Chapter 10 Monitoring Autophagy by Electron Microscopy in Mammalian Cells
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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Double-membraned autophagosomes were identified according to well-established criteria.
“double- membraned autophagosomes were identified according to well established criteria”
From Nilotinib restores memory function by preventing dopaminergic neuron degeneration in a mouse model of Alzheimer’s Disease (2021) · cites this paper as Ylä-Anttila et al., 2009
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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 in higher eukaryotes
Klionsky, D. J., Abeliovich, H., Agostinis, P., Agrawal, D. K., Aliev, G., Askew, D. S., Baba, M., Baehrecke, E. H., Bahr, B. A., Ballabio, A., et al. (2008) · Autophagy · doi:10.4161/auto.5338 · Cited by 2,230 · see citing works
Autophagosome formation: core machinery and adaptations
Xie, Z., & Klionsky, D. J. (2007) · Nature Cell Biology · doi:10.1038/ncb1007-1102 · Cited by 2,165 · see citing works
Principles and Techniques of Electron Microscopy: Biological Applications
Maugel, T. K., & Hayat, M. A. (1979) · Transactions of the American Microscopical Society · doi:10.2307/3226038 · Cited by 1,327 · see citing works
A Unified Nomenclature for Yeast Autophagy-Related Genes
Klionsky, D. J., Cregg, J. M., Dunn, W. A., Emr, S. D., Sakai, Y., Sandoval, I. V., Sibirny, A. А., Subramani, S. E., Thumm, M., Marten Veenhuis, A., et al. (2003) · Developmental Cell · doi:10.1016/s1534-5807(03)00296-x · Cited by 1,267 · see citing works
Unbiased Stereology: Three-Dimensional Measurement in Microscopy
Howard, C. V., & Reed, M. (1998) · Cited by 1,149 · see citing works
Role for Rab7 in maturation of late autophagic vacuoles
Jäger, S., Bucci, C., Tanida, I., Ueno, T., Kominami, E., Säftig, P., & Eskelinen, E. (2004) · Journal of Cell Science · doi:10.1242/jcs.01370 · Cited by 907 · see citing works
Unbiased Stereology. Three‐Dimensional Measurement in Microscopy.
Sandau, K. (1999) · Journal of Microscopy · doi:10.1046/j.1365-2818.1999.0429a.x · Cited by 890 · see citing works
Studies on the mechanisms of autophagy: formation of the autophagic vacuole.
Dunn, W. A. (1990) · The Journal of Cell Biology · doi:10.1083/jcb.110.6.1923 · Cited by 787 · see citing works
Maturation of Autophagic Vacuoles in Mammalian Cells
Eskelinen, E.-L. (2005) · Autophagy · doi:10.4161/auto.1.1.1270 · Cited by 626 · see citing works
Autophagy and related mechanisms of lysosome-mediated protein degradation
Dunn, W. A. (1994) · Trends in Cell Biology · doi:10.1016/0962-8924(94)90069-8 · Cited by 523 · see citing works
Principles and Techniques of Electron Microscopy: Biological Applications
Maugel, T. K., & Hayat, M. A. (1977) · Transactions of the American Microscopical Society · doi:10.2307/3226107 · Cited by 389 · see citing works
Role of LAMP-2 in Lysosome Biogenesis and Autophagy
Eskelinen, E., Illert, A. L., Tanaka, Y., Schwarzmann, G. O. H., Blanz, J., FIGURA, K. V., & Säftig, P. (2002) · Molecular Biology of the Cell · doi:10.1091/mbc.e02-02-0114 · Cited by 365 · see citing works
Studies on the mechanisms of autophagy: maturation of the autophagic vacuole.
