Activation of Toll-like receptors of the immune system studied by atomic force microscopy and confocal microscopy

Toll-like receptors (TLRs) trigger a fast response of the immune system to microbial particles that invade our body. TLRs recognize microbial conserved structures such as CpG-DNA or certain lipopolysaccharides, and are essential for our defense against mi crobes as well as for normal inflammatory reactions. While much progress has been made during the last few years in understanding of TLR signaling, the exact mechanisms of interaction between TLR and various ligands and other interacting partners during activation has not been mapped yet. Recent developments in the field of ultramicroscopy make single-molecule imaging in liquid environment possible. In this project, we focus on activation of TLR4 and TLR9. TLR9 recognizes viral and bacterial CpG-DNA mo tifs. An important research question is if CpG-DNA induces clustering of TLR9. We will visualize receptor complexes and TLR9 distribution before CpG-DNA stimulation, and follow how this is influenced upon stimulation by CpG-DNA. Video-rate AFM enables us to visualize TLR9 cluster formation in membrane fragments with a speed of 10 frames per second, the estimated cluster formation rate, while resolving single receptor molecules. Using FRET, TLR9 cluster formation will be studied in live cells. Potent proin flammatory cellular responses to endotoxin are mediated through activation of the TLR4 receptor complex. We will follow the dynamics of the TLR4 receptor complex upon addition of LPS, in order to visualize cluster formation. By directly measuring the bind ing interaction between LPS and CD14-MD-2-TLR4, between LPS and CD14-TLR4 without MD-2 present, and between LPS and CD14 alone, the contribution of each of the proteins in the complex to the binding interaction will be determined. The direct binding betwe en smooth LPS and CD14, and between rough LPS and CD14 will be measured. Cluster formation of CD14-MD-2-TLR4 receptor complexes in live cells will be visualized with confocal microscopy.