Downscaling the Analysis of Complex Transmembrane Signaling Cascades to Closed Attoliter Volumes

Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity. The vesicles are derived from live mammalian cells using chemicals or optical tweezers. They comprise parts of a cell’s plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells. Using fluorescence microscopies, we measured in individual vesicles the different steps of G-protein-coupled receptor mediated signaling like ligand binding to receptors, subsequent G-protein activation and finally arrestin translocation indicating receptor deactivation. Observing cellular signaling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis, and investigating receptor mediated signaling in multiarray format.

Cell-derived plasma membrane vesicles as minimal cells for analyzing transmembrane signaling

Luigino Grasso, Ghérici Hassaïne, Rudolf Hovius, Joachim Piguet, Horst Vogel, Michael Werner, Romain Wyss

Cellular signalling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show how cell-derived vesicles can be used for anlaysing transmembrane signalling. The vesicles are derived from live mammalian cells by using either chemicals, or by optical tweezers and they comprise parts of the plasma membrane and cytosol of the mother cell. We measured in vesicles derived from individual cells with single molecule sensitivity the different steps of G protein-coupled receptor mediated signalling like ligand binding to receptors, subsequent G protein activation and finally receptor deactivation by interaction with arrestin. Cell-derived plasma membrane vesicles represent the smallest autonomous containers capable of performing cellular signaling reactions thus functioning like minimal cells. Observing cellular signalling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis and investigating receptor mediated signalling in multiarray format.

2012

Transmembrane Signaling Analysis in Model Membrane Systems

Luigino Grasso

Studying the complex mechanisms of transducing an extracellular signal across a plasma membrane to initiate an intracellular responce is of fundamental importance for a cell’s function. Transmembrane signaling is the key allowing cells to sense and communicate with its environment. Signal transduction is primarily mediated by numerous membrane receptors. In this thesis we investigated the function of G-protein-coupled receptors (GPCR). GPCRs represent the largest and most diverse group of membrane proteins, encoded by more than 800 genes. They allow cells to recognize as diverse extracellular stimuli as photons, organic odorants, nucleotides, nucleosides, peptides, lipids and proteins, conferring to GPCRs a fundamental role in signal transduction. They are broadly distributed across the entire body and thus involved in a wide range of physiological and pathological processes, which makes GPCRs one of the main targets of modern medicines. Activation of GPCRs initiate numerous intracellular signaling pathways, involving a large number of proteins and molecules. They act as a central molecular activators and integrators of a complex network of signaling pathways. While most of the studies have been performed on cells, the complexity of cellular systems has raised the need to develop simpler model systems to assess the role of individual signaling com- ponents. In this thesis we present different model systems to study the functionality of GPCR signal transduction from the ligand binding to the activation of downstream cellular reactions. The first part of the thesis concerns the isolation of single receptors from cultured cells by detergent solubilization followed by purification and the subsequent receptor reconstitution into giant unilamellar vesicles (GUV). Detergent micelle receptors were analyzed by fluorescence correlation spectroscopy (FCS) and surface plasmon resonance (SPR) measurements. We could determine the conditions of functional solubilization of GPCRs by characterizing the amount of obtained receptor-detergent micelles, their homogeneity and the capacity of the receptor to selectively bind ligands. The receptors were then reconstituted into GUVs and ligand binding was monitored by confocal microscopy. In the second part of the thesis, we investigated different aspects properties of native plasma membrane vesicles. These vesicles are derived from live mammalian cells using v cytochalasin B or optical tweezers; they comprise parts of a cell’s plasma membrane and cytosol. After the characterization of their overall composition, we investigated their ability to mediated transmembrane activation of intracellular signaling reactions upon agonist binding to a GPCR. We could successfully monitor the ligand binding to receptor by FCS, the subsequent G-protein activation by intermolecular FRET and the receptor desensitization by monitoring the arrestin recruitment to the plasma membrane. These vesicles thus represent the smallest autonomous container performing cellular signaling reactions thus functioning like a minimized cell. Finally, preliminary experiments demonstrated the potential of native vesicles to serve as novel drug delivery system since they meet most of the requirements for an efficient drug delivery platform.

EPFL2012

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.