Mammalian Cell-derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies

Abstract

Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single-molecule imaging, isolated organelle specific vesicles can be employed to study the trafficking and assembly of the embedded proteins. Here we present a method for organelle specific single molecule imaging via isolation of ER and plasma membrane vesicles from HEK293T cells by employing OptiPrep gradients and nitrogen cavitation. The isolation was validated through Western blotting, and the isolated vesicles were used to perform single molecule studies of oligomeric receptor assembly.

Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

“…For imaging and immobilization, we fused the enhanced green fluorescent protein (EGFP) to the cytosolic N-terminus of the CNG channel TAX-4 (GFP-TAX-4). Cell-derived nanovesicles containing GFP-TAX-4 were generated as described for a study of nicotinic acetylcholine receptor stoichiometry 7–9,42 (Fig. 1).…”

“…1). Briefly, HEK-293T cells transfected with GFP-TAX-4 were disrupted using nitrogen cavitation, resulting in the spontaneous formation of cell membrane vesicles with diameters around 200 nm 9,42 (Supplementary Fig. 1).…”

“…Cells were plated in 60 mm dishes and transfected with 1 ug of GFP-TAX4 and 3 ug of PEI-MAX per dish. After 24 hours, cells from four dishes were combined and vesicles were prepared as previously described 9 . Briefly, the cells were subject to nitrogen cavitation at 600 psi.…”

“…However, their application to studies of ligand binding in membrane proteins is challenged by costly sample preparation, dysfunction in nonnative lipid or detergent environments [4][5][6] , lack of solution access to intracellular sites, and nonspecific dye adsorption to imaging surfaces. Here, we overcome these challenges by combining cell-derived nanovesicles [7][8][9] , microfluidics, micro-mirror total internal reflection fluorescence (mmTIRF) 10 , and colocalization SM spectroscopy 11 to optically track binding and unbinding of individual fluorescently-tagged ligands at single membrane proteins. Imaging a ~100 µm × 100 µm field of view much larger than a confocal spot enables high-throughput data acquisition of up to hundreds of molecules simultaneously.…”