Membrane Biophysics of Exo-Endocytosis: from Model …exoendo.org/download/biophee19.pdfSession 1...

Membrane Biophysics of Exo-Endocytosis: from Model Systems to Cells Mandelieu, France, April 3-6th, 2019

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Welcome address from the Organizers

Dear Participants,

The Membrane Study Group (GEM) and Club Exocytose-Endocytose are two French

societies that regroup researchers in the fields of membrane biophysics and membrane trafficking, respectively. With the participation of the Italian societies of Pure and Applied Biophysics (SIBPA), of Chemistry (SCI – Division of Physical Chemistry), and of Biochemistry and Molecular Biology (SIB), for the first time, this joint meeting entitled "Membrane Biophysics of Exo-Endocytosis: from Model Systems to Cells" has been organized. We are pleased to welcome you at the beautiful location of Mandelieu-la-Napoule on the French Riviera. The goal of this international meeting is to illustrate how novel opportunities for discovery in the life sciences arise when the most urgent challenges in the field of exo and endocytic membrane trafficking are addressed from fresh angles based on innovative biophysical tools and concepts. We organized the meeting in a format that encourages interactive discussions of latest research in these areas, with much room for contributions from young scientists. Several scholarships have been awarded to students.

42 abstracts have been selected for oral communications, and 56 for poster presentations in two poster sessions. The poster sessions are assorted with speed-talk presentations as a forum for young and more senior scientists to present and discuss their research. We do hope the speed-talk presentations will be taken as a friendly and enjoyable challenge! The best poster presentations will receive an award by a scientific committee.

150 scientists will attend this meeting. We do hope that you will enjoy all the meeting facets: its scientific program composed of 7 dedicated sessions, the site, food, social events, and informal scientific and social discussions during the 4 days of the meeting.

We would like to thank the internationally renowned speakers who kindly accepted our invitation, our institutions and our sponsors, and all of you, selected speakers and participants, both senior and students, for joining us and contributing to the success of this meeting.

We wish you all a pleasant stay at the French Riviera and a fruitful meeting!

The Organizing Committee


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Organizing Committee

Coordinator Ludger Johannes (Institut Curie, Paris, France) GEM SC member & Exo/Endo Club member [email protected]

GEM Isabelle Mus-Veteau (IPMC, Sophia Antipolis,France) GEM President [email protected] Guillaume Drin (IPMC, Sophia Antipolis,France) GEM SC member [email protected] Giovanna Fragneto (Institut Laue-Langevin, Grenoble, France) GEM SC member [email protected]

Club Exo/Endo Stéphanie Miserey-Lenkei (Institut Curie, Paris, France) Exo/Endo Club President [email protected] Cédric Delevoye (Institut Curie, Paris, France) Exo/Endo Club Treasurer [email protected] Delphine Muriaux (IRIM, Montpellier,France) Exo/Endo Club executive council member [email protected]

Italian Societies Valeria Rondelli (University of Milan,Italy) Steering committee of SIBPA [email protected] Lorenzo Stella (University of Rome Tor Vergata, Italy) Steering committee of SIBPA

and member of SCI [email protected] Vito De Pinto (University of Catania,Italy) President of the membranes group of SIB [email protected] Gerardino D'Errico (University of Naples Federico II, Italy) Steering committee of the [email protected] physical chemistry division of SCI

mailto:[email protected]













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Sponsors Acknowledgements

The organizers would like to thank all of the institutional and private sponsors for their help and their financial supports:

Institutionnal supports:

The IDEX UCA JEDI from University Côte d’Azur & MODELIFE

Centre National de la Recherche Scientifique (CNRS)

LabEx CelTisPhyBio

Labex Qlife

Société Française de Biophysique

Société Française de Microscopie

Société de Biologie Cellulaire de France

Institut Laue-Langevin

ITMO BCDE


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Private foundations and companies:

The Company of Biologist

The Foundation ARC

Intelligent Imaging Innovations

Avanti Polar Lipids Inc.

Sanofi-Pasteur

Applied Photophysics

NIKON

Nanotemper

Hybrigenics Pharma

Covalab

Springer Nature

Scientific Reports

Communications Biology

Communications Physics

Cell Press

Journal of Cell Science

ImageXCell


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Map

Résidence Pierre et Vacances Premium

Route de la Pinéa, 06210 Mandelieu-la-Napoule, France

Phone number 00 33 (0)4 92 97 72 00

Meeting place

Rail station


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Program

Wednesday April 3rd 13:30 – 14:30 Registration and coffee

14:30 Welcome and general information - Ludger Johannes

Keynotes

Chairpersons : Stéphanie Miserey-Lenkei & Isabelle Mus-Veteau

14:45 Antonella de Matteis

(Naples, Italy)

ER-Golgi contact sites

15:30 Lukas Tamm (Charlottesville, USA)

Comparison of molecular mechanisms of calcium triggered fusion of synaptic, dense core and insulin vesicles under control of Munc13, Munc18 and Complexin in a reconstituted single vesicle fusion Assay

16:15 – 16:30 Coffee break

Session 1 Using Biomimetic Systems to Study Membrane Remodeling

Chairpersons : Vito de Pinto & Stéphane Gasman

16:30 Patricia Bassereau (Paris, France)

ESCRT-III filaments have opposite curvature-related orientations on membranes

17:00 Jorge Royes Mir (Paris, France)

Bacterial-based bioproduction of genetically encoded T-responsive nano-capsules

17:15 Naira Ayvazyan (Yerevan, Armenia)

Phospholipid giant unilamellar vesicle deformation under the influence of MACROVIPERA LEBETINA OBTUSA venom components

17:30 Joanna Podkalicka (Paris, France)

How to get sphingolipids to the plasma membrane a lipid sorting story

17:45 Laura Pokorny (London, UK)

Studying vaccinia virus binding and fusion using a minimal model system

18:00 Laura Picas (Montpellier, France)

Probing cell membrane remodeling on nanostructured systems

18:15 Pierre Joseph (Toulouse, France)

Microfluidics to fabricate and probe vesicles mimicking cells

18:30 Poster Session I Flash-talks (Chairperson : Guillaume Drin) 19:30 – 20:00 Welcome aperitif 20:00 – 21:30 Dinner 21:30 – 23:00 Poster Session I


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Thursday April 4th

Session 2 Membrane Organization and Assemblies

Chairpersons : Stéphanie Lebreton & Burkhard Bechinger

8:30 Bruno Antonny (Valbonne, France)

An optimal distribution of polyunsaturated acyl chains in phospholipids for fast membrane deformation and fission by endocytic proteins

9:00 Kaushik Inamdar (Montpellier, France)

A Role for the I-BAR domain IRSp53 protein on HIV-1 assembly

9:15 Evelina Gatti (Marseille, France)

BAD-LAMP controls TLR9 trafficking and signaling in human plasmacytoid dendritic cells

9:30 Gerardo Abbandonato (Milano, Italy)

Trafficking alteration of protein interactors on the HCN2 channel

9:45 Juan Martin D’Ambrosio (Paris, France)

Targeting of the yeast phosphatidylserine transporter Osh6p to membrane contact sites


10:30 Andreas Mayer (Lausanne, Switzerland)

Control of fusion after SNARE complex formation: Tethers in the driver’s seat

10:45 Luca Piantanida (Marseille, France)

Protein mediated structuring of model lipid membrane

11:00 Grégory Lavieu (Paris, France)

Reconstitution of Extracellular Vesicles uptake in a Cell Free Extract

11:15 Fabio Perissinotto (Trieste, Italy)

Extracellular Vesicles interaction with model membranes

11:30 Maria-Isabel Geli (Barcelona, Spain)

Linked sterol synthesis and transfer at highly curved membranes in ER- endocytic contact sites

12:00 – 13:00 Poster Session II- Flash-talks (Chairperson : Cédric Delevoye) 13:00 – 14:30 Lunch

Session 3 In Silico and Theoretical Studies of Exocytosis and Endocytosis

Chairperson : Luca Monticelli

14:30 Herre Jelger Risselada

(Göttingen, Germany) Molecular simulations of protein-mediated membrane fusion

15:00 Nicolas Destainville (Toulouse, France)

