GEM2023

14 Mar 2023, 09:00 → 17 Mar 2023, 20:00 Europe/Paris

Giovanna Fragneto, Christophe Moreau, Anne Martel, Franck Fieschi, Juliette Jouhet, Bruno Demé

The 22nd congress of the French Membrane Group (GEM) will be held for the first time in the French Alps from 14th to 17th March 2023 at the Escandille Village Vacances, Autrans. It is part of a series started in 1987 as GEIMM.

GEM brings together researchers, biologists, biochemists, biophysicists, physicists and chemists interested in biological phenomena associated with membranes at all levels, from the organ to the molecule.

The meeting will focus on the structure, dynamics and function of membranes and will be locally organised by researchers from different institutes situated in Grenoble where several well-known European and national research facilities are located.  

The GEM conference covers the following topics:

• Structural biology
• Host-pathogen interactions
• Nanomedecine
• Computational methods
• Molecular interactions at the membrane surface
• Interaction lipids/polymers/membrane proteins
• Glycobiology

Please note that all abstract submissions received after 15th January 2023 will only be considered as poster submissions

Final registration (and payment) deadline January 31st, 2023.

GEM is a thematic group of the SFB (Societé Francaise de Biophysique).

All participants are highly encouraged to become members of the SFB (Societé Francaise de Biophysique) and GEM in order to attend the GEM conference, in view of the strong financial support of the SFB and the GEM (thesis prize, poster prizes, financial support of the GEM congress, biophysics support at European and National level).

You can become a member for 2023 at the following link  : https://www.sfbiophys.org/adhesion-online.html.

Please choose the option "plein tarif + GEM" for €50 (10€ GEM + €40 SFB)

Please note: Membership is free for students for 2023, but you still need to sign up on the site and choose the "0€" option

We would like to thank the following sponsors for supporting our event:
(for more information on sponsorship please click  on the "Sponsors" tab on the menu on the left hand side)

A special thank you to our Platinum sponsors:

               

                 

 

our Gold sponsors:
 

                  

And our Silver sponsors:
 

                

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Poster-GEM-final improved quality.pdf

Tuesday, 14 March

11:00 Arrivals

12:30 Lunch

Welcome address

Nanomedecine

1 Messenger RNA-based nanomedicines: where are we from now ?

The field of nanomedicine has reached a milestone since the approval of Patisiran the first ever approved siRNA drug designed to treat transthyretin (TTR) amyloidosis and that of Corminaty and Spikevax messenger RNA (mRNA)-based vaccines against COVID-19, both delivered by a lipid nanocarrier. Nucleic acids are now considered as real hope but not only hype to cure unmet medical diseases as well as chronic diseases. As for any types of drugs, nucleic acids-based nanoparticles can be produced using different types of formulations made with polymer, inorganic materials protein/peptide derivatives and lipids. This talk will be focused on mRNA-based formulations mainly those made with lipid- based nanoparticles. Those formulations are quite challenging due to the peculiar nature of mRNA. Others and we have proposed strategies to cross multiple biological barriers including the plasma and intracellular membranes. I will present what is known so far in terms of efficacy of those strategies. Another challenge is to get a targeted delivery which could be reached either by manipulating the lipids composition or using targeting ligands for specific receptors. Issues that we have to face when conducting those two strategies will be discussed. It is becoming clear that we need a multidisciplinary approach to achieve a rational design of nanomedicine. Today, we can apply several available computational methodologies as accelerated modular-orthogonal methodology for formulations design, cell modelling, pharmacokinetic modelling, and computational toxicology associated with relevant high-throughput synthesis and analysis methods.

Speaker: Prof. Chantal PICHON (University of Orléans and Center for Molecular Biophysics CNRS UPR4301)

2 Study of the 2D phase behavior and the supramolecular assemblies of newly synthesized phospholipid-porphyrin conjugates designed to combat bacterial infections

Phospholipid-porphyrin conjugates (PL-Por) are amphiphilic scaffolds that consist of porphyrin derivatives grafted to a lysophosphatidylcholine backbone 1-4. Owing to their structural similarities with phospholipids, several PL-Por conjugates have shown to be able to self-assemble into liposome-like assemblies exhibiting unique photophysical properties compared to their monomeric counterparts. For these reasons PL-Por conjugates are considered nowadays as versatile building blocks to design supramolecular assemblies with multifunctional properties and thus their application in photodynamic therapy, photothermal therapy, photoacoustic imaging and photo-triggerable release properties [1-3]. However, little is known about the impact of their structure on their 2D phase behavior at the air/water interface, their assembling properties, their optical properties as well as on their photothermal and photodynamic activities.

In this work, we synthesized six new PL-Por conjugates exhibiting different alkyl chain lengths in the sn2 position of C16 Lysophosphatidylcholine and linked via peptidic bond to two types of porphyrin derivatives; either pheophorbide-a (PhxLPC) or pyropheophorbide-a (PyrxLPC) 5. By combining a variety of experimental techniques with molecular dynamics simulations, we investigated the 2D phase behavior at the air/water interface, the thermodynamic, the optical properties and the structure of the PL-Por either self-assembled or when incorporated in lipid bilayer membranes which exhibit different fluidity 6. Finally, the photothermal and photodynamic efficiencies of these assemblies were assessed on planktonic bacteria and their biofilms7.

Our results demonstrated that whereas changing the porphyrin moiety controlled the packing of the monolayer and thus the formation of organized domains, the chain length dictated the structure of the formed domains. Finally, all of the conjugates were able to form supramolecular assemblies with bilayers structures and exhibit different photothermal and photodynamic activities against Gram + and Gram - bacterial planktonic cultures and their biofilms depending on their chemical structure of the PL-Por conjugates.

References
[1] J. F. Lovell, C. S. Jin, E. Huynh, H. Jin, C. Kim, J. L. Rubinstein, W. C. Chan, W. Cao, L. V. Wang, G. Zheng Nat. Mater. 2011, 10, 324-332.
[2] J. Massiot, W. Abuillan, O. Konovalov, A. Makky Biochim. Biophys. Acta, Biomembr. 2022, 1864, 183812.
[3] J. Massiot, V. Rosilio, N. Ibrahim, A. Yamamoto, V. Nicolas, O. Konovalov, M. Tanaka, A. Makky Chem. - Eur. J. 2018, 24, 19179-19194.
[4] J. Massiot, V. Rosilio, A. Makky J. Mater. Chem. B. 2019, 7, 1805-1823.
[5] L.-G. Bronstein, P. Cressey, W. Abuillan, O. Konovalov, M. Jankowski, V. Rosilio, A. Makky J Colloid Interf Sci. 2022, 611, 441-450.
[6] L.-G. Bronstein, Á. Tóth, P. Cressey, V. Rosilio, F. Di Meo, A. Makky Nanoscale. 2022, 14, 7387-7407.
[7] P. Cressey, L.-G. Bronstein, R. Benmahmoudi, V. Rosilio, C. Regeard, A. Makky International Journal of Pharmaceutics. 2022, 623, 121915.

