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Immuno-Electron Microscopy of Primary Cell Cultures from Genetically Modified Animals in Liquid by Atmospheric Scanning Electron Microscopy

Published online by Cambridge University Press:  25 February 2014

Takaaki Kinoshita ,
Yosio Mori ,
Kazumi Hirano ,
Shinya Sugimoto ,
Ken-ichi Okuda ,
Shunsuke Matsumoto ,
Takeshi Namiki ,
Tatsuhiko Ebihara ,
Masaaki Kawata  and
Hidetoshi Nishiyama
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Takaaki Kinoshita
Affiliation:
Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan
Yosio Mori
Affiliation:
Department of Virology III, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan
Kazumi Hirano
Affiliation:
Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan
Shinya Sugimoto
Affiliation:
Department of Bacteriology, The Jikei University School of Medicine, Tokyo 105-8461, Japan
Ken-ichi Okuda
Affiliation:
Department of Bacteriology, The Jikei University School of Medicine, Tokyo 105-8461, Japan
Shunsuke Matsumoto
Affiliation:
Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8581, Japan
Takeshi Namiki
Affiliation:
Suntory Global Innovation Center, Research Institute, 5-2-5 Yamazaki, Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan
Tatsuhiko Ebihara
Affiliation:
Biomedical Research Institute and Information Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Masaaki Kawata
Affiliation:
Biomedical Research Institute and Information Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Hidetoshi Nishiyama
Affiliation:
JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558, Japan
Mari Sato
Affiliation:
Biomedical Research Institute and Information Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Mitsuo Suga
Affiliation:
JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558, Japan
Kenichi Higashiyama
Affiliation:
Suntory Global Innovation Center, Research Institute, 5-2-5 Yamazaki, Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan
Kenji Sonomoto
Affiliation:
Laboratory of Microbial Technology, Department of Bioscience and Biotechnology, Division of Applied Molecular Microbiology and Biomass Chemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
Yoshimitsu Mizunoe
Affiliation:
Department of Bacteriology, The Jikei University School of Medicine, Tokyo 105-8461, Japan
Shoko Nishihara*
Affiliation:
Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan
Chikara Sato*
Affiliation:
Biomedical Research Institute and Information Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
*
*Corresponding authors. ti-sato@aist.go.jp; shoko@soka.ac.jp
*Corresponding authors. ti-sato@aist.go.jp; shoko@soka.ac.jp
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Abstract

High-throughput immuno-electron microscopy is required to capture the protein–protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell’s plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future.

Keywords

ASEMimmuno-EMenvironmental cellneuronaxonal segmentationFaslactic acid bacteriarubella virusClairScope
Type
In Situ Special Section
Information
Microscopy and Microanalysis , Volume 20 , Issue 2 , April 2014 , pp. 469 - 483
Copyright
© Microscopy Society of America 2014 

