Live Cell Superresolution Imaging of Protein Conformation

T cells are control centers of our immune system in the recognition of antigens. The key molecule in this process is the T cell receptor, a complex consisting of several proteins that move laterally in the plasma membrane of the T cell. If this receptor binds an antigen molecule at the surface of a so- called antigen-presenting cell, a signaling cascade is induced in the T cell. What is amazing about this process is the combination of sensitivity and specificity: a T cell recognizes a few molecules of the antigen on a background of thousands of other antigen molecules that do not elicit a response. While many of the proteins involved in T cell antigen recognition are known, their role as well as the mechanism of the recognition process is still unclear. For example, it is not known how the extracellular binding of an antigen leads to a specific intracellular signal response. The protein CD3- zeta, which is constitutively associated with the T cell receptor, is important here. This protein has comparatively long, disordered chains that can interact loosely with the inside of the plasma membrane. The basic hypothesis of our project is that the membrane association of CD3-zeta chains is functionally important for signal transduction. To check this, we would like to use high-resolution microscopy to measure for the first time the position of the CD3-zeta chains directly in the T cell. To this end, we will advance a measurement method that has been causing a revolution in the life sciences in the last years: using single-molecular localization microscopy, protein distributions can be determined far below the classic optical resolution limit. This method works great when it comes to studying the lateral distribution of proteins; however, along the optical axis of the microscope, the accuracy is significantly reduced. In our project, we are trying to improve the positional accuracy by observing two focal planes simultaneously so that the distance of the CD3-zeta chains from the plasma membrane can be determined with an accuracy of about 10 nanometers. In addition to being important for our understanding of T cell activation, this method should enable for the first time direct microscopic detection of protein conformational changes in the cell.