Speaker: Ali Makky (Université Paris-Saclay)

3 Small-angle X-ray Scattering on Photo-switchable Lipid Membranes

Light-switchable Azo-PC lipids incorporate a photo-sensitive azobenzene unit in a phosphatidylcholine lipid. Azo-PC undergoes a reversible photo-isomerisation of cis- and trans-state upon irradiation with UV, respectively visible light. The isomerization of such momomers is well understood in good solvents such as methanol. The photo physics of assemblies in water is less well understood and partly inefficient. Here, we use small-angle scattering (SAS) to analyze the structure of Azo-PC membranes in a physiological environment upon switching. We find huge membrane thickness differences of up to 1 nm for suited buffer conditions. Furthermore, high x-ray doses allows to switch Azo-PC into its ground (trans-) state 1.
This way, photolipids enable a novel approach to study and control the properties of membranes. Similar mechanisms exist for photo switchable surfactants, which can stimulate neurons or induce cell lysis in response to isomerisation. Detailed SAS studies by neutrons and x-rays help to establish control of photo-switchable molecules in cell membranes in order to rationalize their potential in nanomedicine, e.g. for membrane perforation, drug release, and cell lysis.
1 SAXS measurements of azobenzene lipid vesicles reveal buffer-dependent photoswitching and quantitative Z->E isomerisation by X-rays, Martina F. Ober, Adrian Müller-Deku, Anna Baptist, Heinz Amenitsch, Oliver Thorn-Seshold, Bert Nickel, Nanophotonics (2022) link

Speaker: Dr Bert Nickel (LMU)

Sponsor presentations

16:00 Coffee Break

Clip Session

Poster Session

Dinner

Wednesday, 15 March

Interaction lipids/polymers/membrane proteins

4 Unraveling the working mechanism of a tumor suppressor lipid

Ceramides attract wide attention as tumor suppressor lipids that can act directly on mitochondria to trigger Bax-mediated cell death. While ceramide engagement in mitochondrial apoptosis is clinically relevant, molecular details of the underlying mechanism are largely unknown. A chemical screen for ceramide binding proteins combined with molecular dynamics simulations and functional studies in cancer cells previously led us to identify the voltage-dependent anion channel VDAC2 as critical effector of ceramide-mediated apoptosis. VDAC residues involved in ceramide binding are also required for mobilizing hexokinase type-I to mitochondria, a potential checkpoint in apoptosis and glycolysis. Our data support a model in which ceramides serve as critical modulators of VDAC-based platforms to control mitochondrial recruitment of pro- and anti-apoptotic machinery. To challenge fundamental aspects of this model, we use molecular dynamics simulations and work towards reconstitution of ceramide-induced apoptotic pore formation in synthetic bilayers. In parallel, we exploit switchable ceramide transfer proteins and mitochondria-specific release of photocaged ceramides in combination with live cell imaging and functional studies. Understanding the molecular principles by which ceramides commit cells to death may facilitate the development of novel strategies to enhance their anti-tumor potential for therapeutic treatment.

Speaker: Prof. Joost Holthuis (Center for Cellular Nanoanalytics Osnabrueck (CellNanOS), University of Osnabrueck | )

5 SANS for membrane proteins

Small angle neutron scattering enables to study the structure of macromolecular complexes in solution, at low resolution. Within last few years new tools dedicated to the study of membrane proteins were developed. An integrated Size exclusion chromatography (SEC, 1) system enables to exchange both the membrane protein detergent for its D2O-invisible counterpart (2, and the buffer for D2O-based low-background buffer.
As a result, when the sample reaches the Neutron measurement cell, only the membrane protein contributes to the scattering signal, which can be analyzed just like SAXS data from soluble protein 3,4.
To go further in this direction, we are developing D2O-invisible nanodiscs using a circularized-solubility enhanced scaffold protein 5 with the goal of studying membrane protein conformation in different lipid environment, and combining with SAXS to image the whole protein-lipid system as well. These new tools will enable to study the conformational impact of specific lipid-protein interaction within a good membrane mimicking environment.

1. Johansen, N. T., Pedersen, M. C., Porcar, L., Martel, A. & Arleth, L. Introducing SEC–SANS for studies of complex self-organized biological systems. Acta Crystallogr D Struct Biol 74, 1178–1191 (2018).
2. Midtgaard, S. R. et al. Invisible detergents for structure determination of membrane proteins by small‐angle neutron scattering. FEBS J 285, 357–371 (2018).
3. Golub, M. et al. “Invisible” Detergents Enable a Reliable Determination of Solution Structures of Native Photosystems by Small-Angle Neutron Scattering. J. Phys. Chem. B 126, 2824–2833 (2022).
4. Johansen, N. T. et al. Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation. eLife 11, e71887 (2022).
5. Johansen, N. T. et al. Circularized and solubility‐enhanced MSP s facilitate simple and high‐yield production of stable nanodiscs for studies of membrane proteins in solution. FEBS J 286, 1734–1751 (2019).

Speaker: Anne Martel (ILL)

6 Membrane permeabilization can be crucially biased by a fusogenic lipid composition: antimicrobial peptides and model membranes

Induced membrane permeabilization or leakage is often taken as an indication for activity of a membrane-active effector molecule, such as antimicrobial peptides (AMPs) or their synthetic mimics. Nevertheless, it is poorly understood, what effect (or combination of effects) is actually required for antimicrobial activity.
What is often missing is the knowledge of the exact leakage mechanism as different mechanisms might be advantageous or irrelevant for a given application.
Here, we illustrate such a leakage mechanism, leaky fusion, that cannot be the explanation of the proven antibacterial activity of the particular example peptide, bacterial membranes are unable to fuse.
We use the self-quenching dye calcein to monitor the vesicle membrane leakage induced by the peptide in commonly used model vesicles, i.e. binary mixtures of anionic and zwitterionic phospholipids. Especially, the ambiguous role of the relatively fusogenic PE-lipids for membrane leakage and fusion is assessed in more detail. With the role of the membrane composition in focus, we point out that leaky fusion is enhanced for the commonly used mixed PG/PE model membranes.
Leakage, both decreases significantly when aggregation and fusion PG/PE vesicles are prevented by PEG-lipids. Furthermore, the mechanism of leakage changes for PG/PC compared to PG/PE.
En route, we discuss the propensity of the model membrane for vesicle aggregation and fusion and illustrate common implications of fusion and aggregation for the reliability of model studies.
In conclusion, choosing the membrane might implicate the effect (here leakage mechanism) that can be observed. In the worst case, as with leaky fusion in PG/PE, this lipid-related effect is not relevant for the application aimed for.

Speaker: Dr Maria Hoernke (Albert-Ludwigs-Universität Freiburg)

7 Spectrin βII suppresses endosomal vesicle trafficking by regulating membrane dynamics

Spectrin is a cytoskeleton protein present in eukaryotic cells, contributing to membrane stability cooperating with actin and ankyrin. Our proteome analysis identified that Spectrin highly associates with Phosphatidylserine (PS) containing vesicles. However, the importance of the interaction between PS and Spectrin remains poorly understand. Therefore, we examined distribution of Spectrin βII, which is the highest expression isoform in HeLa cells. We are found that Spectrin βII localizes on Transferrin-positive endosomes known to have high PS distribution in addition to the plasma membrane. In addition, Spectrin βII deletion affected on the Transferrin transport and endosomal morphology. We provide new insights of Spectrin as regulators of intracellular vesicle trafficking.

