LC3-positive structures are prominent in autophagy-deficient cells
Open accessAbstract
Autophagy is an evolutionarily conserved process across eukaryotes that degrades cargoes like aggregate-prone proteins, pathogens, damaged organelles and macromolecules via delivery to lysosomes. The process involves the formation of double-membraned autophagosomes that engulf the cargoes destined for degradation, sometimes with the help of autophagy receptors like p62, which are themselves autophagy substrates. LC3-II, a standard marker for autophagosomes, is generated by the conjugation of cytosolic LC3-I to phosphatidylethanolamine (PE) on the surface of nascent autophagosomes. As LC3-II is relatively specifically associated with autophagosomes and autolysosomes (in the absence of conditions stimulating LC3-associated phagocytosis), quantification of LC3-positive puncta is considered as a gold-standard assay for assessing the numbers of autophagosomes in cells. Here we find that the endogenous LC3-positive puncta become larger in cells where autophagosome formation is abrogated, and are prominent even when LC3-II is not formed. This occurs even with transient and incomplete inhibition of autophagosome biogenesis. This phenomenon is due to LC3-I sequestration to p62 aggregates, which accumulate when autophagy is impaired. This observation questions the reliability of LC3-immunofluorescence assays in cells with compromised autophagy.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
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?
ATG9A knockout in HeLa cells and mouse embryonic fibroblasts resulted in fewer but larger LC3 puncta that colocalize with ubiquitin and p62.
“ATG9A knockout in HeLa cells and mouse embryonic fibroblasts resulted in fewer but larger LC3 puncta that colocalize with ubiquitin and p62 [50, 55].”
From miRNA family miR-29 inhibits PINK1-PRKN signaling via ATG9A (2026) · cites this paper as [55]
The Results section reports that ATG9-knockout HeLa cells had fewer but larger LC3-positive structures that colocalised with p62; the p62 structures were ubiquitin-positive. The Introduction distinguishes this HeLa finding from earlier reports of LC3-positive structures in mouse embryonic fibroblasts. This paper does not establish the claimed ATG9-knockout phenotype in mouse embryonic fibroblasts. (LLM, conf=0.91)
Experimental — accuracy not yet measured
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.
Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y., & Yoshimori, T. (2000) · The EMBO Journal · doi:10.1093/emboj/19.21.5720 · Cited by 6,605 · see citing works
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
Klionsky, D. J., Abdelmohsen, K., Abe, A., Abedin, M. J., Abeliovich, H., Acevedo Arozena, A., Adachi, H., Adams, C. M., Adams, P. D., Adeli, K., et al. (2016) · Autophagy · doi:10.1080/15548627.2015.1100356 · Cited by 5,894 · see citing works
Self-citationDNA targeting specificity of RNA-guided Cas9 nucleases
Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine, E. J., Wu, X., Shalem, O., et al. (2013) · Nature Biotechnology · doi:10.1038/nbt.2647 · Cited by 4,741 · see citing works
The roles of intracellular protein-degradation pathways in neurodegeneration
Rubinsztein, D. C. (2006) · Nature · doi:10.1038/nature05291 · Cited by 1,625 · see citing works
Self-citationAutophagy-Independent Functions of the Autophagy Machinery
Galluzzi, L., & Green, D. R. (2019) · Cell · doi:10.1016/j.cell.2019.05.026 · Cited by 863 · see citing works
Structural Basis for Sorting Mechanism of p62 in Selective Autophagy
Ichimura, Y., Kumanomidou, T., Sou, Y.-S., Mizushima, T., Ezaki, J., Ueno, T., Kominami, E., Yamane, T., Tanaka, K., & Komatsu, M. (2008) · Journal of Biological Chemistry · doi:10.1074/jbc.m802182200 · Cited by 803 · see citing works
Kuma, A., Matsui, M., & Mizushima, N. (2007) · Autophagy · doi:10.4161/auto.4012 · Cited by 555 · see citing works
Flagged first, then by citations
Research works printed but not linked: 14
- [1]Bento CF Mammalian Autophagy: How Does It Work? Annual Review of Biochemistry 2016 85 685 713 10.1146/annurev-biochem-060815-014556 26865532 · DOI
- [3]Zavodszky E Vicinanza M Rubinsztein DC Biology and trafficking of ATG9 and ATG16L1, two proteins that regulate autophagosome formation FEBS letters 2013 587 1988 1996 10.1016/j.febslet.2013.04.025 23669359 · DOI
- [4]Stolz A Ernst A Dikic I Cargo recognition and trafficking in selective autophagy Nat Cell Biol 2014 16 495 501 10.1038/ncb2979 24875736 · DOI · Paper page
- [5]Moscat J Diaz-Meco MT p62 at the Crossroads of Autophagy, Apoptosis, and Cancer Cell 2009 137 1001 1004 10.1016/j.cell.2009.05.023 19524504 PMC3971861 · DOI · Paper page
- [6]Bjørkøy G p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death The Journal of cell biology 2005 171 603 614 10.1083/jcb.200507002 16286508 PMC2171557 · DOI · Paper page