Dunn, W. A. (1990) · The Journal of Cell Biology · doi:10.1083/jcb.110.6.1935 · Cited by 362 · see citing works
(1968) · PubMed Central · Cited by 332 · see citing works
Biological Specimen Preparation for Transmission Electron Microscopy
Glauert, A. M., & Lewis, P. R. (1998) · Princeton University Press eBooks · doi:10.1515/9781400865024 · Cited by 273 · see citing works
Eskelinen, E., Schmidt, C. K., Neu, S., Willenborg, M., Fuertes, G., Salvador-Montoliu, N., Tanaka, Y., Lüllmann‐Rauch, R., Hartmann, D., Heeren, J., et al. (2004) · Molecular Biology of the Cell · doi:10.1091/mbc.e04-02-0103 · Cited by 266 · see citing works
Angerm�ller, S., & Fahimi, H. D. (1982) · The Histochemical Journal · doi:10.1007/bf01033631 · Cited by 236 · see citing works
Principles and techniques of electron microscopy: Biological applications
Pettitt, J. M. (1974) · Micron (1969) · doi:10.1016/0047-7206(74)90008-9 · Cited by 203 · see citing works
Eskelinen, E.-L. (2008) · Autophagy · doi:10.4161/auto.5179 · Cited by 197 · see citing works
Inhibition of Autophagy in Mitotic Animal Cells
Eskelinen, E., Prescott, A. R., Cooper, J., Brachmann, S. M., Wang, L., Tang, X., Backer, J., & Lucocq, J. M. (2002) · Traffic · doi:10.1034/j.1600-0854.2002.31204.x · Cited by 172 · see citing works
New Insights into the Mechanisms of Macroautophagy in Mammalian Cells
Eskelinen, E. (2008) · International review of cytology · doi:10.1016/s1937-6448(07)66005-5 · Cited by 154 · see citing works
Fine Structure of the Autophagosome
Eskelinen, E. (2008) · Methods in molecular biology · doi:10.1007/978-1-59745-157-4_2 · Cited by 108 · see citing works
Kovács, A. L., Pálfia, Z., Réz, G., Vellai, T., & Kovács, J. (2007) · Autophagy · doi:10.4161/auto.4590 · Cited by 71 · see citing works
Biological Specimen Preparation for Transmission Electron Microscopy
Griffiths, G. (1999) · Journal of Microscopy · doi:10.1046/j.1365-2818.1999.00642.x · Cited by 59 · see citing works
Studies on vinblastine-induced autophagocytosis in mouse liver
Reunanen, H., Punnonen, E., & Hirsim�ki, P. (1985) · Histochemistry and Cell Biology · doi:10.1007/bf00492453 · Cited by 49 · see citing works
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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
“1).(48) The sequential morphological changes during the autophagic process can be followed by TEM.”
Guidelines for the use and interpretation of assays for monitoring autophagy
Klionsky, D. J., Abdalla, F. C., Abeliovich, H., Abraham, R. T., Acevedo-Arozena, A., Adeli, K., Agholme, L., Agnello, M., Agostinis, P., Aguirre-Ghiso, J. A., et al. (2012) · Autophagy · doi:10.4161/auto.19496 · Cited by 3,996 · see citing works
“2A and B).(43,47) It should be emphasized that not all vesicles containing electrondense amorphous material are autolysosomes.”
BackgroundKlionsky, 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-citationAutophagy: assays and artifacts
Barth, S., Glick, D. R., & Macleod, K. F. (2010) · The Journal of Pathology · doi:10.1002/path.2694 · Cited by 788 · see citing works
“Some efforts have been made to quantify autophagy by TEM, for example, by measuring the ratio of early to late autophagic compartments or autophagic volume as a percentage of cytoplasmic volume [9], but again this depends on the trained eye and is arguably subjective.”
