Enlightening G-protein-coupled receptors on the plasma membrane using super-resolution photoactivated localization microscopy

Abstract

The possibility to visualize and image the arrangement of proteins within the cell at the molecular level has always been an attraction for scientists in biological research. In particular, for signalling molecules such as GPCRs (G-protein-coupled receptors), the existence of protein aggregates such as oligomers or clusters has been the topic of extensive debate. One of the reasons for this lively argument is that the molecular size is below the diffraction-limited resolution of the conventional microscopy, precluding the direct visualization of protein super-structures. On the other hand, new super-resolution microscopy techniques, such as the PALM (photoactivated localization microscopy), allow the limit of the resolution power of conventional optics to be broken and the localization of single molecules to be determined with a precision of 10-20 nm, close to their molecular size. The application of super-resolution microscopy to study the spatial and temporal organization of GPCRs has brought new insights into receptor arrangement on the plasma membrane. Furthermore, the use of this powerful microscopy technique as a quantitative tool opens up the possibility for investigating and quantifying the number of molecules in biological assemblies and determining the protein stoichiometry in signalling complexes.

Cryogenic Colocalization Microscopy for Nanometer‐Distance Measurements

“…The aim of this work has been to demonstrate the feasibility of cryogenic colocalization for measuring separations of molecules in the range well below ten nanometers. This method is particularly promising for quantitative distance measurements in systems such as membrane receptor stoichiometry, [40][41][42][43] protein folding, [44,45] protein conformational dynamics using temperature cycling [46] or orientation determination of biomolecules. [47] Moreover, the techniques can be used to obtain information on the position of light emission in multichromophore systems such as light-harvesting complexes or j-aggregates.…”

Nanoscale elucidation of Na,K-ATPase isoforms in dendritic spines

“…An alternative technique utilizing exogenous expression of photoactivatable FPs is known as photoactivated localization microscopy (PALM). It is typically a slower imaging method, but offers high precision without the use of antibodies and provides opportunities for molecular quantification (Greenfield et al 2009, Scarselli et al 2013.…”

Super-resolution fluorescence imaging and correlation spectroscopy: Principles and examples of application

“…The effect of selective agonists, selective antagonists, and non-specific substances (such as lipids, ions and alcohols (ethanol in particular)) on GPCR surface density, oligomerization status, association with lipid rafts and cellular trafficking have also been studied in detail. 64,96 These studies suggested that β 2 -AR is partially pre-associated in nano-scale-sized clusters only in the cardiomyocyte H9C2 cells, but not in HeLa and CHO cells. 95 In comparison, the number of GPCR-dedicated PALM studies is still limited, with only two publications to date, in which the lateral organization of adrenergic receptor β 2 -AR was investigated in HeLa, CHO and cardiomyocyte H9C2 cells.…”