Speaker: Mr Daichi Kitagawa (Toyama university, Japan)

Sponsor presentations

10:30 Coffee Break

Molecular interations at the membrane surface

8 Decorated vesicles - Interactions between α-synuclein and lipid membranes

α-Synuclein is a small neuronal protein that associates with lipid membranes. The membrane interactions are believed to be crucial to both its healthy and disease functions. The healthy functions are associated to synaptic plasticity and neurotransmitter release, while for certain conditions, the protein instead aggregates into amyloid fibrils forming so-called Lewy bodies, which are hallmarks for Parkinson’s disease. Here, the amyloid formation can be triggered by the presence of lipid membranes with associated protein.

In this presentation, I will focus on how α-synuclein associates with lipid membranes, and on the consequences of this association. The protein has a non-uniform charge distribution, and the binding is controlled by anisotropic patchy electrostatic interactions. We further show strong cooperativity of α-synuclein binding to lipid membranes, meaning that the affinity of the protein to a membrane is higher where there is already protein bound compared to a bare membrane. This leads to regions at the membrane with high protein density, which also induces membrane deformations. In cases where there is excess free protein in solution, the vesicles decorated with bound protein may trigger α-synuclein amyloid formation.

Speaker: Prof. Emma Sparr (Lund University)

9 Septin oligomerization induces Membrane Remodeling

Septins are now recognized as a novel component of the cytoskeleton. They are highly conserved proteins in eukaryotes, which form oligomers by self-assembling into linear complexes and higher-order structures, including filaments, bundles and rings. The structure and functions of septins are intimately linked to cell membranes through their interaction with phosphatidylinositol phosphate lipids. They participate in a wide spectrum of cellular processes and the regulation of their expression is also associated with many pathologies.

To better understand the molecular mechanisms associated with septins function in membrane remodeling, we used supported lipid bilayers whose composition mimics the inner leaflet of the eukaryotic plasma membrane. We explored the effects of septins on these model membranes using correlative microscopy combining Atomic Force and fluorescence Microscopy, as well as high-speed atomic force microscopy.

Speaker: Dr Pierre-Emmanuel Milhiet (CBS, University of Montpellier)

10 Effect of trodusquemine on the nanomechanical properties of biomimetic neuronal membranes on solid support

Trodusquemine is an aminosterol which has been proposed as potential drug against neurodegenerative disorders. It exploits its protective action through a direct interaction with the cell membrane, resulting in a modulation of the physico-chemical properties of the lipid bilayer (1). Using atomic force microscopy and force spectroscopy we investigated the effect of trodusquemine on the morphological and nanomechanical properties of supported lipid bilayers (SLBs) mimicking neuronal membranes. These biomimetic SLBs exhibit a phase separation between a disordered, fluid phase and an ordered, condensed and thicker phase. The accurate measurement of the thickness of the two lipid phases allowed us to apply a recently introduced model for the calculation of the intrinsic Young's modulus of thin samples, correcting for the influence of the rigid substrate (2). We found that the membrane Young’s modulus increases in the presence of trodusquemine. This increase in mechanical strength could contribute to an increased resistance of the membranes to the toxic action of misfolded protein oligomers. Moreover, the approach used in the analysis of the force spectroscopy data is shown to be very successful in eliminating artifacts in the bilayer Young’s modulus arising from the presence of the solid substrate on which the lipid membrane is assembled.

1. Errico S., et al., Nanoscale 12, 22596-22614 (2020)
2. Chiodini S., et al., Small 2000269, 1-8 (2020)

Speaker: Beatrice Leonardini (Department of Physics, University of Genoa, Genoa, Italy.)

11 The transport activity of Patched and its role in chemotherapy resistance

The Hedgehog receptor Patched regulates Hedgehog signaling by transporting cholesterol. Hedgehog signaling is aberrantly activated and Patched is overexpressed in many recurrent and metastatic cancers. We showed that Patched pumps chemotherapeutic agents such as doxorubicin out of cancer cells using the proton motive force and contributes to chemotherapy resistance of several cancer cell types, and that cells overexpressing Patched at their plasma membrane have persistent (or cancer stem cell) properties.
We identified small molecules which inhibit the doxorubicin efflux activity of Patched and enhance its cytotoxicity on adrenocortical carcinoma and melanoma cells that endogenously overexpress Patched, and thereby mitigates the resistance of these cancer cells to doxorubicin. We also showed that these Patched drug efflux inhibitors enhance the efficacy of kinase inhibitors such as vemurafenib against melanoma cells resistant to the treatment in cellulo and in vivo on xenografts in mice.
Our data suggest that the use of inhibitors of Patched drug efflux in combination with chemotherapy could be a promising therapeutic option to improve chemotherapy efficiency against cancer cells expressing Patched.
- Feliz Morel, Á.J.; Hasanovic, A.; Morin, A.; Prunier, C.; Magnone, V.; Lebrigand, K.; Aouad, A.; Cogoluegnes, S.; Favier, J.; Pasquier, C.; Mus-Veteau, I. Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched. Pharmaceutics 2022, 14, 988. https://doi.org/10.3390/pharmaceutics14050988
- Kovachka S, Malloci G, Simsir M, Ruggerone P, Azoulay S, Mus-Veteau I. Inhibition of the drug efflux activity of Ptch1 as a promising strategy to overcome chemotherapy resistance in cancer cells. Eur J Med Chem. 2022 Jun 5;236:114306. doi: 10.1016/j.ejmech.2022.114306. Epub 2022 Mar 29. PMID: 35421658.
- Durand N, Simsir M, Signetti L, Labbal F, Ballotti R, Mus-Veteau I. Methiothepin Increases Chemotherapy Efficacy against Resistant Melanoma Cells. Molecules. 2021 Mar 26;26(7):1867.
- Kovachka, G. Malloci, A.V. Vargiu, S. Azoulay, I. Mus-Veteau, P. Ruggerone, Molecular insights into the Patched1 drug efflux inhibitory activity of panicein A hydroquinone: a computational study, Phys. Chem. Chem. Phys. 23 (2021) 8013–8022.
- Simsir M and Mus-Veteau I, RND family efflux pumps: from antibioresistance to chemotherapy resistanceSwed J BioSci Res 2020; 1(1): 51 - 61. Review.DOI: https://doi.org/10.51136/sjbsr.2020.51.61
- Simsir M, Broutin I, Mus-Veteau I, Cazals F. Studying dynamics without explicit dynamics: A structure-based study of the export mechanism by AcrB. Proteins. 2020 Sep 22. doi: 10.1002/prot.26012. Epub ahead of print. PMID: 32960482.
- Hasanovic A, Simsir M, Choveau FS, Lalli E, Mus-Veteau I. Astemizole Sensitizes Adrenocortical Carcinoma Cells to Doxorubicin by Inhibiting Patched Drug Efflux Activity. Biomedicines. 2020;8(8):E251. Published 2020 Jul 29.
- Signetti L, Elizarov N, Simsir M, Paquet A, Douguet D, Labbal F, Debayle D, Di Giorgio A, Biou V, Girard C, Duca M, Bretillon L, Bertolotto C, Verrier B, Azoulay S and Mus-Veteau I. Cancers (Basel), 2020, Inhibition Of Patched Drug Efflux Increases Vemurafenib Effectiveness Against Resistant BRAFV600E Melanoma. PMID: 32526884
- Hasanovic A, Mus-Veteau I. (2018) Targeting the Multidrug Transporter Ptch1 Potentiates Chemotherapy Efficiency. Cells. Aug 14;7(8). pii: E107. doi:10.3390/cells7080107. Review. PubMed PMID: 30110910; PubMed Central PMCID:PMC6115939.
- Hasanovic A, Ruggiero C, Jung S, Rapa I, Signetti L, Ben Hadj M, Terzolo M, Beuschlein F, Volante M, Hantel C, Lalli E, Mus-Veteau I. (2018) Targeting the multidrug transporter Patched potentiates chemotherapy efficiency on adrenocortical carcinoma in vitro and in vivo. Int J Cancer. Jul 1;143(1):199-211. doi:10.1002/ijc.31296. Epub 2018 Feb 23. PubMed PMID: 29411361.
- Fiorini L, Tribalat M-A, Sauvard L, Cazareth J, Lalli E, Broutin I, Thomas O P & Mus-Veteau I. Oncotarget, 2015, Natural paniceins from Mediterranean sponge inhibit the multidrug resistance activity of Patched and increase chemotherapy efficiency on melanoma cells. PMID: 26068979

