Abstract
Autophagy-related organelles, including omegasomes, isolation membranes (or phagophores), autophagosomes, and autolysosomes, are characterized by dynamic changes in lipid membranes including morphology as well as their associated proteins. Therefore, it is critical to define and track membranous elements for identification and detailed morphological analyses of these organelles. However, it is often difficult to clearly observe these organelles with good morphology in conventional electron microscopy (EM), thus hampering 3D analyses and correlative light-electron microscopy (CLEM). Here, we focus on describing fixation procedures using (1) ferrocyanide-reduced osmium for CLEM and (2) aldehyde/OsO4 mixture for detecting omegasome structures and isolation membrane-associated tubules (IMATs). These methods can be easily applied to cultured mammalian cells for conventional and cutting-edge EM analyses, leading to a better understanding of ultrastructural details in autophagosome formation.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Eskelinen EL, Reggiori F, Baba M, Kovacs AL, Seglen PO (2011) Seeing is believing: the impact of electron microscopy on autophagy research. Autophagy 7(9):935–956
Karanasios E, Walker SA, Okkenhaug H, Manifava M, Hummel E, Zimmermann H, Ahmed Q, Domart MC, Collinson L, Ktistakis NT (2016) Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles. Nat Commun 7:12420. https://doi.org/10.1038/ncomms12420
Hayashi-Nishino M, Fujita N, Noda T, Yamaguchi A, Yoshimori T, Yamamoto A (2009) A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat Cell Biol 11(12):1433–1437. https://doi.org/10.1038/ncb1991
Yla-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL (2009) 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy 5(8):1180–1185
Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, Griffiths G, Ktistakis NT (2008) Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 182(4):685–701. https://doi.org/10.1083/jcb.200803137
Uemura T, Yamamoto M, Kametaka A, Sou YS, Yabashi A, Yamada A, Annoh H, Kametaka S, Komatsu M, Waguri S (2014) A cluster of thin tubular structures mediates transformation of the endoplasmic reticulum to autophagic isolation membrane. Mol Cell Biol 34(9):1695–1706. https://doi.org/10.1128/MCB.01327-13
Karnovsky MJ (1971) Use of ferrocyanide-reduced osmium in electron microscopy. In: Proc 14th Annual Meeting Amer Soc Cell Biol
Hoshino Y, Shannon WA, Seligman AM (1976) A study on ferrocyanide-reduced osmium tetroxide as a stain and cytochemical agent. Acta Histochem Cytochem 9:125–136
Langford LA, Coggeshall RE (1980) The use of potassium ferricyanide in neural fixation. Anat Rec 197(3):297–303. https://doi.org/10.1002/ar.1091970304
Schnepf E, Hausmann K, Herth W (1982) The osmium tetroxide-potassium ferrocyanide (OsFeCN) staining technique for electron microscopy: a critical evaluation using ciliates, algae, mosses, and higher plants. Histochemistry 76(2):261–271
Yla-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL (2009) Monitoring autophagy by electron microscopy in Mammalian cells. Methods Enzymol 452:143–164. https://doi.org/10.1016/S0076-6879(08)03610-0
Biazik J, Yla-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL (2015) Ultrastructural relationship of the phagophore with surrounding organelles. Autophagy 11(3):439–451. https://doi.org/10.1080/15548627.2015.1017178
Hua Y, Laserstein P, Helmstaedter M (2015) Large-volume en-bloc staining for electron microscopy-based connectomics. Nat Commun 6:7923. https://doi.org/10.1038/ncomms8923
White DL, Mazurkiewicz JE, Barrnett RJ (1979) A chemical mechanism for tissue staining by osmium tetroxide-ferrocyanide mixtures. J Histochem Cytochem 27(7):1084–1091. https://doi.org/10.1177/27.7.89155
Audrey MG (1975) Fixation, dehydration and embedding of biological specimens. In: Audrey MG (ed) Practical methods in electron microscopy, vol 3. North-Holland Publishing Company, Amsterdam
Hayat M (1981) Fixation for electron microscopy. Academic Press, New York
Yabashi A, Uemura T, Waguri S (2014) Optimal temperature in applying a fixative mixture of aldehyde and osmium tetroxide to the observation of isolation membrane-associated tubules. J Electr Microsc Technol Med Biol 28(1):9–11
Acknowledgments
We thank all the members in our department for helpful discussions. This work was supported by JSPS KAKENHI Grant numbers 24390048 and 15H04670 (to S. Waguri).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Arai, R., Waguri, S. (2019). Improved Electron Microscopy Fixation Methods for Tracking Autophagy-Associated Membranes in Cultured Mammalian Cells. In: Ktistakis, N., Florey, O. (eds) Autophagy. Methods in Molecular Biology, vol 1880. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8873-0_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8873-0_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8872-3
Online ISBN: 978-1-4939-8873-0
eBook Packages: Springer Protocols