Dynamics of intracellular neonatal Fc receptor–ligand interactions in primary macrophages using biophysical fluorescence techniques

The neonatal Fc receptor (FcRn) is responsible for the recycling of endocytosed albumin and IgG, and contributes to their long plasma half-life. We recently identified an FcRn-dependent recycling pathway from macropinosomes in macrophages; however, little is known about the dynamics of intracellular FcRn–ligand interactions to promote recycling. Here we demonstrate a multiplexed biophysical fluorescent microscopy approach to resolve the spatiotemporal dynamics of albumin–FcRn interactions in living bone marrow–derived macrophages (BMDMs). We used the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) to detect the interaction of a FcRn–mCherry fusion protein with endocytosed Alexa Fluor 488–labeled human serum albumin (HSA–AF488) in BMDMs, and raster image correlation spectroscopy (RICS) analysis of single fluorescent-labeled albumin molecules to monitor the diffusion kinetics of internalized albumin. Our data identified a major fraction of immobile HSA–AF488 molecules in endosomal structures of human FcRn-positive mouse macrophages and an increase in FLIM-FRET following endocytosis, including detection of FRET in tubular-like structures. A nonbinding mutant of albumin showed minimum FLIM-FRET and high mobility. These data reveal the kinetics of FcRn–ligand binding within endosomal structures for recruitment into transport carriers for recycling. These approaches have wide applicability for analyses of intracellular ligand–receptor interactions.