Membrane nanotubes drawn by optical tweezers transmit electrical signals between mammalian cells over long distances

Biological cells continuously change shape allowing essential functions such as cell motility, vesicle-mediated release/uptake of soluble and membrane components or nanotube-mediated cell–cell communications. Here we use single cell micromanipulation to induce functional changes of cell shape for nanobiotechnological applications. Optical tweezers are focused on the plasma membrane of living cells to pull membrane nanotubes of similar200 nanometre diameters and 100 micrometre lengths. Upon switching off the laser tweezer membrane nanotubes relax back to the cell surface. Single-exponential relaxation times deliver local mechanical properties of cells' plasma membrane. Nanotubes pulled beyond 100 micrometre tear off and form micrometre-sized vesicles carrying functional membrane receptors and cytoplasmic signaling components. Membrane nanotubes from one cell can be contacted to adjacent cells forming via connexins intercellular electrical connections within seconds in all directions. Our method opens broad applications for multiplexing single-cell analytics to submicrometer/subfemtoliter ranges and for creating artificial intercellular signaling networks, both not attainable by current methodologies.

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

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

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

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

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.