Publications de l'équipe - Microscopie Moléculaire des Membranes (MMM) - Centre de Recherche Institut Curie
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Publications de l’équipe
Microscopie Moléculaire des Membranes (MMM)
Année de publication : 2021
Estelle Dransart, Aurélie Di Cicco, Ahmed El Marjou, Daniel Lévy, Staffan Johansson, Ludger
Johannes, Massiullah Shafaq-Zadah (2021 Jul 14)
Physiological alpha5beta1 integrin transmembrane protein extraction,
purification and reconstitution into proteo-lipidic nanodiscs bilayer
Methods in Molecular Biology on Heterologous Expression of Membrane ProteinsMethods in
Molecular Biology on Heterologous Expression of Membrane Proteins
Résumé
Iv François, Martins C., Castro-Linares G., Taveneau C., Barbier P., Verdier-Pinard P., Camoin L.,
Audebert S., Tsai F.C., Ramond L., Llewellyn A., Belhabib M., Nakazawa K., Di Cicco A., Vincentelli
R., Wenger J., Cabantous S., Koenderink G*. H., Bertin A*., Mavrakis M*. (2021 Jul 8)
Insights into animal septins using recombinant human septin octamers with
distinct SEPT9 isoforms
Journal of Cell Science : DOI : 10.1242/jcs.258484
Résumé
Septin GTP-binding proteins contribute essential biological functions that range from the
establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form
hetero-octameric septin complexes containing the ubiquitously expressed SEPT9. Despite
the established role of SEPT9 in mammalian development and human pathophysiology,
biochemical and biophysical studies have relied on monomeric SEPT9 thus not recapitulating
its native assembly into hetero-octameric complexes. We established a protocol that enabled
the first-time isolation of recombinant human septin octamers containing distinct SEPT9
isoforms. A combination of biochemical and biophysical assays confirmed the octameric
nature of the isolated complexes in solution. Reconstitution studies showed that octamers
with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind
and cross-link actin filaments, raising the possibility that septin-decorated actin structures in
cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will
make it possible to design cell-free assays to dissect the complex interactions of septins with
cell membranes and the actin/microtubule cytoskeleton.
Vial Anthony, Taveneau Cyntia, Costa Luca , Chauvin Brieuc , Nasrallah Hussein , Godefroy
Cédric, Dosset Patrice , Isambert Hervé , Ngo Kien Xuan, Mangenot Stéphanie , Levy Daniel ,
Bertin Aurélie* , Milhiet Pierre-Emmanuel * (2021 Jun 29)
Correlative AFM and fluorescence imaging demonstrate nanoscale membrane
remodeling and ring-like and tubular structure formation by septins
Nanoscale : DOI : 10.1039/D1NR01978C
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 1Publications de l’équipe
Microscopie Moléculaire des Membranes (MMM)
Résumé
Septins are ubiquitous cytoskeletal filaments that interact with the inner plasma membrane
and are
essential for cell division in eukaryotes. In cellular contexts, septins are often localized at
micrometric
gaussian curvatures, where they assemble onto ring-like structures. The behavior of budding
yeast
septins depends on their specific interaction with inositol phospholipids, enriched at the inner
leaflet of
the plasma membrane. Septin filaments are built from the non-polar self-assembly of short
rods into
filaments. However, the molecular mechanisms regulating the interplay with the inner
plasma
membrane and the resulting interaction with specific curvatures are not fully understood. In
this report,
we have imaged dynamical molecular assemblies of budding yeast septins on PIP2-
containing
supported lipid bilayers using a combination of high-speed AFM and correlative AFM-
fluorescence
microscopy. Our results clearly demonstrate that septins are able to bind to flat supported
lipid bilayers
and thereafter induce the remodeling of membranes. Short septin rods (octamers subunits)
can indeed
destabilize supported lipid bilayers and reshape the membrane to form 3D structures such as
rings and
tubes, demonstrating that long filaments are not necessary for septin-induced membrane
buckling.
