Cell‐Derived Vesicles for Single‐Molecule Imaging of Membrane Proteins

Abstract

A new approach is presented for the application of single-molecule imaging to membrane receptors through the use of vesicles derived from cells expressing fluorescently labeled receptors. During the isolation of vesicles, receptors remain embedded in the membrane of the resultant vesicles, thus allowing these vesicles to serve as nanocontainers for single-molecule measurements. Cell-derived vesicles maintain the structural integrity of transmembrane receptors by keeping them in their physiological membrane. It was demonstrated that receptors isolated in these vesicles can be studied with solution-based fluorescence correlation spectroscopy (FCS) and can be isolated on a solid substrate for single-molecule studies. This technique was applied to determine the stoichiometry of α3β4 nicotinic receptors. The method provides the capability to extend single-molecule studies to previously inaccessible classes of receptors.

The Nicotine Metabolite, Cotinine, Alters the Assembly and Trafficking of a Subset of Nicotinic Acetylcholine Receptors

“…3 -Because nAChRs are pentameric, ␣4␤2 can assemble with either an (␣4) 3 (␤2) 2 or an (␣4) 2 (␤2) 3 stoichiometry. We used a technique that our laboratory recently developed to perform single molecule analysis of subunit stoichiometry by spatially isolating nAChRs embedded in cell membrane-derived vesicles (42). We generated vesicles from cells expressing ␣4-gfp and ␤2-wt subunits.…”

“…Control experiments using monomeric receptors showed that there was a low probability of having more than one receptor per isolated vesicle. Of the vesicles generated via nitrogen cavitation, ϳ15% contain a receptor (42). Each field of view was imaged for 500 frames at a frame rate of 200 ms while undergoing continuous 488 nm excitation with an intensity of 60 watt/cm 2 .…”

“…Time traces of 4 ϫ 4-pixel regions of interest were generated using ImageJ with the Time Trace Analyzer V3.0 plugin. Photobleaching steps were determined manually as described previously (42)(43)(44). Previously, a discrete bleaching step was confirmed to correspond to one subunit (42,43).…”

“…Photobleaching steps were determined manually as described previously (42)(43)(44). Previously, a discrete bleaching step was confirmed to correspond to one subunit (42,43). Table 1 includes the number of accepted and rejected spots as well as the complete raw observed distribution of stoichiometry for each condition.…”

“…Time traces showing continuous decrease in fluorescence intensity or indistinct number of bleaching steps were rejected. GFP fails to mature a small percentage of the time, leading to some subunits not exhibiting typical fluorescence, which would result in the undercounting of bleaching events (42,44). To account for this, the probability of observing one, two, or three photobleaching steps in the time traces for each stoichiometry was calculated using a custom MATLAB script by fitting data to a binomial distribution, using the probability of GFP fluorescing equal to 0.90 (44).…”