Mechanisms of Lipid Droplet Formation

项目总结 脂滴(LDs)几乎存在于所有真核细胞中，它们用来储存中性物质 脂类，如甘油三酯(TGS)，作为能量和膜脂的储存库。由于他们的 在细胞新陈代谢、代谢性疾病和工业石油生产中的作用 对LD生物学的研究和进展有了很大的增长。然而，中央 LD生物学问题--这些单层结合细胞器是如何由内质网形成的 双层--仍无人应答。近年来，我们实验室和其他机构的调查 揭示了LD的形成是在不同的步骤中发生的，这些步骤受特定蛋白质的调节。 我们提出的这些步骤是：a)在ER膜中合成TG；b)收集TG透镜 在内质网内；c)TG晶状体从内质网中萌发/分离，形成“新生” LDS(直径小于200 nm)；以及d)新生LDS的生长和成熟为成熟的LDS (直径400-600 nm)。我们提案的首要目标是揭示 形成LD的各个步骤的基础。控制这些步骤的蛋白质似乎是 参与确定LD形成位点的数量，而其他蛋白质参与其中 在促进初级保健服务的萌芽/将其从紧急救济服务中分离方面。此外，我们最近表明， ER蛋白Seipin促进萌芽的初生LDs成熟为成熟的初始LDs， 尽管确切的机制尚不清楚。在这项提案中，我们将使用尖端电池 生物工具(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)和体外测试系统，以解开LD中的特定步骤 队形。在目标1中，我们将研究如何确定LD形成部位，并测试 假设控制内质网形状的特定内质网蛋白，如atlastin，调节LD 急诊室里的队形。目标2侧重于LDS与ER的萌芽(即完全分离)。 在这里，我们将利用体外实验来鉴定促进LD形成的细胞质因子 以及在体外系统中与内质网的萌发/分离。我们将确定这些因素，并 确定它们如何在体外和细胞内促进LD的形成。目标3侧重于人的成熟 从萌芽的新生LDS到成熟的初始LDS，我们最近展示了一个涉及Seipin的过程。 我们将确定Seipin在LD期间导致新生LDS生长的机制 形成，我们将确定除了Seipin外，哪些蛋白质保持LD-ER接触 在LD形成过程中。成功完成我们的目标将对……领域产生重大影响 LD生物学，并揭示了许多过度代谢性疾病的基础生物学 脂滴堆积，如肥胖和相关疾病。

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.