Molecular Microscopy of Membranes
UMR168 – Physico Chimie Curie Lab
Daniel Lévy
Team Leader
[email protected]
Tel: +33 1 56 24 67 82
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.
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.
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
Our most significant projects and findings over the
past few years include:
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 1
Molecular Microscopy of Membranes
UMR168 – Physico Chimie Curie Lab
cryo-tomography.
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.
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 .
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 2
Molecular Microscopy of Membranes
UMR168 – Physico Chimie Curie Lab
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 multitasking 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 posttranslational 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).
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 3
Molecular Microscopy of Membranes
UMR168 – Physico Chimie Curie Lab
432. Bertin et al. (2012), Molecular Biology of the Cell, 23(3), 423-432.
433. 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).
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Molecular Microscopy of Membranes
UMR168 – Physico Chimie Curie Lab
Key publications
Year of publication 2016
A Bertin, E Nogales (2016 Jul 31)
Preparing recombinant yeast septins and their analysis by electron microscopy.
Methods in cell biology : 21-34 : DOI : 10.1016/bs.mcb.2016.03.010
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
Bibiana Peralta, David Gil-Carton, Daniel Castaño-Díez, Aurelie Bertin, Claire Boulogne, Hanna M
Oksanen, Dennis H Bamford, Nicola G A Abrescia (2013 Oct 3)
Mechanism of membranous tunnelling nanotube formation in viral genome
delivery.
PLoS biology : e1001667 : DOI : 10.1371/journal.pbio.1001667
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Molecular Microscopy of Membranes
UMR168 – Physico Chimie Curie Lab
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 6