Protein nanodomains and spontaneous curvature

15:15 Camille Albrecht (Reims, France)

Characterization and evaluation of NEU-1 activity inhibition by interfering peptide approaches

15:30 Pierre Sens (Paris, France)

Non-equilibrium model of the structure and dynamics of the Golgi apparatus



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Session 4 Cytoskeleton and Membrane Dynamics

Chairpersons : Isabelle Alves & Pascale Zimmerman

16:30 Laurent Blanchoin (Grenoble,France)

Reconstitution of the steady state of dynamic actin networks

17:00 Riddhi Jani (Paris, France)

Membrane dynamics and regulators during endosomal tubulation

17:15 Jocelyn Laporte (Illkirch, France)

Modulation of amphiphysin and dynamin rescues severe congenital myopathies

17:30 Florence Niedergang (Paris, France)

Monitoring force generation of phagocytosing macrophages

17:45 Cécile Gauthier-Rouviere (Montpellier, France)

Up-regulation of flotillins, new marker of metastatic development, deregulates endocytosis and vesicular trafficking to induce Epithelio-to-Mesenchymal Transition and cellular invasion

18:00 Laura Salavessa (Paris, France)

Single-molecule analysis of IL-2 receptors reveals their clustering during endocytosis in lymphocytes

18:15 David Perrais (Bordeaux, France)

Exchange dynamics of dynamin measured in living cells during endocytic vesicle formation

GEM PhD thesis prize talk

Chairperson : Isabelle Mus-Veteau

18:30 Raphael Dos Santos Morais (Rennes, France)

Dystrophin-membrane interaction: 3D structure of dystrophin fragments in the presence of phospholipids

Talk sponsored by NanoTemper Techonologies

19:00 – 20:00 General Assembly of GEM 20:00 Dinner 21:30 – 23:00 Poster Session II


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Friday April 5th

Session 5 Biophysical Methods

Chairpersons : Nathalie Sauvonnet & Erick Dufourc

8:30 Debora Berti (Florence, Italy)

Nanostructured materials interacting with synthetic and natural lipid mesophases: challenges and opportunities

9:00 Jose-Jorge Ramirez-Franco (Marseille, France)

An extracellular sphingolipid-binding domain on synaptotagmin: Biochemical characterization and biological function

9:15 Armando Maestro (Grenoble, France)

Endocytosis across scales

9:30 Frank Lafont (Lille, France)

Bacteria internalization: how to discriminate adhesion from entry on line

9:45 Francesco Spinozzi (Ancona, Italy)

SAXS studies of lipid oxidation in mimetic membranes


10:30 Tetiana Mukhina (Grenoble, France)

Out-of-equilibrium active membranes: incorporation of bacteriorhodopsin in a floating lipid bilayer

10:45 Marc Abella Guerra (Marburg, Germany)

Mechanobiology of endocytic vesicle formation analyzed by Sla2 force sensors

11:00 Lionel Porcar (Grenoble, France)

Molecular transport in membranes investigated by neutron scattering: lipid exchange and translocation

11:15 Colin Monks (Denver, USA)

A spectrum of light sheet instruments optimized for different imaging demands

11:30 Wanda Kukulski (Cambridge, UK)

Linking architecture and function of organelle contact sites

12:00 – 13:00 Lunch 13:00 – 16:00 Free Time


Session 6 Structure of Exo-Endocytotic Membrane Systems: From Isolated Molecules to Cells

Chairpersons : Olivier Seksek & Christophe Lamaze

16:30 Georg Pabst (Graz, Austria)

From complex biomembrane mimics to live cells: Lessons learned from scattering techniques

17:00 Jacqueline Cherfils (Paris, France)

Allosteric regulation of small GTPases at the surface of membranes : A structural and biophysical perspective

17:15 Mauro Manno (Palermo, Italy)

The membrane organization of skeletal muscle-derived extracellular vesicles regulated by S-palmitoylation: A comprehensive biochemical and structural characterization

17:30 Oleksiy Kovtun (Cambridge, UK)

Subnanometer structure of the membrane-assembled retromer coat by cryo-electron tomography


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17:45 Jonas Ries (Heidelberg, Germany)

Systematic superresolution analysis of endocytosis reveals an actin nucleation nano-template that drives efficient vesicle

18:00 Lydia Danglot (Paris, France)

Unraveling molecular arrangements and membrane morphogenesis through live and super-resolution microscopy with MemBright probes and ICY SODA plugin (Standard Object Distance Analaysis)

18:15 Gaelle Boncompain (Paris, France)

Targeting CCR5 trafficking to inhibit HIV-1 infection”

18:30 Wolfgang Baumeister (Munich, Germany)

Structural biology in situ: the promise and challenges of cryo-Electron Tomography

19:00 – 20:00 General Assembly of Club Exocytose-Endocytose 20:00

Gala dinner & Party


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Saturday April 6th

Session 7 Therapeutic Delivery

Chairpersons : Marie-Pierre Rols & Claire Desnos

9:30 Ülo Langel (Stockholm, Sweden)

Transfection by cell-penetrating peptides

10:00 Morane Lointier (Strasbourg, France)

Structural and functional investigations of histidine rich-peptides with potent cell penetrating, antimicrobial and lentiviral transduction activities

10:15 Nunzio Iraci (Catania, Italy)

Extracellular vesicles as a novel strategy of cell-to-cell communication

10:30 – 11 :00 Coffee break

11:00 Rosario Oliva (Naple, Italy)

Biophysical studies on the interaction of antimicrobial peptides containing un-natural residues with bacterial model membranes

11:15 Marlene Lúcio (Braga Portugal)

Membrane biophysics and biointerfaces in nanotherapeutics development

11:30 Karidia Konate (Montpellier, France)

Mechanism of cellular internalization of siRNA-loaded WRAP5 nanoparticles

11:45 Leïla Bechtella (Paris,France)

PIP2 contributions for internalization of the cell-penetrating peptide Penetratin. Cell-free reconstitution and model membranes

12:00 Poster prizes and closing remarks

12:30 – 13:00 Lunch & Departure

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ABSTRACTS

Talks abstract are sorted chronologically according to the program. Poster abstracts are sorted alphabetically.

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KEYNOTES

Chairpersons :

Stéphanie Miserey-Lenkei & Isabelle Mus-Veteau

Keynotes Wednesday, April 3rd

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ER-Golgi contact sites Antonella de Matteis

University of Naples Federico II, Naples, Italy

Sites of physical contact between neighbouring organelles are emerging as pivotal pathways in interorganelle communication and cellular homeostasis. Contact sites between the endoplasmic reticulum (ER) and the trans Golgi network have been visualized using electron microscopy but their location in the crowded perinuclear area has rendered their analysis via optical microscopy extremely difficult such that their composition, function and regulation are almost completely unknown. We have set up a FRET-based method suitable for the visualization of the ER-Golgi contact sites (ERGoCS) by optical microscopy. We used this approach to perform a high content screen to investigate ERGoCS structure and function by depleting proteins that possess dual targeting motifs for the Golgi and the ER and thus have the potential to act as tethers at these contact sites. We found that ORP10 is required to maintain the ERGoCS due to its phosphatidylserine transfer activity while OSBP1 and ORP9 have a redundant tethering role, which is independent of their lipid transfer activity. We also show that the ERGoCS, which span on average 12 nm, control the homeostasis of PI4P, a key regulator of Golgi function, via the ER-localized 4-phosphatase Sac1 and the PI4P-binding protein FAPP1, which stimulates the in trans activity of Sac1 on Golgi PI4P. Destabilizing the ERGoCS increases while stabilization (optogenetically or chemically) decreases PI4P levels at the Golgi even under conditions that preclude Golgi-to-ER transfer of PI4P.