Speaker: Isabelle Mus-Veteau (IPMC, CNRS UMR7275, Sophia Antipolis)

12:40 Lunch

FREE TIME/ACTIVITY

17:00 Break/ Discussion

New scientific communications

12 Les publications à l"heure de la science ouverte

La politique de la science ouverte s’inscrit dans une dynamique internationale. Ses objectifs stimulants sont de développer un écosystème de recherche plus efficace et plus juste, avec une science plus cumulative, plus fortement étayée, plus transparente, plus rapidement transmise et d’accès plus universel. Elle constitue un fort levier pour l’intégrité scientifique et renforce l’éthique.
Les voies éditoriales en accès ouvert se sont diversifiées. La voie dite « gold » (chercheur payeur) a des aspects inéquitables : inégalités entre chercheurs, profits indus à certaines maisons d’édition, multiplication des revues prédatrices allant des douteuses aux frauduleuses. La voie dite « verte » de dépôt sur des plates-formes internet (preprints) offre beaucoup d’avantages mais rencontre des réticences et de toute façon ne fournit pas la validation par les pairs. Certains modèles approchent l’idéal de la voie dite « diamant » (gratuit pour le chercheur et pour le lecteur), toutefois inaccessible. De nombreux autres modèles sans éditeurs privés (par exemple les Epi revues ou les Peer community In) se développent et il y a un appel général à la « bibliodiversité ». Ainsi le modèle de eLife propose une possibilité de commentaires sur les preprints avant leur soumission à la revue par les pairs. D’autres possibilités sont à l’étude pour le post peer review ouvert.
Le traitement des données fait l’objet d’une intense recherche interdisciplinaire, l’objectif étant le respect des principes FAIR (findable, accessible, interoperable, reusable). La nature des données est d’une grande variabilité suivant les domaines et souvent très complexe. Les points de vue divergent toujours les concernant entre les différentes parties prenantes sur les questions de valeur, de droit et d’éthique.
Quelques conseils pourront être formulés pour les chercheurs à propos de la propriété intellectuelle et des droits d’auteur, ainsi que des recommandations pour les évaluateurs de la recherche.
Michèle LEDUC
http://www.lkb.upmc.fr/boseeinsteincondensates/leduc/
Directrice de recherche CNRS émérite au Laboratoire Kastler Brossel à l’ENS
Membre du Comité d’éthique du CNRS (COMETS) de 2012 à 2022
Membre du Conseil Français de l’Intégrité Scientifique (CoFIS)
Directrices des collections Savoirs Actuels (CNRS et EDP Sciences) et Introduction à (EPD Sciences).
Corédactrice en chef de la revue Raison Présente

Speaker: Michèle Leduc (CNRS)

19:30 Dinner

Thursday, 16 March

Structural biology

13 Structural biology in the outer membrane of living bacteria

The outer membrane is a crucial barrier that protects Gram-negative bacteria against harsh environments and restricts cellular entry for antibiotics. It consists of lipopolysaccharides (LPS) and phospholipids and is densely packed with outer membrane proteins. Yet it remains largely unknown how these are organised in the outer membrane, with various indications of non-homogeneous distributions of proteins. We use high-resolution atomic force microscopy (AFM) to study the structure and architecture of the outer membrane organisation on living and metabolically active E. coli at molecular resolution. Considering wild-type and mutant bacteria and overcoming the lack of chemical resolution of AFM by novel labelling methods, we reveal that three different, segregated phases can be distinguished at the E. coli surface: An extensive network of proteins in an imperfect lattice spanning the entire bacterial surface; LPS islands of up to several 10s of nm; and, for disrupted outer membranes, phospholipid patches that represent weak chinks in the bacterial armour and that facilitate entry of antibiotics into the periplasm.

Speaker: Prof. Bart Hoogenboom (University College London)

14 Investigation of the Histidine Residues of the Growth Hormone Secretagogue Receptor by Solid-State NMR

The Growth Hormone Secretagogue Receptor (GHSR), a 366 amino acids Rhodopsin-like G proteins-coupled receptor (GPCRs), is involved in signal transduction into the cells and plays an important role in food intake, glucose metabolism and immune response. Its high constitutive activity of 50%, seldom among GPCRs, makes this receptor a particularly attractive pharmacological target. In the last two years, the publications of crystal and cryo-electron microscopy structures of GHSR in complexes with different agonists, inverse agonist, or antagonist, and different G-proteins (Shiimura et al. 2020; Wang et al. 2021; Liu et al. 2021; Qin et al. 2022) have expended our structural knowledge of the receptor’s different conformations and binding mechanisms greatly. In parallel, spectroscopic techniques such as solid-state NMR supported the biophysical investigations of the dynamic properties of the uniformly (Schrottke et al. 2017) and site specific (transmembrane domains, loops, C-terminus) (Pacull et al. 2020) 13C-labelled GHSR in a native-like membrane environment. We now aim to investigate the conformational changes of GHSR upon ligand or agonist binding on single residue level. Here, we monitor the changes in the local chemical environment of the three native histidines of GHSR as a variation of their 13C NMR chemical shift using 13C-13C DARR NMR, for the wild type GHSR and for a physiological GHSR mutant lacking its characteristic constitutive activity. Besides providing the necessary low spectral complexity, these three histidines are located in sensitive receptor sites, namely in the helix 6, known to undergo large motion upon ligand binding, and the extracellular loop 2, involved in binding events. The histidines were 13C labelled through an established cell-free expression system where the labeled GHSR is expressed in the precipitated form with a yield of up to 1.5 mg per 1 mL reaction volume and subsequently functionally reconstituted into DMPC bilayer membranes. Three single histidine deficient mutants H186A (Ueda et al. 2011), H258Y and H280Q (Feighner et al. 1998) were designed to allow the assignment of the NMR signals and the well-described A204E mutant (Pantel et al. 2006) was expressed to characterize the effect of the loss of constitutive activity. Upon ghrelin binding, the two helical histidine residues showed characteristic downfield shifts indicative of the structural alterations in the molecule upon outward movement of helix 6. In contrast, upon inverse agonist binding, this response was much weaker suggesting a different binding mechanism. Finally, the shifts for the constitutive activity mutant highlight the existence of an alternative conformation that can be rescued by ghrelin binding.

