7 research outputs found
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 receptorsâ 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 receptorâsmobility. 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
Single-Molecule Imaging Deciphers the Relation between Mobility and Signaling of a Prototypical G Protein-Coupled Receptor in Living Cells
Lateral diffusion enables efficient interactions between membrane proteins leading to signal transmission across the plasma membrane. An open question is how the spatio-temporal distribution of cell surface receptors influences the transmembrane signaling network. Here we addressed this issue studying the mobility of a prototypical G protein-coupled receptor, the neurokinin-1 receptor (NK1R) during its different phases of cellular signaling. Attaching a single quantum dot to individual NK1Rs enabled us to follow with high spatial and temporal resolution over long time regimes the fate of individual receptors at the plasma membrane. Single receptor trajectories revealed a very heterogeneous mobility distribution pattern with diffusion constants ranging from 0.0005 to 0.1 ÎĽm2/s comprising receptors freely diffusing, confined in 100-600 nm sized membrane domains, as well as immobile ones. A two-dimensional representation of mobility and confinement resolved two major, broadly distributed receptor populations, one showing high mobility and low lateral restriction, the other low mobility and high restriction. We found that about 40% of the receptors in the basal state are already confined in membrane domains and are associated with clathrin. After stimulation with an agonist, 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 clathtrin and is correlated to a significant decrease of the receptors' canonical pathway activity. This shows that the high plasticity of receptor mobility is of central importance for receptor homeostasis and fine regulation of receptor activity
Exchanging ligand-binding specificity between a pair of mouse olfactory receptor paralogs reveals odorant recognition principles
A multi-gene family of ~1000 G protein-coupled olfactory receptors (ORs) constitutes the molecular basis of mammalian olfaction. Due to the lack of structural data its remarkable capacity to detect and discriminate thousands of odorants remains poorly understood on the structural level of the receptor. Using site-directed mutagenesis we transferred ligand specificity between two functionally related ORs and thereby revealed amino acid residues of central importance for odorant recognition and discrimination of the two receptors. By exchanging two of three residues, differing at equivalent positions of the putative odorant binding site between the mouse OR paralogs Olfr73 (mOR-EG) and Olfr74 (mOR-EV), we selectively changed ligand preference but remarkably also signaling activation strength in both ORs. Computer modeling proposed structural details at atomic resolution how the very same odorant molecule might interact with different contact residues to induce different functional responses in two related receptors. Our findings provide a mechanistic explanation of how the olfactory system distinguishes different molecular aspects of a given odorant molecule, and unravel important molecular details of the combinatorial encoding of odorant identity at the OR level