Optical sub-diffraction microscopy of the glycine receptor alpha3 in the plasma membrane

Abstract

In this work, localization based optical sub-diffraction microscopy is applied to measure the nanoscale membrane order of the GlyR α3 in HEK 293 cells. Both lateral membrane diffusion and spatial membrane distribution were measured to determine the effect of the α3K and α3L alternate splice variants. Diffusion measurements were performed by single particle tracking of fluorescently labeled GlyR α3K or α3L in the bottom cell membrane. Ensemble analysis of the trajectories revealed the presence of anomalous sub- and super-diffusion, which was confirmed by local trajectory analysis. Both splice variants showed anomalous sub-diffusion, identified as local receptor confinement in transient confinement zones. The α3L variant receptors also showed a small fraction of super-diffusion. This directed motion was interpreted as transient conveyor belt type motion and was often preceded or followed by confined motion. The spatial distribution of the individual GlyR α3 splice variants in the membrane was studied by using dSTORM to create super-resolution localization maps. Analysis of these localization maps using pair correlation analysis revealed clustering of both variants. The cluster radius was slightly elevated for the α3L variant, but α3L clusters also revealed a higher density of receptors due to an increased affinity for clustering. Furthermore, dual-color dSTORM and pair correlation analysis revealed that, when co-expressed, the splice variant clusters partially colocalize. The co-clusters are of similar size as individual α3L clusters. The aforementioned quantitative measures of diffusion and spatial distribution suggest RNA splicing determines GlyR α3 interaction efficiency in the membrane. More specifically, the measurement parameters suggest interaction with clathrin structures or membrane rafts. In the future, this work can be used as a foundation to uncover the actual molecular nature of GlyR α3 membrane interaction.