Roles of macroautophagy in Drosophila development

巨噬，以下简称自噬（=“自噬”），是一种进化保守的、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。自噬使细胞能够在营养缺乏和其他应激条件下存活，并在分化、组织重塑、衰老和免疫等方面发挥作用。最近的研究也揭示了自噬和脂质代谢之间的联系。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，并且在正常发育过程中，复杂组织和生物体中自噬和脂质代谢之间的相互作用仍然不明确。本研究计划的长期目标是阐明自噬在整个生物体正常发育过程中的功能和调控。为了实现这一目标，我们将采用具有进化保守的自噬相关（Atg）基因的模型系统——黑腹果蝇。本研究的主要目的是研究果蝇自噬与脂质代谢之间的关系。我们最近发现了一种新的自噬和脂质代谢调节因子D-TNFAIP8，在这里我们提出确定其作用机制。我们将验证我们的假设，即D-TNFAIP8功能调节脂噬，即自噬对脂质的降解。此外，我们将利用我们新开发的自噬工具来鉴定新的果蝇基因，这些基因在体外和体内调节脂噬。为了验证我们的假设并实现我们的目标，我们有三个具体目标：目标1：确定D-TNFAIP8在多种细胞类型和组织的自噬和脂质代谢中的作用。在这个目标中，我们将确定D-TNFAIP8在自噬途径的哪一步起作用（例如自噬体形成和/或溶酶体转换），以及D-TNFAIP8是否影响脂质生物发生、储存和/或分解代谢。我们将确定其在这些过程中的功能是否具有细胞类型特异性，我们将研究与自噬和脂质途径已知组分的遗传相互作用。目的2：阐明D-TNFAIP8调控自噬和脂质代谢的分子机制。蛋白-蛋白相互作用和上位分析将用于确定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脂肪吞噬的基因。为此，我们将使用RNAi和遗传方法结合自噬通量报告来识别和表征新的脂噬调节因子。这项研究将为自噬与调节细胞能量储存和营养代谢的其他重要过程之间的关系提供新的认识。我们将了解参与这些过程调节的分子机制，并描述体内脂噬的潜在新调节因子。由于这些细胞过程在进化上是高度保守的，我们在果蝇身上的发现很可能会为包括人类在内的其他生物的自噬/脂噬发育功能提供见解。

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.