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

Published online by Cambridge University Press:  12 August 2016

Diana B. Peckys
Department of Molecular Biophysics, Saarland University, CIPMM, 66421 Homburg, Germany
Dalia Alansary
Department of Molecular Biophysics, Saarland University, CIPMM, 66421 Homburg, Germany
Barbara A. Niemeyer
Department of Molecular Biophysics, Saarland University, CIPMM, 66421 Homburg, Germany
Niels de Jonge*
INM – Leibniz Institute for New Materials, 66123 Saarbrücken, Germany Department of Physics, Saarland University, 66123 Saarbrücken, Germany
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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.

Biological Applications
© Microscopy Society of America 2016 

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