Keynotes Wednesday, April 3rd

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Comparison of Molecular Mechanisms of Calcium Triggered Fusion of Synaptic, Dense Core and Insulin Vesicles Under Control of Munc13, Munc18 and Complexin in a Reconstituted Single Vesicle Fusion Assay

Lukas K. Tamm Kreutzberger A., Kiessling V., Liang B., Castle D. and Tamm L.K.

Center for Membrane and Cell Physiology and Department of Molecular Physiology and Biological Physics University of Virginia, Charlottesville, USA

The rates of regulated secretion vary substantially for different cell types. Neurons release neurotransmitters from synaptic vesicles milli-seconds after stimulation. Release rates from neuroendocrine and endocrine cells are slower despite using similar molecular machineries. These differences are attributed to variations in spatial organizations, molecular machinery to stimulate calcium influx, and the precise molecular state of the secretory vesicles. To compare how the final exocytotic step of membrane fusion that is under the control of multiple regulatory factors differs kinetically among secretory vesicle types, synaptic vesicles from rat brain, dense core vesicles from a rat adrenal chromaffin cell line, and insulin vesicles from a rat endocrine pancreatic beta cell line have been purified and compared by reconstitution into a single vesicle – supported planar membrane fusion assay. Using this approach, we found a striking disparity in fusion rates in response to calcium that scales with the vesicle radius.

All three vesicle types require Munc18 and complexin-1 to restrict fusion of docked vesicles in the absence of calcium, and ensuing calcium injection triggers 60-80% of all docked vesicles to fuse. Synaptic vesicles required a Munc13 fragment consisting of the C1C2MUN domain for fast and robust response to calcium. Dense core vesicles lack this requirement, whereas fusion of insulin vesicles was kinetically enhanced in the presence of the fragment. These differences correlate with the presence of endogenous vesicle associated CAPS (calcium activated protein for secretion), which also contains a SNARE binding MUN domain.

A question of immense interest is how binding of calcium to the calcium sensor synaptotagmin-1 transmits the calcium signal to the neuronal SNARE fusion machinery. We have shown that the lipid bilayer, and specifically lipid order around t- and v-SNAREs, responds to C2B domain binding of synaptotagmin-1 to phosphatidylinositol-4,5-biphosphate and changes the conformation of the SNAREs from an angled to a more upright conformation relative to both membranes. This change in the transmembrane-to-SNARE domain linker region is entirely driven by a synaptotagmin induced change in lipid order and does not require direct interaction of synaptotagmin with the SNAREs.

References [1] Kreutzberger AJB, Kiessling V, Liang B, Seelheim P, Jakhanwal S, Jahn R, Castle JD, Tamm LK (2017) Reconstitution of calcium-mediated exocytosis of dense core vesicles. Science Adv 3:e1603208. Recommended by F1000. [2] Kiessling V, Kreutzberger AJB, Liang B, Nyenhuis SB, Seelheim P, Castle JD, Cafiso DS, Tamm LK (2018) A molecular mechanism for calcium/synaptotagmin-triggered exocytosis. Nature Struct Mol Biol 25:911-917. [3] Kreutzberger AJB, Kiessling V, Liang B, Nakamoto RK, Castle JD, Jahn R, Tamm LK (2019) Triggered Fusion of Synaptic, Dense Core and Insulin Vesicles Under Differential Control of Munc13. Submitted.

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SESSION 1

Using Biomimetic Systems to Study

Membrane Remodeling

Chairpersons : Vito de Pinto & Stéphane Gasman

SESSION 1 | Using Biomimetic Systems to Study Membrane Remodeling Wednesday, April 3rd

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ESCRT-III filaments have opposite curvature-related orientations on membranes

Patricia Bassereau

Laboratoire Physico-Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, 75005 Paris, France.

The multi-proteins ESCRT-III complexes are involved in membrane scission in many different cellular processes. In contrast to dynamin polymers that assemble outside budding vesicle/tubule necks, they assemble inside the bud necks where the membrane has a curvature Gaussian negative. The organization of the proteins of this complex and even more the mechanism of membrane scission remain highly debated. By combining membrane nanotube pulling experiments, CryoEM and high speed AFM on a minimal set of human ESCRT proteins, we have obtained very unexpected results. We have shown that the CHMP proteins assemble in filaments with contrasted affinities for curved membranes. a) CHMP4 form spiral filaments that do not tubulate membranes and rather flatten them. b) CHMP2A with CHMP3 have affinity for positively curved membranes since they form helical structures on the external side of tubules only. c) Together CHMP4, CHMP2A and CHMP3 deform liposomes into helical tubes (with a corkscrew shape) when incubated with liposomes with orthogonal orientations of the filaments; moreover they are not recruited inside tubes pulled from giant vesicles, but only inside their neck, as observed in cells. The peculiar shape of the membrane reveals the mechanical stresses imposed by these ESCRT-III assemblies to the membranes and might provide new insight on the mechanism of scission in the presence of the ATPase Vps4.


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Bacterial-based bioproduction of genetically encoded T-responsive nano-capsules

Jorge Royes Mir

Royes Mir J., Ilioaia O., Duvacheva G., Lubart Q., Bally M., Miroux B. and Tribet C. ENS, Département de chimie, 24 rue Lhomond, 75005 Paris, France

Despite the huge amount of research efforts on the preparation of liposomes or lipidic nano-vesicles, the in vitro insertion of degradation-prone functional proteins in the lipidic bilayer or their inner cavity remain challenging. In this context, a recent interest emerged for the mechanisms of in vivo production of exosomes and outer-membrane bacterial vesicles [1,2]. These nano-sized lipidic vesicles are naturally decorated with membrane proteins and filled with controlled metabolites and/or proteins

Here we present a microbial platform for the production of proteoliposomes with high yield and good control over their properties. At variance with previously described approaches, it is based on proliferation of internal membranes in gram-negative bacteria filled with a dense array of membrane proteins. These proteins can be genetically modified, displaying specific tags for (chemical) post-modification and/or labeling. For this purpose, we used the E.Coli C43(DE3) strain, which was evolved from BL21(DE3) in the past decades for membrane protein production [3,4]. Overexpression of particular proteins in these strains induces proliferation of internal lipid membranes [4], which markedly overcome the expected yield from harvesting outer-membrane peripheral vesicles

We obtained lipidic nano-vesicles monodisperse in size, decorated with a dense, well-defined and customizable corona of transmembrane polypeptides. In addition, this approach enables spontaneous loading of the lipidic nano-vesicles with functional soluble proteins. Due to the mild in cellulo conditions during formation of the assemblies, bacterial production of such capsules appears as a promising eco-friendly platform for production and loading of customizable proteoliposomes.

References

[1] J. P. K. Armstrong, M. N. Holme, M. M. Stevens, ACS Nano 2017, 11, 69–83. [2] A. Kulp, M. J. Kuehn, Annu. Rev. Microbiol. 2010, 64, 163–184. [3] B. Miroux, J. E. Walker, J. Mol. Biol. 1996, 260, 289–298. [4] I. Arechaga, B. Miroux, S. Karrasch, R. Huijbregts, B. de Kruijff, M. J. Runswick, J. E. Walker, FEBS Lett. 2000, 482, 215–219.


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Phospholipid giant unilamellar vesicle deformation under the influence of MACROVIPERA LEBETINA OBTUSA venom components

Naira Ayvazyan

Ayvazyan N., Ghukasyan G., Tadevosyan H.and Kirakosyan G. Orbeli Institute of Physiology, National Academy of Sciences of the Republic of Armenia, Orbeli st. 22, 0019 Yerevan, Armenia Modification of liposomes with proteins from various venoms with their often unique properties and sometimes pronounced biological activity is an informative approach to understanding the topology of protein–membrane complexes and the ways by which these complexes can change the state of lipid membrane (namely, its fluidity, thickness, permeability, ion selectivity, etc.). Membrane lipids exhibit a specific asymmetry, which plays an important role in the activation of membrane proteins and implementation of numerous functions of membranes. The structure of membrane bound sites in proteins determines the mechanism of their interaction with asymmetric membrane and may lead either to the adsorption of proteins on the membrane surface or to their incorporation into the bilayer. The modifications accompanying this process are immediately reflected in the properties of the lipid bilayer. Application of modern methods of fluorescence microscopy and surface-acoustic waves technique, which are currently ones of the most potent technical tools for visualization of supramolecular interactions, makes it possible to study lipid–protein interactions and complex processes, such as the formation of lipid rafts and incorporation of proteins into the bilayer.


