCRs)被运输到初级纤毛，在那里它们携带细胞外信号穿过 启动细胞内信号事件的膜。遗传学研究已将初级纤毛的gpr信号联系起来。 人类代谢性疾病。例如，睫状GPCRs调节不同的代谢过程，如 感觉饱腹感、胰岛素的分泌和脂肪细胞的形成。42%的美国成年人被列为 作为肥胖者，了解初级纤毛如何作为gpr信号中心的功能对于解决 这一重大的人类健康问题。在这项提案中，我将测试初级纤毛膜的组成 控制GPCR信号转导。脂类通过(1)直接结合和(2)对gpr信号产生深远的影响。 通过改变膜双分子层性质的间接调节。通过这些互动，GPCRs发挥了作用 在细胞内脂信号和生物体内脂平衡中的基础作用。理解的一个关键障碍 GPCRs如何调节人类新陈代谢，是因为缺乏研究细胞中脂质的工具。我开发了一种独特的 技能集和一套工具，以可视化，量化和操纵脂肪在初级纤毛，以应对这些挑战。 这项建议测试了与纤毛脂质信号有关的三个基本问题。首先，尽管它 在调节新陈代谢中的重要性，尚不清楚初级纤毛膜是否因饮食而改变。 脂类。外界刺激强烈改变质膜(PM)组成以驱动细胞信号事件 细胞动态平衡所必需的。为了在纤毛上测试这一点，我将用脂肪酸或胆固醇处理细胞，以模拟 人的饮食和询问膜的成分是否改变。将在小鼠身上进一步检查纤毛脂肪 饲喂不同的食物和代谢性疾病的小鼠(目标1)。第二，尽管有一层膜是 初级纤毛在结构上与PM连续，维持一种独特的膜成分，这是至关重要的 通过未知的机制进行GPCR信号传递。我要测试一下首相和纤毛之间的脂类交换 膜，然后通过扰乱膜转运途径来确定脂类如何输送到纤毛 (目标2)。最后，我将确定纤毛FFAR4活动所需的脂类。FFAR4是一种GPCR， 紧密依赖于纤毛膜环境的完整性进行信号传递。通过交叉比较此屏幕 通过我之前进行的一次筛查，找到了睫状GPCRSMO的调节因子，我将确定可推广的 控制纤毛脂质动态平衡的途径。重要的是，这一筛查还将确定特定的脂质 FFAR4的需求，由于其在胰岛素中的作用，FFAR4是治疗代谢性疾病的有希望的靶点 分泌和成脂作用。通过这项研究，我将开发出可靠的方法，可用于 在不同的背景下研究纤毛，我将增进我们对纤毛生物学、膜生物学和 GPCR信号转导。该项目的长期愿景是确定纠正血脂或gpcr的新策略。 人类代谢性疾病中发现的信号缺陷。

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.