Skip to main content Accessibility help

Visualizing Quantum Dot Labeled ORAI1 Proteins in Intact Cells Via Correlative Light and Electron Microscopy

  • Diana B. Peckys (a1), Dalia Alansary (a1), Barbara A. Niemeyer (a1) and Niels de Jonge (a2) (a3)


ORAI1 proteins are ion channel subunits and the essential pore-forming units of the calcium release-activated calcium channel complex essential for T-cell activation and many other cellular processes. In this study, we used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to image plasma membrane expressed ORAI1 proteins in whole Jurkat T cells in the liquid state. Utilizing a stably transfected Jurkat T cell clone expressing human ORAI1 with an extracellular human influenza hemagglutinin (HA) tag we investigated if liquid-phase STEM can be applied to detect recombinant surface expressed protein. Streptavidin coated quantum dots were coupled in a one-to-one stoichiometry to ORAI1 proteins detected by biotinylated anti-HA fragmented antibody fragments. High-resolution electron microscopic images revealed the individual label locations from which protein pair distances were determined. These data were analyzed using the pair correlation function and, in addition, an analysis of cluster size and frequency was performed. ORAI1 was found to be present in hexamers in a small fraction only, and ORAI1 resided mostly in monomers and dimers.


Corresponding author

* Corresponding author.


