| First Author | Pannek A | Year | 2022 |
| Journal | Mol Biol Cell | Volume | 33 |
| Issue | 1 | Pages | ar6 |
| PubMed ID | 34731029 | Mgi Jnum | J:332880 |
| Mgi Id | MGI:7431012 | Doi | 10.1091/mbc.E21-02-0061 |
| Citation | Pannek A, et al. (2022) Dynamics of intracellular neonatal Fc receptor-ligand interactions in primary macrophages using biophysical fluorescence techniques. Mol Biol Cell 33(1):ar6 |
| abstractText | 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 Forster 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. |
