Skip to main content
×
×
Home

Nanogold as a Specific Marker for Electron Cryotomography

  • Yongning He (a1), Grant J. Jensen (a1) (a2) and Pamela J. Bjorkman (a1) (a2)
Abstract

While electron cryotomography (ECT) provides “molecular” resolution, three-dimensional images of unique biological specimens, sample crowdedness, and/or resolution limitations can make it difficult to identify specific macromolecular components. Here we used a 1.4 nm Nanogold® cluster specifically attached to the Fc fragment of IgG to monitor its interaction with the neonatal Fc receptor (FcRn), a membrane-bound receptor that transports IgG across cells in acidic intracellular vesicles. ECT was used to image complexes formed by Nanogold-labeled Fc bound to FcRn attached to the outer surface of synthetic liposomes. In the resulting three-dimensional reconstructions, 1.4 nm Nanogold particles were distributed predominantly along the interfaces where 2:1 FcRn-Fc complexes bridged adjacent lipid bilayers. These results demonstrate that the 1.4 nm Nanogold cluster is visible in tomograms of typically thick samples (∼250 nm) recorded with defocuses appropriate for large macromolecules and is thus an effective marker.

Copyright
Corresponding author
Corresponding author. E-mail: bjorkman@caltech.edu
References
Hide All
Ackerson, C.J., Jadzinsky, P.D., Jensen, G.J. & Kornberg, R.D. (2006). Rigid, specific, and discrete gold nanoparticle/antibody conjugates. J Am Chem Soc 128, 26352640.
Boisset, N., Grassucci, R., Penczek, P., Delain, E., Pochon, F., Frank, J. & Lamy, J.N. (1992). Three-dimensional reconstruction of a complex of human alpha 2-macroglobulin with monomaleimido Nanogold (Au1.4nm) embedded in ice. J Struct Biol 109, 3945.
Burmeister, W.P., Gastinel, L.N., Simister, N.E., Blum, M.L. & Bjorkman, P.J. (1994a). Crystal structure at 2.2 Å resolution of the MHC-related neonatal Fc receptor. Nature 372, 336343.
Burmeister, W.P., Huber, A.H. & Bjorkman, P.J. (1994b). Crystal structure of the complex of rat neonatal Fc receptor with Fc. Nature 372, 379383.
Celia, H., Wilson-Kubalek, E., Milligan, R.A. & Teyton, L. (1999). Structure and function of a membrane-bound murine MHC class I molecule. Proc Natl Acad Sci USA 96, 56345639.
Gan, L., Chen, S. & Jensen, G.J. (2008). Molecular organization of gram-negative peptidoglycan. Proc Natl Acad Sci USA 105, 1895318957.
Hainfeld, J.F. & Furuya, F.R. (1992). A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling. J Histochem Cytochem 40, 177184.
Hainfeld, J.F. & Powell, R.D. (2000). New frontiers in gold labeling. J Histochem Cytochem 48, 471480.
He, W., Kivork, C.K., Machinani, S., Morphew, M.K., Gail, A.M., Tesar, D.B., Tiangco, N.E., McIntosh, J.R. & Bjorkman, P.J. (2007). A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed nanogold-labeled molecules. J Struct Biol 160, 103111.
He, W., Ladinsky, M.S., Huey-Tubman, K.E., Jensen, G.J., McIntosh, J.R. & Bjorkman, P.J. (2008). FcRn-mediated antibody transport across epithelial cells revealed by electron tomography. Nature 455, 542546.
Henderson, G.P., Gan, L. & Jensen, G.J. (2007). 3-D ultrastructure of O. tauri: Electron cryotomography of an entire eukaryotic cell. PLoS ONE 2, e749.
Huber, A.H., Kelley, R.F., Gastinel, L.N. & Bjorkman, P.J. (1993). Crystallization and stoichiometry of binding of a complex between a rat intestinal Fc receptor and Fc. J Mol Biol 230, 10771083.
Jeon, H. & Shipley, G.G. (2000). Localization of the N-terminal domain of the low density lipoprotein receptor. J Biol Chem 275, 3046530470.
Kremer, J.R., Mastronarde, D.N. & McIntosh, J.R. (1996). Computer visualization of three-dimensional data using IMOD. J Struct Biol 116, 7176.
Lucic, V., Forster, F. & Baumeister, W. (2005). Structural studies by electron tomography: From cells to molecules. Annu Rev Biochem 74, 833865.
Martin, F.J. & Papahadjopoulos, D. (1982). Irreversible coupling of immunoglobulin fragments to preformed vesicles. An improved method for liposome targeting. J Biol Chem 257, 286288.
Martin, W.L. & Bjorkman, P.J. (1999). Characterization of the 2:1 complex between the class I MHC-related Fc receptor and its Fc ligand in solution. Biochem 38, 1263912647.
Martin, W.L., West, A.P., Gan, L. & Bjorkman, P.J. (2001). Crystal structure at 2.8 Å of an FcRn/heterodimeric Fc complex: Mechanism of pH dependent binding. Mol Cell 7, 867877.
Montesano-Roditis, L., Glitz, D.G., Traut, R.R. & Stewart, P.L. (2001). Cryo-electron microscopic localization of protein L7/L12 within the Escherichia coli 70 S ribosome by difference mapping and Nanogold labeling. J Biol Chem 276, 1411714123.
Morphew, M., He, W., Bjorkman, P.J. & McIntosh, J.R. (2008). Silver enhancement of nanogold particles during freeze substitution fixation for electron microscopy. J Microsc 230, 263267.
Murphy, G.E. & Jensen, G.J. (2005). Electron cryotomography of the E. coli pyruvate and 2-oxoglutarate dehydrogenase complexes. Structure 13, 17651773.
Raghavan, M., Wang, Y. & Bjorkman, P.J. (1995). Effects of receptor dimerization on the interaction between the class I MHC related Fc receptor and immunoglobulin G. Proc Natl Acad Sci USA 92, 1120011204.
Roopenian, D.C. & Akilesh, S. (2007). FcRn: The neonatal Fc receptor comes of age. Nat Rev Immunol 7, 715725.
Safer, D., Bolinger, L. & Leigh, J.S. Jr. (1986). Undecagold clusters for site-specific labeling of biological macromolecules: Simplified preparation and model applications. J Inorg Biochem 26, 7791.
Weipoltshammer, K., Schofer, C., Almeder, M. & Wachtler, F. (2000). Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography. Histochem Cell Biol 114, 489495.
Zheng, S.Q., Keszthelyi, B., Branlund, E., Lyle, J.M., Braunfeld, M.B., Sedat, J.W. & Agard, D.A. (2007). UCSF tomography: An integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction. J Struct Biol 157, 138147.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

Microscopy and Microanalysis
  • ISSN: 1431-9276
  • EISSN: 1435-8115
  • URL: /core/journals/microscopy-and-microanalysis
Please enter your name
Please enter a valid email address
Who would you like to send this to? *
×

Keywords

Type Description Title
VIDEO
Movie

He Supplementary Movie
Movie

 Video (7.3 MB)
7.3 MB

Metrics

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed