Spatial Intensity Distribution Analysis: Studies of G Protein-Coupled Receptor Oligomerisation

Abstract

Spatial intensity distribution analysis (SpIDA) is a recently developed approach for determining quaternary structure information on fluorophore-labelled proteins of interest in situ. It can be applied to live or fixed cells and native tissue. Using confocal images, SpIDA generates fluorescence intensity histograms that are analysed by super-Poissonian distribution functions to obtain density and quantal brightness values of the fluorophore-labelled protein of interest. This allows both expression level and oligomerisation state of the protein to be determined. We describe the application of SpIDA to investigate the oligomeric state of G protein-coupled receptors (GPCRs) at steady state and following cellular challenge, and consider how SpIDA may be used to explore GPCR quaternary organisation in pathophysiology and to stratify medicines.

Chemokine Receptor Crystal Structures: What Can Be Learned from Them?

“…Chemokine receptor oligomers cause negative cooperativity in the binding of their chemokine ligands and, in the case of some receptors, oligomerization can either enhance or inhibit receptor activation through allosteric communication (Percherancier et al, 2005;Stephens and Handel, 2013;Armando et al, 2014). The current methodologies that are able to probe dimerization are mainly based on fluorescence and bioluminescence resonance energy transfer approaches (Percherancier et al, 2005;Goddard and Watts, 2012;Fumagalli et al, 2019;Heuninck et al, 2019), fluorescence fluctuation spectroscopy (Isbilir et al, 2017;Briddon et al, 2018), and spatial intensity distribution analysis (Pediani et al, 2018). Such biophysical approaches are very sensitive to detect oligomers but are not able to identify the oligomerization interface between protomers, which is key for molecular understanding of the quaternary GPCR structures.…”

Muscarinic receptor oligomerization

“…Recently, to gain further insights into the dimerization of GPCRs and potential effects of ligand binding Milligan and collaborators ( Ward et al., 2015 , Ward et al., 2017 , Pediani et al., 2016 , Marsango et al., 2017 ) have adopted a biophysical technique, Spatial Intensity Distribution analysis (SpIDA), developed by Wiseman and co-workers ( Godin et al., 2011 , Godin et al., 2015 , Barbeau et al., 2013 ). This allows the detection of protein-protein interactions with a spatial resolution of 220 nm; a limitation which is overcome by oversampling the laser spot confocal volume and quantifying the excitation illumination volume for membrane oligomerization measurements as a surface as opposed to a 3-dimensional volume ( Pediani et al., 2017 ) Briefly, SpIDA is based upon the analysis of regions of interest (RoIs) selected within laser scanning confocal images of cells expressing the protein of interest tagged with, for example, an appropriate monomeric fluorescent protein ( Godin et al., 2011 , Godin et al., 2015 , Barbeau et al., 2013 , Ward et al., 2015 , Ward et al., 2017 , Pediani et al., 2016 , Marsango et al., 2017 ). Images are then analysed by constructing fluorescence intensity histograms for the pixels within the RoI and then applying super Poissonian distribution curves.…”

A general method to quantify ligand-driven oligomerization from fluorescence-based images

“…This method nevertheless does not solve the "immobile-fraction" problem and does not resolve the brightness distribution into distinct oligomer sizes. A related method, Spatial Intensity Distribution Analysis (SpIDA) 14,15 , captures oligomer size-related fluctuations from the immobile fraction by converting the problem of sampling the fluorescence in time to sampling it in space, but it does so at the expense of further reduced oligomer-size resolution. In addition, inhomogeneously distributed molecular concentrations may inadvertently appear as oligomer size distributions in SpIDA.…”