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References

Abrams, I.M. & McBrain, J.W. (1944). A closed cell for electron microscopy. J Appl Phys 15, 607–609.Google Scholar
Akita, M., Tanaka, K., Murai, N., Matsumoto, S., Fujita, K., Takaki, T. & Nishiyama, H. (2013). Detection of CD133 (prominin-1) in a human hepatoblastoma cell line (HuH-6 clone 5). Microsc Res Tech 76(8), 844–852.Google Scholar
Al-Bassam, S., Xu, M., Wandless, T.J. & Arnold, D.B. (2012). Differential trafficking of transport vesicles contributes to the localization of dendritic proteins. Cell Rep 2(1), 89–100.Google Scholar
Bellen, H.J., Levis, R.W., He, Y., Carlson, J.W., Evans-Holm, M., Bae, E., Kim, J., Metaxakis, A., Savakis, C., Schulze, K.L., Hoskins, R.A. & Spradling, A.C. (2011). The Drosophila gene disruption project: Progress using transposons with distinctive site specificities. Genetics 188(3), 731–743.Google Scholar
Betzig, E., Patterson, G.H., Sougrat, R., Lindwasser, O.W., Olenych, S., Bonifacino, J.S., Davidson, M.W., Lippincott-Schwartz, J. & Hess, H.F. (2006). Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793), 1642–1645.CrossRefGoogle ScholarPubMed
Cook, K.R., Parks, A.L., Jacobus, L.M., Kaufman, T.C. & Matthews, K.A. (2010). New research resources at the Bloomington Drosophila Stock Center. Fly 4(1), 88–91.Google Scholar
Danilatos, G.D. (1981). The examination of fresh or living plant material in an environmental scanning electron microscope. J Microsc 121, 235–238.Google Scholar
Daulton, T.L., Little, B.J., Lowe, K. & Jones-Meehan, J. (2001). In situ environmental cell-transmission electron microscopy study of microbial reduction of chromium(VI) using electron energy loss spectroscopy. Microsc Microanal 7(6), 470–485.Google Scholar
de Jonge, N., Peckys, D.B., Kremers, G.J. & Piston, D.W. (2009). Electron microscopy of whole cells in liquid with nanometer resolution. Proc Natl Acad Sci USA 106(7), 2159–2164.Google Scholar
de Jonge, N. & Ross, F.M. (2011). Electron microscopy of specimens in liquid. Nat Nanotechnol 6, 695–704.Google Scholar
den Boon, J.A., Diaz, A. & Ahlquist, P. (2010). Cytoplasmic viral replication complexes. Cell Host Microbe 8(1), 77–85.Google Scholar
Dietzl, G., Chen, D., Schnorrer, F., Su, K.C., Barinova, Y., Fellner, M., Gasser, B., Kinsey, K., Oppel, S., Scheiblauer, S., Couto, A., Marra, V., Keleman, K. & Dickson, B.J. (2007). A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448(7150), 151–156.Google Scholar
Donnert, G., Keller, J., Medda, R., Andrei, M.A., Rizzoli, S.O., Luhrmann, R., Jahn, R., Eggeling, C. & Hell, S.W. (2006). Macromolecular-scale resolution in biological fluorescence microscopy. Proc Natl Acad Sci USA 103(31), 11440–11445.Google Scholar
Dukes, M.J., Peckys, D.B. & de Jonge, N. (2010). Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid. ACS Nano 4(7), 4110–4116.CrossRefGoogle ScholarPubMed
Frolova, E.I., Gorchakov, R., Pereboeva, L., Atasheva, S. & Frolov, I. (2010). Functional Sindbis virus replicative complexes are formed at the plasma membrane. J Virol 84(22), 11679–11695.Google Scholar
Froshauer, S., Kartenbeck, J. & Helenius, A. (1988). Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes. J Cell Biol 107(6, Pt 1), 2075–2086.Google Scholar
Galiano, M.R., Jha, S., Ho, T.S., Zhang, C., Ogawa, Y., Chang, K.J., Stankewich, M.C., Mohler, P.J. & Rasband, M.N. (2012). A distal axonal cytoskeleton forms an intra-axonal boundary that controls axon initial segment assembly. Cell 149(5), 1125–1139.CrossRefGoogle ScholarPubMed
Giepmans, B.N., Deerinck, T.J., Smarr, B.L., Jones, Y.Z. & Ellisman, M.H. (2005). Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots. Nat Methods 2(10), 743–749.CrossRefGoogle ScholarPubMed
Gustafsson, M.G. (2005). Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci USA 102(37), 13081–13086.Google Scholar
Hell, S.W. (2007). Far-field optical nanoscopy. Science 316(5828), 1153–1158.Google Scholar
Hirano, K., Kinoshita, T., Uemura, T., Motohashi, H., Watanabe, Y., Ebihara, T., Nishiyama, H., Sato, M., Suga, M., Maruyama, Y., Tsuji, N.M., Yamamoto, M., Nishihara, S. & Sato, C. (in press). Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM. Ultramicroscopy. http://dx.doi.org/10.1016/j.ultramic.2013.10.010 Google Scholar
Hirano, K., Sasaki, N., Ichimiya, T., Miura, T., Van Kuppevelt, T.H. & Nishihara, S. (2012). 3-O-sulfated heparan sulfate recognized by the antibody HS4C3 contribute to the differentiation of mouse embryonic stem cells via Fas signaling. PLoS One 7(8), e43440.Google Scholar
Hirano, K., Van Kuppevelt, T.H. & Nishihara, S. (2013). The transition of mouse pluripotent stem cells from the naive to the primed state requires Fas signaling through 3-O sulfated heparan sulfate structures recognized by the HS4C3 antibody. Biochem Biophys Res Commun 430(3), 1175–1181.Google Scholar
Hirokawa, N. (1998). Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279(5350), 519–526.Google Scholar
Jacobs, J.R. & Goodman, C.S. (1989). Embryonic development of axon pathways in the Drosophila CNS. II. Behavior of pioneer growth cones. J Neurosci 9(7), 2412–2422.CrossRefGoogle ScholarPubMed
