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.
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Parzych, K. R. & Klionsky, D. J. An overview of autophagy: morphology, mechanism, and regulation. Antioxid. Redox Signal. 20, 460–473 (2014).
Russell, R. C., Yuan, H. X. & Guan, K. L. Autophagy regulation by nutrient signaling. Cell Res. 24, 42–57 (2014).
Kuma, A. et al. The role of autophagy during the early neonatal starvation period. Nature 432, 1032–1036 (2004).
Mizushima, N., Yoshimori, T. & Ohsumi, Y. The role of Atg proteins in autophagosome formation. Annu. Rev. Cell Dev. Biol. 27, 107–132 (2011).
Mercer, C. A., Kaliappan, A. & Dennis, P. B. A novel, human Atg13 binding protein, Atg101, interacts with ULK1 and is essential for macroautophagy. Autophagy 5, 649–662 (2009).
Suzuki, H., Kaizuka, T., Mizushima, N. & Noda, N. N. Structure of the Atg101–Atg13 complex reveals essential roles of Atg101 in autophagy initiation. Nat. Struct. Mol. Biol. 22, 572–580 (2015).
Hara, T. et al. FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. J. Cell Biol. 181, 497–510 (2008).
Ganley, I. G. et al. ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy. J. Biol. Chem. 284, 12297–12305 (2009).
Hosokawa, N. et al. Nutrient-dependent mTORC1 association with the ULK1–Atg13–FIP200 complex required for autophagy. Mol. Biol. Cell 20, 1981–1991 (2009).
Jung, C. H. et al. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol. Biol. Cell 20, 1992–2003 (2009).
Lee, E. J. & Tournier, C. The requirement of uncoordinated 51-like kinase 1 (ULK1) and ULK2 in the regulation of autophagy. Autophagy 7, 689–695 (2011).
Gammoh, N., Florey, O., Overholtzer, M. & Jiang, X. Interaction between FIP200 and ATG16L1 distinguishes ULK1 complex-dependent and -independent autophagy. Nat. Struct. Mol. Biol. 20, 144–149 (2013).
Nishimura, T. et al. FIP200 regulates targeting of Atg16L1 to the isolation membrane. EMBO Rep. 14, 284–291 (2013).
Fujita, N. et al. The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy. Mol. Biol. Cell 19, 2092–2100 (2008).
Wu, J. et al. Molecular cloning and characterization of rat LC3A and LC3B—two novel markers of autophagosome. Biochem. Biophys. Res. Commun. 339, 437–442 (2006).
Kabeya, Y. et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720–5728 (2000).
Kimura, S., Fujita, N., Noda, T. & Yoshimori, T. Monitoring autophagy in mammalian cultured cells through the dynamics of LC3. Methods Enzymol. 452, 1–12 (2009).
Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 12, 1–222 (2016).
Jain, A. et al. p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription. J. Biol. Chem. 285, 22576–22591 (2010).
Fujita, K. & Srinivasula, S. M. TLR4-mediated autophagy in macrophages is a p62-dependent type of selective autophagy of aggresome-like induced structures (ALIS). Autophagy 7, 552–554 (2011).
Salminen, A. et al. Emerging role of p62/sequestosome-1 in the pathogenesis of Alzheimer’s disease. Prog. Neurobiol. 96, 87–95 (2012).
Jiang, X. et al. VPS34 stimulation of p62 phosphorylation for cancer progression. Oncogene 36, 6850–6862 (2017).
Clausen, T. H. et al. p62/SQSTM1 and ALFY interact to facilitate the formation of p62 bodies/ALIS and their degradation by autophagy. Autophagy 6, 330–344 (2010).
Kirkin, V., Lamark, T., Johansen, T. & Dikic, I. NBR1 cooperates with p62 in selective autophagy of ubiquitinated targets. Autophagy 5, 732–733 (2009).
Shi, J. et al. NBR1 is dispensable for PARK2-mediated mitophagy regardless of the presence or absence of SQSTM1. Cell Death Dis. 6, e1943 (2015).
Yla-Anttila, P., Vihinen, H., Jokitalo, E. & Eskelinen, E. L. Monitoring autophagy by electron microscopy in mammalian cells. Methods Enzymol. 452, 143–164 (2009).
Roberts, E. A. & Deretic, V. Autophagic proteolysis of long-lived proteins in nonliver cells. Methods Mol. Biol. 445, 111–117 (2008).
Ueno, T. et al. Autolysosomal membrane-associated betaine homocysteine methyltransferase. Limited degradation fragment of a sequestered cytosolic enzyme monitoring autophagy. J. Biol. Chem. 274, 15222–15229 (1999).
Warnes, G. Flow cytometric assays for the study of autophagy. Methods 82, 21–28 (2015).
Guo, S. et al. A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications. Autophagy 11, 560–572 (2015).
Alsaadi, R. M. et al. ULK1-mediated phosphorylation of ATG16L1 promotes xenophagy, but destabilizes the ATG16L1 Crohn’s mutant. EMBO Rep. 20, e46885 (2019).
Diamanti, M. A. et al. IKKɑ controls ATG16L1 degradation to prevent ER stress during inflammation. J. Exp. Med 214, 423–437 (2017).
Koyama-Honda, I., Itakura, E., Fujiwara, T. K. & Mizushima, N. Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site. Autophagy 9, 1491–1499 (2013).
Nguyen, T. N. et al. Atg8 family LC3/GABARAP proteins are crucial for autophagosome–lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation. J. Cell Biol. 215, 857–874 (2016).
Komatsu, M. et al. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 131, 1149–1163 (2007).
Sahani, M. H., Itakura, E. & Mizushima, N. Expression of the autophagy substrate SQSTM1/p62 is restored during prolonged starvation depending on transcriptional upregulation and autophagy-derived amino acids. Autophagy 10, 431–441 (2014).
Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T. & Ohsumi, Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol. Biol. Cell 15, 1101–1111 (2004).
Rosenfeldt, M. T., Nixon, C., Liu, E., Mah, L. Y. & Ryan, K. M. Analysis of macroautophagy by immunohistochemistry. Autophagy 8, 963–969 (2012).
Xi, Y. et al. Knockout of Atg5 delays the maturation and reduces the survival of adult-generated neurons in the hippocampus. Cell Death Dis. 7, e2127 (2016).
Hoffman, M. A. et al. von Hippel-Lindau protein mutants linked to type 2C VHL disease preserve the ability to downregulate HIF. Hum. Mol. Genet. 10, 1019–1027 (2001).
Guo, H. et al. Atg5 disassociates the V1V0-ATPase to promote exosome production and tumor metastasis independent of canonical macroautophagy. Dev. Cell 43, 716–730 e717 (2017).
Mauthe, M. et al. Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation. Autophagy 7, 1448–1461 (2011).
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.
The authors declare no competing interests.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–5 and results.
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.
About this article
Cite this article
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
Journal of Biomedical Science (2021)
p27 controls autophagic vesicle trafficking in glucose-deprived cells via the regulation of ATAT1-mediated microtubule acetylation
Cell Death & Disease (2021)