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How to get sphingolipids to the plasma membrane a lipid sorting story

Joanna Podkalicka

Podkalicka J., Manzi J., Di Cicco A., Levy D., Lamaze C. and Bassereau P. Institut Curie UMR 168, 11 Rue Pierre et Marie Curie, 75231 Paris, France

Lipid and protein sorting are crucial processes that maintain unique biophysical and biological properties of different organelles. Nevertheless, little is known about mechanisms behind lipid sorting. Sphingomyelin (SM) is synthesized at the trans Golgi network (TGN) and transported to the plasma membrane (PM) via an uncharacterized pathway. SM enrichment in transport carriers cannot be explained by a curvature-based mechanism of lipid sorting due to its property to form stiff membranes. A mechanism of protein-mediated lipid sorting has been proposed, taking advantage of protein’s affinity for curved membranes and specific lipids. One of the proteins enriched in the same secretory pathway is caveolin, which shuttles between the Golgi and the PM where it forms cup-shape, SM-cholesterol-enriched domains called caveolae. The assembly process is initiated by the export of caveolin-enriched vesicles from the Golgi but little is known about this step except from a critical role of lipids in caveolin oligomerization.

The central goal of this project is to determine the role of caveolin in SM trafficking from the TGN to the PM and to decipher the molecular mechanisms behind caveolin assembly using a bottom-up approach. For the first time we managed to reconstitute caveolin 1 (Cav1) into small unilamellar vesicles s and analysed their structure with cryo-EM, depending on lipid composition. Furthermore, we established a novel in vitro system in which we reconstituted Cav1-SM complex in giant unilamellar vesicles (GUVs). Protein/lipid sorting was analysed by tube pulling approach with the use of micropipette aspiration coupled with optical tweezers. In parallel ours in cellulo studies showed significant changes in SM distribution within the cell in Cav1 KO cells, indicating that Cav1 play a role in SM trafficking. Currently we are trying to demonstrate SM/Cav1 co-sorting using RUSH technology which enables synchronized release and trafficking of desired molecules.


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Studying vaccinia virus binding and fusion using a minimal model system

Laura Pokorny

Pokorny L. and Albrecht D. MRC Laboratory for Molecular Cell Biology, University College London, London, UK

Poxviruses enter cells using the most complex virus fusion machinery identified. Whilst genetics indicate that poxvirus binding relies on 4 individual proteins and fusion relies on 11, to date the structural organisation of the entry-fusion complex, the fusion mechanism, the fusion peptide(s), and molecular dynamics leading from virus binding to fusion remain a mystery. To investigate these early events in poxvirus entry, we use the prototypic poxvirus, vaccinia virus (VACV), in combination with cell-derived membrane blebs. Membrane blebs serve as a simplified biomimetic model system of cells. Blebs are more easily manipulated than cells and their small size is highly advantageous for imaging studies.

We have developed and characterised this model system for use in combination with VACV to study the early steps of infection. Specifically, we show that VACV can bind and fuse to blebs. This fusion follows similar bulk fusion kinetics as compared to cells. In addition, using electron microscopy in combination with our minimal model system, we show that VACV induces plasma membrane remodelling in the form of curvature. Having demonstrated that viral binding and fusion machineries are organised into spatially distinct functional domains on the virus surface, we are currently investigating the contribution of virus-induced membrane curvature to productive virus-cell membrane fusion.


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Probing cell membrane remodeling on nanostructured systems

Laura Picas

Rathar R., Sanchez-Fuentes D., Desgarceaux R., Charlot B., Carretero-Genevrier A. and Picas L. IRIM, 1919 route de Mende, 34090 Montpellier, France

The remodeling of cellular membranes is a fundamental feature that ensures the viability of vesicle trafficking, cell signaling, and preserves compartment homeostasis. The biogenesis of endocytic vesicles requires the precise spatial and temporal orchestration of a protein machinery to shape cellular membranes. The recruitment of these proteins to remodel the plasma membranes is assisted by different factors such as: (i) the interaction with membrane lipids, such as phosphoinositides, (ii) the sensing of curvature, as observed for BAR (Bin/Amphiphysin/Rvs) domain proteins, and (iii) the interaction between proteins domains. In addition, (iv) the membrane tension that results from membrane-cytoskeleton adhesion sets the load of forming transport vesicles. How all these factors are organized in space and time is not well understood.

Here we have engineered nanostructured systems with tunable shapes and sizes and compatible with super resolution microscopy to manipulate cell membrane remodeling. With this setup we have investigated the nanoscale organization of key endocytic components on cells. Our results show a size-dependent organization of proteins such as clathrin or caveolin-1 and that the main phosphoinositide’s species at the plasma membrane would be sorted by the membrane shape.


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Microfluidics to fabricate and probe vesicles mimicking cells

Pierre Joseph

Joseph P., Elias M., Mesnilgrente F., Laborde A., Berti D., Montis C., Caselli L., Magnani C., Mingotaud A.F. and Lonetti B. LAAS CNRS, 7 avenue du Colonel Roche, BP 54200, 31031 Toulouse Cedex 4, France Being the base element of cell membranes, lipid bilayers are an essential ingredient of many biological processes. In order to understand mechanisms at play for example in molecule translocation through membrane proteins, or interaction of the cell with nanoparticles (NPs), artificial, biomimetic membranes have been developed: supported or suspended bilayers, Giant Unilamellar Vesicles (GUV) which are micron-sized compartments. They constitute simplified models to decompose the elementary events (biochemical, physical, and chemical) at stake in real biological situations [1]. Existing Methods to work with these artificial membranes do not permit reproducible properties or complex configurations (size, chemical composition of the membrane). Microfluidics, the handling of fluids in microfabricated chips, offers new and versatile tools, both to fabricate more elaborated “artificial cells” [2] and to manipulate and study them[3].

We propose to combine microfluidics (LAAS team, https://www.laas.fr/public/en/mile), physico-chemistry with biological relevance (Italian team), and polymeric self-assemblies for nanomedicine (IMRCP team) in order to decipher biological processes occurring at cell membranes, particularly as regards nanoparticles/membrane interaction. Two applications are targeted: nanoparticles toxicity and nanomedicine.

A few results (more or less preliminary) will be discussed: Chips designed to characterize vesicle mechanical properties, in the spirit of on-chip micropipettes [4], show significant stiffening of GUV after their incubation with gold NP. They also permit to test hybrid vesicles, composed of mixtures of lipids and copolymers, promising in particular for nanomedicine [5]. The effects of photoactive drugs included in polymeric nanocarriers are also characterized in real-time on vesicles sensitive to oxidation, thanks to a microfluidic architecture permitting to switch inlet solution. Droplet-based configurations to fabricate GUV are also implemented.

On-going work aims at extracting quantitative information: improving designs, understanding how intrinsic properties (membrane bending modulus) relate to measured properties (such as the critical pressure to release a vesicle), as function of nanoparticles surface chemistry, membrane composition, flow conditions.

References

[1] Montis, C.; Maiolo, D.; Alessandri, I.; Bergese, P.; Berti, D. Interaction of Nanoparticles with Lipid Membranes: A Multiscale Perspective. Nanoscale 2014, 6 (12), 6452–6457. [2] Swaay, D. van; deMello, A. Microfluidic Methods for Forming Liposomes. Lab Chip 2013, 13 (5), 752–767. [3] Robinson, T.; Kuhn, P.; Eyer, K.; Dittrich, P. S. Microfluidic Trapping of Giant Unilamellar Vesicles to Study Transport through a Membrane Pore. Biomicrofluidics 2013, 7 (4), 044105. [4] Guo, Q.; Park, S.; Ma, H. Microfluidic Micropipette Aspiration for Measuring the Deformability of Single Cells. Lab Chip 2012, 12 (15), 2687–2695. [5] Magnani, C.; Montis, C.; Mangiapia, G.; Mingotaud, A.-F.; Mingotaud, C.; Roux, C.; Joseph, P.; Berti, D.; Lonetti, B. Hybrid Vesicles from Lipids and Block Copolymers: Phase Behavior from the Micro- to the Nano-Scale. Colloids and Surfaces B: Biointerfaces 2018, 168, 18–28.


