Abstract
Characterizing the detailed structure of the mammalian synapse is of crucial importance to understand its mechanisms of function. Here I describe a protocol to study synaptic architecture by cryo-electron tomography (cryo-ET), a powerful electron microscopy technique that enables 3D visualization of unstained, fully hydrated cellular structures at molecular resolution. The protocol focuses on purified synaptic terminals (“synaptosomes”), currently the most suitable preparation to analyze mammalian synaptic architecture by cryo-ET.
The original version of this chapter was revised. A correction to this chapter can be found at https://doi.org/10.1007/978-1-4939-8719-1_19
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Change history
24 October 2018
This book was inadvertently published with the incorrect title as Clathrin-Mediated Endoytosis: Methods and Protocols. This has now been corrected throughout the book to Clathrin-Mediated Endocytosis: Methods and Protocols.
References
Asano S, Engel BD, Baumeister W (2015a) In situ Cryo-Electron tomography: a post-reductionist approach to structural biology. J Mol Biol
Fernandez-Busnadiego R, Zuber B, Maurer UE, Cyrklaff M, Baumeister W, Lucic V (2010) Quantitative analysis of the native presynaptic cytomatrix by cryoelectron tomography. J Cell Biol 188:145–156
Zuber B, Nikonenko I, Klauser P, Muller D, Dubochet J (2005) The mammalian central nervous synaptic cleft contains a high density of periodically organized complexes. Proc Natl Acad Sci U S A 102:19192–19197
Asano S, Fukuda Y, Beck F, Aufderheide A, Forster F, Danev R, Baumeister W (2015b) A molecular census of 26S proteasomes in intact neurons. Science 347:439–442
Lucic V, Kossel AH, Yang T, Bonhoeffer T, Baumeister W, Sartori A (2007) Multiscale imaging of neurons grown in culture: from light microscopy to cryo-electron tomography. J Struct Biol 160:146–156
Zuber B, Unwin N (2013) Structure and superorganization of acetylcholine receptor-rapsyn complexes. Proc Natl Acad Sci U S A 110:10622–10627
Fernandez-Busnadiego R, Schrod N, Kochovski Z, Asano S, Vanhecke D, Baumeister W, Lucic V (2011) Insights into the molecular organization of the neuron by cryo-electron tomography. J Electron Microsc 60(Suppl 1):S137–S148
Whittaker VP (1993) Thirty years of synaptosome research. J Neurocytol 22:735–742
Fernandez-Busnadiego R, Asano S, Oprisoreanu AM, Sakata E, Doengi M, Kochovski Z, Zurner M, Stein V, Schoch S, Baumeister W et al (2013) Cryo-electron tomography reveals a critical role of RIM1alpha in synaptic vesicle tethering. J Cell Biol 201:725–740
Dunkley PR, Heath JW, Harrison SM, Jarvie PE, Glenfield PJ, Rostas JA (1988) A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions. Brain Res 441:59–71
Dunkley PR, Jarvie PE, Robinson PJ (2008) A rapid Percoll gradient procedure for preparation of synaptosomes. Nat Protoc 3:1718–1728
Godino MD, Torres M, Sanchez-Prieto J (2007) CB1 receptors diminish both Ca2+ influx and glutamate release through two different mechanisms active in distinct populations of cerebrocortical nerve terminals. J Neurochem 101:1471–1482
Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76
Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51
Tivol WF, Briegel A, Jensen GJ (2008) An improved cry ogen for plunge freezing. Microsc Microanal 14:375–379
Fukuda Y, Schrod N, Schaffer M, Feng LR, Baumeister W, Lucic V (2014) Coordinate transformation based cryo-correlative methods for electron tomography and focused ion beam milling. Ultramicroscopy 143:15–23
Schellenberger P, Kaufmann R, Siebert CA, Hagen C, Wodrich H, Grunewald K (2014) High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers. Ultramicroscopy 143:41–51
Zhang P (2013) Correlative cryo-electron tomography and optical microscopy of cells. Curr Opin Struct Biol 23:763–770
Acknowledgments
I wish to thank Eri Sakata for the critical reading of the manuscript. R. F.-B. is supported by the FP7 GA ERC-2012-SyG_318987–ToPAG grant from the European Commission.
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Fernández-Busnadiego, R. (2018). Cryo-Electron Tomography of the Mammalian Synapse. In: Swan, L. (eds) Clathrin-Mediated Endocytosis. Methods in Molecular Biology, vol 1847. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8719-1_16
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DOI: https://doi.org/10.1007/978-1-4939-8719-1_16
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