Cited by 258Autophagy in Disease and TherapyAdvanced Electron Microscopy Techniques and ApplicationsCellular transport and secretion

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Chapter 10 Monitoring Autophagy by Electron Microscopy in Mammalian Cells

Ylä‐Anttila, Päivi, Vihinen, Helena, Jokitalo, Eija, Eskelinen, Eeva‐Liisa·2009·Methods in Enzymology

DOI 10.1016/s0076-6879(08)03610-0

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Abstract

Electron microscopy remains one of the most accurate methods for the detection of autophagy and quantification of autophagic accumulation. Compared to fluorescence microscopy, the resolution of transmission electron microscopy is superior. In this chapter we describe the fine structure of early and late autophagic compartments in mammalian cells. Instructions are given for the preparation of samples for conventional electron microscopy using three different protocols suitable for cultured cells and animal tissues. We also introduce tomography as a tool to study the three-dimensional morphology of autophagic organelles and show the morphology of a phagophore as an example. Finally, we describe a protocol for the quantification of autophagic compartments by electron microscopy and point counting.

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Eskelinen, 2008, Fine structure of the autophagosome, Methods Mol. Biol., 445, 11, 10.1007/978-1-59745-157-4_2 · DOI

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BackgroundMethod
Disruption of the Autophagy-Lysosome Pathway Is Involved in Neuropathology of the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis
Thelen, M., Daμμe, M., Schweizer, M., Hagel, C., Wong, A. M. S., Cooper, J. D., Braulke, T., & Galliciotti, G. (2012) · PLoS ONE · doi:10.1371/journal.pone.0035493 · Cited by 75
“Quantification of autophagosomes was performed as previously described [48].”
Method
Thioredoxin-interacting protein mediates dysfunction of tubular autophagy in diabetic kidneys through inhibiting autophagic flux
Huang, C., Lin, M. Z., Cheng, D., Braet, F., Pollock, C. A., & Chen, X. (2014) · Laboratory Investigation · doi:10.1038/labinvest.2014.2 · Cited by 71
The protein-vesicle network of autophagy
Ge, L., Baskaran, S., Schekman, R. W., & Hurley, J. H. (2014) · Current Opinion in Cell Biology · doi:10.1016/j.ceb.2014.02.005 · Cited by 70
Neuropathy-causing mutations in HSPB1 impair autophagy by disturbing the formation of SQSTM1/p62 bodies
Haidar, M., Asselbergh, B., Adriaenssens, E., Winter, V. D., Timmermans, J., Auer‐Grumbach, M., Juneja, M., & Timmerman, V. (2019) · Autophagy · doi:10.1080/15548627.2019.1569930 · Cited by 74
“Autophagosomes were identified based on ultrastructural characteristics described in [65] and the total number of autophagosomes was counted by visual inspection at 43,000 magnification of the complete cytoplasmic area in the section of the cell.”
Method
Autophagy Promotes Oligodendrocyte Survival and Function following Dysmyelination in a Long-Lived Myelin Mutant
Smith, C. M., Mayer, J. A., & Duncan, I. D. (2013) · The Journal of Neuroscience · doi:10.1523/jneurosci.0233-13.2013 · Cited by 69
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
Klionsky, D. J., Abdelmohsen, K., Abe, A., Abedin, J., Abeliovich, H., Acevedo‐Arozena, A., Adachi, H., Adams, C. M., Adams, P. D., Adeli, K., et al. (2016) · Kölner Universitäts PublikationsServer (Universität zu Köln) · doi:10.5167/uzh-131237 · Cited by 69
Targeting lipophagy in macrophages improves repair in multiple sclerosis
Haidar, M., Loix, M., Vanherle, S., Dierckx, T., Vangansewinkel, T., Gervois, P., Wolfs, E., Lambrichts, I., Bogie, J. F. J., & Hendriks, J. J. A. (2022) · Autophagy · doi:10.1080/15548627.2022.2047343 · Cited by 71
“Autophagosomes and lipid droplets were identified based on structural characteristics as described previously [51] at 11,000 magnification and normalized to the cytoplasmic area using the imageJ polygon selection tool.”
Method
let‐7 regulates radial migration of new‐born neurons through positive regulation of autophagy
Petri, R., Pircs, K., Jönsson, M. E., Åkerblom, M., Brattås, P. L., Klussendorf, T., & Jakobsson, J. (2017) · The EMBO Journal · doi:10.15252/embj.201695235 · Cited by 68
