Theme 2a : Tetraspanin membrane partition and viral infection
PIs : Pierre-Emmanuel Milhiet & Christine Benistant
People : Selma Dahmane (Thèse), Laurent Fernandez (Thèse), Patrice Dosset (IE)
Collaborations : Markus Thali (Vermont University), Jane McKeating (U. Birmingham, UK), Jean Dubuisson (Pasteur Institute, Lille), Eric Rubinstein (U1004 INSERM, Villejuif), Fedor Berditchevski (UK), Dr Christopher Stipps (USA)
Tetraspanins are ubiquitously expressed transmembrane proteins that form supramolecular assemblies organized in microdomains. They are involved in numerous cell functions and are clearly associated to several pathologies, especially infection diseases and cancer.
Indeed the group of Markus Thali (Vermont University) has shown that Gag protein, which directs viral assembly and release, accumulates at the cell surface tetraspanin microdomains enriched in CD9 and CD81 and HIV-1 egress could be gated through these microdomains. In collaboration with Thali's group, we used single molecule tracking experiments in addition to ensemble labeling techniques to demonstrate the specific recruitment of the tetraspanins CD9 and CD81 at the budding sites of HIV-1 virus-like particles (Krementsov, 2009, we are co-first author and corresponding author of this paper). Our findings support the emerging concept that viral components, instead of clustering at preexisting microdomain platforms, direct the formation of distinct domains for the execution of specific functions. Our results also demonstrated that CD9 and CD81, even often localized in similar areas, display different membrane behavior that was due to an interaction of the C terminal part of CD81 with ERM proteins (Rassam, in preparation).
Tetraspanins promote multiple cancer stages. We used single molecule tracking experiments to study tetraspanin partitioning in cancer cells and to show the high dynamic of interactions in the tetraspanin web and to further indicate that the tetraspanin web is distinct from raft microdomains. (Espenel, 2008). Important components of the tetraspanin web are gangliosides. In collaboration with Fedor Berditchevski (UK), we study how their composition impact on tetraspanin partitioning in breast cancer cells. Integrins are others favorite partners of tetraspanins that play major role in cell motility and invasion. In collaboration with Dr Christopher Stipps (USA), we studied how tetraspanins regulate proteolysis-driven motility through invadopodia.
Theme 2b : Lateral segregation of membrane components using High-speed Atomic Force Microscopy
PI : Pierre-Emmanuel Milhiet
Collaboration : Toshio Ando (Kanazawa, Japan)
With conventional AFMs, it takes more than a minute to capture an image, while biomolecular processes generally occur on a millisecond timescale or less. However major advances have been done by the group of Toshio Ando in Kanazawa (Japan) who developed a new generation of microscope allowing capture successive images up to the video rate in liquid. This improvement has been expected for a long time by the AFM community since with conventional AFMs it takes more than a minute to capture an image. Thanks to collaboration with the Japenese group that created a consortium with two other French laboratories, we mounted and developed a prototype of the HS-AFM allowing image capture up to the video rate under physiological conditions (supported by two ANR grants, (PNANO and PVC). This microscope was first used to image amyloid fibers (Milhiet, 2010) and we then focused on structure and dynamics of membrane assemblies. We investigated membrane structure and diffusion of nanodomains composed of the ganglioside GM1, a component of raft microdomains in cells. Using HS-AFM and artificial bilayers supported on mica, we demonstrated that this lipid form stable domains of 30 nm nanometers that can diffuse within membrane (figure and paper in preparation). Last year, we also started collaborating with Gilles Divita aiming to decipher the molecular mechanism underlying the penetration of amphipathic peptides used for drug targeting (supported by an ANR grant). Beside these activities, we pursued imaging biological samples, especially biological membranes, using standard AFM. In relation with our expertise in manipulating and imaging of transmembrane proteins, we also performed several successful trials of reconstitution within artificial bilayers (see an example in Picas, 2010).
Theme 2c : Structure and Dynamics of the nuclear envelope in eukaryotes
PIs : Christine Doucet and Pierre-Emmanuel Milhiet
Nuclear pore complexes (NPCs) are the only gateways between the nucleus and the cytoplasm and are responsible for the regulated transport of molecules between these compartments. Their assembly occurs in strikingly different cellular contexts. During mitosis, the nuclear envelope (NE) is broken and NPCs assemble on the surface of chromatin whereas, during interphase, NPCs assemble in an intact NE, requiring the formation of a hole across the NE, which implies the local fusion and bending of the inner and outer nuclear membranes (INM and ONM). CD has shown that an essential subcomplex of NPCs is targeted by cell cycle-specific mechanisms to assembly sites and that a membrane curvature sensing motif is required in interphase. However, the fusion mechanisms leading to these highly curved pore membranes are poorly understood, mainly due to the lack of tools to monitor the formation of these sites in real time. Using nuclei purified from cultured cells and in vitro assembled nuclei, we then plan to develop time-lapse AFM to study the topology of nuclear membranes and follow the formation of fusion sites. In addition some candidates potentially involved in the early steps of nuclear membrane fusion (Rtn4 or POM121) have been identified and the effects of depletion or overexpression of these proteins will be assessed. Once key players in hole formation are identified, we will be able, using the PALM-AFM setup together with fluorescent protein candidates, to better characterize the role of these proteins (chronology of events, localization on the INM and/or ONM…).
Theme 2d : Structural dynamics of single metabotropic glutamate receptors dimers
PI : Emmanuel Margeat
People : Fataneh Fatemi (Post-doc), Thi-Phuong-Hanh Cao (Thèse Labex EPIGENMED)
Collaboration with Jean Philippe Pin & Philippe Rondard (IGF Montpellier) and Claus Seidel (U. Dusseldorf)
Metabotropic glutamate receptors (mGluR) are members of the class C G-protein Coupled Receptors (GPCR) family. They are activated by glutamate, the major excitatory neurotransmitter in the central nervous system. They are homodimeric multidomain proteins stabilized by a disulfide bridge. Each subunit is composed of an extracellular domain (ECD) that binds orthosteric ligands such as glutamate, and a heptahelical transmembrane domain (7TM) common to all GPCRs and responsible for G-protein activation. A major re-orientation of the two ECDs within the dimer appears necessary for receptor activation upon agonist binding. We have established a new method for the purification of soluble mGluR2 ECD dimers, fused at their N-terminus with Snap-tags that can be covalently labeled with organic fluoropohores. We then used single molecule Förster Resonance Energy Transfer (smFRET) with multiparametric fluorescence detection (MFD) (Olofsson, 2013) to measure the conformational changes associated with the binding of agonists, antagonists or partial agonists to these dimers. We succeeded in monitoring for the first time the activation of a GPCR using single molecule FRET, and demonstrate as expected from crystallographic and ensemble studies, that agonist binding promotes an increase in distance between the N-terminus of the receptors, as compared to the conformation observed in the presence of antagonist. However, our data indicate that for all types of ligands, the receptor ECD oscillates between two boundary (Resting and Active) conformational states, and that ligand binding solely influences the transition rates between these states, in agreement with the conformational selection theory (Manuscript in preparation).