BackgroundDeacetylation of Nuclear LC3 Drives Autophagy Initiation under Starvation
Huang, R., Xu, Y., Wan, W., Shou, X., Qian, J., You, Z., Liu, B., Chang, C., Zhou, T., Lippincott‐Schwartz, J., et al. (2015) · Molecular Cell · doi:10.1016/j.molcel.2014.12.013 · Cited by 669 · see citing works
3D tomography reveals connections between the phagophore and endoplasmic reticulum
Ylä‐Anttila, P., Vihinen, H., Jokitalo, E., & Eskelinen, E. (2009) · Autophagy · doi:10.4161/auto.5.8.10274 · Cited by 670 · see citing works
Downs, C. A., Kramarsky‐Winter, E., Segal, R., Fauth, J. E., Knutson, S., Bronstein, O., Ciner, F. R., Jeger, R. E., Lichtenfeld, Y., Woodley, C. M., et al. (2015) · Archives of Environmental Contamination and Toxicology · doi:10.1007/s00244-015-0227-7 · Cited by 645 · see citing works
“4a–f, 5b–d; Yla-Antilla et al. 2009).”
BackgroundVijayaraghavan, S., Karakaş, C., Doostan, I., Chen, X., Bui, T. N., Yi, M., Raghavendra, A. S., Zhao, Y., Bashour, S. I., Ibrahim, N. K., et al. (2017) · Nature Communications · doi:10.1038/ncomms15916 · Cited by 294 · see citing works
Chang, C.-M., Su, H., Zhang, D.-H., Wang, Y., Shen, Q.-H., Liu, B., Huang, R., Zhou, T.-H., Peng, C., Wong, C. C. L., et al. (2015) · Molecules and Cells · doi:10.1016/j.molcel.2015.10.037 · Cited by 258 · see citing works
Seeing is believing: The impact of electron microscopy on autophagy research
Eskelinen, E.-L., Reggiori, F., Baba, M., Kovács, A. L., & Seglen, P. O. (2011) · Autophagy · doi:10.4161/auto.7.9.15760 · Cited by 281 · see citing works
Self-citationSharma, S., Mells, J. E., Fu, P. P., Saxena, N. K., & Anania, F. A. (2011) · PLoS ONE · doi:10.1371/journal.pone.0025269 · Cited by 272 · see citing works
“Autophagic vacuoles (including autophagosomes and autophagolysosomes) were counted following the defined protocols [48,49].”
MethodTang, Q., Zheng, G., Feng, Z., Chen, Y., Lou, Y., Wang, C., Zhang, X., Zhang, Y., Xu, H., Shang, P., et al. (2017) · Cell Death and Disease · doi:10.1038/cddis.2017.453 · Cited by 261 · see citing works
“For quantitative analysis of autophagosomes and autolysosomes, 25 fields of each section were randomly selected and captured at a final magnification of 25 000, and the number of autophagosomes and autolysosomes in each captured field was counted as describe dpreviosuly.(53) Autophagosomes had double-membrane structur…”
MethodHMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells
Liu, L., Yang, M., Kang, R., Wang, Z., Zhao, Y., Yu, Y. H., Xie, M., Yin, X.-C., Livesey, K. M., Lotze, M. T., et al. (2010) · Leukemia · doi:10.1038/leu.2010.225 · Cited by 249 · see citing works
Redundancy of human ATG4 protease isoforms in autophagy and LC3/GABARAP processing revealed in cells
Agrotis, A., Pengo, N., Burden, J. J., & Ketteler, R. (2019) · Autophagy · doi:10.1080/15548627.2019.1569925 · Cited by 233 · see citing works
“Using this method, autophagic structures can be identified by their characteristic morphology of cytoplasmic material sequestered within a double or multimembrane compartments [27].”
MethodGuidelines for evaluating myocardial cell death
Mishra, P. K., Adameová, A., Hill, J. A., Baines, C. P., Kang, P. M., Downey, J. M., Narula, J., Takahashi, M., Abbate, A., Piristine, H. C., et al. (2019) · American Journal of Physiology-Heart and Circulatory Physiology · doi:10.1152/ajpheart.00259.2019 · Cited by 238 · see citing works
“The presence of double membranes enclosing electron-dense cytoplasmic material or recognizable organelles is typical of autophagosomes and early autolysosomes (280).”
