Abstract
Field emission scanning electron microscopy (FESEM) is a well-established technique for acquiring three-dimensional surface images of nuclear pore complexes (NPCs). We present an optimized protocol for the exposure of mammalian cell nuclei and direct surface imaging of nuclear envelopes by FESEM, allowing for a detailed morphological comparison of individual NPCs, without the need for averaging techniques. This provides a unique high resolution tool for studying the effects of cellular stress, specific genetic manipulations and inherited diseases on the ultrastructure of human NPCs.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Goldberg MW, Allen TD (1992) High resolution scanning electron microscopy of the nuclear envelope: demonstration of a new, regular, fibrous lattice attached to the baskets of the nucleoplasmic face of the nuclear pores. J Cell Biol 119:1429–1440
Goldberg MW, Allen TD (1993) The nuclear pore complex: three-dimensional surface structure revealed by field emission, in-lens scanning electron microscopy, with underlying structure uncovered by proteolysis. J Cell Sci 106(Pt 1):261–274
Goldberg MW, Allen TD (1995) Structural and functional organization of the nuclear envelope. Curr Opin Cell Biol 7:301–309
Fichtman B, Shaulov L, Harel A (2014) Imaging metazoan nuclear pore complexes by field emission scanning electron microscopy. Methods Cell Biol 122:41–58
Shaulov L, Fichtman B, Harel A (2014) High-resolution scanning electron microscopy for the imaging of nuclear pore complexes and ran-mediated transport. Methods Mol Biol 1120:253–261
Coyne AN, Zaepfel BL, Hayes L, Fitchman B, Salzberg Y, Luo EC, Bowen K, Trost H, Aigner S, Rigo F et al (2020) G4C2 repeat RNA initiates a POM121-mediated reduction in specific nucleoporins in C9orf72 ALS/FTD. Neuron 107:1124–1140.e11
Fichtman B, Harel T, Biran N, Zagairy F, Applegate CD, Salzberg Y, Gilboa T, Salah S, Shaag A, Simanovsky N et al (2019) Pathogenic variants in NUP214 cause "plugged" nuclear pore channels and acute febrile encephalopathy. Am J Hum Genet 105:48–64
Fichtman B, Zagairy F, Biran N, Barsheshet Y, Chervinsky E, Ben Neriah Z, Shaag A, Assa M, Elpeleg O, Harel A et al (2019) Combined loss of LAP1B and LAP1C results in an early onset multisystemic nuclear envelopathy. Nat Commun 10:605
Moreira TG, Zhang L, Shaulov L, Harel A, Kuss SK, Williams J, Shelton J, Somatilaka B, Seemann J, Yang J et al (2015) Sec13 regulates expression of specific immune factors involved in inflammation in vivo. Sci Rep 5:17655
Ravindran E, Juhlen R, Vieira-Vieira CH, Ha T, Salzberg Y, Fichtman B, Luise-Becker L, Martins N, Picker-Minh S, Bessa P et al (2021) Expanding the phenotype of NUP85 mutations beyond nephrotic syndrome to primary autosomal recessive microcephaly and Seckel syndrome spectrum disorders. Hum Mol Genet 30(22):2068–2081
Regmi SG, Lee H, Kaufhold R, Fichtman B, Chen S, Aksenova V, Turcotte E, Harel A, Arnaoutov A, Dasso M (2020) The nuclear pore complex consists of two independent scaffolds. bioRxiv 2020.2011.2013.381947
Acknowledgments
This work was supported by research grants from the Israel Science Foundation (958/15) and the Christians for Israel Chair in Medical Research to A.H.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Fichtman, B., Regmi, S.G., Dasso, M., Harel, A. (2022). High-Resolution Imaging and Analysis of Individual Nuclear Pore Complexes. In: Goldberg, M.W. (eds) The Nuclear Pore Complex. Methods in Molecular Biology, vol 2502. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2337-4_29
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2337-4_29
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2336-7
Online ISBN: 978-1-0716-2337-4
eBook Packages: Springer Protocols