Advanced Fluorescence Microscopy to Study Plasma Membrane Protein Dynamics

Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane.

Investigating molecular interactions of G protein coupled receptors by fluorescence techniques

Jean-Baptiste Perez

G Protein coupled receptors (GPCRs) constitute an abundant family of membrane proteins which play a central role in cellular signaling and are important targets for modern medicine. Despite intensive research, central questions of the molecular mechanism of GPCR mediated signal transduction remain unresolved. This thesis concerns α1b-adrenergic receptor (α1b-AR) as a prototypic GPCR. The question whether ARs form homo- or hetero-oligomers was investigated using fluorescence resonance energy transfer (FRET). An important finding was that both α1a and α1b-AR subtypes form homo-oligomers. Also hetero-oligomers have been observed between the α1b- and the α1a-AR subtypes, but not with less related receptors. Another interesting finding concerns α1-AR co-internalization which correlates with the ability of the receptor to hetero-oligomerize. Investigating particular processes in living cells is often complicated by the complex cellular environment. Here we have developed a rather simple approach to study transmembrane signaling by using supported cell membrane sheets. They were prepared by pressing a glass coverslip on the apical part of cultured cells, ripping off large regions of the native plasma membrane. For the α1b-AR we still could observe ligand binding to such sheets, indicating that GPCR functionality was at least partly conserved. Due to the absence of cytosolic autofluorescence of the cells, supported membrane sheets were also found to be suited for single-molecule microscopy. Because both leaflets of the supported membranes remained fluid, it was possible to follow the diffusion of certain membrane proteins. For example using fluorescence recovery after photobleaching (FRAP) and single-molecule microscopy, we investigated how G proteins diffused along the membrane and compartmentalized. The predominant role of lipid anchors in G protein localization was highlighted by measuring the diffusion of two Gαq proteins fused with citrine at two different positions and citrine based constructs designed to mimic the monomeric and the trimeric form of a G protein. Finally, first results were obtained showing the feasibility to form supported cell membrane sheets on microstructured devices consisting of a planar silicon support perforated with arrays of holes. This geometry provides a full accessibility to both intra- and extracellular sides of the bilayer. This concept could potentially be applied for the development of chip-based screening assays.

EPFL2005

Miniaturized bioanalytics to probe the function of membrane proteins

Pedro Pascoal

G-protein coupled receptors (GPCRs) are the most abundant class of proteins in the cell body. Such receptors are of major interest as potential therapeutic targets. Downscaling and parallelization of bioanalytics opens novel routes to rapidly screen and identify potential drugs with a decrease in regard to the costs, and elucidate novel functions of signaling networks under physiological conditions. Native vesicles are small autonomous biological containers, which are efficiently produced from all cell lines. They are composed of their mother cell plasma membrane and enclose part of their cytoplasm. Membrane receptors are then exposed at their surface and already demonstrate to induce cellular signaling when exposed to receptor ligands. Native vesicles were investigated in this present work, as novel possibilities to downscale receptor investigation in live cells, using neurokinin 1 receptor (NK1R) as a representative model. Here native vesicle production and purification was optimized. The influence of the cell cycle on the production efficiency was demonstrated. Biological proteins were downregulated in order to produce blebbing cells. Native vesicle characterization was achieved. The receptors also demonstrate to be efficiently labeled by agonist and antagonist, allowing to access information about the binding kinetics as well as KD values. The results are in agreement with those obtained in live cells. Native vesicles also demonstrate to internalize agonist after application, demonstrating receptor desensitization and signaling performance similar as live cells. Confocal microscopy shows that cells expressing the NK1R-CFP have two binding affinities for their main agonist, substance P. Similar results could be observed with flow cytometry. The high affinity binding is related to cholesterol content in the cell membrane and was abolished by cholesterol depletion with methyl-β-cyclodextrin. Micro-contact printing (µCP) was used to (bio)functionalize surfaces with proteins, polymers or functionalized nanoparticles. Precise sample positioning by micro-contact printing shows improves nuclear magnetic resonance excitation and detection, when performed with a planar microcoil probe. µCP was used to produce native vesicle arrays by two procedures, and fluorescent binding assay shows the binding of fluorescent ligands to the receptor. Laser tweezers allow manipulating cell membranes without requiring the use of polystyrene beads. From pulled membranes, native vesicles were produced. In addition from pulled membranes, large tethers were produced and artificial connections were established with neighboring cells. Intercellular communication was investigated by whole-cell patch clamp in dissociated primary dorsal root ganglion neurons after optical induced connection, as well as in HEK cells expressing Cx36. The lab-on-chip assay development demonstrates: the high production of native vesicles in microchannels; the efficient purification obtained by negative dielectrophoresis depletion in MEMS chips; perfect trapping and thus immobilization of native vesicles in a new optical multi-tweezer array. Fluorescence labeling was performed with native vesicles trapped in a multi-tweezer array inside microfluidic channel in the presence of two laminar flows. Optical multi-tweezer array setup shows to be the fastest and more efficient technique in order to perform immobilization and labeling of native vesicles in the microfluidic channel. It is presently the only technique to perform fluorescence measurements when maintaining objects trapped.

EPFL2008

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

Miniaturized bioanalytics to probe the function of membrane proteins

Pedro Pascoal

EPFL2008

Functional characterization of a human odorant receptor

Valérie Jacquier

EPFL2005

Investigating molecular interactions of G protein coupled receptors by fluorescence techniques

Jean-Baptiste Perez

EPFL2005