Single Molecule Platform for Protein Interaction Analysis

The plasma membrane of T-cells contains a variety of protein complexes, which are important regulators of T-cell function. During activation, some complexes change their composition by fusing, segregating, or recruiting additional proteins. One prominent example is the T-cell receptor (TCR) complex, in which the T-cell receptor a- and ß-chain are stably linked to CD3, CD3d, CD3e, CD3, but also transiently associated with e.g. Lck, CD2, LAT, or ZAP-70. A second example is the coreceptor CD4, which recruits the kinase Lck to the TCR for tyrosine phosphorylation. In both examples, the stoichiometric composition of the complexes and the variability are only vaguely known. With current technologies it has been difficult to obtain quantitative information on the hetero-oligomeric nature of such complexes. In this project, we will directly approach this need by developing a microscopy-based platform, which allows for quantitative measurements of the composition of individual protein complexes in the cellular plasma membrane. The new method will combine state-of-the-art strategies to nanostructure surfaces and to image single molecules in cells. It is based on a bait-prey technique previously introduced by us for studying protein- protein interactions in the live cell plasma membrane via protein micropatterning. For this, a bait-specific ligand is immobilized on a glass surface in a characteristic micropattern. When cells are grown on such substrates, the bait located in the plasma membrane follows these patterns. Interaction with a fluorescent prey leads to the rearrangement of the prey within the same patterns. We propose now to extend such micropatterning platforms to the nanoscopic regime, and to combine them with single molecule microscopy. Our idea is to produce combined micro- and nanostructured surfaces of capture antibodies to the bait protein which is part of the complex to be investigated. Upon growing cells on such surfaces, the bait proteins and thereby the complexes will be immobilized along the nanopatterns. Combined micro- and nanopatterns will be designed to generate an analysis area, where the protein complexes can be immobilized to 50nm spots at mutual distances of 1m, which provides sufficient separation for single molecule imaging. Additional microscale bulk areas of high capture antibody density will be used to remove the majority of the bait excess from the analysis area. This will allow us to use single molecule microscopy in the analysis area to count the number of different protein molecules contained in each complex. One part of the project aims at the development of the new platforms. In addition, single molecule tools will provide the readout for the second part of the project, the application of the new platform to T-cell biology.

The cellular plasma membrane contains a variety of protein complexes, which are important regulators of cell function. During cell activation, some complexes change their composition by fusing, segregating, or recruiting additional proteins. In this project, we aimed at developing new innovative methods for quantitative analysis of molecular organization directly in the plasma membrane, without the need for biochemical purification steps. Our idea was to capture and immobilize membrane proteins in the plasma membrane by growing cells on micro- and nanostructured surfaces decorated with a specific ligand against a protein of interest. Using fluorescence microscopy, we next wanted to quantify the recruitment of other plasma membrane proteins and lipids. Our ultimate goal was to capture single molecules by this method. We first analyzed the role of lipids for the interaction of plasma membrane proteins. For a long time, it has been believed that lipids can form nanoscopic, highly dynamic entities in the cellular plasma membrane termed lipid rafts, which were thought to be instrumental for protein interactions. Using the micropatterning technique, however, we could rule out the presence of such rafts in the local environment of special lipid-anchored proteins considered to be archetypical raft residents. Particularly, we were interested in understanding protein complexes in the T cell plasma membrane. Novel superresolution microscopy studies indicated that a variety of proteins including the T cell receptor are organized in nanometer-sized domains, which grow larger upon activation. However, we found that the analysis of superresolution images is highly prone to artifacts, which give rise to spurious clusters of molecules. To approach this problem we developed a new technique, which allows for artifact-free analysis of superresolution microscopy data. With this technique, we could rule out substantial nanoclustering of the T cell receptor and a variety of other signaling proteins in non- activated T cells. Finally, we aimed at miniaturizing the micropatterning approach. To this end, we used electron beam lithography to deposit carbon dots on glass coverslips. In a second step, DNA nanostructures were specifically bound to these islands. It is now possible to utilize DNA hybridization in order to specifically decorate the dots with a well-defined number of biomolecules, thereby providing a nanostructured surface for cell biological applications.