Live-cell single-molecule analysis of β₂-adrenergic receptor diffusion dynamics and confinement

Abstract

Abbreviations: single particle tracking (SPT), βarrestin (β-arr), β 2 -adrenergic receptor (β 2 AR) AbstractSignal transduction mechanisms and successive regulatory processes alter the lateral mobility of β 2adrenergic receptors (β 2 AR). In this work we combined modern single particle tracking methods in order to analyze the diffusion dynamics of SNAPtagged β 2 AR in HEK wild-type cells and HEK βarrestin knockout cells before and after agonist stimulation. For analysis of trajectories we first used mean squared displacement (MSD) analysis. Secondly, we applied an advanced variational Bayesian treatment of hidden Markov models (vbSPT) in combination with the recently introduced packing coefficient (Pc), which together provided a detailed model of three discrete diffusive states, state transitioning and spatial confinement. Interesting to note, state switching between S3 (fast-diffusing) and S1 (slow-diffusing) occurred sequentially over an intermediate state S2. After ligand stimulation more SNAP-tagged β 2 AR in HEK wild-type cells switched occupancy into the slow-diffusing state, whereas less receptors were found in the fast diffusive state. Unexpectedly, all three states showed a fraction of confined receptor mobility that increased under stimulation, but confinement sizes were unaffected. Receptor diffusion characteristics were comparable in HEK β-arrestin knockout cells under basal conditions and only minor but non-significant changes occurred upon stimulation, as expected from the depletion of β-arrestin, an important regulatory protein. The data presented here on the occurrence of different diffusion states, their transitioning and variable spatial confinements clearly indicate that lateral mobility of β 2 AR is much more complex than previously thought.

Stoichiometric analysis of protein complexes by cell fusion and single molecule imaging

“…mNG-ADRβ2 expressing cells were co-plated with VSVG-expressing neighbors to dilute membrane receptors from the initial high expression levels. After cell fusion and incubation at 37 °C for 1-h, individual receptor complexes were clearly discernible and mobile within the plasma membrane, displaying similar kinetics to measurements made in living cells 22 (Fig. 1b, SI Appendix Video 1).…”