Single-molecule microscopy to study plasma membrane receptor dynamics

Cell surface receptors allowthe cell to sense and respond to external signals. Receptor malfunctions are associated with many diseases. The diffusional behavior of receptors is of particular interest to understand how the cell modulates receptor function in the complex, heterogenous plasma membrane. Unlike traditional ensemble methods, single-molecule techniques give access to events taking place at the nanometer scale and millisecond time regime, providing unprecedented means to correlate biological functions of the cell membrane with the spatio-temporal organization of its individual components. In this work, advanced fluorescence microscopy techniques were used to track the motion of individual receptors in the plasma membrane of living cells to elucidate principles regulating their function. Two very different neurotransmitter receptors were investigated: the G protein coupled neurokinin-1 receptor (NK1R), and the ionotropic serotonin type 3 receptor (5-HT3R). The first important step towards tracking single receptor molecules is the specific labeling of a small fraction of these receptors with a photostable fluorescent probe. The choice of a suitable labeling strategy is of paramount importance, as it will largely determine the success of the experiment. Single-molecule tracking requires extremely specific, long-lasting, stoichiometric and bright fluorescent labels that do not interfere with the function and diffusion of the receptor. For the NK1R, a two-step labeling strategy based on the strong biotin-streptavidin interaction yielded the best labeling specificity. Enzymatic biotinylation of the receptor followed by binding streptavidin-coated quantum dot (QDot) enabled a straightforward and precise adjustment of the label density to a few QDots per cell. The combination of high brightness and photostability of semi-conductor QDots allowed us to record receptor trajectories with high spatial resolution over long time periods. The investigation of thousands of single NK1R trajectories revealed a very heterogeneous mobility pattern with two major, broadly distributed receptor populations, one showing highmobility and low lateral restriction, the other low mobility and high restriction. We found that 40% of the receptors in the basal state are already confined inmembrane domains. After agonist stimulation, an additional 30% of receptors became further confined. Using inhibitors of clathrin-mediated endocytosis, we showed that the fraction of confined receptors at the basal state depends on the quantity of membrane-associated clathrin and is correlated to a significant decrease of the receptors’ canonical pathway activity. These findings add further insights to the plasticity of receptor signaling. There is a risk that one streptavidin-coated QDot could bind several biotinylated receptors and thereby alter the receptor’smobility. The generation of monovalent StrepTactin-conjugated QDots allowed us to exclude any cross-linking artifacts in our previousmeasurements of NK1R diffusion. In the case of the 5-HT3R, the diffusion of individual receptors was followed using a fluorescent nanobody (VHH15-CF640R). This novel high-affinity label which is small, monovalent and highly photostable enabled us to track native 5-HT3Rs over long time regimes, revealing a surprising and intriguing diffusional behavior of some receptors.

Related material