Direct quantification of autophagic flux by a single molecule-based probe

ABSTRACT

Macroautophagy/autophagy remains a rapidly advancing research topic, and there continues to be a need for constantly evolving methodology to investigate this pathway at each individual step. Accordingly, new assays to measure autophagic flux in a robust and reliable manner are essential to understand the mechanism and physiological roles of autophagy. Kaizuka et al. recently reported a new fluorescence probe, GFP-LC3-RFP-LC3ΔG to directly demonstrate autophagic flux without being combined with lysosomal inhibitors (see the Kaizuka et al. punctum in this issue of the journal). When expressed in cells, the probe is cleaved into a degradable/quenchable part, GFP-LC3, and stable/cytosolic part, RFP-LC3ΔG. The latter serves as an internal control and a decrease of the GFP:RFP ratio indicates the occurrence of autophagy. Since the key index of this probe is the degradation of GFP-LC3, it can be used to measure the cumulative effect of basal autophagy. The assay is applicable to high-throughput drug discovery as well as in vivo analysis.

KEYWORDS:

• autophagic flux
• autophagy assay
• basal autophagy
• fluorescence probe
• LC3

Ever since the first identification of the autophagy pathway in the budding yeast Saccharomyces cerevisiae, a wide range of methods to monitor this pathway have been reported and have helped researchers understand the mechanism of every step of autophagy including initiation, phagophore expansion, autophagosome maturation, fusion with the vacuole/lysosome, cargo degradation and efflux. In return, the elucidated molecular mechanism provides scientists additional tools to further monitor the pathway in a more robust and precise manner in vitro and in vivo. Today, any researcher who wants to examine autophagy activity in his/her favorite system luckily has a handful of established methods available, but new challenges emerge as well: what assays are appropriate and how should the data be interpreted? In 2008, researchers in the autophagy community published the most inclusive guideline up to then to standardize the use and interpretation of autophagy assays,¹ and the updated 3rd version was recently published in 2016.² So far the guideline papers have been of great help to scientists, especially those new to the accelerated expanding autophagy area, and have been cited by thousands of research articles.

Atg8/LC3 is the most widely used marker for autophagosome formation. Upon autophagy induction, lipidated Atg8/LC3, Atg8–phosphatidylethanolamine or LC3-II, is associated with the phagophore and the completed autophagosome. The level of LC3-II can be quantified by western blot and normalized to that of housekeeping proteins. Alternatively, the subcellular distribution of GFP-LC3 can be analyzed by counting the number of GFP-LC3 puncta using fluorescence microscopy. In most cases the increase of LC3-II or GFP-LC3 puncta indicates the accumulation of autophagosomes, but this observation is insufficient to conclude that there is a corresponding increase of autophagic flux. The accumulation of autophagosomes can be a result of either induction of autophagy or a defect in processes such as autophagosome-lysosome fusion or lysosomal degradation. For this reason, it is necessary to include additional treatment with lysosomal inhibitors to block lysosomal degradation. Ideally, the cotreatment with inhibitors should increase the level of LC3-II and the number of GFP-LC3 puncta in the presence of bona fide autophagy inducers. However, suppression of lysosomal function itself may trigger autophagy due to its negative feedback on MTORC1 activity,^3-5 which augments the readout and complicates the analysis, and is challenging in a practical sense for in vivo studies. Moreover, in high-throughput screens of autophagy inducers, it takes excessive effort to exclude false-positive hits using a lysosomal inhibitor cotreatment analysis.

Several methods are available to monitor autophagic flux without using lysosomal inhibitors, such as SQSTM1/p62 turnover and tandem RFP-GFP-LC3 (tfLC3) fluorescence microscopy. Upon autophagy induction SQSTM1 is incorporated within an autophagosome as a receptor of polyubiquitinated proteins and is eventually degraded within an autolysosome.⁶ Thus, the turnover of SQSTM1 can serve as an indicator of autophagic flux. However, SQSTM1 is not exclusively degraded by autophagy; therefore, other proteolytic pathways should be examined as well.⁷ In addition, it is essential to differentiate enhanced degradation from reduced synthesis because the SQSTM1 level is transcriptionally regulated under various stress conditions.⁸ tfLC3 can be used to monitor autophagy induction and flux simultaneously.⁹ The GFP moiety of the fusion protein is sensitive to the acidic conditions of the lysosome lumen, whereas RFP is relatively stable. Hence, compartments that are GFP⁺ RFP⁺ represent phagophores or autophagosomes while GFP⁻ RFP⁺ fluorescence indicates amphisomes or autolysosomes. Still, this widely accepted method has its own limitations. For example, the RFP moiety will eventually be degraded within autolysosomes, so quantification of puncta that are GFP⁻ RFP⁺ tends to underestimate the cumulative effect of autophagy.

