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

Abstract

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 a FcRn-dependent, recycling pathway from macropinosomes in macrophages (Toh et al, 2019), 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-labelled human serum albumin (HSA-AF488) in BMDMs, and Raster Image Correlation Spectroscopy (RICS) analysis of single fluorescent-labelled albumin molecules to monitor the diffusion kinetics of internalised 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 non-binding 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.

The endosomal system of primary human vascular endothelial cells and albumin–FcRn trafficking

“…The acidification of early macropinosomes in Dictyostelium is mediated by WASP regulation of V-ATPase ( Buckley et al, 2016 ) and it is possible that a similar process might occur in mammalian endothelial cells. The emergence of HSA–AF488- and FcRn–mCherry-double-positive tubules was observed after 20–30 min uptake in live BOECs, a period during which acidification had occurred, which would allow for interaction of HSA and FcRn, as we previously demonstrated in BMDMs using biophysical techniques ( Pannek et al, 2022 ). Newly formed macropinosomes then undergo extensive tubulation, which pinches off to form transport carriers that are transported to the periphery of the cell.…”

“…Moreover, FcRn–mCherry showed a similar intracellular distribution as that of endogenous FcRn in BOECs, indicating that the intracellular location was compatible with that of the untagged receptor. Moreover, our previous studies showed that the FcRn–mCherry fusion protein was functionally active in BMDMs ( Pannek et al, 2022 ; Toh et al, 2019 ), indicating that the fluorescently tagged FcRn is correctly located and functional.…”

“…Transient transfection using Lipofectamine 3000 gave only a low level of transfection; hence, lentiviral transduction of BOEC was used. The open reading frames encoding β2 microglobulin and FcRn–mCherry from an internal ribosome entry site (IRES)-containing bicistronic vector ( Pannek et al, 2022 ) were introduced into the plasmid backbone pFUGW under the control of the constitutive UbC promoter, which drives low ectopic gene expression in most mammalian cells ( Qin et al, 2010 ). Lentiviral particles containing the pFUGW-B2M-FcRn-mCherry plasmid were generated and BOECs were transduced with recombinant lentivirus.…”

“…Of the immune cells, macrophages are considered the most important for FcRn-mediated ligand recycling ( Akilesh et al, 2007 ; Montoyo et al, 2009 ; Challa et al, 2019 ). Our previous studies analysed the internalisation and trafficking of human serum albumin (HSA) in genetically modified mouse bone marrow-derived macrophages (BMDMs) that express human FcRn rather than mouse FcRn ( Pannek et al, 2022 ; Toh et al, 2019 ). We showed that primary macrophages internalise HSA by macropinocytosis, a pathway for the uptake of large volumes of extracellular fluid ( Toh et al, 2019 ).…”

“…We showed that primary macrophages internalise HSA by macropinocytosis, a pathway for the uptake of large volumes of extracellular fluid ( Toh et al, 2019 ). Following endocytosis and interaction with FcRn, albumin was rapidly recycled in tubular transport carriers derived from newly formed macropinosomes ( Pannek et al, 2022 ; Toh et al, 2019 ).…”