Characterising C-X-C Chemokine Receptor 4 Dynamics in the Cell Membrane Using Fluorescence Fluctuation Spectroscopy.

The spatial organisation of plasma membrane proteins such as G protein-coupled receptors (GPCRs) plays a critical role in regulating cell signalling, function, and ultimately cell fate. Resolving this organisation requires techniques capable of probing dynamics at the single-molecule level with high spatial and temporal resolution. In this study, we employ the complementary fluorescence fluctuation spectroscopy approaches, Fluorescence Correlation Spectroscopy (FCS), Photon Counting Histogram Analysis (PCH), Raster Image Correlation Spectroscopy (RICS) and Number and Brightness Analysis (N&B), in conjunction with Fluorescence Recovery After Photobleaching (FRAP), to investigate the membrane organisation of the C-X-C chemokine receptor 4 (CXCR4), a GPCR known to undergo ligand-induced reorganisation. At the nanoscale, FCS highlighted opposing effects on diffusion after agonist (CXCL12) and inverse agonist (IT1t) treatment, whilst RICS also showed ligand-mediated changes in particle number. Both single-point and image-based brightness analyses (PCH and N&B) showed increased brightness after CXCL12 treatment, consistent with the pre-internalisation clustering of CXCR4. At the microscale, FRAP showed an increase in immobile CXCR4, not visible to FFS approaches, following CXCL12 stimulation. This integrated approach, performed on a single commercial confocal microscope, provides valuable insight into the reorganisation of CXCR4 in the plasma membrane over a range of temporal and spatial scales, which are not detectable using standard imaging.