Katsuki, T., Ailani, D., Hiramoto, M. & Hiromi, Y. (2009). Intra-axonal patterning: Intrinsic compartmentalization of the axonal membrane in Drosophila neurons. Neuron 64(2), 188–199.Google Scholar
Kujala, P., Ahola, T., Ehsani, N., Auvinen, P., Vihinen, H. & Kaariainen, L. (1999). Intracellular distribution of rubella virus nonstructural protein P150. J Virol 73(9), 7805–7811.Google Scholar
Magliano, D., Marshall, J.A., Bowden, D.S., Vardaxis, N., Meanger, J. & Lee, J.Y. (1998). Rubella virus replication complexes are virus-modified lysosomes. Virology 240(1), 57–63.Google Scholar
Maruyama, Y., Ebihara, T., Nishiyama, H., Suga, M. & Sato, C. (2012). Immuno EM-OM correlative microscopy in solution by atmospheric scanning electron microscopy (ASEM). J Struct Biol 180(2), 259–270.Google Scholar
Nagy, A., Rossant, J., Nagy, R., Abramownewerly, W. & Roder, J.C. (1993). Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci USA 90(18), 8424–8428.Google Scholar
Nairn, A.V., Kinoshita-Toyoda, A., Toyoda, H., Xie, J., Harris, K., Dalton, S., Kulik, M., Pierce, J.M., Toida, T., Moremen, K.W. & Linhardt, R.J. (2007). Glycomics of proteoglycan biosynthesis in murine embryonic stem cell differentiation. J Proteome Res 6(11), 4374–4387.Google Scholar
Nakada, C., Ritchie, K., Oba, Y., Nakamura, M., Hotta, Y., Iino, R., Kasai, R.S., Yamaguchi, K., Fujiwara, T. & Kusumi, A. (2003). Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization. Nat Cell Biol 5(7), 626–632.Google Scholar
Nishiyama, H., Suga, M., Ogura, T., Maruyama, Y., Koizumi, M., Mio, K., Kitamura, S. & Sato, C. (2010). Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film. J Struct Biol 169(3), 438–449.Google Scholar
Patel, N.H., Snow, P.M. & Goodman, C.S. (1987). Characterization and cloning of fasciclin III: A glycoprotein expressed on a subset of neurons and axon pathways in Drosophila. Cell 48(6), 975–988.CrossRefGoogle Scholar
Peckys, D.B., Baudoin, J.P., Eder, M., Werner, U. & de Jonge, N. (2013). Epidermal growth factor receptor subunit locations determined in hydrated cells with environmental scanning electron microscopy. Sci Rep 3, 2626.CrossRefGoogle ScholarPubMed
Powell, R.D., Halsey, C.M., Spector, D.L., Kaurin, S.L., McCann, J. & Hainfeld, J.F. (1997). A covalent fluorescent-gold immunoprobe: Simultaneous detection of a pre-mRNA splicing factor by light and electron microscopy. J Histochem Cytochem 45(7), 947–956.Google Scholar
Rust, M.J., Bates, M. & Zhuang, X. (2006). Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10), 793–795.Google Scholar
Sato, C., Manaka, S., Nakane, D., Nishiyama, H., Suga, M., Nishizaka, T., Miyata, M. & Maruyama, Y. (2012). Rapid imaging of mycoplasma in solution using atmospheric scanning electron microscopy (ASEM). Biochem Biophys Res Commun 417(4), 1213–1218.Google Scholar
Sato, C., Ueno, Y., Asai, K., Takahashi, K., Sato, M., Engel, A. & Fujiyoshi, Y. (2001). The voltage-sensitive sodium channel is a bell-shaped molecule with several cavities. Nature 409(6823), 1047–1051.Google Scholar
Schönborn, J., Oberstrass, J., Breyel, E., Tittgen, J., Schumacher, J. & Lukacs, N. (1991). Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids Res 19(11), 2993–3000.Google Scholar
Schutze, S., Tchikov, V. & Schneider-Brachert, W. (2008). Regulation of TNFR1 and CD95 signalling by receptor compartmentalization. Nat Rev Mol Cell Biol 9(8), 655–662.Google Scholar
Skarnes, W.C., Rosen, B., West, A.P., Koutsourakis, M., Bushell, W., Iyer, V., Mujica, A.O., Thomas, M., Harrow, J., Cox, T., Jackson, D., Severin, J., Biggs, P., Fu, J., Nefedov, M., de Jong, P.J., Stewart, A.F. & Bradley, A. (2011). A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474(7351), 337–342.CrossRefGoogle ScholarPubMed
Spuul, P., Balistreri, G., Kaariainen, L. & Ahola, T. (2010). Phosphatidylinositol 3-kinase-, actin-, and microtubule-dependent transport of Semliki Forest virus replication complexes from the plasma membrane to modified lysosomes. J Virol 84(15), 7543–7557.CrossRefGoogle ScholarPubMed
Suga, M., Nishiyama, H., Konyuba, Y., Iwamatsu, S., Watanabe, Y., Yoshiura, C., Ueda, T. & Sato, C. (2011). The atmospheric scanning electron microscope with open sample space observes dynamic phenomena in liquid or gas. Ultramicroscopy 111(12), 1650–1658.Google Scholar
Thiberge, S., Nechushtan, A., Sprinzak, D., Gileadi, O., Behar, V., Zik, O., Chowers, Y., Michaeli, S., Schlessinger, J. & Moses, E. (2004). Scanning electron microscopy of cells and tissues under fully hydrated conditions. Proc Natl Acad Sci USA 101(10), 3346–3351.CrossRefGoogle ScholarPubMed
White, J.K., Gerdin, A.K., Karp, N.A., Ryder, E., Buljan, M., Bussell, J.N., Salisbury, J., Clare, S., Ingham, N.J., Podrini, C., Houghton, R., Estabel, J., Bottomley, J.R., Melvin, D.G., Sunter, D., Adams, N.C., Tannahill, D., Logan, D.W., Macarthur, D.G., Flint, J., Mahajan, V.B., Tsang, S.H., Smyth, I., Watt, F.M., Skarnes, W.C., Dougan, G., Adams, D.J., Ramirez-Solis, R., Bradley, A. & Steel, K.P. (2013). Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes. Cell 154(2), 452–464.Google Scholar