PIs : Emmanuel Margeat, Nathalie Declerck
People : Caroline Clerte (IR), Soraya Ait-Bara (post-doc)
Collaboration with M. Nollmann (CBS) and M. Boudvillain (CBM Orleans)
Our research focuses on the two mecanisms used by bacteria to terminate transcription of a gene: intrinsic termination and Rho-dependent termination. RNA transcription being a dynamic phenomenon in nature (RNA polymerase is a molecular motor, translocating along DNA while synthesizing RNA), single-molecule studies are particularly well suited for studying the molecular mechanisms involved in this central function of the cell. Most intrinsic terminators contain an inverted and repeated sequence that can base pair to itself once transcribed to create a stable terminator hairpin (7-20 bp long), that will promote RNA release upon folding. LicT and SacY from Bacillus subtilis prevent the premature arrest of transcription by binding to an antiterminator RNA hairpin that overlaps this intrinsic terminator, located in the 5’mRNA leader region of the gene to be regulated. In order to investigate the molecular determinants of this antitermination / termination balance, we have developed a fluorescence-based nucleic acids system that mimics the competition between the LicT or SacY antiterminator targets and the overlapping terminators. Using Förster Resonance Energy Transfer on single diffusing RNA hairpins, we have monitored directly their opening or closing state, and thus investigated the effects on this equilibrium of the binding of antitermination proteins or terminator-mimicking oligonucleotides. We show that the antiterminator hairpins adopt spontaneously a closed structure and that their structural dynamics is mainly governed by the length of their basal stem. The induced stability of the antiterminator hairpins determines both the affinity and specificity of the antitermination protein binding (Clerte, 2013).
The second part of this research theme focuses on the mechanisms of transcription termination by Rho, using a combination of fluorescence measurements and nanomanipulation. Rho is a hexameric helicase that uses the energy derived from ATP hydrolysis to translocate directionally along the RNA transcript, and to dissociate the transcription elongation complex. To understand the structure /function relationships giving rise to these activities, it is necessary to understand the physical basis of this translocation. Through a collaboration with Marc Boudvillain (CBM, Orleans) we showed that upon opening of a DNA / RNA hybrid model, Rho establishes specific contacts with 2’OH groups of RNA at positions separated by 7 bases (Schwartz 2009, 2012, Boudvillain 2010), and established biochemical evidence for an assymetric configuration of the hexamer upon translocation (Rahbi 2011).
Theme 1b : Transcriptional regulation in single cells
PI : Nathalie Declerck
People : Caroline Clerte (IR), Elodie Le Monnier (IE CDD)
Collaboration with the group of S. Aymerich (INRA, AgroParisTech) et O. Radulescu (DIMNP, UM2 Montpellier).
This project results from a long standing collaboration between the CBS (ND) and the team of S. Aymerich at INRA in Grignon investigating on regulatory proteins and networks in Bacillus subtilis. The aim of these researches is to unravel, from the atomic and single molecule level to the single cell and population level, the molecular bases of the transcriptional control mechanisms that underlie bacterial adaptation to environmental changes. Several transcriptional regulators have already been well characterized in vitro by combining many different approaches and technics available at the CBS or through external collaborations (e.g. with S. Sanglier in Strasbourg for mass spectrometry, G. Rivas in Madrid for analytical ultra-centrifugation, J. Stulke in Gottingen for B. subtilis engineering) (Déméné 2008, Doan 2008, Zorrilla 2008, Chaix 2010, Atmanéné 2010, Hubner 2011, Clerté 2013). Recently, we have started the characterization of these regulatory systems in live bacterial cells using state-of-the-art fluorescence microscopy methods based on multiple scanning fluctuation analysis. In the frame of the ANR grant “QuantinBaCell” (Oct 2009-June 2013), we have adapted a highly sensitive method called “2-photon scanning number and brightness” (2psN&B) analysis for counting the number of fluorescent protein molecules produced from different inducible promoters in B. subtilis reporter strains, in hundreds of individual cells under both permissive and repressive conditions (Ferguson 2011, Declerck & Royer 2013). In a case study with O. Radulescu (UM2), the stochastic activity of glycolytic and gluconeogenic gene promoters quantified in vivo by 2psN&B could be modeled and related to the repression mechanisms proposed from in vitro studies (Ferguson 2012). We also applied 2psN&B as well as “Raster Imaging Correlation Spectroscopy” (RICS) for monitoring in vivo the change in the oligomeric state and diffusion properties of fluorescent-labeled repressors upon a nutritional switch (manuscript in preparation). Finally, we have also performed in vitro studies under conditions that mimic the cellular environment. In the case of the CggR repressor, this led us to the discovery of a second effector metabolite that, in contrast to the previously identified inducer metabolite (FBP), positively modulates the DNA binding activity of this protein, probably explaining the observed discrepancy between its in vivo and in vitro behavior (manuscript in preparation).
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).