Daniel Lévy, Aurélie Di Cicco, Aurélie Bertin, Manuela Dezi (2021 Jun 7)
[Cryo-electron microcopy for a new vision of the cell and its components]
Medecine/Sciences : 379-385 : DOI : 10.1051/medsci/2021034
Résumé
Cryo-electron microscopy (cryo-EM) is a technique for imaging biological samples that plays
a central role in structural biology, with high impact on research fields such as cell and
developmental biology, bioinformatics, cell physics and applied mathematics. It allows the
determination of structures of purified proteins within cells. This review describes the main
recent advances in cryo-EM, illustrated by examples of proteins of biomedical interest, and
the avenues for future development.
Eugenio de la Mora, Manuela Dezi, Aurélie Di Cicco, Joëlle Bigay, Romain Gautier, John Manzi,
Joël Polidori, Daniel Castaño Díez, Bruno Mesmin, Bruno Antonny, Daniel Lévy. (2021 Jun 7)
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 2Publications de l’équipe
Microscopie Moléculaire des Membranes (MMM)
Nanoscale architecture of a VAP-A-OSBP tethering complex at membrane
contact sites
Nature Communications : DOI : 10.1038/s41467-021-23799-1
Résumé
Membrane contact sites (MCS) are subcellular regions where two organelles appose their
membranes to exchange small molecules, including lipids. Structural information on how
proteins form MCS is scarce. We designed an in vitro MCS with two membranes and a pair of
tethering proteins suitable for cryo-tomography analysis. It includes VAP-A, an ER
transmembrane protein interacting with a myriad of cytosolic proteins, and oxysterol-binding
protein (OSBP), a lipid transfer protein that transports cholesterol from the ER to the trans
Golgi network. We show that VAP-A is a highly flexible protein, allowing formation of MCS of
variable intermembrane distance. The tethering part of OSBP contains a central, dimeric, and
helical T-shape region. We propose that the molecular flexibility of VAP-A enables the
recruitment of partners of different sizes within MCS of adjustable thickness, whereas the T
geometry of the OSBP dimer facilitates the movement of the two lipid-transfer domains
between membranes.
Szuba Agata, Bano Fouzia, Castro Linares Gerard , Iv Francois, Mavrakis Manos*, Richter Ralf P*,
Bertin Aurélie*, Koenderink Gijsje H* (2021 Apr 13)
Membrane binding controls ordered self-assembly of animal septins
eLifeeLife : eLife 2021;10:e63349 : DOI : 10.7554/eLife.63349
Résumé
Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The
mechanisms of their interactions with the plasma membrane remain poorly understood. Here
we show by cell-free reconstitution that binding to flat lipid membranes requires electrostatic
interactions of septins with anionic lipids and promotes the ordered self-assembly of fly
septins into filamentous meshworks. Transmission electron microscopy reveals that both fly
and mammalian septin hexamers form arrays of single and paired filaments. Atomic force
microscopy and quartz crystal microbalance demonstrate that the fly filaments form
mechanically rigid, 12 to 18 nm thick, double layers of septins. By contrast, C-terminally
truncated septin mutants form 4 nm thin monolayers, indicating that stacking requires the C-
terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is
required for fly septins to form ordered arrays of single and paired filaments and provides
new insights into the mechanisms by which septins may regulate cell surface mechanics.
Tsai Feng-Ching, Simunovic Mijo, Sorre Benoit , Bertin Aurélie, Manzi John, Callan-Jones Andrew,
Bassereau Patricia (2021 Apr 6)
Comparing physical mechanisms for membrane curvature-driven sorting of BAR-
domain proteins
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 3Publications de l’équipe
Microscopie Moléculaire des Membranes (MMM)
Soft Matter : DOI : 10.1039/D0SM01573C
Résumé
Protein enrichment at specific membrane locations in cells is crucial for many cellular
functions. It is well-recognized that the ability of some proteins to sense membrane
curvature contributes partly to their enrichment in highly curved cellular membranes. In the
past, different theoretical models have been developed to reveal the physical mechanisms
underlying curvature-driven protein sorting. This review aims to provide a detailed discussion
of the two continuous models that are based on the Helfrich elasticity energy, (1) the
spontaneous curvature model and (2) the curvature mismatch model. These two models are
commonly applied to describe experimental observations of protein sorting. We discuss how
they can be used to explain the curvature-induced sorting data of two BAR proteins,
amphiphysin and centaurin. We further discuss how membrane rigidity, and consequently
the membrane curvature generated by BAR proteins, could influence protein organization on
the curved membranes. Finally, we address future directions in extending these models to
describe some cellular phenomena involving protein sorting.