BackgroundYu, Y. H., Yang, L., Zhao, M., Zhu, S., Kang, R., Vernon, P. J., Tang, D., & Cao, L. (2012) · Leukemia · doi:10.1038/leu.2012.65 · Cited by 198 · see citing works
Wang, F., Salvati, A. L., & Boya, P. (2018) · Open Biology · doi:10.1098/rsob.170271 · Cited by 182 · see citing works
Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation
Liu, B., Fang, M., Hu, Y., Huang, B., Li, N., Chang, C., Huang, R., Xu, X., Yang, Z., Chen, Z., et al. (2013) · Autophagy · doi:10.4161/auto.27286 · Cited by 184 · see citing works
Insulin secretory granules control autophagy in pancreatic β cells
Goginashvili, A., Zhang, Z., Erbs, E., Spiegelhalter, C., Kessler, P., Mihlan, M., Pasquier, A., Krupina, K., Schieber, N., Cinque, L., et al. (2015) · Science · doi:10.1126/science.aaa2628 · Cited by 160 · see citing works
Autophagy precedes apoptosis during the remodeling of silkworm larval midgut
Franzetti, E., Huang, Z.-J., Shi, Y.-X., Xie, K., Deng, X.-J., Li, J.-P., Li, Q.-R., Yang, W.-Y., Zeng, W.-N., Casartelli, M., et al. (2012) · Apoptosis · doi:10.1007/s10495-011-0675-0 · Cited by 172 · see citing works
“1 M imidazole buffer at room temperature [49], dehydrated in an ethanol series and embedded in Epon”
MethodThe Role of Oxidative Stress and Autophagy in Atherosclerosis
Perrotta, I. D., & Aquila, S. (2015) · Oxidative Medicine and Cellular Longevity · doi:10.1155/2015/130315 · Cited by 147 · see citing works
Ultrastructural relationship of the phagophore with surrounding organelles
Biazik, J. M., Ylä‐Anttila, P., Vihinen, H., Jokitalo, E., & Eskelinen, E. (2015) · Autophagy · doi:10.1080/15548627.2015.1017178 · Cited by 145 · see citing works
Assays to monitor autophagy in Drosophila
Mauvezin, C., Ayala, C. I., Braden, C. R., Kim, J., & Neufeld, T. P. (2014) · Methods · doi:10.1016/j.ymeth.2014.03.014 · Cited by 147 · see citing works
“Methods for unbiased sampling, volume estimation and point counting have been described [8] and are generally applicable for studies of Drosophila samples.”
BackgroundWang, Z., Cao, L., Kang, R., Yang, M., Liu, L., Zhao, Y., Yu, Y., Xie, M., Yin, X.-C., Livesey, K. M., et al. (2011) · Autophagy · doi:10.4161/auto.7.4.14397 · Cited by 140 · see citing works
Li, N., Tang, B., Jia, Y.-P., Zhu, P., Zhuang, Y., Fang, Y., Li, Q., Wang, K., Zhang, W., Guo, G., et al. (2017) · Frontiers in Cellular and Infection Microbiology · doi:10.3389/fcimb.2017.00417 · Cited by 139 · see citing works
“The method of counting autophagosomes’ numbers was followed as described previously by Yla-Anttila et al. (2009).”
MethodZhang, Y., Wu, Q., Zhang, L., Wang, Q., Yang, Z.-X., Liu, J., & Feng, L. (2019) · Pharmacological Research · doi:10.1016/j.phrs.2019.104538 · Cited by 139 · see citing works
Roy, S., & Debnath, J. (2010) · Seminars in Immunopathology · doi:10.1007/s00281-010-0213-0 · Cited by 138 · see citing works
“The detection of autophagosomes (with intact cytoplasmic materials and organelles) and autolysosomes (with partially degraded materials) can be used to identify the different stages of autophagy by electron microscopy [97].”