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31

SESSION 2

Membrane Organization and Assemblies

Chairpersons : Stéphanie Lebreton & Burkhard Bechinger

SESSION 2 | Membrane Organization and Assemblies Thursday, April 4th

33

An optimal distribution of polyunsaturated acyl chains in phospholipids for fast membrane deformation and fission by endocytic proteins Bruno Antonny

Tiberti M., Manni M., Gautier R. and Antonny B. Université Côte d’Azur et CNRS, IPMC, 660 route des lucioles, 06560 Valbonne, France

We previously showed that polyunsaturated phospholipids facilitate membrane deformation and fission by endocytic proteins [1], suggesting a link between the striking enrichment of neuronal membranes in these lipids and the very fast speed at which endocytosis occurs in neurons [2]. To further explore the impact of polyunsaturated phospholipids on membrane deformation and fission, we perform experiments and simulations on membranes in which we control the distribution of polyunsaturated acyl chains in model membranes in two ways. First, we systematically change the acyl chain profiles of membranes by introducing an acyl chain of defined length and unsaturation level at position 1 and/or 2 of the phospholipids [3]. Second, we restrict the distribution of the polyunsaturated phospholipids in the inner or in the outer leaflet of the membrane. These approaches indicate that the combination of one saturated and one polyunsaturated acyl chain in phospholipids strikes a balance between impermeable and flexible membranes and that polyunsaturated phospholipids boost inward membrane deformation and fission when present in the inner (i.e. cytosolic) but not outer membrane leaflet. These two observations are in agreement with the distribution of polyunsaturated acyl chains in cellular membranes. References [1] Pinot M, Vanni S, Pagnotta S, Lacas-Gervais S, Payet LA, Ferreira T, Gautier R, Goud B, Antonny B, Barelli H. Lipid cell biology. Polyunsaturated phospholipids facilitate membrane deformation and fission by endocytic proteins. Science. 2014, 345:693-7. [2] Barelli H, Antonny B. Lipid unsaturation and organelle dynamics. Curr Opin Cell Biol. 2016, 41:25-32. [3] Manni MM, Tiberti ML, Pagnotta S, Barelli H, Gautier R, Antonny B. Acyl chain asymmetry and polyunsaturation of brain phospholipids facilitate membrane vesiculation without leakage. Elife. 2018, 7. pii: e34394.


34

A Role for the I-BAR domain IRSp53 protein on HIV-1 assembly

Kaushik Inamdar

Inamdar K., Tsai FC., dePoret A., Merida P., Lappalainen P., Roingeard P., Favard C., Bassereau P. and Muriaux D. RIM, CNRS UMR9004, CNRS & University of Montpellier, 34293 Montpellier, France

The I-BAR domain proteins are a family of proteins known to sense and induce negative curvature at the cell plasma membrane. They also act as scaffold proteins for several signalling pathways at the cell membrane. Our research team has revealed that IRSp53, an I-BAR domain protein, is involved in HIV-1 assembly via the activation of an actin-modulating small GTPase Rac1 signalling pathway1. Hence, the specific role of IRSp53 in HIV-1 assembly was examined. During HIV-1 assembly and budding, the viral structural Gag proteins are targeted to the plasma membrane where it multimerizes, leading to HIV-1 particle budding from the host cell plasma membrane. Here, we first studied the effect of siRNA mediated gene inhibition of IRSp53 on HIV-1 particle production. Our results show that inhibition of IRSp53, but not IRTKS, leads to a significant reduction in HIV-1 Gag particle production. Second, we examined the specificity of IRSp53 incorporation in HIV-1 particles, across other I-BAR family of proteins. Our results show that IRSp53 is specifically incorporated into Gag virus-like-particles as compared to other proteins of the I-BAR family. We further examined the presence of IRSp53 at the virus budding site by immuno-electron microscopy and dual colour super resolution fluorescence microscopy. Imaging analysis indicated the possible presence of IRSp53 at Gag assembly sites. Then, we analysed the possible interactions between the structural viral Gag proteins and IRSp53. We reveal that Gag and IRSp53 are in the same intracellular complex, and report an increased cell membrane localization of IRSp53 upon Gag expression. Finally, the dynamic recruitment of IRSp53 at HIV-1 Gag assembling site was examined in live cells by TIRF-Microscopy. Furthermore, by setting up a protein-membrane model system, we could characterize the interaction between IRSp53 I-BAR domain and Gag in the context of Giant Unilamellar Vesicles (GUVs). Molecular studies in cell free conditions on GUVs indicated that binding of purified HIV-1 Gag protein to GUV membranes was enhanced by the I-BAR domain of IRSp53. Finally, we found that Gag binds preferentially at the curved tips of tubules induced by the IRSp53 I-BAR domain. Overall, our results highlight that IRSp53 has a definite and specific role at the HIV assembly site by helping Gag binding to the membrane while possibly providing the initial curvature necessary for bud formation.

Reference

[1] Thomas,A., Mariani-Floderer,C., López-Huertas,M.R., Gros,N., Hamard-Péron,E., Favard,C., Ohlmann,T., Alcamí,J. and Muriaux,D. (2015) J Virol, 89, 8162–8181.


35

BAD-LAMP controls TLR9 trafficking and signaling in human plasmacytoid dendritic cells

Evelina Gatti

Combes A., Bendriss-Vermare N., Caux C. and Gatti E. CIML – CNRS, 13010 Marseille, France

Toll-like receptors (TLR) are essential components of the innate immune system. Several accessory proteins, such as UNC93B1, are required for transport and activation of nucleic acid sensing Toll-like receptors in endosomes

We have demonstrated1 that BAD-LAMP (LAMP5) controls TLR9 trafficking to LAMP1+ late endosomes in human plasmacytoid dendritic cells (pDC), leading to NF-κB activation and TNF production upon DNA detection. An inducible VAMP3+/LAMP2+/LAMP1− endolysosome compartment appears in pDCs, from which TLR9 activation occurs and triggers type I interferon expression upon recruitment of signaling complex components. BAD-LAMP-silencing enhances TLR9 retention in this compartment and consequent downstream signaling events. Conversely, sustained BAD-LAMP expression in pDCs contributes to their lack of type I interferon production after exposure to a TGF-β-positive microenvironment or isolation from human breast tumours.

Hence, BAD-LAMP limits interferon expression in pDCs indirectly, by promoting TLR9 sorting to late endosome compartments at steady state and in response to immunomodulatory cues.


36

Trafficking alteration of protein interactors on the HCN2 channel

Gerardo Abbandonato

Abbandonato G., Saponaro A., Porro A., Brocca L., Santoro B. and Moroni A. Department of Biosciences, University of Milan, Via Celoria 26 , 4th Floor, Building C 20133 Milan, Italy

Hyperpolarization-activated Cyclic Nucleotide-gated ion channels (HCN) are the molecular correlate of an inward current activated by a plasma membrane hyperpolarization which control the pacemaker activity both in hearth and in brain [1]. Four mammalian isoforms have been discovered with different localization, kinetics and steady-state voltage dependence [1]. We focused our attention on the HCN2. Single-point mutations of this channel are often associated with loss-of.-function of the channel leading to generalized epilepsy[2,3]. This different behavior could reasonable be attributed both to structural modifications, resulting in altered or null channel functionality, and/or to a trafficking defect leading to lack of channels at the plasma membrane. Besides, there is a variety of proteins, whose physiological role is not well understood yet, that have been shown to be associated with HCN channels: TRIP8b[4], filamin A[5], caveolin-3[6], KCR1[7], KCNE2[8], MINT2[9], tamalin[9], S-SCAM[9], and several protein kinases[10]. Similarly to single-point mutations, protein assembly contributes to distribution, trafficking and clustering of ion channels as well as coupling of ion channels to intracellular signaling cascades[11]. In order to understand the effect of these interacting proteins on the trafficking of HCN2, by using fluorescence microscopy, we performed FRET measurements between labeled protein interactors and the labeled ion channel and specific quantitative colocalizations. In this way, we analyzed the different distributions of the channel and the effect of specific interactors on the membrane localization.

