Riccio Victoria, Vissa Miluska, McQuibban Angus, Kim Peter Kijun
Cell Biology Department, The Hospital for Sick Children, Toronto, Canada.
Department of Biochemistry, University of Toronto, Toronto, Canada.
Bio Protoc. 2019 Dec 20;9(24):e3455. doi: 10.21769/BioProtoc.3455.
The process of autophagy is an essential cellular mechanism, required to maintain general cell health through the removal of dysfunctional organelles, such as the ER, peroxisomes and mitochondria, as well as protein aggregates, and bacteria. Autophagy is an extremely dynamic process, and tools are constantly being developed to study the various steps of this process. This protocol details a method to study the end steps of autophagy-lysosomal fusion and the formation of the autolysosome. Many techniques have been used to study the various steps of the autophagy process. Here we describe the RedGreen-assay (RG-assay), an immunofluorescence-based technique used to visualize the targeting of substrates to the autolysosome in live cells. This technique takes advantage of the low lysosomal pH and over-expression of a tandem GFP-mCherry tagged protein targeted to an organelle of interest. While in the neutral cytosol or autophagosome, both GFP and RFP will fluoresce. However, within the autolysosome, the GFP signal is quenched due to the low pH environment and the RFP emission signal will predominate. This technique is readily quantifiable and amenable to high throughput experiments. Additionally, by tagging the GFP-RFP tandem fluorescent protein with organelle specific targeting sequences, it can be used to measure a wide range of substrates of autophagy.
自噬过程是一种重要的细胞机制，通过清除功能失调的细胞器（如内质网、过氧化物酶体和线粒体）、蛋白质聚集体以及细菌来维持细胞的整体健康。自噬是一个极其动态的过程，研究该过程各个步骤的工具也在不断开发。本方案详细介绍了一种研究自噬 - 溶酶体融合的最终步骤以及自噬溶酶体形成的方法。许多技术已被用于研究自噬过程的各个步骤。在这里，我们描述了红绿分析法（RG分析法），这是一种基于免疫荧光的技术，用于在活细胞中可视化底物靶向自噬溶酶体的过程。该技术利用了溶酶体的低pH值以及靶向感兴趣细胞器的串联GFP - mCherry标记蛋白的过表达。在中性细胞质或自噬体中时，GFP和RFP都会发出荧光。然而，在自噬溶酶体内，由于低pH环境，GFP信号被淬灭，RFP发射信号将占主导。该技术易于量化且适用于高通量实验。此外，通过用细胞器特异性靶向序列标记GFP - RFP串联荧光蛋白，它可用于测量多种自噬底物。