References:
Feighner SD, Howard AD, Prendergast K, Palyha OC, Hreniuk DL, Nargund R, Underwood D, Tata JR, Dean DC, Tan CP, et al. 1998. Structural requirements for the activation of the human growth hormone secretagogue receptor by peptide and nonpeptide secretagogues. Mol Endocrinol. 12:137–145. eng. doi:10.1210/mend.12.1.0051

Liu H, Sun D, Myasnikov A, Damian M, Baneres J-L, Sun J, Zhang C. 2021. Structural basis of human ghrelin receptor signaling by ghrelin and the synthetic agonist ibutamoren. Nat Commun. 12:6410. eng. doi: 10.1038/s41467-021-26735-5

Pacull EM, Sendker F, Bernhard F, Scheidt HA, Schmidt P, Huster D, Krug U. 2020. Integration of cell-free expression and solid-state NMR to investigate the dynamic properties of different sites of the growth hormone secretagogue receptor. Front. Pharmacol. 11:562113. eng. doi:10.3389/fphar.2020.562113

Pantel J, Legendre M, Cabrol S, Hilal L, Hajaji Y, Morisset S, Nivot S, Vie-Luton M-P, Grouselle D, de Kerdanet M, Kadiri A, Epelbaum J, Le Bouc Y, Amselem S. 2006. Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature. J Clin Invest. 116(3):760-8. doi: 10.1172/JCI25303.

Qin J, Cai Y, Xu Z, Ming Q, Ji S-Y, Wu C, Zhang H, Mao C, Shen D-D, Hirata K, Ma Y, Yan W, Zhang Y, Shao Z. 2022. Molecular mechanism of agonism and inverse agonism in ghrelin receptor. Nat Commun. 13: 300. eng. doi:10.1038/s41467-022-27975-9

Schrottke S, Kaiser A, Vortmeier G, Els-Heindl S, Worm D, Bosse M, Schmidt P, Scheidt HA, Beck-Sickinger AG, Huster D. 2017. Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes. Sci Rep. 7:46128. eng. doi:10.1038/srep46128.

Shiimura Y, Horita S, Hamamoto A, Asada H, Hirata K, Tanaka M, Mori K, Uemura T, Kobayashi T, Iwata So, et al. 2020. Structure of an antagonist-bound ghrelin receptor reveals possible ghrelin recognition mode. Nat Commun. 11:4160. eng. doi:10.1038/s41467-020-17554-1

Ueda T, Matsuura B, Miyake T, Furukawa S, Abe M, Hiasa Y, Onji M. 2011. Mutational analysis of predicted extracellular domains of human growth hormone secretagogue receptor 1a. Regul Pept. 166:28–35. eng. doi:10.1016/j.regpep.2010.08.002

Wang Y, Guo S, Zhuang Y, Yun Y, Xu P, He X, Guo J, Yin W, Xu HE, Xie X, Jiang Y. 2021 Molecular recognition of an acyl-peptide hormone and activation of ghrelin receptor. Nat Commun. 12:5064. eng. doi:10.1038/s41467-021-25364-2

Speaker: Ms Emelyne Pacull (University of Leipzig)

15 Solid-state NMR investigation of the synergistic action of magainin antimicrobial peptides

Solid-state NMR investigation of the synergistic action of magainin antimicrobial peptides
Ahmad Saad, Jesus Raya, Elise Glattard, Burkhard Bechinger
Membrane Biophysics and NMR, Institut of Chemistry (UMR-7177), 1, rue Blaise Pascal Universite de Strasbourg/CNRS, 67000 Strasbourg, FRANCE

Abstract:
Magainin and PGLa are cationic, amphipathic antimicrobial peptides isolated from the skin of Xenopus Laevis African frog, known to interfere with the barrier function of the bacterial membrane and thereby cause bacterial killing. They adopt alpha-helical structures in membrane environments and can disrupt the lipid bilayer organization and ordering. When added as a mixture, they show enhanced synergistic activities in both antimicrobial assays as well as biophysical experiments1,2. Several models have been proposed to understand the synergistic behavior between PGLa and magainin 2, however, the mechanism behind this is still elusive. Recently we observed the formation of mesophase arrangements for PGLa and magainin 2 along the membrane surface. Therefore, our objective is to elaborate a high-resolution study at molecular level of the peptide-lipid assemblies to understand the interactions behind these supramolecular structures, and to decipher the mechanism of synergism between the two peptides.
In the present work, we show a structural analysis of the interaction between magainin and a liposome model that mimics bacterial membranes, composed of POPE:POPG (3/1 molar) lipids using MAS solid-state NMR. We study the secondary structure, insertion, and dynamic of the uniformly 13C-15N labeled peptides on specific positions in the lipid bilayer. Experimental chemical shift analysis of 13C indicates an alpha-helical conformation of magainin. While the lack of correlations between peptide and lipid acyl chain signals in HETCOR experiment reveals that the peptide is not inserted in the hydrophobic core of the membrane and relies on the water lipid interface. Furthermore, signal intensities analysis of CP and INEPT experiments show that the peptides undergo isotropic motion in the liquid crystalline phase, while the system becomes more rigid in presence of the equimolar peptides mix.
References:
1. A. Marquette, B. Bechinger ; Biomolecules, 2018, 8, 18
2. B. Bechinger, D. Juhl, E. Glattrad, C. Aisenbrey; Front. Med. Technol., 2020,2,615494

Speaker: Dr Ahmad Saad (University of Strasbourg)

16 Model, structure and function of a bacterial virulence factor

Bacterial virulence factors are essential for pathogens to disrupt host defense mechanisms and survive in the host. Our study focuses on a specific virulence factor, a membrane protein of unknown structure and function, common to several major bacterial pathogens.
First, combining structure prediction using Alphafold and evolutionary trace using Consurf has not only revealed an original fold but also a particular multimery. This peculiar multimeric organization was subsequently confirmed by native mass spectrometry. Cryo-electron microscopy has been finally used to solve the first-ever structure of the full-length protein.
These data revealed, for the first time, an originally channel organization opening new insight on the real function of this virulence factor. Understanding the structure and molecular details to evaluate the dynamic and the function shall guide the design of novel anti-infectious strategies.

Speaker: Charline Mary (CNRS)

17 Molecular-Level Architecture of a Microalgal Cell Wall

A. Poulhazan 1, A. A. Arnold 1, F. Mentink-Vigier 2, T. Wang 3, I. Marcotte 1, D. E. Warschawski 4

1 Department of Chemistry, University of Quebec at Montreal, Montreal, QC, Canada
2 National High Magnetic Field Laboratory, Tallahassee, FL, USA
3 Department of Chemistry, Michigan State University, East Lansing, MI, USA
4 Laboratoire des Biomolécules, CNRS UMR 7203, Sorbonne Université, Paris, France

Keywords:  Microalga, Cell wall, Solid-State NMR, Dynamic Nuclear Polarization

Abstract:
Recent progress in in-cell solid-state NMR (ssNMR) has allowed the in situ structure determination of starch (1), but also order determination of membranes in living bacteria (2) or red blood cell ghosts (3). Studying the cell wall of microorganisms brings new challenges to structural biology, especially since polysaccharides are organized as polymers with no unique sequence or structure.

Precise glycan composition of Chlamydomonas reinhardtii has been determined using mostly ssNMR, following a protocol recently described (4). A similar protocol was applied to the amino-acid composition determination of the microalga. The first Dynamic Nuclear Polarization study of a microalga also allowed us to detect preferential contacts within and between amino acids and glycans.

We further identified a strong heterogeneity in flexibility and hydration, depending on the nature of the glycan or amino-acid, providing hints towards molecular organization and biophysical properties of C. reinhardtii polysaccharides and glycoproteins in the cell wall, that can be compared to extensins in higher plants.

