Characterization of nanostructures in the live cell plasma membrane utilizing advanced single molecule fluorescence techniques

Unrevealing the detailed structure of the cellular plasma membrane at a nanoscopic length scale is the key for understanding the regulation of various signaling pathways or interaction mechanism. Hypotheses postulate the existence of nanoscopic lipid platforms in the cell membrane which are termed lipid- or membrane rafts. Based on biochemical studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition and heterogeneity. In this thesis I present an ultra-sensitive fluorescence based method which allows for the first time the direct imaging of single mobile rafts in the live cell plasma membrane. The method senses rafts by their property to assemble a characteristic set of fluorescent marker-proteins or lipids on a time-scale of seconds. A special photobleaching protocol was developed and used to reduce the surface density of labeled mobile rafts down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per raft was determined by single molecule brightness analysis. For demonstration, I used the consensus markers Bodipy-GM1, a fluorescent lipid analogue, and glycosylphosphatidyl-inositol-anchored monomeric GFP. For both markers I found cholesterol-dependent association in the plasma membrane of living CHO and Jurkat T cells in the resting state, indicating the presence of mobile, stable rafts hosting these probes. I further characterized these structures by taking cell-to-cell variations under consideration. By comparing Bodipy-GM1 with mGFP-GPI homo-association upon temperature variation, two different states - a non-equilibrated and an equilibrated state - could be identified. I conclude that rafts are loaded non-randomly; the characteristic load is maintained during its lifetime in the plasma membrane of a non-activated cell. Beside these lipid-lipid or protein-lipid interactions, protein-protein interactions play of mayor role for the regulation of cell metabolism and function. In this thesis I further characterized the interaction between human CD4, the major co-receptor in T cell activation, and human Lck, the protein tyrosine kinase essential for early T cell signaling using an ultra-sensitive fluorescence-based method. Interaction dynamics were studied in detail by performing photobleaching experiments and single molecule brightness analysis. This enabled a combined mobility and stoichiometry analysis of Lck-molecules interacting with the captured CD4 protein. In the last part of my thesis I present a single molecule fluorescence study using a variant of an oxidized phospholipid - which is known to induce apoptosis - to probe the structure of the cellular plasmamembrane. The cells were illuminated using a recently introduced technique which utilizes a highly inclined and laminated optical sheet (HILO) to reduce background signal arising from intracellular fluorophores or from cellular autofluorescence. Our data demonstrate the relevance of plasma membrane properties for uptake of oxidized phospholipids, and indicate a novel indirect mechanism for the control of endocytosis. (author)