CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation
Dionne C G Klein1, Astrid Skjesol1, Esther D Kers-Rebel2,3
1Department of Cancer Research and Molecular Medicine, Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway.
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.
Insights
Toll-like receptor 4 (TLR4) forms immobile complexes with CD14/LPS upon stimulation, preparing for endocytosis. TRAM is localized to endosomes independently of CD14/LPS, suggesting distinct recruitment pathways.
Area of Science:
- Immunology
- Cell Biology
- Microbiology
Background:
- Toll-like receptor 4 (TLR4) initiates innate immune responses to Gram-negative bacteria.
- TLR4 signaling involves two main pathways: Mal-MyD88 for pro-inflammatory cytokines and TRAM-TRIF for type I interferons.
- Understanding TLR4 and adaptor protein dynamics during lipopolysaccharide (LPS) stimulation is crucial for deciphering immune signaling.
Purpose of the Study:
- To investigate the dynamic behavior of TLR4 and TRAM during LPS stimulation.
- To elucidate the mechanisms of TLR4 and TRAM recruitment to cellular compartments.
- To understand the spatial and temporal regulation of TLR4-mediated immune signaling.
Main Methods:
- Utilized Total Internal Reflection Fluorescence (TIRF) microscopy to observe TLR4 and TRAM dynamics.
- Employed cell lines HEK293 and U373-CD14, a macrophage model.
- Investigated the roles of CD14, clathrin, and RAB11A in receptor complex formation and trafficking.
Main Results:
- LPS stimulation induced a CD14-dependent immobile fraction of TLR4 in the plasma membrane.
- TLR4 clustered into punctate structures with CD14/LPS and clathrin, indicating preparation for endocytosis.
- RAB11A mediated TRAM localization to endosomes, but CD14/LPS and TRAM were recruited independently.
Conclusions:
- TLR4 receptor complexes become laterally immobilized and prepared for endocytosis upon LPS stimulation.
- TRAM and CD14/LPS can be recruited to endosomes through distinct mechanisms.
- These findings provide insights into the spatiotemporal regulation of TLR4 signaling pathways.
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