Single-particle tracking (SPT)
In two recent studies, the authors tracked fluorophore-labelled molecules of the M1 muscarinic receptor and the N-formyl peptide receptor, and concluded that both receptors exist as a 70:30 mixture of monomers and dimers in live cells. We used a similar single-particle tracking approach to measure the oligomeric size of the M2 muscarinic receptor, which has been reported to exist as a tetramer. The integral membrane proteins CD86 and CD28 were used as monomeric and dimeric controls, respectively, and each construct was fused with a SNAP tag at its N– terminus and was labelled with SNAP-Surface 488. Chinese hamster ovary cells expressing each construct were imaged on a total internal reflection fluorescence (TIRF) microscope to obtain time-lapse movies of labelled proteins moving along the cell membrane. Those movies were analysed using publicly available software to obtain a list of intensities and trajectories for all molecules in the movie. Three different algorithms were used to detect and measure the intensities of single molecules, but in each case the resulting distributions of intensities showed single asymmetric peaks that could be fit well by a log-logistic distribution. The main factors determining the mean and standard deviation of the distributions were the level of expression and the average intensity of the images, and cells expressing monomeric CD86, dimeric CD28 and the M2 receptor at a comparable level showed single-molecule intensity distributions that were nearly the same. These and other results illustrate the difficulty in using single-particle tracking to measure the oligomeric size of transmembrane proteins.