In this highlighted study, Kaizuka and colleagues developed a new fluorescence probe, GFP-LC3-RFP-LC3ΔG, to measure autophagic flux.¹⁰ When expressed in cells, the fusion protein is efficiently cleaved into GFP-LC3 and RFP-LC3ΔG by the ATG4 family of proteases. Upon autophagy induction, GFP-LC3 is packaged into phagophores and gets degraded (and quenched) after fusion of the corresponding autophagosomes with lysosomes; in contrast, RFP-LC3ΔG remains in the cytosol due to its C-terminal glycine deletion (i.e., it cannot be conjugated to phosphatidylethanolamine). Because the initial amount of RFP-LC3ΔG is equimolar to that of GFP-LC3, the former can be used as an internal control for GFP-LC3 degradation. Kaizuka et al. showed that the GFP:RFP ratio dramatically decreases in response to multiple autophagy stimuli both in vitro and in vivo, but remains relatively unchanged in autophagy-deficient systems, suggesting it can be used as a reliable index of autophagic flux without using any lysosomal inhibitors. Since the essential readout in this assay is the fluorescence intensity of GFP and RFP, theoretically it is not necessary to acquire high-resolution microscopy images in order to count accurate numbers of GFP-LC3 puncta. Thus, this probe should be applicable to high-throughput microscopy, flow cytometry and microplate readers for large-scale screening. In addition, when this fluorescence probe is used as a primary assay to screen for autophagy inducers, there is no need to perform a secondary screen together with lysosomal inhibitors in order to exclude false-positive hits as aforementioned.

As described by Kaizuka et al., the GFP-LC3-RFP-LC3ΔG probe provides a strong advantage in the quantification of basal autophagy in vivo. Basal autophagy performs various homeostatic and quality control functions under normal conditions. Because of its low activity, the readout of LC3-II level or GFP-LC3 puncta number is usually complicated by huge variations in response to nutrient supply. The GFP-LC3-RFP-LC3ΔG probe, instead, can serve as a cumulative index for basal autophagy activity. Using this new tool, Kaizuka et al. observed higher levels of basal autophagy in the lens and skeletal muscle of 1-d post-fertilization zebrafish compared to those in the retina and spinal cord. One of the many challenges during in vivo study is that tissue samples contain a population of different cell types with asynchronous autophagy activation. This difference may mask the significance of autophagic flux in a certain cell type if the tissue samples are only tested by western blot. Given this new single-molecule probe, researchers may collect autophagic flux data within certain types of cells by combining it with cell type-specific markers.

This new method certainly has its own limitations. Kaizuka et al. described homologous recombination between 2 LC3 sequences of GFP-LC3-RFP-LC3ΔG in retrovirally transfected cells, resulting in GFP-LC3ΔG that cannot be incorporated into phagophores. An alternative option is to use GFP-LC3-RFP instead, and the processed RFP moiety can be used as an internal control (with the disadvantage that RFP alone may be recognized and/or degraded differently than RFP-LC3ΔG). Also, this assay is not applicable to investigate certain steps of autophagy. The reduction of the GFP:RFP ratio is a result of autophagic degradation and quenching, so the temporal resolution of this assay is not high enough to detect early events of autophagosome formation. tfLC3 should be a better tool to track the autophagosome-lysosome fusion step of individual autophagosomes, although its overall sensitivity to monitor autophagic flux is lower than that of GFP-LC3-RFP-LC3ΔG. Furthermore, the accuracy of the GFP:RFP ratio change depends on the expression level of the probe, and caution has to be taken when comparing autophagic flux in different cell lines or tissues. Last but not least, it should be noted that so far there is no single autophagy assay that can be applied to all experimental systems. Which method(s) to choose depends on the question being asked and the model system being used.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by NIH grant GM053396 to DJK.