Cell-to-cell variability in the diffusion constants of the plasma membrane proteins CD59 and CD147

In recent years increasing evidence has been reported for the inherent heterogeneity of cell populations. Cell-to-cell variability was particularly found when analyzing protein expression patterns or the responses of cells to different stimuli. Yet, structural features - in particular below the resolution limit of light microscopy - have so far eluded in-depth statistical analysis. We report here for the first time a detailed investigation of the variations in protein mobility between nominally identical cells. Our reasoning was that mobility probes nanometer-sized properties of the moving protein and its local environment, which may be subject to cell-to-cell variability. Single-molecule tracking was employed to characterize the diffusion constant of the glycosylphosphatidylinositol- (GPI-) anchored protein CD59 and the transmembrane protein CD147 in the plasma membrane of T24 cells. Automated and tailored data analysis routines allowed for the analysis of the required large data sets: ∼200000 trajectories obtained on ∼350 cells were analyzed in total. We found up to five-fold higher variability of the diffusion constant between cells compared to the uncertainty for the determination of the diffusion constant on a single cell.