Roles of macroautophagy in Drosophila development

巨自噬在果蝇发育中的作用

• 批准号: RGPIN-2014-05029
• 负责人: Gorski, Sharon
• 金额: $ 2.99万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566903
• 关键词: Roles; macroautophagy; Drosophila; development

Macroautophagy, hereafter referred to as autophagy (= “self-eating”), is an evolutionarily conserved and ubiquitous cellular catabolic process responsible for the degradation and recycling of long-lived proteins and organelles. Autophagy enables cell survival during nutrient starvation and other stress conditions, and plays a role in differentiation, tissue remodeling, aging, and immunity. Recent studies have also revealed links between autophagy and lipid metabolism. However, the majority of studies to date have been limited to single cell or cell culture model systems, and the interactions between autophagy and lipid metabolism in complex tissues and organisms during normal development remain ill defined. The long term objective of this research program is to elucidate the functions and regulation of autophagy in normal development in the context of a whole organism. To achieve this goal, we will employ the model system, Drosophila melanogaster, which has evolutionarily conserved autophagy related (Atg) genes. The primary objective of this proposal is to investigate the relationships between autophagy and lipid metabolism in Drosophila. We recently discovered D-TNFAIP8, a novel regulator of both autophagy and lipid metabolism, and here we propose to determine its mechanism of action. We will test our hypothesis that D-TNFAIP8 functions to regulate lipophagy, the degradation of lipids by autophagy. In addition we will utilize our newly developed autophagy tools to identify new Drosophila genes that modulate lipophagy in vitro and in vivo. Directed toward testing our hypothesis and achieving our objectives, we have three specific aims: Aim 1: Determine the roles of D-TNFAIP8 in autophagy and lipid metabolism in multiple cell types and tissues. In this aim we will determine at what step in the autophagy pathway D-TNFAIP8 functions (e.g. autophagosome formation and/or lysosomal turnover) and if D-TNFAIP8 affects lipid biogenesis, storage and/or catabolism. We will determine whether its function in these processes is cell-type-specific, and we will investigate genetic interactions with known components of autophagy and lipid pathways. Aim 2: Elucidate the molecular mechanisms underlying D-TNFAIP8 regulation of autophagy and lipid metabolism. Protein-protein interactions and epistasis analyses will be used to determine if DTNFAIP8 regulates lipid degradation through autophagy or other mechanisms. Aim 3: Identify genes that modulate lipophagy in Drosophila cells and tissues. In this aim, we will use RNAi and genetic approaches coupled with autophagy flux reporters to identify and characterize new lipophagy regulators. This study will provide new knowledge about the relationship between autophagy and other processes important for regulating cellular energy stores and nutrient metabolism. We will gain an understanding for molecular mechanisms involved in the regulation of these processes and characterize potential new regulators of lipophagy in vivo. Since these cellular processes are highly conserved evolutionarily, it is likely that our findings in Drosophila will provide insights into developmental functions of autophagy/lipophagy in other organisms, including humans.

巨自噬，以下简称自噬，是一种进化上保守且普遍存在的细胞分解代谢过程，负责长寿命蛋白质和细胞器的降解和再循环。自噬使细胞在营养饥饿和其他应激条件下存活，并在分化、组织重塑、衰老和免疫中发挥作用。最近的研究也揭示了自噬和脂肪代谢之间的联系。然而，到目前为止，大多数研究都局限于单细胞或细胞培养模型系统，而复杂组织和生物在正常发育过程中自噬和脂代谢之间的相互作用仍不清楚。这项研究计划的长期目标是阐明自噬在整个生物体的正常发育中的功能和调节。为了实现这一目标，我们将使用模式系统，果蝇黑腹果蝇，它具有进化保守的自噬相关(ATG)基因。这项建议的主要目的是调查果蝇自噬和脂肪代谢之间的关系。我们最近发现了D-TNFAIP8，一种新的自噬和脂代谢调节因子，我们建议在这里确定它的作用机制。我们将验证我们的假设，即D-TNFAIP8功能调节脂噬，即通过自噬来降解脂类。此外，我们将利用我们新开发的自噬工具，在体外和体内鉴定调节脂噬作用的新的果蝇基因。为了验证我们的假设和实现我们的目标，我们有三个具体的目标：目标1：确定D-TNFAIP8在多种细胞类型和组织中的自噬和脂质代谢中的作用。为此，我们将确定D-TNFAIP8在自噬途径中的哪一步发挥作用(例如，自噬小体形成和/或溶酶体周转)，以及D-TNFAIP8是否影响脂质的生物生成、储存和/或分解代谢。我们将确定它在这些过程中的功能是否是细胞类型特有的，我们将研究与自噬和脂质途径的已知组件的遗传相互作用。目的2：阐明D-TNFAIP8调节自噬和脂代谢的分子机制。蛋白质-蛋白质相互作用和上位性分析将被用来确定DTNFAIP8是否通过自噬或其他机制调节脂质降解。目的3：鉴定果蝇细胞和组织中调节吞脂性的基因。在这一目标中，我们将使用RNAi和遗传方法结合自噬通量报告程序来识别和表征新的脂噬调节因子。这项研究将为自噬和其他对调节细胞能量储存和营养代谢至关重要的过程之间的关系提供新的知识。我们将对参与调控这些过程的分子机制有一个了解，并确定体内潜在的新的脂噬调节因子。由于这些细胞过程在进化上是高度保守的，我们在果蝇中的发现很可能会为包括人类在内的其他生物的自噬/脂噬的发育功能提供见解。