One of the most important functions of immune cell is to detect biochemical signatures of harmful organisms and muster the appropriate response. Lipopolysaccharides from the outer membrane of gram-negative bacteria is a complex mixture that can elicit specific signaling, beginning with the activation of toll-like receptors. While it is intuitive that this would be local to the insult, neither the level of spatial regulation nor the dynamics of these activation over time can be clearly resolved at present. To help us understand the location, transience, and specificity of lipopolysaccharide stimulated initial immune cascade, we will examine several protein engineering designs for novel toll-like receptor biosensors that can fluorescently visualize toll-like receptor activation in living single cells. We will employ FRET biosensing for the development of these biosensors. Successful biosensor candidates will be co-opted using FLINC and refSOFI to reveal receptor activation at super-resolution.
– Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution.
– RefSOFI for Mapping Nanoscale Organization of Protein-Protein Interactions in Living Cells.