title: CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation.
abstract: Toll-like receptor 4 (TLR4) is responsible for the immediate response to Gram-negative bacteria and signals via two main pathways by recruitment of distinct pairs of adaptor proteins. Mal-MyD88 [Mal (MyD88-adaptor-like) - MYD88 (Myeloid differentiation primary response gene (88))] is recruited to the plasma membrane to initiate the signaling cascade leading to production of pro-inflammatory cytokines while TRAM-TRIF [TRAM (TRIF-related adaptor molecule)-TRIF (TIR-domain-containing adapter-inducing interferon-β)] is recruited to early endosomes to initiate the subsequent production of type I interferons. We have investigated the dynamics of TLR4 and TRAM during lipopolysaccharide (LPS) stimulation. We found that LPS induced a CD14-dependent immobile fraction of TLR4 in the plasma membrane. Total internal reflection fluorescence microscopy (TIRF) revealed that LPS stimulation induced clustering of TLR4 into small punctate structures in the plasma membrane containing CD14/LPS and clathrin, both in HEK293 cells and the macrophage model cell line U373-CD14. These results suggest that laterally immobilized TLR4 receptor complexes are being formed and prepared for endocytosis. RAB11A was found to be involved in localizing TRAM to the endocytic recycling compartment (ERC) and to early sorting endosomes. Moreover, CD14/LPS but not TRAM was immobilized on RAB11A-positive endosomes, which indicates that TRAM and CD14/LPS can independently be recruited to endosomes.
authors.affiliation: Department of Cancer Research and Molecular Medicine, Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Dionne C G
authors.initials: DC
authors.lastname: Klein
authors.affiliation: Department of Cancer Research and Molecular Medicine, Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Astrid
authors.initials: A
authors.lastname: Skjesol
authors.affiliation: Graduate School of Life Sciences, University of Utrecht, Utrecht, The Netherlands.|Present address: Radboud university medical center, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands.
authors.forename: Esther D
authors.initials: ED
authors.lastname: Kers-Rebel
authors.affiliation: Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Sherstova
authors.affiliation: Department of Cancer Research and Molecular Medicine, Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Bjørnar
authors.initials: B
authors.lastname: Sporsheim
authors.affiliation: Department of Cancer Research and Molecular Medicine, Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Kjartan W
authors.initials: KW
authors.lastname: Egeberg
authors.affiliation: Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Bjørn T
authors.initials: BT
authors.lastname: Stokke
authors.affiliation: Department of Cancer Research and Molecular Medicine, Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Terje
authors.initials: T
authors.lastname: Espevik
authors.affiliation: Department of Cancer Research and Molecular Medicine, Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Harald
authors.initials: H
authors.lastname: Husebye
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D021381:Protein Transport; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002966:Clathrin; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051197:Toll-Like Receptor 4; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C480228:TICAM2 protein, human; C495345:TLR4 protein, human
keywords: CD14; FRAP; MyD88; TIRF microscopy; TLR4; TRAM; confocal microscopy; diffusion coefficient; half-life; mobile fraction