G-protein coupled receptors (GPCRs) are the largest family of cell surface receptors in eukaryotic cells. These seven-transmembrane receptors have influence in physiological events such as cell to cell communication, immune responses, nerve transmission and even hunger and sleep regulation. The role of GPCRs in diseases such as rheumatoid arthritis, heart disease, cancer, obesity, and neurodegenerative disorders accentuates the need to investigate this family of receptors further. More than a third of all drugs approved by the FDA target GPCRs but often such drugs have a variety of poorly understood mechanisms, as a recent example surrounding opioid receptor agonists illustrates.
GPCRs have been shown to form dimers and/or oligomers, where the receptor is present in groups of two or more receptors of the same or different kind. There are several reports of oligomers affecting ligand-binding pharmacology, function, trafficking, and internalisation compared to their monomeric form
. Several experimental techniques, ranging from traditional biochemical approaches to biophysical ensemble methods based on resonance energy transfer (RET), such as fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET), have been key to observing the formation of dimers and oligomers
. However, one of the main issues with these techniques is that they cannot provide information on the size of the oligomeric complexes, nor on the location of the GPCRs in the cell. Over the years, different optical microscopy approaches, based on molecular brightness analysis, have been proposed to tackle some of these shortcomings
. However, they provide average oligomerization information, and it is not possible to extract the precise stoichiometry of specific molecular complexes. A suitable technique to provide information on the oligomeric state of GPCRs and their dynamics down to the single-molecule level is single-molecule tracking
. Yet, this typically requires receptor densities that are orders of magnitude lower than those occurring in natural settings. The relevance of this shortcoming is particularly important when investigating GPCR oligomerization in certain cancer cell lines, where the receptors are overexpressed.
Here, we present a novel single-molecule based approach to study the oligomerization of a rhodopsin-like GPCR, the purinergic receptor Y2 (P2Y2
), in a pancreatic cancer cell line with high expression of the receptor
. Our approach is based on the most recently developed single-molecule localization microscopy (SMLM) method named DNA-PAINT
(a variation of point accumulation for imaging in nanoscale topography) in combination with an imaging analysis pipeline suitable for protein quantification. DNA-PAINT is an SMLM technique that relies on the repetitive binding between two short complementary single-stranded DNAs, one conjugated to a fluorescent dye (imager strand) and the other chemically coupled to either a primary or secondary antibody targeting the protein of interest (docking strand). These short-lived transient events (millisecond to second range) create the necessary blinking required for SMLM, allowing the localization of the position of single molecules with nanometer precision
. Over the course of the experiment, these cumulative DNA binding events form a cluster of single molecule localizations within the true position of the biological target, as illustrated in Scheme 1
. Depending on the DNA pair’s binding kinetics in particular the association rate, kon
, and the imager DNA strand concentration, ci
, each docking strand is visited by an imager at a frequency given by (kon *ci
), which corresponds to the inverse of the dark time for a single docking strand (i.e., length of time that a docking strand is not bound to an imager strand between binding events)
. The frequency of the imagers binding to their docking strand scales linearly with the number of docking strands, and this is the principle of the quantitative analysis known as qPAINT (Scheme 1
). The predictable binding kinetics between imager and docking strands make qPAINT suitable to accurately correlate the frequency of single-molecule events with the underlying number of labelled molecular targets
, overcoming ‘overcounting’ artifacts observed with other SMLM techniques.
Scheme 1. Overview of qPAINT analysis pipeline (created with BioRender.com, BioRender, Toronto YTO, Canada).
To demonstrate the potential of qPAINT to study GPCRs oligomerization status, we investigated the nanoscale distribution of the P2Y2
receptor, a member of the d subgroup of the family A of GPCRs, in the cancer cell line AsPC-1, as it endogenously expresses high levels of this receptor and single cells are easily imaged due to its low levels of cell grouping
has been related to immune regulation, bone mineralisation, intraocular pressure, HIV-1 infection, and cancer metastasis and proliferation
has recently gained traction due to its role in several cancers such as breast, head and neck, prostate, and pancreatic 
. This receptor, which can interact with adenosine triphosphate (ATP) and uridine triphosphate (UTP), is known to homo-dimerise and homo-oligomerise
. When applied to P2Y2
, qPAINT revealed the molecular densities and nano- and micro-scale spatial arrangements of GPCRs down to the single molecule level, as well as their modulation in response to agonist and antagonist treatment. Understanding the natural oligomerisation state of P2Y2
and the effect of agonists and antagonists could inform the mechanist action of P2Y2
and aid in targeting it therapeutically.
2. Super-Resolution Imaging of P2Y2 Receptors in AsPC-1 Cells Using DNA-PAINT
To unravel the molecular organization of P2Y2
receptors in and near the plasma membrane of AsPC-1 cells, we used DNA-PAINT imaging under total internal reflection (TIR) excitation. Figure 1
a shows a representative super-resolution image of P2Y2
obtained via DNA-PAINT. TIR excitation allows investigation of samples at or near the cell membrane by optically sectioning light illumination to only the most superficial ~100 nm of the sample. This is extremely beneficial in the study of GPCRs located at the plasma membrane as the receptors are typically not only at the cell membrane, but also at intracellular sites such as endosomes, endoplasmic reticulum, and the Golgi complex
and TIRF imaging minimizes their intracellular visualisation.
