Molecular Microscopy of Membranes

Daniel Levy

Daniel Lévy Chef d'équipe Tel:

Membrane proteins are involved in major cellular processes e.g. cell homeostasis, bioenergetics, cell division and communication. Nearly 25% of genes encode for a membrane protein and these include protein targets for over about 50 % of all drugs in use today.

Figure 1: Research areas of interest and strategies. Our team focuses on the functional and structural analysis of membrane proteins and membrane associated proteins. Their functions are analyzed after purification and reconstitution into proteoliposomes. Their structures are determinate by cryo-electron microscopy and cryo-tomography.
Figure 1: Research areas of interest and strategies. Our team focuses on the functional and structural analysis of membrane proteins and membrane associated proteins. Their functions are analyzed after purification and reconstitution into proteoliposomes. Their structures are determinate by cryo-electron microscopy and cryo-tomography.

Their knowledge at the molecular level that is needed for the conception of new pharmacological tools lacks far beyond those of the cytoplasmic proteins. This is due to their amphiphilic character that complicates their handling from the overexpression, the purification to the structural analysis. Our team combines membrane biochemistry, physico-chemistry and cell biology to tackle specific biological questions involving transmembrane or membrane bound proteins. Our specificity is to develop new biomimetic membrane systems that are further used for the analysis of membrane proteins in a native like-environment (1,2). Our favorite tool is cryo-electron microscopy and cryo-tomography for building 3D models of proteins in their membrane environment. The membrane proteins under studies are involved in cellular multidrug resistance and detoxification and in cell division and multicellularisation.

Our most significant projects and findings over the past few years include:

New methods of reconstitution of membrane proteins in lipidic membrane

Following the pioneered work of the team of J.L.Rigaud on the reconstitution of membrane proteins in liposomes (A), we developed new membrane systems with transmembrane and/or membrane associated proteins for functional or structural studies: – reconstitution of membrane proteins in Giant Unilamellar Vesicles (C); 2D crystallization by the lipid layer; Reconstitution in planar lipid bilayer for AFM analysis (B). The main interest of these last two methods is to decrease to the picomole level the amount of proteins needed for the structural analysis and thus to get access to human membrane proteins that are poorly expressed.

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Multidrugs Resistant transporters

ABC (ATP-binding cassette) transporters are membrane transporters that hydrolyse ATP for the transmembrane transport of various xenobiotics and the cell detoxification. Several ABC’s confers a multidrug resistance phenotype (MDR) to bacteria against antibiotics and to human against drugs use in anticancer treatments. Our project includes the structural analysis of bacterial homolog and human MDR transporters involved in the transport of anticancer drugs with the final aim to contribute to the conception of new inhibitors. We recently described the molecular architecture of ABCG2 (Breast Cancer Resistance Protein), a human ABC transporter,  of BmrA and of BmrC/BmrD, MDR transporters homologs to the human Pgp and MRP1, respectively .levy3

Septins

Septins are cytoskeletal filamentous proteins which are bound to the inner cell membrane and are involved in membrane remodeling processes (constriction, invagination). Septins are multi-tasking proteins and have a prominent role in cell division, neuron morphogenesis, bacterial invasion, cell motility, membrane rigidity. This “so called” fourth cytoskeletal member can make scaffolds to recruit other factors and is implicated in establishing diffusion barriers between cellular compartments. Septins interact specifically with phosphoinositides. As opposed to other cytoskeletal proteins (actin, tubulin) septins polymerize in a non-polar fashion into paired filaments. Septins further self-assemble into variable organizations (rods, filaments, rings, gauzes) both in vitro and in situ (1-3), most likely depending on the proteic content and the post-translational modification state within the septin complexes.

We focus our interest in understanding how septins from distinct organisms interact with specific partners: membrane and proteins (cytoskeletal proteins (actin) and transmembrane proteins). To this end we are using a set of complementary microscopy methods (cryo-electron microscopy, fluorescence microcopy and atomic force microscopy).

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  1. Bertin et al. (2012), Molecular Biology of the Cell, 23(3), 423-432.
  2. Bertin et al. (2010), J. Mol. Biol., 404(4), 711-31.
    A.Bertin et al. (2008), Proc.Natl. Acac. Sci USA, 105, 8274-8279

 

Cryo-electron microscopy and cryo tomography of biomimetic systems/PICT IBISA

Within the PICT-IBISA of Cell imaging and in collaboration, we also analyse different biomimetic systems by cryo-electron microscopy (see Publications).

Key publications

Year of publication 2016

Aurélie Bertin, Eva Nogales (2016 Jan 15)

Characterization of Septin Ultrastructure in Budding Yeast Using Electron Tomography.

Methods in molecular biology (Clifton, N.J.) : 113-23 : DOI : 10.1007/978-1-4939-3145-3_9

Year of publication 2014

Guillaume van Niel, Ptissam Bergam, Aurelie Di Cicco, Ilse Hurbain, Alessandra Lo Cicero, Florent Dingli, Roberta Palmulli, Cecile Fort, Marie Claude Potier, Leon J Schurgers, Damarys Loew, Daniel Levy, Graça Raposo (2014 Nov 13)

Apolipoprotein E Regulates Amyloid Formation within Endosomes of Pigment Cells.

Cell reports : 43-51 : DOI : 10.1016/j.celrep.2015.08.057
Lin Jia, Di Cui, Jérôme Bignon, Aurelie Di Cicco, Joanna Wdzieczak-Bakala, Jianmiao Liu, Min-Hui Li (2014 May 19)

Reduction-responsive cholesterol-based block copolymer vesicles for drug delivery.

Biomacromolecules : 2206-17 : DOI : 10.1021/bm5003569
Ayako Yamada, Alexandre Mamane, Jonathan Lee-Tin-Wah, Aurélie Di Cicco, Coline Prévost, Daniel Lévy, Jean-François Joanny, Evelyne Coudrier, Patricia Bassereau (2014 Apr 7)

Catch-bond behaviour facilitates membrane tubulation by non-processive myosin 1b.

Nature communications : 3624 : DOI : 10.1038/ncomms4624

Year of publication 2013

Pierre Frederic Fribourg, Mohamed Chami, Carlos Oscar S Sorzano, Francesca Gubellini, Roberto Marabini, Sergio Marco, Jean-Michel Jault, Daniel Lévy (2013 Aug 7)

3D cryo-electron reconstruction of BmrA, a bacterial multidrug ABC transporter in an inward-facing conformation and in a lipidic environment.

Journal of molecular biology : 2059-69 : DOI : 10.1016/j.jmb.2014.03.002
Manos Mavrakis, Yannick Azou-Gros, Feng-Ching Tsai, José Alvarado, Aurélie Bertin, Francois Iv, Alla Kress, Sophie Brasselet, Gijsje H Koenderink, Thomas Lecuit (2013 Jun 10)

Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles.

Nature cell biology : 322-34 : DOI : 10.1038/ncb2921
All publications