Mechanisms of Lipid Droplet Formation

项目摘要 脂滴（LD）存在于几乎所有的真核细胞中，它们用于储存中性 脂质，例如甘油三酸酯（TG），作为能量和膜脂的储存库。由于其 在细胞代谢、代谢性疾病和工业石油生产中的作用， LD生物学的研究大量增加并取得进展。但中央 LD生物学的问题-这些单层结合的细胞器是如何从ER形成的 双层-仍然没有答案。近年来，我们实验室和其他机构的研究 已经揭示了LD的形成发生在由特定蛋白质调节的不同步骤中。 我们提出这些步骤是：a）在ER膜中合成TG; B）收集TG透镜 在ER膜内; c）TG晶状体从ER出芽/分离以形成“新生的” LD（直径小于200 nm）;和d）新生LD生长和成熟为成熟iLD （400-600 nm直径）。我们提案的首要目标是揭示 LD形成的各个步骤的基础。控制这些步骤的蛋白质似乎是 参与决定LD形成位点的数量，而其他蛋白质参与 促进LD从ER的出芽/分离。此外，我们最近表明， ER蛋白seipin促进出芽的新生LD成熟为成熟的初始LD， 尽管确切的机制尚不清楚。在这个建议中，我们将利用尖端细胞 生物学工具（细胞的CRISPR工程; LD形成的蛋白质标记物;共聚焦，晶格 光片和FIB-SEM显微镜）和体外测定系统，以阐明LD中的特定步骤 阵在目标1中，我们将研究如何确定LD形成位点并测试 假设控制内质网形状的特定内质网蛋白，如atlastin， 在急诊室里形成。目标2侧重于萌芽（即，完全分离）。 在这里，我们将利用体外试验，以确定细胞质因子，促进LD的形成 以及在体外系统中从ER出芽/分离。我们将确定这些因素， 确定它们如何在体外和细胞中促进LD形成。目标3侧重于 出芽的新生LD到成熟的初始LD，我们最近展示了一个涉及seipin的过程。 我们将确定机制，其中seipin导致生长的新生LD在LD 形成，我们将确定哪些蛋白质，除了seipin，保持LD-ER接触 在LD形成期间。成功完成我们的目标将对以下领域产生重大影响： LD生物学和揭示基础的生物学，众多的代谢疾病的过度 脂滴积聚，如肥胖及相关疾病。

PROJECT SUMMARY Lipid droplets (LDs) are found in virtually all eukaryotic cells where they serve to store neutral lipids, such as triglycerides (TGs), as reservoirs of energy and membrane lipids. Owing to their roles in cellular metabolism, metabolic diseases, and industrial oil production, there has been a huge increase in investigation of LD biology and progress is being made. However, the central question of LD biology – how these monolayer-bound organelles are formed from the ER bilayer – remains unanswered. In recent years, investigations by our laboratory and others have revealed that LD formation occurs in distinct steps that are regulated by specific proteins. We propose these steps are: a) TG synthesis in the ER membrane; b) collection of TG lenses within the ER membrane; c) budding/separation of TG lenses from the ER to form “nascent” LDs (less than 200 nm diameter); and d) growth and maturation of nascent LDs to mature iLDs (400-600 nm diameter). The overarching goal of our proposal is to reveal the mechanistic bases for the individual steps of LD formation. Proteins governing these steps appear to be involved in determining the number of LD formation sites, whereas other proteins are involved in promoting the budding/separation of LDs from the ER. Moreover, we recently showed that the ER protein seipin promotes the maturation of budded nascent LDs to mature initial LDs, although the precise mechanism is unclear. In this proposal, we will utilize cutting edge cell biology tools (CRISPR engineering of cells; protein markers of LD formation; confocal, lattice light-sheet and FIB-SEM microscopy) and in vitro assay systems to unravel specific steps in LD formation. In Aim 1, we will study how LD formation sites are determined and test the hypothesis that specific ER proteins that control ER shape, such as atlastin, regulate LD formation in the ER. Aim 2 focuses on budding (i.e., complete separation) of LDs from the ER. Here we will utilize an in vitro assay to identify cytoplasmic factors that promote LD formation and budding/separation from the ER in an in vitro system. We will identify these factors and determine how they promote LD formation in vitro and in cells. Aim 3 focuses on maturation of budded nascent LDs to mature initial LDs, a process we showed recently that involves seipin. We will determine the mechanism by which seipin causes growth of nascent LDs during LD formation, and we will determine which proteins, in addition to seipin, maintain LD-ER contact during LD formation. Successful completion of our aims will have a major impact on the field of LD biology and reveal the basic biology that underlies numerous metabolic diseases of excess lipid droplet accumulation, such as obesity and related diseases.