Determine steric exclusion at the plasma membrane

Univ. Prof. Dr. Stefan Howorka, Institute of Biophysics Johannes Kepler University Linz, Austria Univ. Prof. D.I. Dr. Gerhard J. Schütz, Institute of Applied Physics TU Wien, Austria The plasma membrane not only forms a protective coat around cells, it also contains a variety of important proteins, including receptors, transporters, and scaffold proteins. Knowing the spatial organization of these proteins is crucial for understanding molecular interactions that encompass signalling processes. Particularly, the high surface density of proteins at the plasma membrane leads to excluded area effects, which change the diffusional paths and hence the mutual encounter rates of any two membrane proteins. In this project, we will for the first time determine the principles that govern membrane protein interactions by quantifying molecular steric exclusion effects at the cell surface. As probe, we will insert fluorescent DNA nanostructures of adjustable size into the plasma membrane. Their diffusional paths will be recorded at nanometer resolution in situ, which allows for identifying membrane regions of hindered accessibility. The approach will be applied to study protein crowding effects within the immunological synapse between T cells and activating surfaces. Currently, it is believed that size exclusion effects are decisive for the initiation of the T cell signal. With this method we can quantify the physical dimensions of void areas at the cell surface at the various phases of T cell activation. The results will enable a better understanding of T cell antigen recognition, which is one of the key steps during our immune response against pathogens. In the future, also the engineering of artificial chimeric antigen receptors will strongly benefit from an improved mechanistic understanding of the T cell response, thereby helping novel strategies of immune therapy.

The plasma membrane forms a protective cover around biological cells and also contains a large number of important functional proteins, such as receptors, transporters and scaffold proteins. The spatial 3D organization of these proteins is key for a large number of molecular processes involved in initiating an immune response. Crucially, the density of the proteins influences their mutual functional interaction. In this project we have developed an approach to determine for the first time the proteins' spatial movement on the cell surface with unprecedented precision below 20 nm in all three dimensions. As essential probe to develop and validate this approach, we used highly defined fluorescent DNA nanostructures of adjustable properties. Our approach is being used to uncover key steps during the formation of the immunological synapse to create an immune response against pathogens.