References

[1] Robinson R.B., et al., Hyperpolarization-activated cation currents: from molecules to physiological function, Annu. Rev. Physiol., 2003, 65, 453-80. [2] DiFrancesco J.C., et al., Recessive Loss-of-Function Mutation in the Pacemaker HCN2 Channel Causing Increased Neuronal Excitability in a Patient with Idiopathic Generalized Epilepsy, The J. Neurosci., 2011, 31(48), 17327–17337. [3] DiFrancesco J.C., et al., Dysfunctional HCN ion channels in neurological diseases, Front. Cell. Neurosci., 2015, 6, 174-184. [4] Saponaro A., et al., Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function, PNAS, 2014, 111(40), 14577-14582. [5] Gravante B., et al., Interaction of the pacemaker channel HCN1 with filamin A, J. Biol. Chem, 2004, 279, 43847–43853. [6] Barbuti A., et al., Localization of f-channels to caveolae mediates specific β2-adrenergic receptor modulation of rate in sinoatrial myocytes. J. Mol. Cell Cardiol. 2007, 42, 71–78. [7] Michels G., et al., K+ channel regulator KCR1 suppresses heart rhythm by modulating the pacemaker current If, PLoS One, 2008, 3, e1511. [8] Decher N. et al., KCNE2 modulates current amplitudes and activation kinetics of HCN4: influence of KCNE family members on HCN4 currents, Pflugers Arch., 2003, 446, 633–640. [9] Kimura K., et al., Hyperpolarization-activated, cyclic nucleotide-gated HCN2 cation channel forms a protein assembly with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction, Genes Cells, 2004, 9, 631–640. [10] Hammelmann V., et al., The cGMP-dependent protein kinase II Is an inhibitory modulator of the hyperpolarization-activated HCN2 channel, PLoS One 2011, 6, e17078. [11] Garnera C.C., et al., PDZ domains in synapse assembly and signaling, Trends Cell Biol., 2000, 10(7), 274-280.


37

Targeting of the yeast phosphatidylserine transporter Osh6p to membrane contact sites

Juan Martin D’Ambrosio

D’Ambrosio J.M.1, Albanèse V.

1, Lipp NF.

2, Drin G.

2 and Čopič

A.

1

1 Institut Jacques Monod, CNRS, Université Paris Diderot, Sorbonne Paris Cité, 75013 Paris, France.

2 Institut de Pharmacolo ie Moléculaire et Cellulaire, UCA and CNRS, 660 route des lucioles, 06560 Valbonne,

France.

Membrane lipid composition varies between the compartments of eukaryotic cells. The anionic phospholipid phosphatidylserine (PS) is synthetized at the endoplasmic reticulum (ER) and then transported to other cellular compartments. In yeast, PS accumulates at the plasma membrane (PM), where it is important for recruiting and activating signaling proteins. Two yeast proteins, Osh6p and Osh7p, members of the oxysterol-binding protein family, transport PS from the ER to the PM in exchange for phosphatidylinositol-4-phosphate. In contrast to many other members of this evolutionarily-conserved family, Osh6p and Osh7p consist of a single domain that can carry alternately the two lipids, but it is not known how these proteins are targeted to the ER-PM contact sites. We show that their cellular targeting depends on another yeast protein, Ist2p, which can tether ER and PM using a long cytosolic tail, but whose function is poorly understood. Using protein interaction assays in yeast and structural predictions, we have been able to generate mutations in Ist2p and in Osh6p that abrogate their interaction. We are using these mutants to test how the interaction between the two proteins affects PS transport.


38

Control of fusion after SNARE complex formation: Tethers in the driver’s seat

Andreas Mayer

UNIL, Faculty of Biology and Medicine, Department of Biochemistry, chemin des Boveresses 155 - CP 51 - CH-1066 Epalinges, Switzerland

Distinct tasks in intracellular membrane fusion have been assigned to conserved protein systems. Whereas tether proteins mediate initial recognition and attachment of membranes, SNARE complexes are considered as the core fusion engine, which mechanically distorts membranes and drives them through a hemifusion intermediate towards the formation of a fusion pore[1-3]. This last step is highly energy-demanding[4,5]. We combined the fusion of yeast vacuoles with molecular simulations to show that tether proteins are critical to overcome the final energy barrier to fusion pore formation[6]. SNAREs alone drive vacuoles only into hemifusion. But associated tether proteins greatly increase the volume of SNARE complexes and deform the site of hemifusion, which lowers the energy barrier for pore opening. SNAREs and tether proteins should hence be considered as a single, non-dissociable device to drive fusion.

Although the expansion of nanoscopic fusion pores faces a major energy barrier and might hence be a rate-limiting and regulated step, corresponding states with non-expanding pores are difficult to assay and have remained elusive. We found that vacuoles in living yeast are connected by a metastable, non-expanding, nanoscopic fusion pore[7]. This is their default state, from which full fusion is regulated. Expansion of the nanoscopic pore to full fusion can thus be triggered by osmotic pressure gradients, providing a simple mechanism to rapidly adapt organelle volume to increases in its content. Metastable, nanoscopic fusion pores are then not only a transient intermediate but can be a long-lived, physiologically relevant state of SNARE-dependent membrane fusion, at which the reaction can be regulated.

References

[1] Gao, Y. et al. Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science 337, 1340–1343 (2012). [2] Zhang, X. et al. Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex. Proc. Natl. Acad. Sci. U.S.A. 113, E8031–E8040 (2016). [3] Reese, C., Heise, F. & Mayer, A. Trans-SNARE pairing can precede a hemifusion intermediate in intracellular membrane fusion. Nature 436, 410–414 (2005). [4] Chernomordik, L. V. & Kozlov, M. M. Protein-lipid interplay in fusion and fission of biological membranes. Annu Rev Biochem 72, 175–207 (2003). [5] Cohen, F. S. & Melikyan, G. B. The energetics of membrane fusion from binding, through hemifusion, pore formation, and pore enlargement. J Membr Biol 199, 1–14 (2004). [6] D'Agostino, M., Risselada, H. J., Lürick, A., Ungermann, C. & Mayer, A. A tethering complex drives the terminal stage of SNARE-dependent membrane fusion. Nature 551, 634–638 (2017). [7] D'Agostino, M., Risselada, H. J., Endter, L. J., Comte-Miserez, V. & Mayer, A. SNARE-mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle. EMBO J 37, e99193 (2018).


39

Protein mediated structuring of model lipid membrane

Luca Piantanida

Lafargue E., Piantanida L., Zuttion F. and Casuso I. INSERM Bâtiment TPR2 , Campus de Luminy, 163 avenue de Luminy, 13288 Marseille, France

The variety of molecules interacting with cellular membrane make it a crowded place where spatial organization and segregation is essential for proteins to fulfil their functions. In order to allow the needed dynamism and adequately respond to stimuli, the cell membrane can actively remodel itself [1] and existing lipid phase separations partition the membrane in different domains (lipid rafts-like), where proteins are selectively and transiently recruited for mutual interaction and function. The classic view is that lipids promote the domains formation in which proteins can get transiently trapped, but the role of proteins as actors on the lipids segregation is seldom considered [2]. We found membrane partitioning induced by a bacterial membrane protein (MurG). On monovalent model bilayer, self-induced domains formed when MurG protein was added. Until the date how the protein contribute in lipid segregation is still not yet clear [3].