“Autophagic structures were defined in GFP-positive gold immunolabeled cytoplasmic areas based on size, cellular contents, and morphology (Eskelinen, 2008; Yla-Anttila et al , 2009; Eskelinen & Kovacs, 2011; Klionsky et al , 2016).”
Division and Adaptation to Host Environment of Apicomplexan Parasites Depend on Apicoplast Lipid Metabolic Plasticity and Host Organelle Remodeling
Amiar, S., Katris, N. J., Berry, L., Dass, S., Duley, S., Arnold, C.-S., Shears, M. J., Brunet, C. D., Touquet, B., McFadden, G. I., et al. (2020) · Cell Reports · doi:10.1016/j.celrep.2020.02.072 · Cited by 70
“The gMVBs are distinct from host autophagosome, as they lack the typical double/multiple surrounding membranes and the cytosolic material defining autophagosomes (Ylä-Anttila et al., 2009).”
Background
Excess sphingomyelin disturbs ATG9A trafficking and autophagosome closure
Corcelle-Termeau, E., Vindeløv, S. D., Hämälistö, S., Mograbi, B., Keldsbo, A., Bräsen, J. H., Favaro, E., Adam, D., Szyniarowski, P., Hofman, P., et al. (2016) · Autophagy · doi:10.1080/15548627.2016.1159378 · Cited by 64
“Autophagic structures were classified as described previously.(59)”
Systematic Transmission Electron Microscopy‐Based Identification and 3D Reconstruction of Cellular Degradation Machinery
Neikirk, K., Vue, Z., Katti, P., Rodriguez, B. I., Omer, S., Shao, J., Christensen, T. A., Lopez, E. G., Marshall, A. G., Palavicino‐Maggio, C. B., et al. (2023) · Advanced Biology · doi:10.1002/adbi.202200221 · Cited by 66
Methods to Assess Autophagy In Situ—Transmission Electron Microscopy Versus Immunohistochemistry
Martinet, W., Timmermans, J., & Meyer, G. R. Y. D. (2014) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/b978-0-12-801329-8.00005-2 · Cited by 64
Curvature of Double-Membrane Organelles Generated by Changes in Membrane Size and Composition
Knorr, R. L., Dimova, R., & Lipowsky, R. (2012) · PLoS ONE · doi:10.1371/journal.pone.0032753 · Cited by 61
“The membrane sheets can have continuous membrane connections with other organelles via one or even multiple contacts [22,49,50], and a shape transformation can be sterically hindered, for example, by large substrates or unrelated organelles.”
Background
A study of autophagy in hemocytes of the Pacific oyster, Crassostrea gigas
Picot, S., Morga, B., Faury, N., Chollet, B., Dégremont, L., Travers, M.-A., Renault, T., & Arzul, I. (2019) · Autophagy · doi:10.1080/15548627.2019.1596490 · Cited by 54
“The presence of ultrastructural modifications and autophagic structures of the hemocytes was examined based on the criteria described in the literature [13,36,46].”
BackgroundMethod
The autophagy research in electron microscopy
Jung, M., Choi, H., & Mun, J. Y. (2019) · Han-guk hyeonmigyeong hakoeji/Applied microscopy · doi:10.1186/s42649-019-0012-6 · Cited by 59
Autophagosomal IκBα Degradation Plays a Role in the Long Term Control of Tumor Necrosis Factor-α-induced Nuclear Factor-κB (NF-κB) Activity
Colleran, A., Ryan, A. E., O'Gorman, A., Mureau, C., Liptrot, C. H., Dockery, P., Fearnhead, H. O., & Egan, L. J. (2011) · Journal of Biological Chemistry · doi:10.1074/jbc.m110.199950 · Cited by 60
Autophagy regulates UBC9 levels during viral-mediated tumorigenesis
Mattoscio, D., Casadio, C., Miccolo, C., Maffini, F. A., Raimondi, A., Tacchetti, C., Gheit, T., Tagliabue, M., Galimberti, V. E., Lorenzi, F. D., et al. (2017) · PLoS Pathogens · doi:10.1371/journal.ppat.1006262 · Cited by 58
Hydroxysafflor yellow a protects brain microvascular endothelial cells against oxygen glucose deprivation/reoxygenation injury: Involvement of inhibiting autophagy via class I PI3K/Akt/mTOR signaling pathway
Yang, G., Wang, N., Seto, S. W., Chang, D., & Liang, H. (2018) · Brain Research Bulletin · doi:10.1016/j.brainresbull.2018.05.011 · Cited by 58
Targeting Glioblastoma Stem Cells with 2-Deoxy-D-Glucose (2-DG) Potentiates Radiation-Induced Unfolded Protein Response (UPR)
Shah, S. S., Rodriguez, G. A., Musick, A., Walters, W. M., de Cordoba, N., Barbarite, E., Marlow, M. M., Marples, B., Prince, J. S., Komotar, R. J., et al. (2019) · Cancers · doi:10.3390/cancers11020159 · Cited by 60
“Autophagic vesicle were defined as a membrane bound vesicle containing either intact or degraded cytoplasmic material or organelles [51].”
