title: Visualizing Quantum Dot Labeled ORAI1 Proteins in Intact Cells Via Correlative Light and Electron Microscopy.
abstract: 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.
authors: 1Department of Molecular Biophysics,Saarland University,CIPMM,66421 Homburg,Germany.
authors: Diana B
authors: DB
authors: Peckys
authors: 1Department of Molecular Biophysics,Saarland University,CIPMM,66421 Homburg,Germany.
authors: Dalia
authors: D
authors: Alansary
authors: 1Department of Molecular Biophysics,Saarland University,CIPMM,66421 Homburg,Germany.
authors: Barbara A
authors: BA
authors: Niemeyer
authors: 2INM - Leibniz Institute for New Materials,66123 Saarbrücken,Germany.
authors: Niels
authors: N
authors: de Jonge
mesh_terms: D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure*; D006801:Humans; D017348:Microscopy, Electron, Scanning Transmission; D000071740:ORAI1 Protein / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D045663:Quantum Dots / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D000071740:ORAI1 Protein; C510746:ORAI1 protein, human
keywords: ESEM; HA tag; Jurkat T cell; STEM; pair correlation function