UMR168 – Physico-Chimie Curie Lab

Team Publications

Year of 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 Learn more

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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 Learn more

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.

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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 Learn more

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.

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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 Learn more

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.

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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)

Nanoscale architecture of a VAP-A-OSBP tethering complex at membrane contact sites

Nature Communications : DOI : 10.1038/s41467-021-23799-1 Learn more

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.

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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 Learn more

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.

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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

Soft Matter : DOI : 10.1039/D0SM01573C Learn more

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.

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Year of 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 Learn more

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 structural and regulatory role. Overall, our work shed light on the allosteric mechanism of transcription employed by this GntR repressor

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R G Alonso, M Tobin, P Martin, A J Hudspeth (2020 Nov 17)

Fast recovery of disrupted tip links induced by mechanical displacement of hair bundles.

Proceedings of the National Academy of Sciences of the United States of America : 30722-30727 : DOI : 10.1073/pnas.2016858117 Learn more

Hearing and balance rely on the capacity of mechanically sensitive hair bundles to transduce vibrations into electrical signals that are forwarded to the brain. Hair bundles possess tip links that interconnect the mechanosensitive stereocilia and convey force to the transduction channels. A dimer of dimers, each of these links comprises two molecules of protocadherin 15 (PCDH15) joined to two of cadherin 23 (CDH23). The “handshake” that conjoins the four molecules can be disrupted in vivo by intense stimulation and in vitro by exposure to Ca chelators. Using hair bundles from the rat’s cochlea and the bullfrog’s sacculus, we observed that extensive recovery of mechanoelectrical transduction, hair bundle stiffness, and spontaneous bundle oscillation can occur within seconds after Ca chelation, especially if hair bundles are deflected toward their short edges. Investigating the phenomenon in a two-compartment ionic environment that mimics natural conditions, we combined iontophoretic application of a Ca chelator to selectively disrupt the tip links of individual frog hair bundles with displacement clamping to control hair bundle motion and measure forces. Our observations suggest that, after the normal Ca concentration has been restored, mechanical stimulation facilitates the reconstitution of functional tip links.

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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 Learn more

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.

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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 Learn more

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
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.

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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 Learn more

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.

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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 Learn more

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 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.

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Year of publication 2019

Rumiana Dimova, Carlos Marques M. Garten, D. Lévy, P. Bassereau (2019 Oct 7)

The Giant Vesicle Book

The Giant Vesicle Book : 37-51 Learn more

Giant vesicles are widely used as a model membrane system, both for basic biological systems and for their promising applications in the development of smart materials and cell mimetics, as well as in driving new technologies in synthetic biology and for the cosmetics and pharmaceutical industry. The reader is guided to use giant vesicles, from the formation of simple membrane platforms to advanced membrane and cell system models. It also includes fundamentals for understanding lipid or polymer membrane structure, properties and behavior. Every chapter includes ideas for further applications and discussions on the implications of the observed phenomena towards understanding membrane-related processes. The Giant Vesicle Book is meant to be a road companion, a trusted guide for those making their first steps in this field as well as a source of information required by experts. Key Features • A complete summary of the field, covering fundamental concepts, practical methods, core theory, and the most promising applications • A start-up package of theoretical and experimental information for newcomers in the field • Extensive protocols for establishing the required preparations and assays • Tips and instructions for carefully performing and interpreting measurements with giant vesicles or for observing them, including pitfalls • Approaches developed for investigating giant vesicles as well as brief overviews of previous studies implementing the described techniques • Handy tables with data and structures for ready reference

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Attner MA*, Keil W*, Benavidez JM, Greenwald I (2019 Sep 23)

HLH-2/E2A Expression Links Stochastic and Deterministic Elements of a Cell Fate Decision during C. elegans Gonadogenesis

Current Biology : 29 : 1-7 : DOI : Learn more

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