Immunology at a Nanoscopic View: A Single-Molecule Approach

Current scientific research throughout the natural sciences aims at the exploration of the Nanocosm, the collectivity of structures with dimensions between 1 and 100 nm. There is a strong demand for technologies offering access to these dimensions, for structuring, manipulating, or measuring at high resolution. In the life sciences, the diversity of this Nanocosm attracts more and more researchers and investors to the emerging field of Nanobiotechnology - biotechnology at or for the nanoscopic dimension. This proposal aims at establishing single molecule microscopy as a novel tool to study nanostructures and molecular dynamics in living biological cells. The method is in general applicable to a manifold of different biological systems; here, I selected a hot topic in immunology - the formation of the immunological synapse in early T cell signaling. The proper formation of this stable contact zone between antigen presenting cell and T cell represents one crucial step in the activation of our immune response. T cell activation critically depends on the prolonged and orchestrated interaction of ligands and receptors on the two opposing cells. Currently, however, researchers have to rely on kinetical parameters obtained in highly artificial experiments, using purified substances. In this project, we will use the novel methodology of single molecule microscopy to measure interactions between two different proteins directly in the cellular context. Moreover, we will explore the substructure of the developing synapse at a dimension of a few tens of nanometers, using the excellent localization precision of the technique. Thereby we hope to unravel the role of lipid domains in the activation of T cells. So far, there are a lot of indications for the relevance of such nanoscopic lipid platforms during T cell activation, however, due to their small size of a few tens of nanometers scientists so far failed to directly observe them. It is interesting that only a few antigen molecules are required to induce the formation of an immunological synapse; the extreme sensitivity of T cell activation makes ultra-sensitive detection schemes the proper choice for unraveling the accompanying molecular rearrangements.

Behind the title Immunology at a nanoscopic view stood a wordplay: I aimed at replacing classical microscopy which was limited by diffraction to dimensions above ~1m by a method, which allowed for obtaining information on the nanometer scale via the imaging of single molecules. In the meantime, the term nanoscopy has been accepted by the scientific community, and optical imaging at the nanometer length scale has become state of the art, to which we contributed with this project.Besides developing new microscopy methodologies, the central aim of this project was their application to problems in immunology, particularly the antigen-specific activation of CD4-positive T cells. It is mediated by the binding of the T cell receptor to antigenic peptide presented via MHCII on an opposing antigen-presenting cell (APC). Together with our partners, we could replace this cell-to-cell interface by an artificial system, in which the APC was replaced by a supported lipid bilayer, which was functionalized with the activating proteins in particular the peptide loaded MHCII. Dye molecules attached to the proteins of interest allowed us to specifically watch the T cell receptor during the recognition of the antigenic peptide. In this experiment we found that the interaction lifetimes were substantially shortened compared to previous in vitro experiments; we could show that the cell exerts a force to the bond, which accelerates the unbinding process. In addition to this central result we could gain numerous additional insights into the structure of cell membranes at the nanometer length scale. Previous experiments indicated that proteins and lipids do not diffuse freely in the plasma membrane, but experience periodic hindrances due to the underlying membrane skeleton. This question is biophysically highly interesting, as it involves the coupling between the two bilayer leaflets. We could clarify it using microscopy at extremely high spatiotemporal resolution: by tracking single lipid-anchored proteins at a time resolution below 1ms we did not find any indication for periodic hindrances. Subsequent measurement by others confirmed our results. Moreover, we were interested whether lipid-mediated interactions of proteins exist in the cell membrane. Such interactions were postulated based on biochemical data as the raft hypothesis, however, raft-like structures have never been directly observed. It was suspected that they were either too small, too short-lived, or simply not existing. We developed a novel microscopy modality based on single molecule observations, which was especially suited to tackle this question. We could show that small lipid-platforms with an upper size limit of ~20nm do exist in the cell membrane. We further used this method for quantifying subunit stoichiometries of other protein complexes. In summary, the START project enabled us to develop a tailored set of single molecule methods, and their application to study central processes on the plasma membrane.