If one were to ask autophagy researchers around the globe about the most frustrating aspects of their research, failed autophagy assays would probably come up as one of the top answers. Two autophagy proteins, LC3B and p62, are used ubiquitously as autophagy markers in assays measuring autophagic activity. However, it is not always a simple task to correctly set up experimental conditions or interpret results utilizing these two markers. From experienced PI’s to novice undergraduate students, we have all experienced cases where highly-established autophagy inducing conditions either failed or produced varying results among biological replicates due to minor variations including: confluency, FBS or media variations or unknown factors (Figure 1). As a result, research is slowed and money wasted as unwieldy markers for autophagy must be brought from time-to-time back to their previous kinetics.
We first considered the development of an alternative autophagy marker when our group discovered a new signaling event in early autophagy initiation (Alsaadi et al, 2019). When characterizing this new phosphorylation event of ATG16L1 by ULK1, we noticed that it was strongly linked with starvation-induced autophagy. In addition, immunofluorescent microscopy showed that pATG16L1 was highly associated with early stage autophagosomes. These two characteristics of pATG16L1 potentially makes it an ideal marker of autophagy induction, while not bound by the same caveats as LC3 or p62, as pATG16L1 seemed to be only be found on early stage autophagosomes. It was then when we first formulated a plan for a method paper revolving around the use of pATG16L1 as a new marker for autophagy assays.
The first step of our plan was to develop a highly-reliable phospho-antibody against pATG16L1 in order to demonstrate any usage of pATG16L1 as an autophagy marker. Luckily, after first identifying this new phosphorylation site and before we had any idea on its exact functions, we had already been in contact with Abcam to develop a phospho-antibody for future research. The entire antibody development process took almost 2 years, with multiple rounds of hybridomas screened and multiple clones tested. Finally, nearing the time of publication of our EMBO Reports study, a single most reactive clone of the antibody was identified as functional and specific for both denatured and native protein.
Now that we had access to the core reagent for our study. I was eager to start the project. However, as it is often the case in research, things often do not work out as simple and clean-cut in practice as envisioned. Since we were working with a brand new antibody, we had to optimize staining conditions for all methods of detection we chose to employ: western blot, immunofluorescence microscopy (IF), and immunohistochemistry (IHC). For a period of 6 months I struggled with experimenting with different tedious western blotting protocols that produced at best semi-decent blots. The saving grace came when we discovered a commercial blocking buffer from Abcam, which markedly reduced background and made acquiring quality western blots much less labour intensive (Figure 2). We also went through a similar long process before finally optimizing staining conditions for both IF and IHC analysis.
The need for a new method of autophagy measurement is echoed by many autophagy researchers today. We believe that pATG16L1 would prove to be a potent tool in the arsenals of researchers wishing to monitor autophagy in their research. However, we do not envision pATG16L1 as a replacement for the conventional autophagy markers LC3 and p62, but instead as an alternative method with unique advantages over the latter. For example: in challenging experimental systems where target cell population is rare, or systems where usage of drugs (autophagy inhibitors) is impractical. Ultimately it is up to individual investigators to determine what methods best fit their needs. By broadening their selection with a reliable new tool, we hope our method can serve to further enable novel discoveries and advances of the field.
Alsaadi, R. M. et al. ULK1-mediated phosphorylation of ATG16L1 promotes xenophagy, but destabilizes the ATG16L1 Crohn’s mutant. EMBO Rep. 20, e46885 (2019).