a Department of Biochemistry, Faculty of Natural Sciences , University of Stellenbosch , Stellenbosch , South Africa.
b Department of Laboratory Medicine and Pathology , University of Minnesota , Minneapolis , MN , USA.
Autophagy. 2018;14(6):1060-1071. doi: 10.1080/15548627.2018.1469590. Epub 2018 Jul 20.
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.
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; nA, number of autophagosomes; nAL, number of autolysosomes; nL, 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.
自噬是一种蛋白水解途径，既参与细胞质蛋白的批量降解，也参与细胞质细胞器的选择性降解。自噬流通常被定义为自噬降解活性的衡量标准，有许多技术可用于评估自噬流。尽管这些技术为自噬系统提供了宝贵的信息，但人们仍在继续寻求开发不仅能提高灵敏度并提供定量手段，而且还能准确反映途径动态特性的方法。基于代谢控制分析的理论框架，自噬体通量是沿途径流动的速率的定量描述，我们将自噬系统视为多步骤途径。我们描述了一种基于单细胞荧光活细胞成像的方法，可准确测量自噬体通量。该方法根据完整的自噬体和自溶酶体池大小、自噬体通量 J 和自噬体和自溶酶体在稳态下的转变时间τ来描述自噬。这种方法提供了一种灵敏的定量方法，可以测量细胞和组织中自噬体通量、池大小和转变时间。
ATG5/APG5，自噬相关 5；GFP，绿色荧光蛋白；LAMP1，溶酶体相关膜蛋白 1；MAP1LC3/LC3，微管相关蛋白 1轻链 3；J，通量；MEF，小鼠胚胎成纤维细胞；MTOR，雷帕霉素靶蛋白激酶；nA，自噬体数量；nAL，自溶酶体数量；nL，溶酶体数量；p-MTOR，磷酸化雷帕霉素靶蛋白激酶；RFP，红色荧光蛋白；siRNA，小干扰 RNA；τ，转变时间；TEM，透射电子显微镜。