PKD is a kinase of Vps34 that mediates ROS-induced autophagy downstream of DAPk
Eisenberg‐Lerner, A., & Kimchi, A. (2011) · Cell Death and Differentiation · doi:10.1038/cdd.2011.149 · Cited by 136 · see citing works
Goussetis, D. J., Gounaris, E. G., Wu, E. J., Vakana, E., Sharma, B., Bogyo, M. S., Altman, J. K., & Platanias, L. C. (2012) · Blood · doi:10.1182/blood-2012-01-402578 · Cited by 133 · see citing works
Zhang, X., Ramírez, C. M., Aryal, B., Madrigal‐Matute, J., Liu, X., Díaz, A., Torrecilla-Parra, M., Suárez, Y., Cuervo, A. M., Sessa, W. C., et al. (2020) · Arteriosclerosis Thrombosis and Vascular Biology · doi:10.1161/atvbaha.120.314291 · Cited by 127 · see citing works
Zhang, F., Kumano, M., Beraldi, E., Fazli, L., Du, C., Moore, S. C., Sorensen, P. H. B., Zoubeidi, A., & Gleave, M. E. (2014) · Nature Communications · doi:10.1038/ncomms6775 · Cited by 125 · see citing works
New Insights into the Pathology of Podocyte Loss
Liapis, H., Romagnani, P., & Anders, H. (2013) · American Journal Of Pathology · doi:10.1016/j.ajpath.2013.06.033 · Cited by 127 · see citing works
“An obvious possibility is lysosomal storage diseases, but little work has been performed in this area.(63,64) In random renal biopsy specimens, podocyte cytoplasmic vacuoles and lysosomes may represent routine cytoplasmic debris removal and homeostatic maintenance.”
BackgroundSteele, S., Brunton, J., Ziehr, B., Taft-Benz, S., Moorman, N., & Kawula, T. (2013) · PLoS Pathogens · doi:10.1371/journal.ppat.1003562 · Cited by 123 · see citing works
Huang, W., Cao, Z., Zhang, J., Ji, Q., & Li, Y. (2019) · Environmental Pollution · doi:10.1016/j.envpol.2019.113317 · Cited by 117 · see citing works
Downs, C. A., Kramarsky‐Winter, E., Fauth, J. E., Segal, R., Bronstein, O., Jeger, R. E., Lichtenfeld, Y., Woodley, C. M., Pennington, P. L., Kushmaro, A., et al. (2013) · Ecotoxicology · doi:10.1007/s10646-013-1161-y · Cited by 115 · see citing works
“The majority of other cell types in the epidermis exhibited classic signs of autophagic cell death (Fig. 5a–c; Samara et al. 2008; Tasdemir et al. 2008; Yla-Antilla et al. 2009; Eskelinin et al. 2011).”
BackgroundLeidal, A. M., Cyr, D. P., Hill, R. J., Lee, P. W. K., & McCormick, C. (2012) · Cell Host & Microbe · doi:10.1016/j.chom.2012.01.005 · Cited by 113 · see citing works
“The criteria outlined in (Ylä-Anttila et al., 2009) were employed to enumerate the number of autophagic structures per cell section, in 30 cells in two independent experiments.”
MethodInhibition of 6-phosphofructo-2-kinase (PFKFB3) induces autophagy as a survival mechanism
Klarer, A. C., O’Neal, J., Imbert-Fernandez, Y., Clem, A. L., Ellis, S. R., Clark, J. A., Clem, B. F., Chesney, J. A., & Telang, S. (2014) · Cancer & Metabolism · doi:10.1186/2049-3002-2-2 · Cited by 102 · see citing works
“An increase in intracellular structures including double-membrane bound vesicles consistent with autophagosomes was observed only in cells transfected with PFKFB3 siRNA (Figure 2E) [46].”
