A Guide to Autophagy Markers

Autophagy, the biological pathway for degrading and recycling cellular components, is crucial for maintaining cellular homeostasis, especially under stress conditions. It is characterized by the formation of autophagosomes, which engulf cytoplasmic material destined for degradation. This process drives the recycling of macromolecules and energy and adaptation to starvation. Autophagy is important not only in the normal physiological state, but it is also involved in many pathological conditions, including cancer, tissue injuries, and neurodegeneration. 

In order to make qualitative and quantitative measurements of the state of autophagy in cells and tissues, it is essential to have reliable markers. Specific markers must also be used to determine if observed experimental effects are due to changes in autophagy rather than other cellular processes, especially in the context of disease. In current autophagy research the most effective markers, which can be application-specific, are still being actively investigated. Here we review some of the most commonly mentioned protein markers directly associated with autophagy. 

Autophagosome Markers

A defining structure in macroautophagy is the autophagosome, an intermediate organelle formed by an isolation membrane (phagophore) that sequesters cytoplasmic materials and organelles.¹ Certain proteins associated with the phagosome have been widely used as marker proteins to monitor the autophagic process.² 

The LC3 autophagosome marker

The LC3 protein is one of the most commonly used markers for autophagosomes. LC3 is conjugated to phosphatidylethanolamine to form LC3-II and is localized to isolation membranes and autophagosomes. The quantity of LC3-II or LC3-PE thus serves as an indicator for the number of autophagosomes and autophagy-related structures.³ A homolog of the yeast ATG8 gene, LC3 in mammals is known as MAP1LC3 and is encoded by three isoform genes: MAP1LC3A, MAP1LC3B, and MAP1LC3C. The MAP1LC3B isoform is the most well-studied isoform, but MAP1LC3A is also reported to be a major marker of autophagic flux.²   

There are also other ATG8 homologs in mammals aside from MAP1LC3. Closely related proteins GABARAP (ATG8A) and GABARAPL2 (GATE-16) are essential for autophagosome formation but are believed to act at a later stage. 

This diagram highlights proteins expressed during various stages in autophagy.

ATG proteins

The ATG (autophagy-related) proteins are highly conserved in mammals and govern autophagy-related pathways, including autophagosome formation. Notable examples include ULK1 (Atg1), which forms a complex with ATG13, FIP200, and ATG101 to promote autophagy induction.¹ Beclin 1 (ATG6), which forms a complex with ATG14, AMBRA1, VPS15, and VPS34 to regulate the initiation of the isolation membrane, is another notable protein marker. The complex consisting of ATG12, ATG5, and ATG16L1 functions in the elongation and closure of the isolation membrane.

The autophagy proteins ATG7 and ATG3 also function in the conjugation of phosphatidylethanolamine to form LC3-II. ATG9A, a membrane lipid carrier protein for the expansion of the phagophore, is suspected to be an early marker of autophagosome assembly based on studies in yeast. Other notable ATG proteins involved in autophagosome formation include WIPI1 (ATG18) and WIPI2 (ATG18B). 

p62 in the autophagosome

Another notable protein involved in the autophagosome is p62, encoded by the gene SQSTM1. P62, which binds LC3 and serves as an autophagic cargo adaptor, is selectively incorporated into autophagosomes and then degraded. Low levels of the protein represents autophagy induction, while its accumulation can indicate inhibition of autophagy.² When using SQSTM1 as a marker of autophagic flux, it should be noted that the protein is also involved in a variety of other cellular pathways. 

Other phagosome markers

Syntaxin 17 is an autophagosomal SNARE that localizes to the outer membrane of completed autophagosomes. It has been reported to exhibit promising staining patterns in starvation-induced autophagy.² DFCP1 (ZFYVE1) is a characteristic marker of the omegasome, a ring-shaped structure from which isolation membranes emerge.³ 

Selective autophagy markers

In contrast to non-selective (or bulk) macroautophagy, selective autophagy involves the targeted delivery of specific substrates to autophagosomes. This is mediated by selective receptors that directly bind to LC3, ensuring the degradation of particular cellular components, such as in mitophagy, ribosomal autophagy, ER-phagy, pexophagy, and others. Elements of selective autophagy may represent potential biomarkers in neurodegeneration and may even serve as an effective strategy for cancer treatment.^5,8