We used unique macromolecular sub-second visualization High Speed Atomic Force Microscopy (HS-AFM). The membrane protein MurG is a N-acetilglucosamine transferase involved in the synthesis of peptidoglycan precursor Lipid II in bacterial membrane that has been reported to co-localize in bacteria with the Laurdan membrane fluidity marker [4]. Thanks to the HS-AFM label free imaging we found that MurG not only self-induces the formation of lipid domains but it diffuses in them suggesting a mutual efficient interaction between protein and phospholipids. Moreover, our analysis indicate that MurG enzymatic substrate (UDP-GlcNAc) influences the arrangements of so-formed domains. A panel of phospo-glycerol (PG) alkyl chains have been used for highest medical relevance. This membrane is mimetic of the clinical pathogen Clostridium difficile (C. difficile) [5]. Protein-induced domains formation is a new paradigm in membrane biology, and could help elucidating the protein role on bacteria membrane synthesis tailoring innovative antibiotics strategies.

References

[1] Raviv, D. H. a. U. 2014. Liposomes, Lipid Bilayers and Model Membranes, CRC Press. 3-30. [2] Segzin, E. et al. 2017. The mystery of membrane organization: composition, regulation and roles of lipid rafts, Nat. Reviews 18, 361-374. [3]Schmid Friederike. 2017. Physical mechanisms of micro- and nanodomain formation in multicomponent lipid membranes. Biochimica et Biophisica Acta 509-528. [4] Müller A, et al. 2016. Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains. Proc Natl Acad Sci.;113(45):E7077-E7086. [5] Drucker, D.B., et al. 1996. Phospholipid profiles of Clostridium difficile, J. Bacteriol, 178(19): 5844–5846.


40

Reconstitution of Extracellular Vesicles uptake in a Cell Free Extract

Grégory Lavieu

Bonsergent E., Thery C. and Lavieu G. Institut Curie, 26 rue d’Ulm, 75005 Paris, France

Intercellular communication is an absolute requirement to establish and maintain the proper organization and integrity of multicellular organisms. Extracellular Vesicles (EVs) recently emerged as an important vector for intercellular communication by sampling the biochemical content (in a donor cell and transferring the sample to an acceptor cell). Tremendous progresses have been made in understanding the physiology and physiopathology of EVs. However, our knowledge of the cell biology of EVs remains far behind, especially the delivery process within the acceptor cell. This is not satisfying when considering the high translation impact that EVs could offer.

To gain insight in the EVs uptake process, we used a classical in vitro cell free approach. We developed a content mixing assay: briefly, purified EVs containing a tagged cargo were mixed with purified plasma membrane sheets. After incubation, samples were submitted to protease digestion. EVs cargo that is normally protected from protease digestion became degraded only when PM sheets and EVs were exposed at pH5.5. This suggests that EVs content release requires PM-derived membranes and an endosome-like environment. Importantly, pre-treatment with protease that stripped off proteins from the surface of EV/PM sheets, prevented the EV content release.

Altogether our results suggest that EVs content release requires proteins present at the surface of the acceptor cell, followed by endocytosis/acidification that triggers the content release. Analogy with certain viruses suggests that the delivery process could correspond to a membrane fusion event.


41

Extracellular Vesicles interaction with model membranes

Fabio Perissinotto

Perissinotto F., Senigagliesi B., Rondelli V., Len A., Amenitsch H., Pachler K., Gimona M., Rohde E., Casalis L. and Parisse P. University of Trieste,Department of Physics, Via Alfonso Valerio 2, 34127 Trieste TS, Italy

Extracellular vesicles (EVs) are a potent intercellular communication system. Within a lipidic bilayer, such small vesicles (diameter ranging from 30 to few hundreds nanometers) transport biomolecules between cells and throughout the body, strongly influencing the fate of recipient cells. They have been proposed as biomarkers for several diseases and as optimal candidates for therapeutic applications, due to their small size and specific biological functions. Nonetheless, since their isolation, quantification and biophysical and biochemical characterization are challenging tasks, the understanding of the complex network of EVs/cell interaction is still incomplete. Here we propose a combination of Atomic Force Microscopy and Small Angle X-Ray and Neutron Scattering (SAXS and SANS) for the preliminary characterization of isolated vesicles and for the analysis of their interaction with model membranes, in form of liposomes and supported lipid bilayers. Our analysis reveals a strong interaction of EVs with model membranes mimicking lipid rafts (DOPC, SM), pointing out the importance of rafts-like structure in the uptake processes.


42

Linked sterol synthesis and transfer at highly curved membranes in ER- endocytic contact sites Maria-Isabel Geli

Institute for Molecular Biology of Barcelona (CSIC), Baldiri Reixac 15, 08028 Barcelona, Spain.

We have recently shown using Time Resolved Electron Microscopy (TREM) that endocytic sites become associated with the rims of the cortical endoplasmic reticulum (cER) tubules, shortly before actin polymerization starts. We also demonstrated that the molecular link between the cER and the endocytic sites is composed of the yeast VAPs (Scs2/22), the ORP Osh2, and the endocytic myosin-I Myo5. The data also indicated that the VAP/ORP/Myosin-I complex has the capacity to locally transfer sterols to the plasma membrane (PM) and that this activity is required to properly initiate actin polymerization. We now analyze the mechanisms that might assist sterol transport by Osh2 against the steep ER-PM gradient. Mutational analysis suggest that similar to Osh4, Osh2 uses the PI4P counter transport to facilitate ER to PM sterol transfer, and that the type II PI-kinase Lsb6, a suspected palmitoylated and sterol-activated enzyme, is responsible for the generation of the PI4P pool used by Osh2 at endocytic sites. However, PI4P counter transport does not seem to be essential in the process, suggesting that other mechanisms might cooperate. Interestingly, we found that a subset of enzymes involved in the late ergosterol biosynthesis, form a stable complex localized at the highly curved cER rims, where they transiently contact Osh2 and the endocytic machinery. Staining of sterols with the D4H domain of PFO fused to GFP indicates that indeed, sterols accumulate and are exposed at these sites. Based on the available literature, we propose that the enforced synthesis of sterols at highly curved membranes, coupled to the sterol transport machinery, might be one of the essential mechanisms supporting vectorial sterol transport out of the ER at ER-contact sites.

43

SESSION 3

In Silico and Theoretical Studies of

Exocytosis and Endocytosis

Chairperson : Luca Monticelli

SESSION 3 | In Silico and Theoretical Studies of Exocytosis and Endocytosis Thursday, April 4th

45

Molecular simulations of protein-mediated membrane fusion

Herre Jelger Risselada1,2 1Georg-August University, Dept. of Theoretical Physics, Göttingen, Germany 2Leiden University, Leiden Institute of Chemistry, The Netherlands

It has been widely accepted that the opening of the productive fusion pore is actively mediated by proteins. Unlocking the molecular features on how molecules either accelerate or inhibit membrane remodeling both selectively and efficiently may help in generating novel therapeutic strategies. To this aim, we perform state-of-the-art molecular simulations and free energy modeling techniques (e.g., [1]) to explore how proteins alter membrane remodeling reaction pathways. Based on coarse-grained molecular simulations and free energy calculations, we will illustrate a few examples of how proteins alter the free energy of distinct membrane remodeling steps. In particular, we will illustrate how the presence of voluminous SNARE associated proteins such as tether complexes may accelerate opening of the fusion pore in yeast vacuoles [2,3], and how docking proteins can subsequently contribute to the expansion of the fusion pore up-to a microscopic size.

References

[1] Smirnova Y, Risselada HJ, Mueller, MM Thermodynamically reversible paths of the first fusion intermediate reveal an important role for membrane anchors of fusion proteins, PNAS, in press, 2019 [2] D’A ostino M, Risselada HJ, Lürick A, Un ermann C, Mayer A A tetherin complex drives the terminal stage of SNARE-dependent membrane fusion. Nature 551, 7682, 2017 [3] D’A ostino M, Risselada HJ, Endter LJ, Comte‐Miserez V, Mayer A SNARE‐mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle. EMBO J. 37, e99193, 2018


46

Protein nanodomains and spontaneous curvature

Nicolas Destainville

Destainville N., Cornet J. and Manghi M. IRSAMC, Université Paul Sabatier, 118 route de Narbonne, 31400 Toulouse, France

It is now widely agreed that plasma membrane component spatial repartition is not

homogenous but that they are organized into nanodomains. Different mechanisms have been proposed by physicists to account for the finite size of membrane protein/lipid nanodomains. Among them, spontaneous curvature imposed by membrane inclusions or membrane leaflet asymmetry is able to explain most of nanodomain features, and typical size. In this talk, we describe the membrane with a composition/curvature coupling mechanism and, using the tools of statistical mechanics (both analytical and numerical), we characterize the properties of nanodomain assemblies promoted by curvature (e.g., phase diagram and size distribution). A connection with membrane budding as a promoter of endocytosis is proposed.