References : 
(1) A. Poulhazan, A. A. Arnold, D. E. Warschawski and I. Marcotte "Unambiguous Ex Situ and in Cell 2D 13C Solid-State NMR Characterization of Starch and Its Constituents" Int. J. Mol. Sci. 19:3817 (2018)
(2) Z. Bouhlel, A. A. Arnold, J.-S. Deschênes, J.-L. Mouget, D. E. Warschawski, R. Tremblay and I. Marcotte "Investigating the action of the microalgal pigment marennine on Vibrio splendidus by in vivo 2H and 31P solid-state NMR" Biochim. Biophys. Acta Biomembr. 1863:183642 (2021)
(3) K. Kumar, M. Sebastiao, A. A. Arnold, S. Bourgault, D. E. Warschawski and I. Marcotte "In situ solid-state NMR study of antimicrobial peptide interactions with erythrocyte membranes" Biophys. J. 121:1512-1524 (2022)
(4) A. Poulhazan, M. C. Dickwella Widanage, A. Muszyński, A. A. Arnold, D. E. Warschawski, P. Azadi, I. Marcotte and T. Wang "Identification and Quantification of Glycans in Whole Cells: Architecture of Microalgal Polysaccharides Described by Solid-State Nuclear Magnetic Resonance" J. Am. Chem. Soc. 143:19374-19388 (2021)

Contact:
dror.warschawski@sorbonne-universite.fr

Speaker: Dror WARSCHAWSKI (CNRS)

10:30 Coffee Break

Host-pathogen interactions

18 Studying Vaccina virus assembly using cellular cryo electron tomography

Poxviruses are large enveloped dsDNA viruses that infect a wide range of eukaryotes and can spread in human populations. Vaccinia virus (VACV) is the type species of the Poxviridae family and serves as a well-established laboratory model to study virus-host interactions. Notably, the complex infectious particles of VACV are enveloped and brick-shaped. On the inside, there are two lateral bodies and the viral core, which encases the viral genome within a proteinaceous core wall. These infectious particles are produced after a complex maturation process of spherical immature particles that involves protein phosphorylation and proteolytic cleavage. The immature particle, in turn, is formed from typical open-ended membrane precursors termed VACV crescents. The mechanism of viral membrane assembly is hence unique for poxviruses and different from budding or envelopment observed for other enveloped viruses. However, fundamental questions remain open in the field, particularly regarding the formation of membrane precursors, the growth of crescents and the maintenance of open membrane ends in the host cytoplasm. In my group, we aim at gaining a better mechanistic understanding of VACV membrane assembly using cellular cryo electron tomography (cryoET) and correlative approaches. We therefore developed an optimized workflow to target the viral assembly sites directly within infected cells with a fluorescently labelled virus (from Michael Way). These sites can hence be identified and then targeted with focused ion beam (FIB)-milling under cryo conditions to produce thin sections for cellular cryoET that contain crescents and other assembly intermediates. With such advanced workflow, we could observe viral assembly intermediates at various stages in the native cellular context and gain access to the structure of viral components with unprecedented details. Altogether, our new findings already provide significant insights into the unique mechanism of VACV membrane assembly and its coordination with viral genome packaging in particular.

Speaker: Emmanuelle Quemin (Department of Virology, Institute for Integrative Biology of the Cell (I2BC), CNRS UMR 9198)

19 Lipoprotein modification enzyme Lgt is an essential integral membrane protein in proteobacteria and a promising target for novel antibiotics.

Nienke Buddelmeijer

Institut Pasteur, Université de Paris, CNRS UMR6047, INSERM U1306, Unité Biologie et Génétique de la Paroi Bactérienne, 25-28 rue du docteur Roux, 75724 Paris cedex 15

The post-translational modification of bacterial lipoproteins is catalyzed by a sequential action of three integral membrane enzymes. Lgt transfers a diacylglyceryl moiety from phospholipid phosphatidylglycerol onto pre-prolipoprotein resulting in the formation of prolipoprotein and glycerol-1-phosphate in the first reaction of the pathway. In proteobacteria this pathway is essential for viability due to the important role of lipoproteins in cell envelope biogenesis and cell shape maintenance. The most abundant protein in E. coli is Lpp, a lipoprotein located in the outer membrane and covalently linked to the cell wall. Mislocalization of Lpp to the cytoplasmic membrane due to inhibition of the lipoprotein modification pathway or depletion of the enzymes leads to cell death when the protein is cross-linked to the cell wall. Small molecules and cyclic peptides have been described as inhibitors targeting lipoprotein modification through signal peptidase II (Lsp) or the lipoprotein outer membrane localization machinery (Lol). Resistance against these compounds is always related to Lpp via compensatory mutations in the promoter or its inability to interact with the cell wall. We showed that Lgt is still essential in the absence of Lpp and that alterations in metabolism occur to compensate for a growth defect when Lgt levels are too low. Lgt is highly conserved in bacteria; even species that lack a cell wall possess the enzyme. The X-ray crystal structure of Lgt from E. coli has been reported and we showed that the overall structure is conserved among Lgt homologues although slight differences could be observed. Our preliminary results from complementation analyses suggest that the periplasmic exposed so-called head domain plays a role in substrate specificity. We hypothesize that Lgt is a promising target for the development of both broad-spectrum and narrow-spectrum novel antibiotics against proteobacteria.

Speaker: Dr Nienke Buddelmeijer (Institut Pasteur)

PhD Prize presentation

PhD Prize presentation

Sponsor presentations

12:40 Lunch

Structural biology

20 The puzzling activity of the antidepressant Vortioxetine at 5-HT3 receptors

Uriel López-Sánchez, Lachlan Munro, Anders S. Kristensen, Jacques Neyton, Guy Schoehn, Hugues Nury.

The serotonin 5-HT3 receptors are pentameric ligand-gated ion channels that play important roles in fast neurotransmission within the central and peripheral nervous systems. They are the targets of therapeutic compounds to treat chemotherapy-induced nausea, and also the targets of the multimodeal antidepressant Vortioxetine. Vortioxetine is an atypical ligand with both agonistic and antagonistic effect at human homomeric receptors while at rodent ones, not agonist response has been shown. The mouse and human 5-HT3A receptor subunits share 85% sequence identity. The differential activity of Vortioxetine those two receptors constitutes a pharmacological oddity. In this study we sought to better understand the molecular determinants for the intriguing properties of Vortioxetine at both mouse and human 5-HT3A receptors using structural and functional analysis.
We firstly determined the structure of vortioxetine in complex with the mouse homomeric 5-HT3A receptor (m5-HT3A) by Cryo-EM. A 3.1 Å resolution 3D reconstruction was obtained showing that the mouse receptor adopts an inhibited conformation, largely similar to the conformations previously determined in the presence of antagonist drugs fighting nausea and emesis. The drug is clearly seen at the five orthosteric sites located at clefts between subunits.
Furthermore, we expressed, purified and imaged the human homomeric 5-HT3A (h5-HT3A) receptors in the presence of vortioxetine. A 3D reconstruction was obtained at 3.8 Å resolution for the whole human receptor solubilized in detergent showing a very well resolved extracellular domain (ECD) but a distorted transmembrane domain (TMD). We replaced the detergent environment by a lipidic one seeking to restore the structure integrity. We obtained a 3.6 Å resolution reconstruction also featuring a distorted TMD, probably an indication of intrinsic fragility of the receptor.
We focused our comparative structural analysis at the level of the extracellular domain, where the drugs bind. Looking specifically at the subunit/subunit organization, we found that all antagonist-bound structures clustered with m5-HT3A receptors while all agonist-bound structures clustered with h5-HT3A receptors. The comparison of both human ECD structures with the mouse one supports the idea of vortioxetine stabilizing an agonist-bound like conformation at the human receptors. In addition, we have identified key amino acids that seems to play an important role in the differential activity of vortioxetine between rodent and human receptors.