Hide All
Alansary, D., Bogeski, I. & Niemeyer, B.A. (2015). Facilitation of Orai3 targeting and store-operated function by Orai1. Biochim Biophys Acta 1853, 15411550.
Arant, R.J. & Ulbrich, M.H. (2014). Deciphering the subunit composition of multimeric proteins by counting photobleaching steps. Chem Phys Chem 15, 600605.
Bogner, A., Thollet, G., Basset, D., Jouneau, P.H. & Gauthier, C. (2005). Wet STEM: A new development in environmental SEM for imaging nano-objects included in a liquid phase. Ultramicroscopy 104, 290301.
Cambi, A. & Lidke, D.S. (2012). Nanoscale membrane organization: Where biochemistry meets advanced microscopy. ACS Chem Biol 7, 139149.
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 U S A 106, 21592164.
Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S.H., Tanasa, B., Hogan, P.G., Lewis, R.S., Daly, M. & Rao, A. (2006). A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441, 179185.
Field, J., Nikawa, J., Broek, D., MacDonald, B., Rodgers, L., Wilson, I.A., Lerner, R.A. & Wigler, M. (1988). Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol 8, 21592165.
Fiksel, T. (1988). Edge-corrected density estimators for points processes. Statistics 19, 6775.
Georgieva, M.V., Yahya, G., Codo, L., Ortiz, R., Teixido, L., Claros, J., Jara, R., Jara, M., Iborra, A., Gelpi, J.L., Gallego, C., Orozco, M. & Aldea, M. (2015). Inntags: small self-structured epitopes for innocuous protein tagging. Nat Methods 12, 955958.
Gwack, Y., Srikanth, S., Feske, S., Cruz-Guilloty, F., Oh-hora, M., Neems, D.S., Hogan, P.G. & Rao, A. (2007). Biochemical and functional characterization of Orai proteins. J Biol Chem 282, 1623216243.
Hermannsdörfer, J., Tinnemann, V., Peckys, D.B. & de Jonge, N. (2016). The effect of electron beam irradiation in environmental scanning transmission electron microscopy of whole cells in liquid. Microsc Microanal 20, 656665, doi:
Hodeify, R., Selvaraj, S., Wen, J., Arredouani, A., Hubrack, S., Dib, M., Al-Thani, S.N., McGraw, T. & Machaca, K. (2015). A STIM1-dependent “trafficking trap” mechanism regulates Orai1 plasma membrane residence and Ca(2)(+) influx levels. J Cell Sci 128, 31433154.
Hou, X., Pedi, L., Diver, M.M. & Long, S.B. (2012). Crystal structure of the calcium release-activated calcium channel Orai. Science 338, 13081313.
Inayama, M., Suzuki, Y., Yamada, S., Kurita, T., Yamamura, H., Ohya, S., Giles, W.R. & Imaizumi, Y. (2015). Orai1-Orai2 complex is involved in store-operated calcium entry in chondrocyte cell lines. Cell Calcium 57, 337347.
Kilch, T., Alansary, D., Peglow, M., Dorr, K., Rychkov, G., Rieger, H., Peinelt, C. & Niemeyer, B.A. (2013). Mutations of the 2+ -sensing stromal interaction molecule STIM1 regulate 2+ influx by altered oligomerization of STIM1 and by destabilization of the 2+ channel Orai1. J Biol Chem 288, 16531664.
Kinoshita, T., Mori, Y., Hirano, K., Sugimoto, S., Okuda, K., Matsumoto, S., Namiki, T., Ebihara, T., Kawata, M., Nishiyama, H., Sato, M., Suga, M., Higashiyama, K., Sonomoto, K., Mizunoe, Y., Nishihara, S. & Sato, C. (2014). Immuno-electron microscopy of primary cell cultures from genetically modified animals in liquid by atmospheric scanning electron microscopy. Microsc Microanal 20, 469483.
Kourkoutis, L.F., Plitzko, J.M. & Baumeister, W. (2012). Electron microscopy of biological materials at the nanometer scale. Annu Rev Mater Res 42, 3358.
Laganowsky, A., Reading, E., Hopper, J.T.S. & Robinson, C.V. (2013). Mass spectrometry of intact membrane protein complexes. Nat Protoc 8, 639651.
Lippincott-Schwartz, J. & Manley, S. (2009). Putting super-resolution fluorescence microscopy to work. Nat Methods 6, 2123.
Lis, A., Peinelt, C., Beck, A., Parvez, S., Monteilh-Zoller, M., Fleig, A. & Penner, R. (2007). CRACM1, CRACM2, and CRACM3 are store-operated 2+ channels with distinct functional properties. Curr Biol 17, 794800.
Liv, N., Lazic, I., Kruit, P. & Hoogenboom, J.P. (2014). Scanning electron microscopy of individual nanoparticle bio-markers in liquid. Ultramicroscopy 143, 9399.
Luik, R.M., Wu, M.M., Buchanan, J. & Lewis, R.S. (2006). The elementary unit of store-operated 2+ entry: Local activation of CRAC channels by STIM1 at ER-plasma membrane junctions. J Cell Biol 174, 815825.
Maruyama, Y., Ogura, T., Mio, K., Kato, K., Kaneko, T., Kiyonaka, S., Mori, Y. & Sato, C. (2009). Tetrameric Orai1 is a teardrop-shaped molecule with a long, tapered cytoplasmic domain. J Biol Chem 284, 1367613685.
