Structure and Dynamics of Nucleoproteic and Membrane Assemblies

Structure and Dynamics of Nucleoproteic Complexes

Our projects aim at bringing a quantitative and integrative view on molecular mechanisms of transcription regulation in bacteria, combining in vivo and in vitro approaches.

 


Prokaryotic transcription termination

N. Declerck, E. Margeat, C. Clerté, R. Vishwakarma

This work focuses on the two mechanisms used by bacteria to terminate transcription of a gene (intrinsic termination and Rho-dependent termination.

We use a combination of single molecule observation (smFRET, SPT-PALM & correlation spectroscopies), nano-manipulation and structural biology to study the dynamics events that lead to transcription termination in bacteria. Specifically, our research focuses on:

1. LicT/SacY antitermination proteins from Bacillus subtilis, that prevent the premature arrest of transcription by binding to an antiterminator RNA hairpin that overlaps an intrinsic terminator.
2. The transcription terminator Rho, an ATP-fueled hexameric helicase and translocase, implicated in the dissociation of transcription elongation complexes.

lict1

Cartoon representation of the LicT antitermination protein dimer, bound to its target RNA hairpin that has been labeled with a pair of fluorophores to monitor its conformational dynamics in a single pair FRET experiment.

Collaborations: H. Demene, F. Pedaci, M. Nollmann (CBS), M. Boudvillain (CBM, Orleans), N. Figueroa-Bossi (I2BC, Gif/Yvette), K. Brodolin (IRIM, Montpellier)

 


Bacterial adaptation to environmental changes

N. Declerck, A. Bourges, C. Clerté

Our aim is to unravel the molecular mechanisms that underlie the adaptation of bacteria to rapid changes in their environment, from the atomic and single molecule resolution to the level of single cells and bacterial populations.


We particularly focus on the control of glycolysis/gluconeogenesis by the CggR and CcpN repressors from B. subtilis. We use highly sensitive and quantitative fluorescence fluctuation-based microscopy methods to measure directly the intracellular concentration, oligomerization state and diffusion properties of these regulatory proteins in live bacterial cells. Combined with in vitro experimental and modelling approaches, our studies provide an integrated picture of the adaptive response to a nutritional shift. We apply similar approaches for characterizing the Mrr endonuclease responsible for the activation of the SOS response in E. coli cells exposed to high pressure.

declerck2

Live cell imaging of a B. subtilis strain expressing a PccgR-GFP transcriptional fusion under repression on malate.

Collaborations: S. Aymerich (INRA, Jouy-en-Josas), C. Royer (RPI, NY), O. Radulescu (UM, Montpellier)

 


DNA nano-engineering

G. Bellot, E. Margeat, N. Aissaoui, A. Mills

We use DNA self-assembly methods that offer 3D-shape control at nanoscale resolution and programmable actuation. Our aim is to develop new bio-inspired intelligent systems to help solve problems of fundamental and medical interest.


DNA nanotechnology. DNA origami represents a major landmark as the first method to self-assemble megadalton scale nanostructures with arbitrarily-defined morphology. Our main objective is to explore new design principles for self-assembling molecular architectures for de novo fabrication of high-performing materials.


Membrane proteins. The understanding of the molecular mechanisms of the cell membrane requires new experimental tools at the nanometre level. We aim to develop DNA nanostructures to facilitate structural studies of membrane proteins by cryo-EM and single-molecule fluorescence microscopy.


Bio-inspired nanomechanics. Inspired by the biomechanical features of protein assemblies, our goal is to build nanometer-sized structures that exhibit controllable motions and functions. These man-made nanodevices can be used as sensors that respond to specific stimuli and may find applications in areas such as biosensing, biophysics and on-demand drug delivery.

 

bellot

DNA self-assembly of 3D nanostructures from 1.5 million of nucleobases.

Collaborations: Y. Ke, (Georgia Tech, USA), S. Granier (IGF, Montpellier), S. Bidault, (ESPCI, Paris).

Structure and Dynamics of Membrane Assemblies

We explore the structure and dynamics of membrane assemblies, including mechanical properties, membrane proteins structure and interaction, and membrane remodeling mechanisms, on living cells or artificial membrane models, using single molecule experimental approaches such as atomic force (AFM) and fluorescence/super resolution microscopies.

 


Remodeling and partition of membrane components

L. Costa, P.E. Milhiet, C. Bénistant, P. Dosset, C. Doucet, C. Godefroy, L. Fernandez

We are interested in the molecular mechanisms of lipid-protein interaction within membranes and their spatiotemporal recruitment in different contexts, especially cancer and infection.