Figure 1. qPAINT calibration to determine qi. (a) Representative rendered DNA-PAINT image of P2Y2 proteins in an AsPC-1 cell. Inset shows a schematic representation of DNA docking and imager strand sequences displaying the 3× repeat binding motif (created with BioRender.com). White box indicates ROI for subsequent cluster analysis. (b) DBSCAN cluster analysis output for ROI indicated in (a). Non-clustered points are shown in grey. Box represents clusters used for subsequent single molecule on-off time series. (c) Histogram of internal calibration qPAINT indexes per cluster pooled from all data samples fit with a multi-Gaussian function. (d) Histogram of calibration sample qPAINT indexes per cluster from DNA coupled anti P2Y2 antibody deposited on glass fit with a multi-Gaussian function.
receptors were labelled with a primary antibody which we validated using the immortalised human pancreatic stellate cell line PS-1 as it expresses traces amounts of the protein of interest
. For DNA-PAINT imaging, the anti-P2Y2
receptor antibody was chemically coupled to an optimised docking strand sequence design to increase imaging speed. The docking strand features a repetitive (ACC)n sequence motif that provides 3× overlapping binding sites for the imager strand (Figure 1
. The benefit of such docking sequence is not only the increase of imaging speed, but also the possibility to use relatively low imager strand concentrations achieving a high signal-to-noise ratio and single-molecule localisation precision. In our experiments we achieved an overall localisation precision of ~10 nm using a 1 nM concentration of imager strand fluorescently labelled with ATTO643, as determined by the nearest-neighbour-based analysis.
Over the years, multiple optical microscopy techniques have been applied to the study of GPCR oligomerization, with one of the first single molecule imaging studies done by Kasai et al.
. Subsequently, both Spatial Intensity Distribution Analysis (SpIDA)
and molecular brightness approaches
were also developed and applied to study a variety of GPCRs. In recent years, single-molecule tracking and FRET imaging have also been applied to identify key factors in the regulation of GPCRs dynamic interactions in living cells
. While these techniques have been paramount to investigate the oligomeric organisation of GPCRs and the dynamic interactions that control GPCRs signalling, many challenges still remain; including the limitation of applicability to endogenous settings (i.e., many of these methods require labelled receptors or fusion proteins) and the possibility of multi-colour imaging to simultaneously study different types of hetero-oligomers. Most recently, dSTORM (direct stochastic optical reconstruction microscopy), a single-molecule super-resolution microscopy method, was employed to investigate the nanoscale organisation of a GPCR in presynaptic active zones
, presenting a step-forward in visualising the organization of these receptors in endogenous conditions with nanoscale resolution. However, this was possible at the cost of increased experimental complexity, as the approach required transiently transfecting cells under controlled conditions to accurately quantify the number of receptors per cluster of single-molecule localizations.
As a novel method for quantitative analysis and nano-scale visualisation of the oligomeric state of GPCRs in single cells, here we employed a quantitative single molecule based super-resolution imaging technique based on DNA-PAINT, named qPAINT. Our approach allows the study of GPCR oligomerization in an endogenous setting, while avoiding the difficulties experienced with other SMLM techniques, such as dSTORM or PALM, to quantify the protein copy number
. During the last four years, examples of the use of qPAINT to quantify protein clustering in different biological systems have started to emerge
. This work is the first to employ qPAINT towards the study of GPCRs, a technique that can certainly help resolve contentious issues associated with GPCR oligomerization in different settings. Furthermore, due to the comparative advantages of DNA-PAINT over other SMLM techniques—it can routinely deliver lateral resolution in the 5–10 nm range and it allows multiplexing with a single laser source—it is straightforward to expand the applicability of qPAINT to detect and quantify GPCR hetero-oligomers. We note, however, that like any other technique that relies on antibody labelling, its applicability will depend on the availability of validated antibodies and that the final accuracy towards the absolute protein copy number is affected by the overall antibody-target binding efficiency, which can lead to potential undercounting. Still, by keeping data acquisition and analysis the same it is possible to draw conclusions about changes in the nanoscale distribution of the receptors in the different conditions. Furthermore, the presented approach is not suitable for live-cell imaging.
By leveraging the capabilities of qPAINT to its maximum potential, our study provides a detailed quantitative characterization of the density, spatial organization, and stoichiometry of the antibody labelled purinergic receptor Y2 oligomers in the pancreatic cancer cell line AsPC-1. Specifically, our data indicate that P2Y2
receptors are highly expressed in AsPC-1 cells with an average density of ca. 40 receptors per µm2
. We find that P2Y2
receptors are mainly organised in nanodomains containing three or more sub-units, with only 20% of the receptors forming dimers and 10% distributed as monomers. Our results also show that while the oligomerization status of P2Y2
receptors does not change upon agonist treatment, there is a marked reduction of the percentage of P2Y2
receptors forming oligomers in antagonistic conditions. Our results are in line with current models for the P2Y2
receptor, which imply that homo-oligomeric assemblies of P2Y2
receptors are required for receptor internalisation and the effect of the antagonist prevents the formation of these complexes. Similar antagonist effects have been observed for CXCR4 and dopamine D3 GPCR receptors
. The fact that we did not observe significant difference in the oligomerisation of P2Y2
receptors in control and agonistic conditions suggest that oligomeric assemblies could be part and parcel of the natural activation of the receptor.
In conclusion, we demonstrated the accuracy and ease of implementation of qPAINT to quantitatively characterise the oligomeric state of GPCRs, alongside achieving single receptor visualisation. qPAINT analysis of the oligomeric state of P2Y2
revealed consistency with currently proposed models for this receptor