ABSTRACT
Title
Single-molecule Analysis of G Protein-Coupled Receptors Localization and Clustering on the Cell Membrane using the Photo-activated Localization Microscopy (PALM)
Authors
M. Scarselli, P. Annibale and A. Radenovic
Laboratory of Nanoscale Biology, Swiss Federal Institute of Technology (EPFL), Lausanne
Laboratory of Nanoscale Biology, Swiss Federal Institute of Technology (EPFL), Lausanne
Abstract
The established model of G protein-coupled receptor (GPCR) signaling is based on a tight and regulated interaction with its partners, particularly with the heterotrimeric G protein. Besides, some evidences propose that cell membrane proteins could be organized in signaling platform as clusters or domains to maintain the correct fidelity and efficacy in the transduction of the signal. Conventional fluorescence microscopy application is precluded by its diffraction limited resolution of about 200nm when the cluster size is supposed to be below this resolution. To overcome such limitations, we decided to apply the novel super-resolution microscope technique PALM (Photo-Activated Localization Microscopy) that improves the resolution of 10-20 times with respect to conventional fluorescence microscopy and it is able to determine the localization of single molecules with a precision of 10-20nm.
For GPCRs, using atomic force microcopy, previous studies revealed that rhodopsin receptors assembly in defined clusters. However, for the other GPCRs, the formation of clusters is still controversial. If, on one hand, endogenous Beta2 adrenergic receptors were imaged as clusters in cardiac myocytes using near field scanning optical microscopy (NSOM), on the other hand, M1 muscarinic receptors seen by total internal reflection fluorescence (TIRF) looked randomly distributed on CHO cells. Here, using the new super-resolution technique PALM, we analyzed the localization and distribution of two prototypical class I GPCRs, the Beta2 adrenergic and M3 muscarinic receptors on the cell membrane. We investigated the assembly in clusters (size and density) of these membrane proteins, and the effect of agonists upon these domains. We found that the Beta2 adrenergic receptor is partially preassociated in nanoscale-sized clusters but only in specific cell types. The application of this powerful microscopy opens up the possibility to investigate and quantify the number of molecules in biological assemblies and to determine the protein stochiometry in signaling complexes.
For GPCRs, using atomic force microcopy, previous studies revealed that rhodopsin receptors assembly in defined clusters. However, for the other GPCRs, the formation of clusters is still controversial. If, on one hand, endogenous Beta2 adrenergic receptors were imaged as clusters in cardiac myocytes using near field scanning optical microscopy (NSOM), on the other hand, M1 muscarinic receptors seen by total internal reflection fluorescence (TIRF) looked randomly distributed on CHO cells. Here, using the new super-resolution technique PALM, we analyzed the localization and distribution of two prototypical class I GPCRs, the Beta2 adrenergic and M3 muscarinic receptors on the cell membrane. We investigated the assembly in clusters (size and density) of these membrane proteins, and the effect of agonists upon these domains. We found that the Beta2 adrenergic receptor is partially preassociated in nanoscale-sized clusters but only in specific cell types. The application of this powerful microscopy opens up the possibility to investigate and quantify the number of molecules in biological assemblies and to determine the protein stochiometry in signaling complexes.