Measuring autophagosome flux

Macroautophagy/autophagy is a proteolytic pathway that is involved in both bulk degradation of cytoplasmic proteins as well as in selective degradation of cytoplasmic organelles. Autophagic flux is often defined as a measure of autophagic degradation activity, and many techniques exist to assess autophagic flux. Although these techniques have generated invaluable information about the autophagic system, the quest continues for developing methods that not only enhance sensitivity and provide a means of quantification, but also accurately reflect the dynamic character of the pathway. Based on the theoretical framework of metabolic control analysis, where the autophagosome flux is the quantitative description of the rate a flow along a pathway, here we treat the autophagy system as a multi-step pathway. We describe a single-cell fluorescence live-cell imaging-based approach that allows the autophagosome flux to be accurately measured. This method characterizes autophagy in terms of its complete autophagosome and autolysosome pool size, the autophagosome flux, J, and the transition time, τ, for autophagosomes and autolysosomes at steady state. This approach provides a sensitive quantitative method to measure autophagosome flux, pool sizes and transition time in cells and tissues of clinical relevance. <b>Abbreviations</b>: ATG5/APG5, autophagy-related 5; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; J, flux; MEF, mouse embryonic fibroblast; MTOR, mechanistic target of rapamycin kinase; <i>nA</i>, number of autophagosomes; <i>nAL</i>, number of autolysosomes; <i>nL</i>, number of lysosomes; p-MTOR, phosphorylated mechanistic target of rapamycin kinase; RFP, red fluorescent protein; siRNA, small interfering RNA; τ, transition time; TEM, transmission electron microscopy.

巨自噬（Macroautophagy）/自噬（autophagy）是一类蛋白水解通路，既参与细胞质蛋白的批量降解，也介导细胞质细胞器的选择性降解。自噬流（autophagic flux）通常被用作自噬降解活性的量化指标，目前已有多种技术可用于评估自噬流。尽管此类技术已为自噬系统研究提供了宝贵的研究信息，但学界仍在持续研发既能提升灵敏度、实现定量检测，又能准确反映该通路动态特性的方法。
基于代谢控制分析的理论框架——其中自噬体通量（autophagosome flux）是对通路内物质流动速率的定量描述——本研究将自噬系统视为多步通路。我们描述了一种基于单细胞荧光活细胞成像的方法，可精准测定自噬体通量。该方法通过完整自噬体与自溶酶体池的大小、自噬体通量J以及稳态下自噬体与自溶酶体的转换时间τ来表征自噬水平。此方法可为临床相关的细胞与组织中的自噬体通量、池大小及转换时间提供灵敏的定量检测手段。
缩写：ATG5/APG5（autophagy-related 5）：自噬相关蛋白5；GFP（green fluorescent protein）：绿色荧光蛋白；LAMP1（lysosomal-associated membrane protein 1）：溶酶体相关膜蛋白1；MAP1LC3/LC3（microtubule-associated protein 1 light chain 3）：微管相关蛋白1轻链3；J（flux）：通量；MEF（mouse embryonic fibroblast）：小鼠胚胎成纤维细胞；MTOR（mechanistic target of rapamycin kinase）：雷帕霉素靶蛋白激酶；*nA*（number of autophagosomes）：自噬体数量；*nAL*（number of autolysosomes）：自溶酶体数量；*nL*（number of lysosomes）：溶酶体数量；p-MTOR（phosphorylated mechanistic target of rapamycin kinase）：磷酸化雷帕霉素靶蛋白激酶；RFP（red fluorescent protein）：红色荧光蛋白；siRNA（small interfering RNA）：小干扰RNA；τ（transition time）：转换时间；TEM（transmission electron microscopy）：透射电子显微镜。