BackgroundDysregulated autophagy contributes to caspase-dependent neuronal apoptosis
Chung, Y., Lee, J., Jung, S., Lee, Y., Cho, J. W., & Oh, Y. J. (2018) · Cell Death & Disease · doi:10.1038/s41419-018-1229-y · Cited by 99 · see citing works
Ribosome Abundance Control Via the Ubiquitin–Proteasome System and Autophagy
An, H., & Harper, J. W. (2019) · Journal of Molecular Biology · doi:10.1016/j.jmb.2019.06.001 · Cited by 98 · see citing works
“Indeed, ribosomes are frequently detected within double membranous structures with the conserved density between interior and exterior of the lipid membranes in starved cells [11], leading to a general notion that ribosomes are non-selectively engulfed into growing autophagosomal structures during bulk autophagy.”
BackgroundLa Barbera, L., Vedele, F., Nobili, A., Krashia, P., Spoleti, E., Latagliata, E. C., Cutuli, D., Cauzzi, E., Marino, R., Viscomi, M. T., et al. (2021) · Progress in Neurobiology · doi:10.1016/j.pneurobio.2021.102031 · Cited by 98 · see citing works
“In each cytoplasmic fields, double- membraned autophagosomes were identified according to well established criteria (Eskelinen, 2008; Klionsky et al., 2021; Ylä-Anttila et al., 2009), and their density calculated.”
MethodNecroptosis promotes autophagy-dependent upregulation of DAMP and results in immunosurveillance
Lin, S., Hsieh, S., Fan, Y.-T., Wei, W., Hsiao, P., Tsai, D.-H., Wu, T., & Yang, N. (2017) · Autophagy · doi:10.1080/15548627.2017.1386359 · Cited by 96 · see citing works
“In addition, such SK-treated 4T1luc2 cells were examined by TEM to observe the double-membrane structure, which is the critical ultrastructure of autophagosomes.(37,39) The result shown in Fig.”
BackgroundZhu, P., Xue, J., Zhang, Z.-J., Jia, Y.-P., Tong, Y.-N., Han, D., Li, Q., Xiang, Y., Mao, X., & Tang, B. (2017) · Cell Death and Disease · doi:10.1038/s41419-017-0011-x · Cited by 96 · see citing works
What to Eat: Evidence for Selective Autophagy in PlantsF
Floyd, B. E., Morriss, S. C., MacIntosh, G. C., & Bassham, D. C. (2012) · Journal of Integrative Plant Biology · doi:10.1111/j.1744-7909.2012.01178.x · Cited by 95 · see citing works
“Ribosomes have been frequently observed in large numbers in the interior of autophagosomes (Ylä-Anttila et al. 2009; Eskelinen et al. 2011).”
BackgroundAltman, J. K., Szilard, A., Goussetis, D. J., Sassano, A., Colamonici, M., Gounaris, E. G., Frankfurt, O., Giles, F. J., Eklund, E. A., Beauchamp, E. M., et al. (2014) · Clinical Cancer Research · doi:10.1158/1078-0432.ccr-13-3218 · Cited by 94 · see citing works
“Electron microscopy Such studies were performed as previously described (33, 35).”
MethodJiang, X., Lü, W., Shen, X., Wang, Q., Lv, J., Liu, M., Cheng, F., Zhao, Z., & Pang, X. (2018) · JCI Insight · doi:10.1172/jci.insight.98921 · Cited by 90 · see citing works
“vested and fixed following a standard protocol (63).”
