Abstract
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.