Année de publication : 2020
Armelle Vigouroux, Thibault Meyer Anaïs Naretto, Pierre Legrand, Magali Aumont-Nicaise, Aurélie
Di Cicco, Sébastien Renoud, Jeanne Doré, Daniel Lévy, Ludovic Vial, Céline Lavire, Solange
Moréra (2020 Nov 19)
Characterization of the first tetrameric transcription factor of the GntR
superfamily with allosteric regulation from the bacterial pathogen
Agrobacterium fabrum
Nucleic Acids Reseach : DOI : 10.1093/nar/gkaa1181
Résumé
A species-specific region, denoted SpG8-1b allowing hydroxycinnamic acids (HCAs)
degradation is important for the transition between the two lifestyles (rhizospheric versus
pathogenic) of the plant pathogen Agrobacterium fabrum. Indeed, HCAs can be either use as
trophic resources and/or as induced-virulence molecules. The SpG8-1b region is regulated by
two transcriptional regulators, namely, HcaR (Atu1422) and Atu1419. In contrast to HcaR,
Atu1419 remains so far uncharacterized. The high-resolution crystal structures of two
fortuitous citrate complexes, two DNA complexes and the apoform revealed that the
tetrameric Atu1419 transcriptional regulator belongs to the VanR group of Pfam PF07729
subfamily of the large GntR superfamily. Until now, GntR regulators were described as
dimers. Here, we showed that Atu1419 represses three genes of the HCAs catabolic
pathway. We characterized both the effector and DNA binding sites and identified key
nucleotides in the target palindrome. From promoter activity measurement using defective
gene mutants, structural analysis and gel-shift assays, we propose N5,N10-
methylenetetrahydrofolate as the effector molecule, which is not a direct product/substrate
of the HCA degradation pathway. The Zn2+ ion present in the effector domain has both a
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 4Publications de l’équipe
Microscopie Moléculaire des Membranes (MMM)
structural and regulatory role. Overall, our work shed light on the allosteric mechanism of
transcription employed by this GntR repressor
Pernier Julien, Morchain Antoine, Caorsi Valentina, Bertin Aurélie, Bousquet Hugo, Bassereau
Patricia, Coudrier Evelyne (2020 Sep 7)
Myosin 1b Flattens and Prunes Branched Actin Filaments.
Journal of Cell Science : DOI : 10.1242/jcs.247403
Résumé
Abstract
Motile and morphological cellular processes require a spatially and temporally
coordinated branched actin network that is controlled by the activity of various regulatory
proteins including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously
reported that myosin 1b regulates the density of the actin network in the growth cone. Using
in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we
show in this report that this molecular motor flattens the Arp2/3-dependent actin branches
up to breaking them and reduces the probability to form new branches. This experiment
reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated
actin junction. Together with the former in vivo studies, this work emphasizes the essential
role played by myosins in the architecture and in the dynamics of actin networks in different
cellular regions.