Speaker: Hugues Nury

21 Probing the coupled dynamics between lipids and membrane proteins in nanodiscs by high-pressure NMR spectroscopy

Cell membranes represent a complex and variable environment in time and space of lipids and proteins. Their physico-chemical and functional properties are determined by lipid and protein components and their respective interactions. Here, we used NMR spectroscopy under hydrostatic pressure to study the close dynamic relationships between lipids and membrane proteins in ~10 nm nanodiscs. We demonstrate that nanodisc particles can reversibly accommodate high-pressure, in absence or in presence of embedded membrane proteins such as the beta-barrel OmpX porin and the alpha-helical BLT2 G Protein-Coupled Receptor [1]. Yet, the lipid fluid-to-gel phase transition triggered by pressure, as monitored by 1H NMR (Fig 1), is delayed to higher pressure in presence of OmpX or BLT2, suggesting that proteins tend to preserve the fluid nature of their surrounding lipids. Pressure can also modify the conformational landscape of the membrane protein per se. Indeed, we observed a population redistribution in the complex conformational landscape of BLT2 [2], and a dynamic change at the micro-millisecond timescale leading to a dramatic 4-5x NMR signal increase for BLT2. In OmpX, we observed at high pressure a distortion of the H-bond network in the beta-barrel and a specific dynamic change for methyls groups exposed to the lipid bilayer, suggesting concerted motion of methyls surrounded by lipids. The strategy proposed [1] herein opens new perspectives to scrutinize the dynamic interplay between membrane proteins and their surrounding lipids in nanodiscs.

[1] Pozza A, Giraud F, Cece Q, Casiraghi M, Point E, Damian M, Le Bon C, Moncoq K, Banères JL, Lescop E, Catoire LJ. (2022). Exploration of the dynamic interplay between lipids and membrane proteins by hydrostatic pressure. Nat Commun. 13(1):1780. doi: 10.1038/s41467-022-29410-5.
[2] Casiraghi M, Damian M, Lescop E, Point E, Moncoq K, Morellet N, Levy D, Marie J, Guittet E, Banères JL, Catoire LJ. (2016) Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer. J Am Chem Soc. 138(35):11170-5. doi: 10.1021/jacs.6b04432.

Speaker: Ewen Lescop (CNRS)

22 DNA Origami-Based Protein Manipulation Systems : From structural biology to mechanical regulation

The introduction of DNA origami, which uses many staple strands to fold one long scaffold strand into a rationally designed nanostructure has dramatically improved the complexity and scalability of DNA nanostructures with unprecedented capabilities. Our goal is to explore the bottom-up structural DNA origami nanotechnology to build artificial molecular systems and machines sufficiently sophisticated to recapitulate and decipher fundamental aspects of biology. In this presentation, I will first, discuss the DNA origami method. Second, I will introduce the V-shape design as a modular imaging scaffold for single-particle electron microscopy (EM). I will also present our latest progress in constructing a nano-machine that can be programmed to actuate autonomously as a “robot” for the mechanical activation of membrane proteins.

Speaker: Dr Nesrine Aissaoui (Laboratoire CiTCoM, Faculté de Santé, Université Paris Cité, CNRS, Paris, 75006 France)

Clip Session

15:50 Coffee Break

Poster Session

18:00 Poster Prizes and General Assembly

Friday, 17 March

Glycobiology

23 Glycobiology

Speaker: Prof. Hans Wandall (University of Copenhagen, Copenhagen, DK)

Hans WENDELL

24 Use of membrane mimetics to explore complex glycolipids recognition mechanisms at the surface of gram-negative bacteria

The outer membrane of Gram-negative bacteria is compositionally asymmetric with LipoPolySaccharides (LPS) covering most of the outer membrane surface, while phospholipids compose the inner leaflet. LPSs form a highly impermeable barrier and are an important factor in bacterial virulence; their high structural variability and tight assembly protect bacteria against the uptake of antimicrobials and host defences. These complex glycolipids are detected by the immune system: the TLR4 system recognizes the lipid A moiety of LPS, while C-type Lectin Receptors (CLRs) and antibodies interact with the glycan part. They are also exploited by phages, bacteriocins and antibiotics to reach their main target. Glycolipids are difficult molecules to study due to their hydrophobic nature and their arrangment in solution that does not resemble to their natural environment. We have exploited recent advances in the technology of amphiphilic nanodiscs that enable the formation of nanodiscs either from purified LPS or directly from the outer membrane of pathogenic and non-pathogenic bacteria. To validate that the LPS nanodiscs constitute a viable model for the study of structure, dynamics and interactions of LPS, we report their study by several biophysical methods. We have shown that LPS nanodiscs can be formed routinely and form stable objects with relatively good monodispersity. They can be studied at atomic scale by Magic Angle Spinning NMR spectroscopy, and immobilised on different media to study their interaction with proteins and antibiotics by Quartz Crystal microbalance and Biolayer Interferometry. LPS nanodiscs are so far the best platform to study interactions interactions occuring at the cell surface of gram-negative bacteria and open interesting perspectives for the study of multiresistant gram negative bacteria.

Speaker: Cedric LAGURI (CNRS)

25 Photoswitchable azobenzene-glycoconjugates induce bidirectional switching in phospholipid monolayers and vesicles

The role of the lipid environment within a biomembrane is of fundamental interest to understand intercellular and cell-cell interaction. The lipid molecules define not only the cell structure and geometry but also influence the functionality and self-assembly of membrane proteins and channels.[1,2] Dynamic switching behaviour of the membrane associated proteins and channels regulates the transportation of ions and metabolites of immense cellular importance through the lipid membrane into the cell. However, the in-depth molecular mechanisms involved in the switching are poorly understood. Thus, molecular switches in biomembranes are currently under intense investigation owing to their diverse applications in biosensor engineering and drug delivery. To understand the interaction between lipid and functional molecules, we investigate photoswitchable glycoconjugates embedded in a 1,2-dipalmitoyl-phosphatidylcholine (DPPC) model systems in the form of Langmuir films and vesicles. Here, the structure of photoswitchable glycoconjugates change reversibly between their trans- and cis-conformation by illumination with visible and UV light[3] inducing a reversible change in the molecular arrangement within the surrounding membrane[4]. The resulting structural changes, their evolution and time scales are characterised with multiple measurement techniques including absorption spectroscopy, surface pressure-area isotherm, X-ray reflectivity, grazing incident X-ray diffraction and also X-ray and neutron small angle scattering. Studies performed on mixed monolayers with varying glycoconjugates identify an additional phase transition, which unexpectedly represents a crossover point. The surface pressure and monolayer thickness change in opposite directions above and below the phase transition indicate that the photoswitchable glycoconjugates change orientation within the monolayer. In vesicles the switching induces a reversible transition between lamellar and cubic phase at room temperature. Therefore, photoswitchable glycoconjugates can act as a bidirectional switch in DPPC monolayers and vesicles.
In addition, as this work is part of a pilot project within the DAPHNE4NFDI consortium [5], the best practice for scientific data handling to ensure that our data is findable, accessible, interoperable and re-usable (FAIR), will be discussed.