Mitophagy markers

The selective removal of damaged mitochondria, or mitophagy, can occur as a response to the loss of mitochondrial membrane potential. In mammals, this is mediated by several stress signals. Notable mitophagy receptors on the outer mitochondrial membrane (OMM) surface include BNIP3 and NIX. These receptors contain MAP1LC3-interacting regions that can direct the mitochondria to the autophagosomes for degradation. The kinase PINK1 accumulates in the OMM of damaged mitochondria and recruits Parkin, a ubiquitin ligase. Parkin-mediated ubiquitination then leads to autophagosome recruitment and degradation. 

Additional considerations

It is important to remember that much of what is known about autophagy protein expression comes from studies in organisms such as yeast and rodents. When considering using a certain marker in a new model, such as in human tissue, and for a certain application, users should validate that marker before experimentation. For example, in the case of autophagy marker antibodies for immunohistochemistry, users should verify that the reagents are validated by the supplier, in addition to performing independent validation tests. 

The use of autophagy assay kits may also serve as a useful complement in addition to specific markers. These kits generally fluorescently label autophagic vacuoles or other components involved in autophagy for detection via microscopy or flow cytometry. Labeling is mediated using autophagy-specific cell-permeant dyes (often called probes) or antibodies against markers like LC3 or Beclin-1. 

Table of Autophagy Markers

The table below highlights notable proteins involved in autophagy compiled from recent literature. In addition, links to relevant antibodies and ELISA kits are provided as these immunodetection tools are routinely used for cell characterization via flow cytometry and immunostaining. The associated products are offered by a variety of manufacturers and, when used in combination with other phenotypic markers, can serve as a useful reference for assessing autophagy in cells and tissues across normal and disease conditions. The marker types can be classified as associated with the Autophagosome, Regulation of autophagy, neurodegenerative (Neuro), cancer, selective autophagy, mitophagy, traumatic brain injury (TBI), and ER-phagy (ER). 

Note: *Some markers are protein isoforms, multi-subunit protein complexes, or protein families composed of several distinct genes. Information on Protein Type, Localization, and Size (kDa) obtained from UniProt.org (for human genes only)

References

1. Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell. 2011;147(4):728-741. doi:10.1016/j.cell.2011.10.026

2. Martinet W, Roth L, De Meyer GRY. Standard Immunohistochemical Assays to Assess Autophagy in Mammalian Tissue. Cells. 2017;6(3):17. Published 2017 Jun 30. doi:10.3390/cells6030017

3. Yoshii SR, Mizushima N. Monitoring and Measuring Autophagy. Int J Mol Sci. 2017;18(9):1865. Published 2017 Aug 28. doi:10.3390/ijms18091865 Monitoring and Measuring Autophagy. Int J Mol Sci. 2017;18(9):1865. Published 2017 Aug 28. doi:10.3390/ijms18091865

4. Vishnupriya S, Priya Dharshini LC, Sakthivel KM, Rasmi RR. Autophagy markers as mediators of lung injury-implication for therapeutic intervention. Life Sci. 2020;260:118308. doi:10.1016/j.lfs.2020.118308

5. Giorgi C, Bouhamida E, Danese A, Previati M, Pinton P, Patergnani S. Relevance of Autophagy and Mitophagy Dynamics and Markers in Neurodegenerative Diseases. Biomedicines. 2021;9(2):149. Published 2021 Feb 4. doi:10.3390/biomedicines9020149

6. Gundamaraju R, Lu W, Paul MK, et al. Autophagy and EMT in cancer and metastasis: Who controls whom?. Biochim Biophys Acta Mol Basis Dis. 2022;1868(9):166431. doi:10.1016/j.bbadis.2022.166431

7. Livieri T, Cuttaia C, Vetrini R, et al. Old and Promising Markers Related to Autophagy in Traumatic Brain Injury. Int J Mol Sci. 2022;24(1):72. Published 2022 Dec 21. doi:10.3390/ijms24010072

8. Liu J, Wu Y, Meng S, et al. Selective autophagy in cancer: mechanisms, therapeutic implications, and future perspectives. Mol Cancer. 2024;23(1):22. Published 2024 Jan 24. doi:10.1186/s12943-024-01934-y