Influenza A viruses use multivalent sialic acid clusters for cell binding and receptor activation

Influenza A virus (IAV) binds its host cell using the major viral surface protein hemagglutinin (HA). HA recognizes sialic acid, a plasma membrane glycan that functions as the specific primary attachment factor (AF). Since sialic acid alone cannot fulfill a signaling function, the virus needs to activate downstream factors to trigger endocytic uptake. Recently, the epidermal growth factor receptor (EGFR), a member of the receptor-tyrosine kinase family, was shown to be activated by IAV and transmit cell entry signals. However, how IAV's binding to sialic acid leads to engagement and activation of EGFR remains largely unclear. We used multicolor super-resolution microscopy to study the lateral organization of both IAV's AFs and its functional receptor EGFR at the scale of the IAV particle. Intriguingly, quantitative cluster analysis revealed that AFs and EGFR are organized in partially overlapping submicrometer clusters in the plasma membrane of A549 cells. Within AF domains, the local AF concentration reaches on average 10-fold the background concentration and tends to increase towards the cluster center, thereby representing a multivalent virus-binding platform. Using our experimentally measured cluster characteristics, we simulated virus diffusion on a flat membrane. The results predict that the local AF concentration strongly influences the distinct mobility pattern of IAVs, in a manner consistent with live-cell single-virus tracking data. In contrast to AFs, EGFR resides in smaller clusters. Virus binding activates EGFR, but interestingly, this process occurs without a major lateral EGFR redistribution, indicating the activation of pre-formed clusters, which we show are long-lived. Taken together, our results provide a quantitative understanding of the initial steps of influenza virus infection. Co-clustering of AF and EGFR permit a cooperative effect of binding and signaling at specific platforms, thus linking their spatial organization to their functional role during virus-cell binding and receptor activation. Author summary The plasma membrane is the major interface between a cell and its environment. This complex and dynamic organelle needs to protect, as a barrier, but also transmit subtle signals into and out of the cell. For the enveloped virus IAV, the plasma membrane represents both a major obstacle to overcome during infection, and the site for the assembly of progeny virus particles. However, the organisation of the plasma membrane-a key to understanding how viral entry works-at the scale of an infecting particle (length scales < 100 nm) remains largely unknown. Sialylated glycans serve as IAV attachment factors but are not able to transmit signals across the plasma membrane. Receptor tyrosine kinases were identified to be activated upon virus binding and serve as functional receptor. How IAV engages and activates its functional receptors while initially binding glycans still remains speculative. Here, we use super resolution microscopy to study the lateral organization of plasma membrane-bound molecules involved in IAV infection, as well as their functional relationship. We find that molecules are organized in submicrometer nanodomains and, in combination with virus diffusion simulations, present a mechanistic model for how IAV first engages with AFs in the plasma membrane to subsequently engage and trigger entry-associated membrane receptors.

Enlightening G-protein-coupled receptors on the plasma membrane using super-resolution photoactivated localization microscopy

Paolo Annibale, Aleksandra Radenovic, Marco Scarselli

The possibility to visualize and image the arrangement of proteins within the cell at the molecular level has always been an attraction for scientists in biological research. In particular, for signalling molecules such as GPCRs (G-protein-coupled receptors), the existence of protein aggregates such as oligomers or clusters has been the topic of extensive debate. One of the reasons for this lively argument is that the molecular size is below the diffraction-limited resolution of the conventional microscopy, precluding the direct visualization of protein super-structures. On the other hand, new super-resolution microscopy techniques, such as the PALM (photoactivated localization microscopy), allow the limit of the resolution power of conventional optics to be broken and the localization of single molecules to be determined with a precision of 10-20 nm, close to their molecular size. The application of super-resolution microscopy to study the spatial and temporal organization of GPCRs has brought new insights into receptor arrangement on the plasma membrane. Furthermore, the use of this powerful microscopy technique as a quantitative tool opens up the possibility for investigating and quantifying the number of molecules in biological assemblies and determining the protein stoichiometry in signalling complexes.

Portland Press2013

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

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.