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

Joachim Piguet, Luc Veya, Horst Vogel

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.

American Society for Biochemistry and Molecular Biology2015

Single molecule detection and fluorescence correlation spectroscopy on surfaces

Kai Hassler

In this thesis a new approach for single molecule detection and analysis is explored. This approach is based on the combination of two well established methods, fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRFM). In contrast to most existing fluorescence spectroscopy techniques, the subject of primary interest in FCS is not the fluorescence intensity itself but the random intensity fluctuation around the mean value. Intensity fluctuations are induced by thermal noise in a minute observation volume, which is in classical FCS the confocal volume of a confocal microscope. E.g. FCS is commonly utilized to investigate diffusion. In this case, diffusing fluorescent molecules entering or leaving the observation volume cause intensity fluctuations, which are analyzed by calculating the temporal autocorrelation of the observed signal. The autocorrelation is a measure for the self-similarity of a signal and contains information about the average fluctuation strength and duration. The confocal observation volume, i.e. the measurement volume that is actually seen by the detector is approximately given by the product of the optical transfer function with the fluorescence exciting intensity distribution of a focused laser beam. To achieve a high signal-to-background ratio a small observation volume is absolutely essential, first of all because the background from e.g. scattered light increases with the size of the observation volume. Second, a small volume assures for a small average number of fluorophores inside the observation volume and therefore for a high fluctuation amplitude i.e. FCS signal. This thesis proposes and discusses an alternative to confocal FCS specially conceived for measurements on surface-bound molecular systems, such as biological receptors or immobilized enzymes. In contrast to confocal FCS, fluorescence is excited within an evanescent field generated by total internal reflection (TIR) of a laser beam at the interface between a microscope coverslip and the sample. This is achieved by focusing the laser beam off-axis at the back focal plane of a high NA oil-immersion objective. The collimated beam that emerges from the objective is incident at an oblique angle at the coverslip-sample interface and totally internal reflected. In contrast to confocal FCS, the generated observation volume is completely confined to the surface and background fluorescence as well as scattered light from the bulk is efficiently suppressed. Our method, called objective-type TIR-FCS in the following, features an increased collection efficiency compared to existing techniques that combine evanescent wave excitation and FCS. Existing techniques use total internal reflection on the surface of a prism to generate an evanescent field. This leads to a configuration where the choice of objectives is limited to air or water-immersion objectives. In our system we use a high NA oil-immersion objective, specially conceived for TIR applications, which collects light efficiently. The collection efficiency is further enhanced by a naturally occurring change of the emission properties of fluorophores close to interfaces between dielectric media. The presence of the interface favors emission into the optically denser medium so that about 60% of the emitted light can be collected. These factors, together with a reduced observation volume lead to a very sensitive method with a high potential for applications in single molecule detection and analysis. The performance of the proposed method was experimentally shown for measurements on molecules subject to Brownian motion and binding to modified coverslips. In particular, it was experimentally shown that objective-type TIR-FCS features high signal-to-background ratio on a single molecule level. In this thesis, concise derivations of analytical expressions for autocorrelation functions for diffusion and most important, the case of ligands reversibly binding to a single and localized binding site are presented. The derived model allows for the quantitative determination of binding rates for a single receptor. We strongly believe that the application of these results in the context of investigations of receptor-ligand binding kinetics will allow for deeper understanding of cellular signaling. Moreover, this thesis discusses the applicability of the proposed method in enzymology. Enzymes, as most proteins are subject to continuous changes of their structure or conformation. These conformational changes are correlated with the function of the enzyme. In the discussed example the enzyme catalyzes an oxidation where the product is fluorescent but the substrate is not. The function of the enzyme i.e. the recurring product formation leads to observable intensity fluctuations. Since function and conformational states are correlated, conformational fluctuations can be investigated by means of FCS as was already shown for confocal FCS. A technique closely related to FCS is fluorescence lifetime spectroscopy (where lifetime refers to the mean lifetime of the electronic excited state). Whereas in FCS the relaxation after random deviations from thermal equilibrium is investigated, relaxation of excited fluorophores towards their electronic ground-state is investigated in lifetime spectroscopy. The technique is used for e.g. discrimination between fluorophores with different lifetimes. Lifetime spectroscopy combined with imaging is used in many domains of life-science, including microarray reading and medical diagnostics. In preliminary work we developed a novel approach to perform lifetime imaging that is based on a multiplexing technique. The proposed method requires no mechanical scanning stage and only a single-point detector. Furthermore, noise is reduced under certain circumstances if the signal is low. Characteristics of this technique as well as advantages and disadvantages are shortly discussed.

EPFL2006

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.