Molecular Cell Biology of Autophagy, The Francis Crick Institute; MRC Laboratory of Molecular Biology.
Molecular Cell Biology of Autophagy, The Francis Crick Institute.
J Vis Exp. 2023 Jun 16(196). doi: 10.3791/65349.
Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive pathological conditions. A set of proteins, called ATG proteins, comprise the core autophagy machinery and work together in a defined hierarchy. Studies in recent years have improved our knowledge of the autophagy pathway. Most recently, it has been proposed that ATG9A vesicles are at the heart of autophagy, as they control the rapid de novo synthesis of an organelle called the phagophore. The study of ATG9A has proven challenging, since ATG9A is a transmembrane protein, and it is present in different membrane compartments. As such, understanding its trafficking is an important element for understanding autophagy. Here, detailed methods are presented that can be used to study ATG9A and, in particular, its localization using immunofluorescence techniques, which can be assessed and quantified. The pitfalls of transient overexpression are also addressed. The correct characterization of ATG9A function and the standardization of techniques to analyze its trafficking are crucial to further characterize the events governing autophagy initiation.
自噬是一种高度保守的途径，细胞利用它来维持体内平衡、降解受损的细胞器、抵御入侵的病原体并在病理条件下存活。一组称为 ATG 蛋白的蛋白质构成了核心自噬机制，并在一个明确的层次结构中协同工作。近年来的研究提高了我们对自噬途径的认识。最近，有人提出 ATG9A 小泡是自噬的核心，因为它们控制着一种称为吞噬体的细胞器的快速从头合成。对 ATG9A 的研究证明具有挑战性，因为 ATG9A 是一种跨膜蛋白，并且存在于不同的膜隔室中。因此，理解其运输是理解自噬的一个重要因素。本文详细介绍了可以用于研究 ATG9A 的方法，特别是使用免疫荧光技术评估和定量其定位的方法。还解决了瞬时过表达的陷阱问题。正确表征 ATG9A 的功能和标准化技术来分析其运输对于进一步表征控制自噬起始的事件至关重要。
