Background

Macroautophagy (herein referred to as autophagy) is an essential cellular process, required for the removal of large cellular material. It can be both general and selective, either removing bulk material or specific cytoplasmic material, respectively. The method described in this protocol can be used in the study of either process, but the focus here will be on selective autophagy. In brief, selective autophagy in mammalian cells begins with the tagging of damaged organelles or other cellular material, generally with the small protein ubiquitin. The collection of ubiquitinated proteins on the substrate allows the recruitment of autophagy receptors which interact with the ubiquitin protein and with the ATG8 family of autophagic factors on developing phagophores through their LC3 interacting region (LIR). This interaction results in the engulfment of the cargo by the phagophore, producing an autophagosome. Finally, the autophagosome fuses with the lysosome, producing an autolysosome and degrading the cargo within. Selective autophagy is responsible for degrading cargo such as peroxisomes (pexophagy), mitochondria (mitophagy), protein aggregates (aggrephagy) and bacteria (xenophagy). For a review, refer to Lahiri et al., 2019 and Morishita and Mizushima, 2019. As autophagy proceeds in many steps, different assays have been developed to study the various stages. Historically, the most popular assay to detect changes in autophagy induction is the activation of the ATG8 family of proteins, particularly LC3B. Another common assay for detecting targeting of substrate to the autolysosome is the co-localization between the cargo with either the autophagosome marker LC3, or with various lysosomal markers such as Lamp1. Co-localization studies are technically easy to perform, can be performed in a high throughout manner and can detect specific cargo within the lysosome easily through the use of fluorescent tagging. However, it is limited by the microscope resolution, and the cargo can be difficult to detect due to degradation. For a review describing these and other methods for detecting autophagy, refer to Klionsky et al., 2016.

The protocol described here, lysosome targeting Red-Green (RG) assay, can be used to study the end stages of autophagy, namely the localization of the cargo to the autolysosome. This assay takes advantage of the acidic pH of the lysosome (~4.6) and its ability to quench GFP (pKa ~6) fluorescence without affecting RFP (pKa = 4.5) fluorescence. Through the use of a tandem RFP-GFP fusion protein targeted to an organelle of choice, cargo that is cytosolic or within an autophagosome, and therefore exposed to neutral pH, will appear yellow when using pseudo colors green for GFP and red for RFP due to the fluorescence of both GFP and RFP. Upon lysosomal fusion, the labeled cargo will be exposed to an acidic pH, quenching GFP fluorescence and resulting in mainly RFP fluorescence (Figure 1). Again, this can be detected by microscopy and the ratio of cargo with RFP-only fluorescence can be compared to the total RFP fluorescence, to obtain a measure of autophagy. This method was first used to study aggrephagy (Pankiv et al., 2007). By tagging LC3 with a tandem GFP-mCherry, autophagic vesicles and autolysosomes were able to be readily visualized and distinguished. We have adopted the principle used with LC3 to visualize the targeting of peroxisomes to autolysosomes. This is done by tagging the peroxisome targeting transmembrane domain of PEX26 with the tandem fluorophore, allowing it to be localized to the cytosolic face of the peroxisomal membrane (Deosaran et al., 2013; Riccio et al., 2019). This allows for the visualization of pexophagy specifically. Others have also used a similar technique to study pexophagy, where the fluorescent proteins were targeted to the peroxisome matrix through the use of a matrix targeting sequence (Nazarko et al., 2014). Importantly, our method, targeting the fluorophore to the cytosolic face of the peroxisome membrane, allows for increased sensitivity of the tandem fluorophore to the acidic environment of the lysosome. Additionally, our quantification method differs, taking into account the relative GFP and mCherry fluorescence in order to determine the quantity of peroxisomes targeted to the lysosome. In addition to our use of the tandem fluorophore in pexophagy, we and others have used this method to quantify mitophagy (Allen et al., 2013; Kim et al., 2014; Wang et al., 2015).

We have shown that the use of the tandem fluorophore is a reproducible method for observing and quantifying selective autophagy. As displayed above, the tandem mCherry-GFP lysosome targeting assay is extremely flexible, and can be used to measure various forms of selective autophagy, as well as general autophagy. Through the pH sensitivity of the fluorophores, it is a reliable method to detect lysosomal targeting. Additionally, because GFP and mCherry are expressed at equal levels, comparing the fluorescent intensities of mCherry to GFP allows for a reproducible method to quantify pexophagy.