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An antibody for analysis of autophagy induction

Abstract

Autophagy is a degradative program that maintains cellular homeostasis. Autophagy defects have been described in numerous diseases. However, analysis of autophagy rates can be challenging, particularly in rare cell populations or in vivo, due to limitations in currently available tools for measuring autophagy induction. Here, we describe a method to monitor autophagy by measuring phosphorylation of the protein ATG16L1. We developed and characterized a monoclonal antibody that can detect phospho-ATG16L1 endogenously in mammalian cells. Importantly, phospho-ATG16L1 is only present on newly forming autophagosomes. Therefore, its levels are not affected by prolonged stress or late-stage autophagy blocks, which can confound autophagy analysis. Moreover, we show that ATG16L1 phosphorylation is a conserved signaling pathway activated by numerous autophagy-inducing stressors. The described antibody is suitable for western blot, immunofluorescence and immunohistochemistry, and measured phospho-ATG16L1 levels directly correspond to autophagy rates. Taken together, this phospho-antibody represents an exciting tool to study autophagy induction.

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Fig. 1: Phosphorylation of ATG16L1 is a conserved indicator of autophagy induction, which is activated by multiple stimuli.
Fig. 2: pATG16L1 can be analyzed by immunofluorescence and is recruited to the expanding autophagosomal membrane.
Fig. 3: pATG16L1 level provides a reliable measurement of autophagy rates independent of late-stage autophagy block and directly reflects autophagic vesicle formation.
Fig. 4: pATG16L1 is compatible with IHC staining of tissue samples to measure autophagic activities in vivo.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank the uOttawa PALM-Histology Core Facility for processing the IHC tissue samples, the Cell Biology and Image Acquisition Core (CBIA) for assistance in three-dimensional reconstruction and rendering of brain imaging, N. Vernoux for technical assistance in electron microcopy, J.A. Lunde for technical assistance in collecting mouse skeletal muscle samples and members of the Russell laboratory for advice and critical reading of this manuscript. This work was supported by a Canadian Institutes of Health Research Project Grant awarded to R.C.R. (grant no. PJT153034), funding from the Canada Foundation for Innovation to the CBIA core and a Canada Research Chair Tier 2 to M.T.

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Authors

Contributions

W.T. and R.C.R. wrote the manuscript. W.T. was primarily responsible for data production in all figures except for immunofluorescence and IHC panels in Figs. 2 and 4. R.A. assisted with immunofluorescence experiments and data analysis. Z.G. was responsible for optimizing pATG16L1 antibody protocols for immunofluorescence and IHC. A.K. and D.L. provided the mice used for the study and assisted with IHC experimental planning and tissue staining. M.C. and M.E.T. imaged EM samples. B.L. provided expertise and guidance for EM sample preparation. R.C.R. oversaw manuscript planning and conceived of the study.

Corresponding author

Correspondence to Ryan C. Russell.

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The authors declare no competing interests.

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Peer review information Nicole Rusk and Rita Strack were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–5 and results.

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Supplementary Video

Brain hippocampus tissue sections of wild-type and Atg5flox/flox mice were stained with pATG16L1S278 GFP and RFP signals were enhanced with anti-GFP/RFP antibodies. GFP expression is indicative of cells knocked out of Atg5. 3D model of the cell was constructed using the 3D reconstruction function in Imaris based on GRP and RFP signals. N = 2 animals per group. Representative cell from one conditional Atg5 KO mouse sample is shown in the video.

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Tian, W., Alsaadi, R., Guo, Z. et al. An antibody for analysis of autophagy induction. Nat Methods 17, 232–239 (2020). https://doi.org/10.1038/s41592-019-0661-y

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