Background
Autophagy: novel insights into therapeutic target of electroacupuncture against cerebral ischemia/ reperfusion injury
Huang, Y.-G., Wei, T., Yang, S.-B., Wang, J.-F., Mei, Z., & Feng, Z.-T. (2019) · Neural Regeneration Research · doi:10.4103/1673-5374.250569 · Cited by 55
“Mature autophagosomes are double-membrane limited vacuoles containing undegraded cytoplasm such as ER membranes and mitochondria but no lysosomal proteins (Ylä-Anttila et al., 2009; Ma et al., 2012).”
Background
Microglial derived extracellular vesicles activate autophagy and mediate multi‐target signaling to maintain cellular homeostasis
den Broek, B. V., Pintelon, I., Hamad, I., Kessels, S., Haidar, M., Hellings, N., Hendriks, J. J. A., Kleinewietfeld, M., Brône, B., Timmerman, V., et al. (2020) · Journal of Extracellular Vesicles · doi:10.1002/jev2.12022 · Cited by 56
Inhibiting autophagy potentiates the anticancer activity of IFN1@/IFNα in chronic myeloid leukemia cells
Zhu, S., Cao, L., Yu, Y., Yang, L., Yang, M., Liu, K., Huang, J. X., Kang, R., Livesey, K. M., & Tang, D. (2012) · Autophagy · doi:10.4161/auto.22923 · Cited by 54
Methodologies to investigate intracellular barriers for nucleic acid delivery in non-viral gene therapy
Vermeulen, L. M. P., Brans, T., Smedt, S. C. D., Remaut, K., & Braeckmans, K. (2018) · Nano Today · doi:10.1016/j.nantod.2018.06.007 · Cited by 54
Allethrin Promotes Apoptosis and Autophagy Associated with the Oxidative Stress-Related PI3K/AKT/mTOR Signaling Pathway in Developing Rat Ovaries
Jalouli, M., Mofti, A., Elnakady, Y. A., Nahdi, S., Feriani, A., Alrezaki, A., Sebei, K., Bizzarri, M., Alwasel, S. H., & Harrath, A. H. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms23126397 · Cited by 54
“In agreement with these results, the findings of the current study revealed the appearance of large numbers of autophagosomes, the gold-standard markers of autophagy, in the allethrin-treated ovaries [42].”
Result
The use of electron microscopy for the detection of autophagy in human atherosclerosis
Perrotta, I. D. (2013) · Micron · doi:10.1016/j.micron.2013.03.007 · Cited by 52
Measurement of Autophagic Activity in Mammalian Cells
Menzies, F. M., Moreau, K., Puri, C., Renna, M., & Rubinsztein, D. C. (2012) · Current Protocols in Cell Biology · doi:10.1002/0471143030.cb1516s54 · Cited by 50
“To determine these parameters, it is normal to use the point-hit method (Yla-Anttila et al., 2009b) or software such as Image Analysis (Olympus; Ravikumar et al., 2010a; Moreau et al., 2011).”
Method
Autophagy controls the pathogenicity of OPA 1 mutations in dominant optic atrophy
Kane, M. S., Alban, J., Desquiret‐Dumas, V., Guéguen, N., Ishak, L., Ferré, M., Amati‐Bonneau, P., Procaccio, V., Bonneau, D., Lenaers, G., et al. (2017) · Journal of Cellular and Molecular Medicine · doi:10.1111/jcmm.13149 · Cited by 51
“cell area was estimated using square grid openings, which were selected according to the rules of uniform random sampling [31].”
Method
RAB24 facilitates clearance of autophagic compartments during basal conditions
Ylä‐Anttila, P., Mikkonen, E., Happonen, K. E., Holland, P., Ueno, T., Simonsen, A., & Eskelinen, E. (2015) · Autophagy · doi:10.1080/15548627.2015.1086522 · Cited by 53
“Quantitative counting of autophagic compartments was performed using a transmission electron microscope (JEOL 1200 EX II or JEOL 1400 EX, Electron microscopy unit, Institute of Biotechnology, University of Helsinki) as described previously.(40) For immuno-electron microscopy NRK or HeLa cells were grown on 3.”
Method
The midgut of the silkmoth Bombyx mori is able to recycle molecules derived from degeneration of the larval midgut epithelium
Franzetti, E., Romanelli, D., Caccia, S., Cappellozza, S., Congiu, T., Rajagopalan, M., Grimaldi, A., de Eguileor, M., Casartelli, M., & Tettamanti, G. (2015) · Cell and Tissue Research · doi:10.1007/s00441-014-2081-8 · Cited by 50
“Bars (a) 4 mm, (b) 50μm, (c, f) 20μm, (d) 10μm, (e, g, h) 2 μm, (i) 500 nm some specimens were post-fixed in a solution of 2 % osmium tetroxide in 0.1 M imidazole buffer for 30 min at room temperature (Yla-Anttila et al. 2009).”
Influential
Platycodin D triggers autophagy through activation of extracellular signal-regulated kinase in hepatocellular carcinoma HepG2 cells
