Modular transient nanoclustering of activated β2-adrenergic receptors revealed by single-molecule tracking of conformation-specific nanobodies
Rachel S. Gormal, Pranesh Padmanabhan, Ravikiran Kasula, Adekunle T. Bademosi, Sean Coakley, Jean Giacomotto, Ailisa Blum, Merja Joensuu, Tristan P. Wallis, Harriet P. Lo, Srikanth Budnar, James Rae, Charles Ferguson, Michele Bastiani, Walter G. Thomas, Els Pardon, Jan Steyaert, Alpha S. Yap, Geoffrey J. Goodhill, Massimo A. Hilliard, Robert G. Parton, Frédéric A. Meunier
Abstract
Significance Proteins moving freely on the plasma membrane can become transiently trapped in functionally essential clusters. This capability is likely to be influenced by subtle conformational states of the protein promoting or preventing such confinement. The downside of conventional imaging of overexpressed tagged proteins is that it precludes selective tracking of inherently minor albeit functionally essential conformer populations. Intracellular expression of single-chain nanobodies allowed us to track endogenous proteins in highly specific conformational states in live cells and small organisms. We unveiled the full scope of nanoclustering behavior of β 2 -adrenergic receptors in various conformations, along with their transient nature. This technique is broadly applicable to other proteins and will help unravel essential dynamics and organization of nanoclusters.