Nanoscale Imaging of T-Cell Membrane Proteins
The plasma membrane has a specific protein composition that plays pivotal roles in signaling transduction. In this talk, I will present two super-resolution imaging techniques that enable direct visualization of the spatial and temporal organization of membrane proteins in T cell lymphocytes. In the first part of the talk, I will demonstrate the development of prism-coupled light-sheet microscopy. Through a prism-coupled mechanism, light-sheet microscopy delivers sub-cellular thin-plane optical illumination at various sample depths. Combined with direct stochastic optical reconstruction microscopy, the light-sheet system revealed pre-existing nanoscale organizations of T-cell receptors (TCRs) and their rearrangements into microclusters in mouse lymph nodes after antigenic challenge. This demonstration is an important step toward studying many biological systems in which the cellular architecture controls their physiological functions at the nanometer scale. In the second part of my talk, I will discuss a newly developed technique termed antibody fragments for point accumulation in imaging nanoscale topology (Fab-PAINT). Fab-PAINT utilizes transient binding of antibody fragments to break the “labeling barrier” for low-copy-number proteins and circumvent the clustering artifact in single-molecule localization microscopy. We employed optimized Fab-PAINT to visualize the organization of TCR and linker for activation of T cell (LAT) molecules on the plasma membrane of activated T cells. Integrating dual-color Fab-PAINT with single molecule tracking, we uncovered dynamic translocation of a proximal kinase between the upstream TCR and downstream LAT molecules during T cell activation. Taken together, novel nanoscale imaging tools present new opportunities for mechanistic studies of membrane-associated signaling pathways.