Structure and dynamics of tetraspanins. Tetraspanins are transmembrane proteins forming a network of protein-protein-lipid interactions at the plasma membrane of eukaryotic cells. They are involved in numerous cell functions and associated with several diseases. Using single molecule fluorescence microscopy and AFM we are exploring tetraspanin lateral segregation into micro- or nanodomains. We are especially investigating the dynamics and partitioning of the metastasis suppressor CD82 and its rela tionship to cell migration, and the role of tetraspanins CD9 and CD81 in cell infection by HIV-1 and Influenza viruses.

pem1

Actin and paxillin labeling of micropatterned cells expressing CD82

Septin organization during membrane remodeling. Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. Their ability to form functional filaments depends on their binding to PIP2 lipids. Using supported lipid bilayers observed with high-speed AFM and correlative AFM-fluorescence microscopy, we explore at the meso and nanoscale how these proteins polymerize, recruit lipids and deform biological membranes.

pem2

AFM micrograph of yeast septin imaged in air

Collaborations: E. Rubinstein (Villejuif), D. Levy (Institut Curie), F. Berditchevski (Birmingham, Royaume-Uni), O. Piétrement (Institut Gustave Roussy), G. Drin (IPMC, Nice), M. Mougel (IRIM, Montpellier), Sanofi-Pasteur.

 


Nanomechanics of cancer metastasis

C. Bénistant, L. Costa, C. Godefroy, P.E. Milhiet

Physical properties of the cancer cell microenvironment play important roles in promoting cell invasive migration phenotypes and can constitute an obstacle for therapy accessibility. We aim at analyzing how biomechanics impact the formation and progression of cancer by measuring physical properties of cancer cells and tissues, using AFM (force spectroscopy and force mapping).

Mechanical properties of colorectal cancers in transition to metastasis. We measure and correlate the rigidity (Young Modulus) of a bank of colorectal cancer tissues with genomic and patient's survival data, in collaboration with clinicians of the Montpellier Cancer Research Center.
CD82 metastasis suppressor functions. CD82 regulates several membrane remodeling events such as adhesion, migration, endocytosis... by unknown mechanisms. As membrane organizers, tetraspanins could influence membrane nanomechanics. We test the influence of CD82 on membrane tension, a key parameter in membrane remodeling.

Collaborations: E. Crapez and A. Turtoi (IRCM, Montpellier)

 


Mechanisms of nuclear pore complex assembly

C. Doucet, L. Costa, P. Dosset, P.E. Milhiet, A. Vial

Nuclear Pore Complexes (NPCs) are the only gateways between the nucleus and cytoplasm. In dividing cells, the number of NPCs doubles during interphase. New pore insertion in the nuclear envelope requires the local fusion of inner and outer nuclear membranes. This implies exquisite coordination between membrane remodeling and nucleoporins recruitment and assem bly. We aim at understanding the mechanisms involved in early steps of interphase NPC formation, in particular nuclear membrane deformation, following 2 main axes:


1. Identify the sequential involvement of molecular players in membrane deformation. We combine AFM and Single Molecule Localization Microscopy on isolated nuclei, to correlate the recruitment of specific proteins with nuclear membrane deformation and pore assembly.

2. Understand the peculiar deformation of the nuclear envelope. Membrane bending agents suspected to intervene in pore formation induce convex deformations on synthetic liposomes while pore intermediates are concave. We investigate determinants of the pore membrane leading to this discrepancy.

doucet

Correlative AFM and fluorescence microscopy

 


Structural dynamics of single metabotropic glutamate receptors

E. Margeat, C. Clerté, R. Quast

Using a combination of advanced single molecule microscopy methods, we investigate the activation mechanism of G-protein coupled receptors.

Metabotropic glutamate receptors (mGluR) are members of the class C G-protein coupled Receptors (GPCR family). Each subunit is composed of an extracellular domain that binds orthosteric ligands such as glutamate, and a heptahelical transmembrane domain responsible for G-protein activation. We study mGluR structural dynamics by a combination of site-specific labeling, receptor purification and solubilization, and smFRET with multiparametric fluorescence detection (MFD) .

margeat

Cartoon representation of a metabotropic glutamate receptor dimer in a lipid bilayer

Collaborations: J.P. Pin (IGF, Montpellier), C. Seidel (Düsseldorf, Allemagne), D. Levy, M. Dahan (Institut Curie, Paris)

 


 Methodological developments

L. Costa, E. Margeat, P.E. Milhiet, C. Clerté

 Our research requires the development of new methodological approaches in the fields of fluorescence spectroscopy, microscopy and atomic force microscopy, such as:

  • Correlative Atomic Force with super-resolution fluorescence microscopy (STORM and Light Sheet Microscopy (LSM))
  • Non-contact, photonic-based near-field probe microscopy
  • One- and two-photon scanning fluorescence fluctuation microscopies
  • Two-spot correlation spectroscopy coupled with rheometry
  • Photonic structures for high resolution single molecule FRET

Collaborations: M. Nollmann, M. Abkarian (CBS), T. Ando (Kanazawa, Japon), C. Seidel (Düsseldorf, Allemagne), J. Wenger (Institut Fresnel, Marseille), E. Lesniewska (Institut Carnot, Dijon)

 

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