BackgroundRegulatory effects of autophagy on spermatogenesis
Yin, J., Ni, B., Tian, Z., Yang, F., Liao, W.-G., & Gao, Y. (2017) · Biology of Reproduction · doi:10.1095/biolreprod.116.144063 · Cited by 89 · see citing works
Autophagy in traumatic brain injury: a new target for therapeutic intervention
Zhang, L., & Wang, H. (2018) · Frontiers in Molecular Neuroscience · doi:10.3389/fnmol.2018.00190 · Cited by 82 · see citing works
Martins‐Marques, T., Catarino, S., Zuzarte, M., Marques, C., Matafome, P. N., Pereira, P., & Girão, H. (2015) · Biochemical Journal · doi:10.1042/bj20141370 · Cited by 80 · see citing works
Song, C., Song, C., & Tong, F. (2014) · Cytotherapy · doi:10.1016/j.jcyt.2014.04.006 · Cited by 78 · see citing works
“Transmission electron microscopy is currently one of themost reliable and accurate methods to monitor autophagy (26,27).”
MethodWang, X., Zhang, X., Chu, E. S., Chen, X., Kang, W., Wu, F., To, K., Wong, V. W., Chan, H. L., Chan, M. T. V., et al. (2017) · The FASEB Journal · doi:10.1096/fj.201601393r · Cited by 77 · see citing works
“5 h and epoxy resin for 1 h at 37°C, andpolymerized at 60°C overnight (12).”
MethodLin, H., Li, B., Liu, M., Zhou, H., He, K., & Fan, H. (2020) · Veterinary Microbiology · doi:10.1016/j.vetmic.2020.108684 · Cited by 77 · see citing works
“Quantitative electron microscopy is one of the most sensitive methods to detect the presence of Autophagosome-like vesicles (Yla-Anttila et al., 2009).”
MethodWang, H., Wang, R., Carrera, I., Xu, S., & Lakshmana, M. K. (2016) · eNeuro · doi:10.1523/eneuro.0042-16.2016 · Cited by 76 · see citing works
“Due to its high resolution, electron microscopy remains a gold standard for the accurate detection of autophagy, and therefore we used transmission electron microscopy (TEM) to directly visualize and detect morphological evidence of autophagy (Ylä-Anttila et al., 2009).”
MethodMethods for Studying Autophagy Within the Tumor Microenvironment
Murugan, S., & Amaravadi, R. K. (2016) · Advances in experimental medicine and biology · doi:10.1007/978-3-319-26666-4_9 · Cited by 76 · see citing works
“Methods detailing this process can be found in [39].”
BackgroundEnergy deprivation by silibinin in colorectal cancer cells
Raina, K., Agarwal, C., Wadhwa, R., Serkova, N. J., & Agarwal, R. (2013) · Autophagy · doi:10.4161/auto.23960 · Cited by 75 · see citing works
An antibody for analysis of autophagy induction
Tian, W., Alsaadi, R. M., Guo, Z., Kalinina, A., Carrier, M., Tremblay, M., Lacoste, B., Lagace, D. C., & Russell, R. C. (2019) · Nature Methods · doi:10.1038/s41592-019-0661-y · Cited by 75 · see citing works
Zeng, Y., Yang, X., Wang, J., Fan, J., Kong, Q., & Yu, X. (2012) · PLoS ONE · doi:10.1371/journal.pone.0030312 · Cited by 74 · see citing works
“Despite some autophagy research were done in other renal diseases [5,21,22], information about autophagy in AAs-induced Figure 4.”
BackgroundDual role of autophagy in HIV-1 replication and pathogenesis
Killian, M. S. (2012) · AIDS Research and Therapy · doi:10.1186/1742-6405-9-16 · Cited by 73 · see citing works
“The extraordinary magnification of intracellular constituents enabled by electron microscopy allows for the direct visualization of the double membrane autophagosome structures [29].”
BackgroundISG20L1 is a p53 family target gene that modulates genotoxic stress-induced autophagy
Eby, K. G., Rosenbluth, J. M., Mays, D. J., Marshall, C. B., Barton, C. E., Sinha, S., Johnson, K. N., Tang, L., & Pietenpol, J. A. (2010) · Molecular Cancer · doi:10.1186/1476-4598-9-95 · Cited by 72 · see citing works
Autophagy: an emerging therapeutic target in vascular diseases
Vindis, C. (2014) · British Journal of Pharmacology · doi:10.1111/bph.13052 · Cited by 72 · see citing works
“…a multi-membrane source model for the biogenesis of autophagosome in mammalian cells: the ER (Axe et al., 2008; Hayashi-Nishino et al., 2009; Yla-Anttila et al., 2009a), the outer membrane of the mitochondrion (Hailey et al., 2010), the ER-mitochondrial junction (Hamasaki et al., 2013),…”
BackgroundMethodThelen, 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 73 · see citing works
“Quantification of autophagosomes was performed as previously described [48].”