[1] Laganowsky, A., Reading, E., Allison, T. M., Ulmschneider, M. B., Degiacomi, M. T., Baldwin, A. J., Robinson C. V. (2014). Nature 510, 172.
[2]. Phillips, R., Ursell, T., Wiggins, P., Sens, P. (2009). Nature 459, 379.
[3] Reise, F., Warias, J. E., Chatterjee, K., Krekiehn, N. R., Magnussen, O., Murphy, B. M., Lindhorst, T. K. (2018). Chemistry – A European Journal 24, 17497-17505.
[4] Warias, J. E., Reise, F., Hövelmann, S. C., Giri, R. P., Röhrl, M., Kuhn, J., Jacobsen, M., Chatterjee, Arnold, T., Shen, C., Festersen, S., Sartori, A., Jordt, P., Magnussen, O., Lindhorst, T. K., Murphy, B. M. submitted.
[5] DAPHNE4NFDI is a National Research Data Infrastructure consortium funded by the German German Research Foundation (DFG). https://www.daphne4nfdi.de/

Speaker: Ms Svenja C. Hövelmann (Kiel University, Deutsches Elektronen Synchrotron Hamburg)

09:50 Coffee Break

Computational methods

26 Free energy landscape of pore and stalk formation are greatly modified by lipid composition and lipid-protein interactions

Topological transitions of membranes, such as pore formation or membrane fusion, play key roles biophysical processes including exocytosis, viral infection, intracellular trafficking, proton transport, and many others. In biological cells these transitions are carried out by complex, asymmetric membranes composed of thousands of lipid species and crowded by membrane proteins. How the lipid composition and lipid-protein interactions control the free energy landscape of topological transitions is poorly understood.

We have developed computationally efficient methods for screening the effects of lipids and membrane-associated proteins on the energetics of the formation of membrane pores or membrane stalks at atomic or coarse-grained resolution. The simulations reveal that the membrane composition may bias such topological transitions by tens or even hundreds of kilojoule per mole, suggesting that the complexity of membranes may have evolved to control the kinetics of such events. For instance, we found that the inner leaflet of a typical plasma membrane is far more fusogenic than the outer leaflet, which may be an adaptation for allowing efficient exocytosis while resisting viral infection. Among our recent work, the calculations provide a critical view on widely accepted continuum models for membrane electroporation, and they reveal how membrane anchors of fusion proteins facilitate membrane fusion.

Speaker: Jochen HUB

27 Birth of an organelle, in silico: molecular mechanism of lipid droplet budding

Lipid droplets (LDs) are organelles regulating lipid storage and metabolism in cells. LD biogenesis takes place mostly in the endoplasmic reticulum (ER), and starts with the synthesis of neutral lipids, such as triglycerides. When the concentration of neutral lipids reaches a certain threshold, oil droplets form by phase-separation, yielding a lens-shaped nascent lipid droplet – a process known as nucleation. As more neutral lipids are synthesized, the lens grows and eventually buds out of the ER membrane, generally towards the cytosol, at sites marked by a specific protein named seipin. Several open questions remain regarding the mechanism of LD budding, because the initial steps of LD biogenesis are extremely difficult to observe experimentally. Notably, the roles of ER topology, leaflet asymmetry, and membrane composition in the budding mechanism are substantially unknown.
Here we develop a new, simple methodology allowing to simulate the generation of lipid droplets in membranes mimicking the topology and composition of the ER, and we explore possible mechanisms of LD budding. Molecular dynamics simulations at the coarse-grained level are performed out of equilibrium, as in real life experiments. First, we build ER tubular membranes with realistic size and different compositions, including a realistic ER composition, then we progressively increase the number of triglyceride molecules in the system to mimic the effect of triglyceride synthesis and induce budding. We explore different possibilities for the driving forces of the budding process, including LD volume and ER leaflet asymmetry, and observe LD budding with different mechanisms depending on the specific conditions imposed on the system. The simulations allow us to make prediction on the role of seipin, the localization of phospholipid synthesis, and the stability of the ER-LD connection. Our methodology pushes the limit of biological simulations allowing, for the first time, simulations of organelle biogenesis.

Speaker: Dr Luca Monticelli (MMSB, CNRS)

28 Small-Angle Neutron Scattering of a Pentameric Ligand-Gated Ion Channel Reveals a Dynamic Regulatory Domain

Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in organisms ranging from bacteria to humans. Among the prokaryotic pLGICs there is an architectural diversity involving N-terminal domains (NTDs) not found for the eukaryotic relatives, exemplified by the calcium-sensitive channel DeCLIC. Here, we characterized DeCLIC structure using cryogenic electron microscopy (cryo-EM), small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. In both the presence and absence of calcium, cryo-EM reconstructions were similar to a previously reported calcium-bound X-ray structure. The NTD exhibited lower local resolution than the canonical unit, consistent with this domain being relatively mobile. The small-angle scattering profile revealed a feature not explained by the available structures, indicating that further conformational diversity is available to DeCLIC. MD simulations indicated that this profile is largely attributable to rigid-body motions of the NTD relative to the protein core, including conformations similar to those in experimental structures, as well as more expanded and asymmetric conformations. Using these expanded conformations, it was possible to fit the previously unexplained SANS feature, indicating the presence of such conformations under solution conditions. This work reveals the range of motion available to the DeCLIC NTD, expanding the conformational landscape of the pLGIC family; and demonstrates the power of combining low-resolution, high-resolution, and simulations data in the study of protein structure.

Speaker: Marie Lycksell (Stockholm University)

29 Deciphering the structural defects from SNP variants of ABCB4 transporter by means of molecular dynamics simulations

Bile secretion is an essential function of the liver for the elimination of xenobiotics and endogenous metabolites. This function mostly relies on membrane transporters located at the canalicular membrane of hepatocytes, such as the ATP-binding cassette (ABC) transporter ABCB4 responsible for phosphatidylcholine (PC) secretion into bile. Variations in the ABCB4 gene were reported from patients with rare liver diseases. Most of them are missense variations which were associated with defects of (i) protein expression, (ii) intracellular trafficking, (iii), secretion activity, or (iv) transporter stability. In this context, the present study aims to provide insights into structural defects of selected genetic variants of ABCB4 by means of molecular dynamics (MD) simulations, supported by cellular and molecular biology experiments.
Taking advantages of the recent cryogenic electron microscopy (cryo-EM) resolutions of ABCB4 in different conformations, $\mu$s-scaled MD simulations were performed considering: (i) different bound states (i.e., ATP- and/or PC-bound ABCB4), and (ii) different lipid bilayer models (symmetric and asymmetric). The overall ABCB4 dynamics was monitored to decipher key structural features along transport cycle. Furthermore, local destabilization events arising from genetic variations were assessed by mutating wild type ABCB4 using alchemical calculations. Particular attention was paid to the local structural impact of mutations in order to provide hints associated with function impairment.

Speaker: Ms Veronica Crespi (Inserm U1248 Pharmacology & Transplantation, University of Limoges, Limoges, France)

Closing Speech and Poster Prizes

12:15 Lunch

14:00 Departures