Taveneau Cyntia, Blanc Rémi, Péhau-Arnaudet Gérard, Di Cicco Aurélie, Bertin Aurélie (2020 Jul
29)
Synergistic role of nucleotides and lipids for the self-assembly of Shs1 septin
oligomers
Biochemical Journal : 477 : 2697-2714 : DOI : 10.1042/BCJ20200199
Résumé
Budding yeast septins are essential for cell division and polarity. Septins assemble as
palindromic linear octameric complexes. The function and ultra-structural organization of
septins are finely governed by their molecular polymorphism. In particular, in budding
yeast, the end subunit can stand either as Shs1 or Cdc11. We have dissected, here, for
the first time, the behavior of the Shs1 protomer bound to membranes at nanometer
resolution, in complex with the other septins. Using electron microscopy, we have shown
that on membranes, Shs1 protomers self-assemble into rings, bundles, filaments or
twodimensional
gauzes. Using a set of specific mutants we have demonstrated a synergistic
role of both nucleotides and lipids for the organization and oligomerization of budding
yeast septins. Besides, cryo-electron tomography assays show that vesicles are
deformed by the interaction between Shs1 oligomers and lipids. The Shs1–Shs1 interface
is stabilized by the presence of phosphoinositides, allowing the visualization of micrometric
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 5Publications de l’équipe
Microscopie Moléculaire des Membranes (MMM)
long filaments formed by Shs1 protomers. In addition, molecular modeling experiments
have revealed a potential molecular mechanism regarding the selectivity of septin
subunits for phosphoinositide lipids.
Cao Luyan, Yonis Amina, Vaghela Malti, Barriga Elias, Chugh Priyamvada, Smith Matthew,
Maufront Julien, Lavoie Geneviève, Méant Antoine, Ferber Emma, Bovellan Miia, Alberts Art,
Bertin Aurélie, Mayor Roberto, Paluch Eva, Roux Philippe, Jégou Antoine, Romet-Lemonne
Guillaume, Charras Guillaume (2020 Jun 22)
SPIN90 associates with mDia1 and the Arp2/3 complex to regulate cortical actin
organization
Nature Cell BiologyNature Cell Biology : DOI : 10.1038/s41556-020-0531-y
Résumé
Cell shape is controlled by the submembranous cortex, an actomyosin network mainly
generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in
relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we
investigate how nucleation-promoting factors mediate interactions between nucleators. In
vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments
by Arp2/3, a process thought to provide the initial filament for generation of dendritic
networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in
vitro experiments reveal that this feature stems mainly from two mechanisms: efficient
recruitment of mDia1 to SPIN90–Arp2/3 nucleated filaments and formation of a ternary
SPIN90–Arp2/3–mDia1 complex that greatly enhances filament nucleation. Both mechanisms
yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90–Arp2/3 at
their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the
proportion of long filaments elongated by mDia1.
Aurélie Bertin , Nicola de Franceschi , Eugenio de la Mora , Sourav Maiti, Maryam Alqabandi,
Nolwen Miguet, Aurélie di Cicco, Wouter H. Roos, Stéphanie Mangenot , Winfried Weissenhorn,
Patricia Bassereau (2020 May 29)
Human ESCRT-III polymers assemble on positively curved membranes and
induce helical membrane tube formation
Nature Communications : 11 : 2663 : DOI : 10.1038/s41467-020-16368-5
Résumé
Endosomal sorting complexes for transport-III (ESCRT-III) assemble in vivo onto membranes
with negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization
and how ESCRT-III shapes membranes is yet unclear. Human core ESCRT-III proteins,
CHMP4B, CHMP2A, CHMP2B and CHMP3 are used to address this issue in vitro by combining
membrane nanotube pulling experiments, cryo-electron tomography and AFM. We show that
CHMP4B filaments preferentially bind to flat membranes or to tubes with positive mean
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 6Publications de l’équipe
Microscopie Moléculaire des Membranes (MMM)
curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane
tubes. Combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the
neck of pulled membrane tubes and reshape vesicles into helical “corkscrewlike” membrane
tubes. Sub-tomogram averaging reveals that the ESCRT-III filaments assemble parallel and
locally perpendicular to the tube axis, highlighting the mechanical stresses imposed by
ESCRT-III. Our results underline the versatile membrane remodeling activity of ESCRT-III that
may be a general feature required for cellular membrane remodeling processes.