MethodHuang, 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 · see citing works
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 69 · see citing works
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 70 · see citing works
“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.”
MethodSmith, C. M., Mayer, J. A., & Duncan, I. D. (2013) · The Journal of Neuroscience · doi:10.1523/jneurosci.0233-13.2013 · Cited by 69 · see citing works
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 · see citing works
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 69 · see citing works
“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.”
Methodlet‐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 65 · see citing works
“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).”
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 66 · see citing works
“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).”
BackgroundExcess 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. M., et al. (2016) · Autophagy · doi:10.1080/15548627.2016.1159378 · Cited by 63 · see citing works
“Autophagic structures were classified as described previously.(59)”
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 · see citing works
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 · see citing works
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 · see citing works
“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.”
BackgroundA study of autophagy in hemocytes of the Pacific oyster, Crassostrea gigas
Picot, S., Morga, B., Faury, N., Chollet, B., Dégremont, L., Travers, M., Renault, T., & Arzul, I. (2019) · Autophagy · doi:10.1080/15548627.2019.1596490 · Cited by 59 · see citing works
“The presence of ultrastructural modifications and autophagic structures of the hemocytes was examined based on the criteria described in the literature [13,36,46].”
BackgroundMethodThe 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 · see citing works
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 58 · see citing works
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 · see citing works
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 · see citing works
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 58 · see citing works
“Autophagic vesicle were defined as a membrane bound vesicle containing either intact or degraded cytoplasmic material or organelles [51].”
BackgroundHuang, 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 · see citing works
“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).”
Backgroundden 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 54 · see citing works
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 53 · see citing works
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 · see citing works
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 52 · see citing works
“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].”
ResultThe 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 · see citing works
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 · see citing works
“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).”
MethodAutophagy 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 · see citing works
“cell area was estimated using square grid openings, which were selected according to the rules of uniform random sampling [31].”
MethodRAB24 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 50 · see citing works
“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.”
MethodFranzetti, 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 · see citing works
“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).”
InfluentialLi, 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 · see citing works
“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).”
MethodPseudorabies 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 · see citing works
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 · see citing works
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 · see citing works
“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).”
Liao, C.-C., Ho, M., Liang, S., & Liang, C. (2012) · Autophagy · doi:10.4161/auto.22379 · Cited by 45 · see citing works
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 · see citing works
“8A), indicative of autophagosomes [43].”
BackgroundLien, J., Lin, M., Chang, S., Lai, K., Huang, A., Yu, F., & Chung, J. (2016) · Environmental Toxicology · doi:10.1002/tox.22238 · Cited by 45 · see citing works
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 · see citing works
Eid, N. A. S., Ito, Y., & Otsuki, Y. (2011) · Journal of Molecular Histology · doi:10.1007/s10735-011-9372-0 · Cited by 45 · see citing works
“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).”
MethodCollins, 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 44 · see citing works
“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.”
MethodZhang, 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 44 · see citing works
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 44 · see citing works
- Authors
- Ylä‐Anttila, P., Vihinen, H., Jokitalo, E., & Eskelinen, E.
- Affiliations
- University of Helsinki (FI)
- Venue
- Methods in Enzymology
- Publisher
- Elsevier
- Published
- 2009
- Volume
- 452
- Pages
- 143-164
- ISSN
- 0076-6879 (ISSN-L)
- 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
- Health Sciences › Medicine › Epidemiology › 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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