Dynamics of toll-like receptor 4 and its adaptor protein TRAM

The pattern-recognition receptor Toll-like receptor 4 (TLR4) is crucial in the defence against gram-negative bacteria. Upon challenge with lipopolysaccharide (LPS), TLR4 initiates one signaling pathway from the plasma membrane and another pathway from endosomes. By photobleaching a small region (circular spot, diameter 5 µm) in the plasma membrane or perinuclear region in HEK293 cells expressing TLR4, we were able to address the mobility of TLR4 and how it is affected by its adaptor proteins and by stimulation with LPS. Our FRAP results indicate that stimulation with LPS decreased the mobile fraction of TLR4 in HEK cells, both on the plasma membrane and on intracellular compartments. We found that this effect of LPS is smaller on TRAM mobility in the plasma membrane, while this effect was larger for TRAM mobility in the endocytic recycling compartment (ERC). Cytochalasin D treatment resulted in an increase in the mobility (reduction of the halftime) of TLR4 in the plasma membrane. Conclusion: We observed that the mobile fraction of TLR4 and also of its adaptor TRAM both in the plasma membrane and in the ERC decreased upon stimulation with LPS. This result indicates a restriction in the lateral movement of the receptor and its adaptor. Actin polymerization inhibitor Cytochalasin D decreased the halftime of TLR4 recovery in the plasma membrane significantly, suggesting that the actin network has an important role in the mobility of TLR4.