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 μm2/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.

Single-molecule microscopy to study plasma membrane receptor dynamics

Luc Veya

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.

EPFL2016

Functional characterization of a human odorant receptor

Valérie Jacquier

Olfaction appears to be mediated by a large family of odorant receptors (ORs) that accounts for ~ 1% of the human genome [1–4]. The functional characterization of these receptors has so far been hindered by their poor functional expression in the plasma membrane of heterologous cells. In order to investigate the problem of inefficient OR membrane targeting, sequential stages in the life cycle of the human OR17-40 were monitored. The modification of the receptor by different tags enabled the direct visualization of its biogenesis and trafficking in HEK293 cells. A multi-color labelling approach, which involved fluorescent subcellular markers as well as the spectral separation of membrane-inserted and intracellularly located receptors, indicated that high overexpression led to the accumulation of the receptor in the ER, whereas low expression levels improved its membrane targeting. To increase the efficiency of OR-mediated signalling, fluorescence-activated cell sorting of a functional OR17-40 – GFP fusion protein was used to separate low from high expressing cells, thus increasing the fraction of odorant-responsive cells up to 80% of the total cell population. Selectively labelled cell surface receptors accumulated over time in intracellular compartments, indicating constitutive receptor internalization. This process, which was independent of receptor activation, occurred along the clathrin-mediated pathway. Moreover, the imaging of single receptors in the plasma membrane indicated that, in absence of ligand, part of the receptors are already confined within small domains of 195 ± 10 nm. This fraction, which increased upon agonist or antagonist binding, probably corresponds to receptors located in clathrin-coated prepits. Odorant molecules structurally related to the cognate agonist helional were tested for their ability to activate OR17-40. It was found that an aldehyde group connected to an aromatic ring via a carbon chain of defined length and containing a methyl group in alpha or beta position is necessary for activating the receptor. In addition, first evidence for an OR17-40-specific antagonism was provided. Finally, OR17-40 activation was monitored in cell-derived native vesicles, opening new ways for assay miniaturization and the development of odorant screenings in a micro-array format.

EPFL2005

Activation and Spatial Organization of the Adenosine A2A Receptor in Supported Plasma Membrane Sheets

Sophie Roizard

G protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. The main mechanisms involving GPCR activation and signal transduction to the cell interior are understood but many of the processes which fine-tune GPCRs signalling need to be unraveled. The work performed in this thesis intended to develop new methods to study GPCRs in their native environment but in a simplified system compared to live cells. In this context, the tethering GPCR-containing membrane fragments on solid surfaces appeared to be interesting: it enabled studying the receptors in their real native environment with several high resolution imaging techniques and with the possibility to engineer and control the downstream intracellular signaling components. Two procedures have been established to produce plasma membrane supported on micrometer-sized porous beads or on microscope grids for fluorescence or electron microscopy studies. They have been applied in this thesis to study the Adenosine A2A receeptor (A2A) as a prototypical GPCR. The first procedure enabled the transfer of native membrane patches from live cells with an inside-out orientation onto lectin-coated beads. Using heterologously expressed A2ARs carrying a yellow fluorescent protein (A2AR-Citrine) we showed that the tethered membranes were simultaneously accessible from both sides and comprised fully functional receptors in terms of ligand and G protein binding. These beads were investigated by confocal (Chapter 2) and single molecule (Chapter 3) fluorescence microscopies to study agonist and antagonist binding to the A2AR and the assembly and disassembly of the ternary complexes agonist-A2AR-Gαβγ proteins. The interactions between the different signalling partners could be observed in real time using multicolor fluorescence microscopy. These beads were also used in collaboration with colleagues for screening ligand binding to other transmembrane receptors. The second procedure produced GPCRs-containing plasma membrane fragments ripped-off on electron microscope grids for investigation by transmission electron microscopy (TEM) (Chapter 4). This procedure was improved from an existing protocol in order to produce reproducibly and with a high yield large membrane sheets and to preserve the membrane-associated cytoskeleton during the ripping-off. A2AR-Citrine were specifically gold immuno-labelled on their extracellular terminus using the FLAG-tag or their intracellular side by targeting the protein Citrine. The high resolution of TEM showed that the gold immuno-labelled A2AR-Citrine clustered and compartmentalized on actin-based cytoskeletton. This approach provided the ability to sample multiple single cells on a single grid and presented excellent potential to probe and unravel the organization of protein signalling networks in direct association with the plasma membrane-associated cytoskeleton.

EPFL2013