Dissecting diffusion and oligomerization dynamics of GPCRs in live cells using single-particle tracking.

Abstract

Crucial insights about the signaling and trafficking mechanisms of G Protein Coupled Receptors (GPCRs) can be derived from their membrane diffusion properties. Previous studies in our lab (Shivnaraine et al., JACS 2016, Li at al., Biophys. J. 2018) have shown that the M2 muscarinic receptor (M2R) can be purified as oligomers, yet the oligomerization dynamics and the implications for signalling in live cells remain elusive. Here we used single-molecule fluorescence techniques, such as single-particle tracking (SPT) and single-molecule photobleaching (smPB), to characterize the dynamic distribution of M2R in HEK293 cells. To that end, receptors have been co-expressed at their N-terminus either with a green fluorescent protein (eGFP), or with a HaloTag for labelling with HaloTag ligand (HTL) dyes, such as JF549 HTL. For comparison, we applied the same techniques to μ-opioid receptors (MORs) in live U2OS cells. For both cases, measured diffusion maps are spatially and temporally heterogeneous, with receptors transitioning between normal and anomalous diffusion regimes. The analysis of experimental data was reinforced by simulations and by control measurements on monomeric (CD86) and dimeric (CD28) membrane proteins. Intensity traces of immobile, single receptor complexes and of monomeric/dimeric controls in the membrane of fixed cells were analyzed using an in-house smPB code based on a Bayesian algorithm (Garry et al., J. Chem. Phys. 2020). The results confirm the presence, correlated with expression levels, of higher order M2R oligomers in cells, and lay out the foundation for subsequent studies of their functional role.