PMID- 34731029
OWN - NLM
STAT- MEDLINE
DCOM- 20220321
LR  - 20220321
IS  - 1939-4586 (Electronic)
IS  - 1059-1524 (Print)
IS  - 1059-1524 (Linking)
VI  - 33
IP  - 1
DP  - 2022 Jan 1
TI  - Dynamics of intracellular neonatal Fc receptor-ligand interactions in primary 
      macrophages using biophysical fluorescence techniques.
PG  - ar6
LID - 10.1091/mbc.E21-02-0061 [doi]
LID - ar6
AB  - 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.
FAU - Pannek, Andreas
AU  - Pannek A
AD  - Department of Biochemistry and Pharmacology and Bio21 Molecular Science and 
      Biotechnology Institute.
AD  - Institute of Experimental Immunology, University of Bonn, Venusberg Campus, 
      D-53127, Germany.
FAU - Houghton, Fiona J
AU  - Houghton FJ
AD  - Department of Biochemistry and Pharmacology and Bio21 Molecular Science and 
      Biotechnology Institute.
FAU - Verhagen, Anne M
AU  - Verhagen AM
AD  - CSL Limited, Research, Bio21 Molecular Science and Biotechnology Institute, 
      Victoria 3010, Australia.
FAU - Dower, Steven K
AU  - Dower SK
AD  - CSL Limited, Research, Bio21 Molecular Science and Biotechnology Institute, 
      Victoria 3010, Australia.
FAU - Hinde, Elizabeth
AU  - Hinde E
AD  - School of Physics and Bio21 Molecular Science and Biotechnology Institute, and.
AD  - Department of Biochemistry and Pharmacology, The University of Melbourne, 
      Victoria 3010, Australia.
FAU - Gleeson, Paul A
AU  - Gleeson PA
AD  - Department of Biochemistry and Pharmacology and Bio21 Molecular Science and 
      Biotechnology Institute.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20211103
PL  - United States
TA  - Mol Biol Cell
JT  - Molecular biology of the cell
JID - 9201390
RN  - 0 (Albumins)
RN  - 0 (Histocompatibility Antigens Class I)
RN  - 0 (Ligands)
RN  - 0 (Receptors, Fc)
RN  - TW3XAW0RCY (Fc receptor, neonatal)
SB  - IM
MH  - Albumins/metabolism
MH  - Animals
MH  - Endocytosis/physiology
MH  - Endosomes/metabolism
MH  - Female
MH  - Fluorescence
MH  - Fluorescence Resonance Energy Transfer/methods
MH  - Half-Life
MH  - HeLa Cells
MH  - Histocompatibility Antigens Class I/*metabolism/physiology
MH  - Humans
MH  - Kinetics
MH  - Ligands
MH  - Macrophages/*metabolism
MH  - Male
MH  - Mice
MH  - Mice, Knockout
MH  - Mice, Transgenic
MH  - Microscopy, Fluorescence/methods
MH  - Protein Binding
MH  - Receptors, Fc/*metabolism/physiology
PMC - PMC8886815
EDAT- 2021/11/04 06:00
MHDA- 2022/03/22 06:00
PMCR- 2022/03/16
CRDT- 2021/11/03 17:16
PHST- 2021/11/04 06:00 [pubmed]
PHST- 2022/03/22 06:00 [medline]
PHST- 2021/11/03 17:16 [entrez]
PHST- 2022/03/16 00:00 [pmc-release]
AID - E21-02-0061 [pii]
AID - 10.1091/mbc.E21-02-0061 [doi]
PST - ppublish
SO  - Mol Biol Cell. 2022 Jan 1;33(1):ar6. doi: 10.1091/mbc.E21-02-0061. Epub 2021 Nov 
      3.