Johnson Courtney R. , Steingesser Marc G., Khan Anum, Gladfelter Amy, Bertin Aurélie, McMurray
Michael A. (2020 Jan 28)
Guanidine hydrochloride reactivates an ancient septin hetero-oligomer
assembly pathway in budding yeast
eLife : eLife 2020;9:e54355 : DOI : DOI: 10.7554/eLife.54355
Résumé
Septin proteins evolved from ancestral GTPases and co-assemble into hetero-oligomers and
cytoskeletal filaments. In Saccharomyces cerevisiae, five septins comprise two species of
hetero-octamers, Cdc11/Shs1–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11/Shs1. Slow
GTPase activity by Cdc12 directs the choice of incorporation of Cdc11 vs Shs1, but many
septins, including Cdc3, lack GTPase activity. We serendipitously discovered that guanidine
hydrochloride rescues septin function in cdc10 mutants by promoting assembly of non-native
Cdc11/Shs1–Cdc12–Cdc3–Cdc3–Cdc12–Cdc11/Shs1 hexamers. We provide evidence that in S.
cerevisiae Cdc3 guanidinium occupies the site of a ‘missing’ Arg side chain found in other
fungal species where (i) the Cdc3 subunit is an active GTPase and (ii) Cdc10-less hexamers
natively co-exist with octamers. We propose that guanidinium reactivates a latent septin
assembly pathway that was suppressed during fungal evolution in order to restrict assembly
to octamers. Since homodimerization by a GTPase-active human septin also creates
hexamers that exclude Cdc10-like central subunits, our new mechanistic insights likely apply
throughout phylogeny.
Année de publication : 2019
Jamecna D, Polidori DJ, Mesmin B, Dezi M, Lévy D, Bigay J, Antonny B (2019 Mar 22)
An intrinsically disordered region in OSBP acts as an entropic barrier to control
protein dynamics and orientation at membrane contact sites
Developmental cell * : * highlighted Trend in Cell Biology 2019 : DOI :
10.1016/j.devcel.2019.02.021
Résumé
Lipid transfer proteins (LTPs) acting at membrane contact sites (MCS) between the ER and
other organelles contain domains involved in heterotypic (e.g. ER to Golgi) membrane
tethering as well as domains involved in lipid transfer. Here, we show that a long ≈ 90 aa
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 7Publications de l’équipe
Microscopie Moléculaire des Membranes (MMM)
intrinsically unfolded sequence at the N-terminus of oxysterol binding protein (OSBP)
controls OSBP orientation and dynamics at MCS. This Gly-Pro-Ala-rich sequence, whose
hydrodynamic radius is twice as that of folded domains, prevents the two PH domains of the
OSBP dimer from homotypically tethering two Golgi-like membranes and considerably
facilitates OSBP in-plane diffusion and recycling at MCS. Although quite distant in sequence,
the N-terminus of OSBP-related protein-4 (ORP4) has similar effects. We propose that N-
terminal sequences of low complexity in ORPs form an entropic barrier that restrains protein
orientation, limits protein density and facilitates protein mobility in the narrow and crowded
MCS environment.
Simon C*, Kusters R*, Caorsi V*, Allard A, Abou-Ghali M, Manzi J, Di Cicco A, Lévy D, Lenz M,
Joanny J-F, Campillo C, Plastino J, Sens P*, Sykes C* (2019 Mar 18)
Actin dynamics drive cell-like membrane deformation
Nature Physics : DOI : 10.1038/s41567-019-0464-1
Résumé
Cell membrane deformations are crucial for proper cell function. Specialized protein
assemblies initiate inward or outward membrane deformations that the cell uses respectively
to uptake external substances or probe the environment. The assembly and dynamics of the
actin cytoskeleton are involved in this process, although their detailed role remains
controversial. We show here that a dynamic, branched actin network is sufficient to initiate
both inward and outward membrane deformation. The polymerization of a dense actin
network at the membrane of liposomes produces inward membrane bending at low tension,
while outward deformations are robustly generated regardless of tension. Our results shed
light on the mechanism cells use to internalize material, both in mammalian cells, where
actin polymerization forces are required when membrane tension is increased, and in yeast,
where those forces are necessary to overcome the opposing turgor pressure. By combining
experimental observations with physical modelling, we propose a mechanism that explains
how membrane tension and the architecture of the actin network regulate cell-like
membrane deformations.
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 8You can also read
