CoChat
Sign Up
Cited by 72

The DOI matches its Crossref record; 9 of the 9 references it cites were found in a registry or an index.

An antibody for analysis of autophagy induction

Tian, Wensheng, Alsaadi, Reham, Guo, Zhihao, Kalinina, Alena, Carrier, Micaël, Tremblay, Marie-Eve, Lacoste, Baptiste, Lagace, Diane, Russell, Ryan C.·2020·Nature Methods
DOI 10.1038/s41592-019-0661-y
Open this paper in CoChat — free account

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.

Overview

Integrity signals

Registry

Crossref · 10.1038/s41592-019-0661-y

Cites

9 of 9 references verified0 citation errors · 0 not checkable

Statistics

Recomputing p-values needs the paper’s full text.

Verification

Claims the paper makes

This paper’s full text isn’t openly available, so its claims and self-check weren’t run. The registry and citation checks still apply.

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Citations

Cites

Flagged first, then by citations

Show all 9 references

Research works printed but not linked: 34

  1. Parzych, K. R. & Klionsky, D. J. An overview of autophagy: morphology, mechanism, and regulation. Antioxid. Redox Signal. 20, 460–473 (2014). · DOI · Paper page
  2. Russell, R. C., Yuan, H. X. & Guan, K. L. Autophagy regulation by nutrient signaling. Cell Res. 24, 42–57 (2014). · DOI
  3. Kuma, A. et al. The role of autophagy during the early neonatal starvation period. Nature 432, 1032–1036 (2004). · DOI · Paper page
  4. 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). · DOI
  5. 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). · DOI
  6. Hara, T. et al. FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. J. Cell Biol. 181, 497–510 (2008). · DOI · Paper page
  7. Ganley, I. G. et al. ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy. J. Biol. Chem. 284, 12297–12305 (2009). · DOI · Paper page
  8. Jung, C. H. et al. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol. Biol. Cell 20, 1992–2003 (2009). · DOI · Paper page
  9. 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). · DOI
  10. Nishimura, T. et al. FIP200 regulates targeting of Atg16L1 to the isolation membrane. EMBO Rep. 14, 284–291 (2013). · DOI
  11. 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). · DOI
  12. 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). · DOI
  13. Kabeya, Y. et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720–5728 (2000). · DOI · Paper page
  14. 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). · DOI
  15. Salminen, A. et al. Emerging role of p62/sequestosome-1 in the pathogenesis of Alzheimer’s disease. Prog. Neurobiol. 96, 87–95 (2012). · DOI · Paper page
  16. Jiang, X. et al. VPS34 stimulation of p62 phosphorylation for cancer progression. Oncogene 36, 6850–6862 (2017). · DOI
  17. 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). · DOI
  18. Kirkin, V., Lamark, T., Johansen, T. & Dikic, I. NBR1 cooperates with p62 in selective autophagy of ubiquitinated targets. Autophagy 5, 732–733 (2009). · DOI
  19. 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). · DOI
  20. Roberts, E. A. & Deretic, V. Autophagic proteolysis of long-lived proteins in nonliver cells. Methods Mol. Biol. 445, 111–117 (2008). · DOI
  21. Warnes, G. Flow cytometric assays for the study of autophagy. Methods 82, 21–28 (2015). · DOI
  22. 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). · DOI · Paper page
  23. Alsaadi, R. M. et al. ULK1-mediated phosphorylation of ATG16L1 promotes xenophagy, but destabilizes the ATG16L1 Crohn’s mutant. EMBO Rep. 20, e46885 (2019). · DOI
  24. Diamanti, M. A. et al. IKKɑ controls ATG16L1 degradation to prevent ER stress during inflammation. J. Exp. Med 214, 423–437 (2017). · DOI
  25. 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). · DOI
  26. 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). · DOI · Paper page
  27. Komatsu, M. et al. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 131, 1149–1163 (2007). · DOI · Paper page
  28. 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). · DOI · Paper page
  29. 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). · DOI · Paper page
  30. Rosenfeldt, M. T., Nixon, C., Liu, E., Mah, L. Y. & Ryan, K. M. Analysis of macroautophagy by immunohistochemistry. Autophagy 8, 963–969 (2012). · DOI
  31. 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). · DOI
  32. 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). · DOI · Paper page
  33. 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). · DOI
  34. 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). · DOI · Paper page

Cited by

Sorted by citations

See all 72

Metadata

Authors
Tian, W., Alsaadi, R., Guo, Z., Kalinina, A., Carrier, M., Tremblay, M.-E., Lacoste, B., Lagace, D., & Russell, R. C.
ORCID
Venue
Nature Methods
Publisher
Springer Science and Business Media LLC
Published
2019-11-25
Volume
17
Issue
2
Pages
232-239
ISSN
  • 1548-7091
  • 1548-7105
DOI
10.1038/s41592-019-0661-y
Cited by
72
References
9
Language
en
Licence
Funders
Gouvernement du Canada | Canadian Institutes of Health Research (PJT153034)

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review