Moraes, I., Evans, G., Sanchez-Weatherby, J., Newstead, S. & Stewart, P.D.S. (2014). Membrane protein structure determination—The next generation. Biochim Biophys Acta 1838, 7887.
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, 438449.
Nolz, J.C., Gomez, T.S., Zhu, P., Li, S., Medeiros, R.B., Shimizu, Y., Burkhardt, J.K., Freedman, B.D. & Billadeau, D.D. (2006). The WAVE2 complex regulates actin cytoskeletal reorganization and CRAC-mediated calcium entry during T cell activation. Curr Biol 16, 2434.
Orci, L., Ravazzola, M., Le Coadic, M., Shen, W.-w., Demaurex, N. & Cosson, P. (2009). STIM1-induced precortical and cortical subdomains of the endoplasmic reticulum. Proc Natl Acad Sci U S A 106, 1935819362.
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. 2621–2626.
Peckys, D.B. & de Jonge, N. (2014). Liquid scanning transmission electron microscopy: Imaging protein complexes in their native environment in whole eukaryotic cells. Microsc Microanal 20, 346365.
Peckys, D.B. & de Jonge, N. (2015). Studying the stoichiometry of epidermal growth factor receptor in intact cells using correlative microscopy. J Vis Exp 103, e53186.
Peckys, D.B., Korf, U. & de Jonge, N. (2015). Local variations of HER2 dimerization in breast cancer cells discovered by correlative fluorescence and liquid electron microscopy. Sci Adv 1, e1500165.
Piston, D.W. & Kremers, G.J. (2007). Fluorescent protein FRET: The good, the bad and the ugly. Trends Biochem Sci 32, 407414.
Prakriya, M., Feske, S., Gwack, Y., Srikanth, S., Rao, A. & Hogan, P.G. (2006). Orai1 is an essential pore subunit of the CRAC channel. Nature 443, 230233.
Prakriya, M. & Lewis, R.S. (2015). Store-operated calcium channels. Physiol Rev 95, 13831436.
Quintana, A., Pasche, M., Junker, C., Al-Ansary, D., Rieger, H., Kummerow, C., Nunez, L., Villalobos, C., Meraner, P., Becherer, U., Rettig, J., Niemeyer, B.A. & Hoth, M. (2011). Calcium microdomains at the immunological synapse: How ORAI channels, mitochondria and calcium pumps generate local calcium signals for efficient T-cell activation. EMBO J 30, 38953912.
Ring, E.A., Peckys, D.B., Dukes, M.J., Baudoin, J.P. & de Jonge, N. (2011). Silicon nitride windows for electron microscopy of whole cells. J Microsc 243, 273283.
Sauc, S., Bulla, M., Nunes, P., Orci, L., Marchetti, A., Antigny, F., Bernheim, L., Cosson, P., Frieden, M. & Demaurex, N. (2015). STIM1L traps and gates Orai1 channels without remodeling the cortical ER. J Cell Sci 128, 15681579.
Schuh, T. & de Jonge, N. (2014). Liquid scanning transmission electron microscopy: Nanoscale imaging in micrometers-thick liquids. C R Phys 15, 214223.
Shine, J., Fettes, I., Lan, N.C., Roberts, J.L. & Baxter, J.D. (1980). Expression of cloned beta-endorphin gene sequences by Escherichia coli. Nature 285, 456463.
Soboloff, J., Rothberg, B.S., Madesh, M. & Gill, D.L. (2012). STIM proteins: Dynamic calcium signal transducers. Nat Rev Mol Cell Biol 13, 549565.
Stoyan, D., Bertram, U. & Wendrock, H. (1993). Estimation variances for estimators of product densities and pair correlation functions of planar points processes. Ann Inst Statist Math 45, 211221.
Stoyan, D. & Stoyan, H. (1996). Estimating pair correlation functions of planar cluster processes. Biom J 38, 259271.
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, 3346.
Vig, M., Peinelt, C., Beck, A., Koomoa, D.L., Rabah, D., Koblan-Huberson, M., Kraft, S., Turner, H., Fleig, A., Penner, R. & Kinet, J.P. (2006). CRACM1 is a plasma membrane protein essential for store-operated 2+ entry. Science 312, 12201223.
Wojcik, M., Hauser, M., Li, W., Moon, S. & Xu, K. (2015). Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells. Nat Commun 6, 7384.
Wu, M.M., Buchanan, J., Luik, R.M. & Lewis, R.S. (2006) Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane. J Cell Biol 174, 803813.
Zhang, S.L., Yeromin, A.V., Zhang, X.H., Yu, Y., Safrina, O., Penna, A., Roos, J., Stauderman, K.A. & Cahalan, M.D. (2006). Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity. Proc Natl Acad Sci USA 103, 93579362.


Type Description Title
Supplementary materials

Peckys supplementary material
Figures S1-S3

 Word (979 KB)
979 KB

Visualizing Quantum Dot Labeled ORAI1 Proteins in Intact Cells Via Correlative Light and Electron Microscopy

  • Diana B. Peckys (a1), Dalia Alansary (a1), Barbara A. Niemeyer (a1) and Niels de Jonge (a2) (a3)


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