Li, T., Tang, Z.-H., Xu, W., Wu, G., Wang, Y., Chang, L.-L., Zhu, H., Chen, X., Wang, Y., Chen, Y., et al. (2015) · European Journal of Pharmacology · doi:10.1016/j.ejphar.2015.01.003 · Cited by 50
“Transmission electron microscopy analysis can qualitatively reveal the presence of autophagosomes in cells and has been considered the most conventional method for monitoring autophagy (Eskelinen et al., 2011; Yla-Anttila et al., 2009).”
Method
Pseudorabies virus induces autophagy to enhance viral replication in mouse neuro-2a cells in vitro
Xu, C.-M., Wang, M., Song, Z., Wang, Z., Liu, Q., Jiang, P., Bai, J., Li, Y., & Wang, X. (2018) · Virus Research · doi:10.1016/j.virusres.2018.02.004 · Cited by 49
Wnt3a ligand facilitates autophagy in hippocampal neurons by modulating a novel GSK-3β-AMPK axis
Ríos, J. A., Godoy, J. A., & Inestrosa, N. C. (2018) · Cell Communication and Signaling · doi:10.1186/s12964-018-0227-0 · Cited by 48
A subcellular analysis of genetic modulation of PINK1 on mitochondrial alterations, autophagy and cell death.
Lenzi, P., Marongiu, R., Falleni, A., Gelmetti, V., Busceti, C. L., Michiorri, S., Valente, E. M., & Fornai, F. (2012) · PubMed · doi:10.4449/aib.v150i2/3.1417 · Cited by 46
“In our experimental settings we took advantage from cellular pellets because ultrathin sections contain cells randomly oriented, which is an appropriate condition for quantitative calculation (Yla-Anttila et al., 2009).”
Recombinant protein rVP1 upregulates BECN1-independent autophagy, MAPK1/3 phosphorylation and MMP9 activity via WIPI1/WIPI2 to promote macrophage migration
Liao, C.-C., Ho, M., Liang, S., & Liang, C. (2012) · Autophagy · doi:10.4161/auto.22379 · Cited by 45
Chlamydia trachomatis vacuole maturation in infected macrophages
Sun, H., Eng, E. W. Y., Jeganathan, S., Sin, A. T. ‐., Patel, P. C., Gracey, E., Inman, R. D., Terebiznik, M. R., & Harrison, R. E. (2012) · Journal of Leukocyte Biology · doi:10.1189/jlb.0711336 · Cited by 45
“8A), indicative of autophagosomes [43].”
Background
Tetrandrine induces programmed cell death in human oral cancer CAL 27 cells through the reactive oxygen species production and caspase‐dependent pathways and associated with beclin‐1‐induced cell autophagy
Lien, J., Lin, M., Chang, S., Lai, K., Huang, A., Yu, F., & Chung, J. (2016) · Environmental Toxicology · doi:10.1002/tox.22238 · Cited by 45
Inhibition of autophagy-attenuated calcium oxalate crystal-induced renal tubular epithelial cell injury in vivo and in vitro
Liu, Y., Li, D., He, Z., Liu, Q., Wu, J., Guan, X., Tao, Z., & Deng, Y. (2017) · Oncotarget · doi:10.18632/oncotarget.23383 · Cited by 45
Enhanced mitophagy in Sertoli cells of ethanol-treated rats: morphological evidence and clinical relevance
Eid, N. A. S., Ito, Y., & Otsuki, Y. (2011) · Journal of Molecular Histology · doi:10.1007/s10735-011-9372-0 · Cited by 45
“The gold standard technique for analyzing autophagy, specifically the morphology of AVs in mammalian cells, remains TEM (Yla-Anttila et al. 2009; Holt et al. 2011).”
Method
Mitochondrial Morphology and Mitophagy in Heart Diseases: Qualitative and Quantitative Analyses Using Transmission Electron Microscopy
Collins, H. E., Kane, M. S., Litovsky, S. H., Darley-Usmar, V. M., Young, M. E., Chatham, J. C., & Zhang, J. (2021) · Frontiers in Aging · doi:10.3389/fragi.2021.670267 · Cited by 47
“TEM has been heavily utilized for the examination of autophagy due to the ability to visualize autophagosomes and vacuolar structures (Yla-Anttila et al., 2009; Backues et al., 2014) and has been a recommended methodology (Klionsky et al., 2016) for visualizing autophagosomes.”
Method
Endothelial Heterogeneity in the Response to Autophagy Drives Small Vessel Muscularization in Pulmonary Hypertension
Zhang, Q., Yaoita, N., Tabuchi, A., Liu, S., Chen, S.-H., Li, Q., Hegemann, N., Li, C., Rodor, J., Timm, S., et al. (2024) · Circulation · doi:10.1161/circulationaha.124.068726 · Cited by 47
The Autophagoproteasome a Novel Cell Clearing Organelle in Baseline and Stimulated Conditions
Lenzi, P., Lazzeri, G., Biagioni, F., Busceti, C. L., Gambardella, S., Salvetti, A., & Fornai, F. (2016) · Frontiers in Neuroanatomy · doi:10.3389/fnana.2016.00078 · Cited by 45