- [7]Menzies FM Moreau K Puri C Renna M Rubinsztein DC Measurement of autophagic activity in mammalian cells Current protocols in cell biology/editorial board, Juan S. Bonifacino… [et al.] Chapter 15, Unit 15.16-Unit 2012 15 16 10.1002/0471143030.cb1516s54 22422474 · DOI
- [9]Komatsu M Homeostatic Levels of p62 Control Cytoplasmic Inclusion Body Formation in Autophagy-Deficient Mice Cell 2007 131 1149 1163 10.1016/j.cell.2007.10.035 18083104 · DOI · Paper page
- [10]Szeto J ALIS are stress-induced protein storage compartments for substrates of the proteasome and autophagy Autophagy 2006 2 189 199 10.4161/auto.2731 16874109 · DOI
- [12]Shvets E Elazar Z Autophagy-independent incorporation of GFP-LC3 into protein aggregates is dependent on its interaction with p62/SQSTM1 Autophagy 2008 4 1054 1056 10.4161/auto.6823 18776740 · DOI
- [13]Bento, C. F., Ashkenazi, A., Jimenez-Sanchez, M. & Rubinsztein, D. C. The Parkinson’s disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway. Nature Communications7, 11803, 10.1038/ncomms11803 https://www.nature.com/articles/ncomms11803#supplementary-information (2016). PMC4906231 27278822 · DOI
- [14]Tanida I Ueno T Kominami E Human Light Chain 3/MAP1LC3B Is Cleaved at Its Carboxyl-terminal Met121 to Expose Gly120 for Lipidation and Targeting to Autophagosomal Membranes Journal of Biological Chemistry 2004 279 47704 47710 10.1074/jbc.M407016200 15355958 · DOI
- [16]Zaffagnini, G. et al. p62 filaments capture and present ubiquitinated cargos for autophagy. The EMBO Journal (2018). PMC5830917 29343546
- [18]Tung YT Hsu WM Lee H Huang WP Liao YF The evolutionarily conserved interaction between LC3 and p62 selectively mediates autophagy-dependent degradation of mutant huntingtin Cellular and molecular neurobiology 2010 30 795 806 10.1007/s10571-010-9507-y 20204693 PMC11498752 · DOI
- [19]Wurzer B Oligomerization of p62 allows for selection of ubiquitinated cargo and isolation membrane during selective autophagy eLife 2015 4 e08941 10.7554/eLife.08941 26413874 PMC4684078 · DOI
Cited by
Sorted by citations
Klionsky, D. J., Abdel-Aziz, A. K., Abdelfatah, S., Abdellatif, M., Abdoli, A., Abel, S., Abeliovich, H., Abildgaard, M. H., Abudu, Y. P., Acevedo-Arozena, A., et al. (2021) · Autophagy · doi:10.1080/15548627.2020.1797280 · Cited by 2,622 · see citing works
Self-citationStructure of Human ATG9A, the Only Transmembrane Protein of the Core Autophagy Machinery
Guardia, C. M., Tan, X.-F., Lian, T., Rana, M. S., Zhou, W., Christenson, E. T., Lowry, A. J., Faraldo-Gómez, J. D., Bonifacino, J. S., Jiang, J., et al. (2020) · Cell Reports · doi:10.1016/j.celrep.2020.107837 · Cited by 163 · see citing works
The contribution of altered neuronal autophagy to neurodegeneration
Filippone, A., Esposito, E., Mannino, D., Lyssenko, N., & Praticò, D. (2022) · Pharmacology & Therapeutics · doi:10.1016/j.pharmthera.2022.108178 · Cited by 55 · see citing works
Lin, T.-K., Lin, K.-J., Lin, K.-L., Liou, C.-W., Chen, S.-D., Chuang, Y.-C., Wang, P.-W., Chuang, J.-H., & Wang, T.-J. (2020) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2020.607392 · Cited by 19 · see citing works
Balsamo, J. A., Penton, K. E., Zhao, Z., Hayes, M. J., Lima, S. M., Irish, J. M., & Bachmann, B. O. (2022) · Journal of Biological Chemistry · doi:10.1016/j.jbc.2022.102300 · Cited by 12 · see citing works
miRNA family miR-29 inhibits PINK1-PRKN signaling via ATG9A
Markham, B. N., Ramnarine, C., Kim, S., Grever, W. E., Soto-Beasley, A. I., Heckman, M. G., Ren, Y., Osborne, A. C., Kehili, M., Bhagwate, A. V., et al. (2026) · Molecular Neurodegeneration Advances · doi:10.1186/s44477-026-00029-w
- Authors
- Runwal, G., Stamatakou, E., Siddiqi, F. H., Puri, C., Zhu, Y., & Rubinsztein, D. C.
- Affiliations
- University of Cambridge (GB)
- Corresponding author
- Rubinsztein, David C.
- Venue
- Scientific Reports
- Published
- 2019-07-12
- Volume
- 9
- Issue
- 1
- Pages
- 10147-10147
- ISSN
- 2045-2322 (ISSN-L)
- PMC
- PMC6625982
- OpenAlex
- W2957721022
- Cited by
- 413
- References
- 7
- Citations by year
- 2026: 44 · 2025: 70 · 2024: 71 · 2023: 62 · 2022: 84 · 2021: 56 · 2020: 24 · 2019: 2
- FWCI
- 31.45
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Scientific Reports (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Apollo (University of Cambridge) (submittedVersion)
- Apollo (University of Cambridge) (submittedVersion, cc-by)
- Apollo
- Apollo (cc-by)
- Funders
- Commonwealth Scholarships Commission, UK
- UK Dementia Research Institute
- Cambridge Trust and China Scholarship Council
- Topics
- Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Keywords
- autophagy, LC3, LC3-II, autophagosomes, p62, autophagy receptors, autophagosome formation, autophagy markers
- MeSH terms
- Autophagy-Related Proteins, Autophagy-Related Protein 7, Autophagy, HeLa Cells, Humans, Microtubule-Associated Proteins, RNA-Binding Proteins, Ubiquitin, Vesicular Transport Proteins, Gene Knockdown Techniques
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
