Dynamics of intracellular neonatal Fc receptor-ligand interactions in primary macrophages using biophysical
Andreas Pannek1,2, Fiona J Houghton1, Anne M Verhagen3
1Department of Biochemistry and Pharmacology and Bio21 Molecular Science and Biotechnology Institute.
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 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.
Insights
The neonatal Fc receptor (FcRn) recycles albumin in macrophages. This study reveals FcRn-albumin binding dynamics within cells, crucial for understanding protein recycling and long half-lives.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- The neonatal Fc receptor (FcRn) is critical for maintaining the long plasma half-life of albumin and IgG by mediating their recycling.
- While an FcRn-dependent pathway from macropinosomes in macrophages is known, the intracellular dynamics of FcRn-ligand interactions remain poorly understood.
Purpose of the Study:
- To investigate the spatiotemporal dynamics of albumin-FcRn interactions within living macrophages.
- To elucidate the kinetics of FcRn-ligand binding in endosomal compartments to promote protein recycling.
Main Methods:
- Utilized a multiplexed biophysical fluorescent microscopy approach in bone marrow-derived macrophages (BMDMs).
- Employed phasor fluorescence lifetime imaging microscopy (FLIM) with Förster resonance energy transfer (FRET) to detect FcRn-albumin interactions.
- Applied raster image correlation spectroscopy (RICS) to monitor the diffusion kinetics of internalized albumin.
Main Results:
- Identified a significant population of immobile albumin molecules within endosomal structures in FcRn-positive macrophages.
- Observed an increase in FLIM-FRET signals post-endocytosis, indicating FcRn-albumin binding.
- Detected FRET signals in tubular-like structures and minimal binding with a nonbinding albumin mutant.
Conclusions:
- Revealed the kinetics of FcRn-ligand binding within endosomes, essential for recruiting cargo into recycling transport carriers.
- Demonstrated the utility of FLIM-FRET and RICS for analyzing intracellular ligand-receptor dynamics.
- Provided insights into the molecular mechanisms underlying albumin homeostasis and FcRn-mediated recycling.