Authors: Ylä‐Anttila, P., Vihinen, H., Jokitalo, E., & Eskelinen, E.
Affiliations: University of Helsinki (FI)
ORCID: Ylä‐Anttila, Päivi
Vihinen, Helena
Jokitalo, Eija
Eskelinen, Eeva‐Liisa
Venue: Methods in Enzymology
Publisher: Elsevier
Published: 2009
Volume: 452
Pages: 143-164
ISSN: 0076-6879 (ISSN-L)
DOI: 10.1016/s0076-6879(08)03610-0
OpenAlex: W1594293927
Cited by: 257
References: 26
Citations by year: 2026: 4 · 2025: 7 · 2024: 13 · 2023: 6 · 2022: 14 · 2021: 14 · 2020: 19 · 2019: 27 · 2018: 17 · 2017: 25 · 2016: 19 · 2015: 18 · 2014: 20 · 2013: 17 · 2012: 19
FWCI: 37.92
Type: book chapter
Language: en
Open access: closed
Locations: Methods in enzymology on CD-ROM/Methods in enzymology (publishedVersion)
PubMed (publishedVersion)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Keywords: autophagy, phagophore, electron microscopy specimen preparation, electron tomography, three-dimensional morphology, point counts, mammalian cells
MeSH terms: Animals, Autophagy, Cells, Cultured, Microscopy, Electron, Transmission, Mice

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Chapter 10 Monitoring Autophagy by Electron Microscopy in Mammalian Cells

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