References

[1] S. Weitz, N. Destainville, Attractive asymmetric inclusions in elastic membranes under tension : cluster phases and membrane invaginations,Soft Matter 9, 7804 (2013). [2] G. Gueguen, N. Destainville, M. Manghi, Mixed lipid bilayers with locally varying spontaneous curvature and bending, Eur. Phys. J. E. 37,76 (2014). [3] N. Destainville, M. Manghi, J. Cornet, A rationale for mesoscopic domainformation in biomembranes, Biomolecules 8, 104 (2018).


47

Characterization and evaluation of NEU-1 activity inhibition by interfering peptide approaches

Camille Albrecht

Albrecht C., Dhaideh Z., Kuznecov A., Dauchez M., Duca L., Efremov RG., Maurice P., Bennasroune A. and Guéroult M. MEDyC UMR-CNRS 7369, University of Reims, 51 rue Cognacq-Jay, 51096 Reims, France

Elastin undergoes various modifications during vascular aging. The elastin network is degraded by proteases generating elastin-derived peptides (EDP) that contribute to the development of age-linked vascular diseases. The ERC (Elastin Receptor Complex) is a heterotrimeric complex composed of three proteins, the Elastin-Binding Protein (EBP) which binds EDP and tropoelastin, PPCA/CathepsinA that ensures the integrity of the ERC and a catalytic subunit Neuraminidase 1 (NEU-1) hydrolyzing terminal sialic acid residues from glycoproteins. Binding of EDP to the ERC activates NEU-1 sialidase activity which modulates ERC signaling and the development of diseases such as cancer, diabetes and atherosclerosis.

Previous studies of our laboratory have shown that membrane NEU-1 catalytic activity is linked to its dimerization. Thus, this subunit constitutes a key pharmacological target to fight against deleterious effects of EDP. The aim of this work is to develop by molecular dynamic (MD) simulations, in conjunction with experimental biological/biochemical data, a specific inhibitor of NEU-1 sialidase activity using transmembrane interfering peptides able to block its dimerization. The hypothesis is to form an inactive heterodimer composed of TM2 interfering peptide and NEU-1 leading to homodimerization disruption. Preliminary MD simulation studies show that both interfering peptide and the transmembrane domain of NEU-1 are stable and helix integrity is conserved all along MD simulations. Moreover, NEU-1 and the interfering peptide form a spontaneous dimer in a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membrane model. Further analyses underline the biological relevance of our membrane model. The MTT viability test shows no cellular toxicity both for interfering peptides delivered to COS-7 cells by Lithium Dodecyl Sulfate micelles and TAT peptides. Moreover, for both strategies, the interfering peptide is able to reduce sialidase activity in COS-7 cells overexpressing NEU-1. Co-immunoprecipitation experiments highlight the interaction between the interfering peptide and NEU-1. Altogether, these results show the ability of these interfering peptides to bind to their target, NEU-1, and to inhibit its sialidase activity at the plasma membrane.


48

Non-equilibrium model of the structure and dynamics of the Golgi apparatus

Pierre Sens

Institut Curie, Paris, France

The Golgi apparatus performs many essential functions, including the transport, sorting and biochemical maturation of cellular components. Understanding what controls the structure and dynamics of this organelle is an outstanding problem of great interest for cell biologists, but also for physicists. I will discuss a theoretical model in which the Golgi is viewed as a dynamical system in non-equilibrium steady-state. I will show that the structure of this organelle can be varied in a controlled manner by modifying a limited number of coarse-grained kinetic parameters: the maturation rate of membrane components, the budding rate of transport vesicles, and the rate of fusion between compartments. One consequence of this is that the structure and dynamics of the organelle are intimately linked. In particular, we will see that the two archetypal models of Golgi dynamics: vesicular transport and cisternal maturation, can be seen as extreme cases of a much richer and tunable dynamics.

49

SESSION 4

Cytoskeleton and Membrane Dynamics

Chairpersons : Isabel Alves & Pascale Zimmerman

SESSION 4 | Cytoskeleton and Membrane Dynamics Thursday, April 4th

51

Reconstitution of the steady state of dynamic actin networks

Laurent Blanchoin

Laboratoire Physiologie Cellulaire & Végétale, Institut de Biosciences et de Biotechnologies de Grenoble, CEA-Grenoble, 17 avenue des Martyrs, 38054 Grenoble Cedex 9, France

The dynamic assembly and turnover of actin networks in cells control shape changes, migration and organelle function, as well as communication with extracellular substrates or neighbors. We studied, in vitro and in silico, how actin organization controls the rate, the dimensions and the steering during lamellipodium growth. We use a high-resolution surface structuration assay combined with mathematical modeling to describe the growth of a reconstituted lamellipodium. We demonstrate that local monomer depletion at the site of assembly negatively impacts the network growth rate. At the same time, network architecture tunes the protrusion efficiency, and regulates the rate of growth. One consequence of this interdependence between monomer depletion and network architecture effects is the ability of heterogeneous network to impose steering during motility. We found that in the presence of ADF/Cofilin, networks reached equilibrium and became treadmilling. A mathematical model predicts the network length as a function of width and actin and ADF/Cofilin concentrations. Our results establish general principles on how the dynamic equilibrium state of actin network emerges from biochemical and structural feedbacks.


52

Membrane dynamics and regulators during endosomal tubulation

Riddhi Jani

Jani R., Di Cicco A., Kaplan K., Yueyao Z, Tsai FC., Bonifacino J., Raposo G., Marks MS., Bassereau P., Lévy D. and Delevoye C. Institut Curie, 12 Rue Lhomond, 75005 France, Paris

The endosomal system is a major sorting station in the cell consisting of highly dynamic membrane compartment controlling many intracellular transport and signaling events. The sorting nature of endosomes is by the virtue of the emergence of dynamic narrow and highly curved tubular network (comprisin of recyclin endosomes ≈60 nm) that enables it to sort specific cargoes en route to pre-destined target membranes. Our group implicated the role of BLOC-1 (Biogenesis of Lysosome Related Organelle complex-1), an 8-subunit complex that acts as a “choirmaster” to or anize the morpho enesis and the scission of the recycling tubules by co-operating with kinesin motor (KIF13A) and the actin polymerizing machineries (ANXA2 and ARP2/3). We hereby show that BLOC-1 has affinity for PI4P (phosphoinositide 4 phosphate) positive membranes in-vitro and depleting PI4P by specific inhibitors or by inactivating the PI4P synthesizing kinases perturbs the formation of recycling endosomal tubules. Further, our data indicates that BLOC-1 co-operates with PI4P and its binding cargo-sorting AP-1 complex (Adaptor Protein-1) and for the morphogenesis of these tubules. Investigating putative BAR (Bin/Amphiphysin/Rvs) like proteins needed to form recycling tubules illustrated that BLOC-1 could be a new type of membrane curvature-sensor/inducer, in contrast to the BAR-domain containing APPL1 endosomal protein. Together, BLOC-1 can orchestrate the PI4P rich membrane remodeling events by co-operating with the motor proteins, actin machinery and adaptor proteins.


53

Modulation of amphiphysin and dynamin rescues severe congenital myopathies

Jocelyn Laporte

Lionello V., Cowling B., Nicot AS., Tasfaout H., Buono S., Kretz C., Bitoun M. and Laporte J. IGBMC, 1 rue Laurent Fries, 67404 Illkirch, France

Centronuclear and myotubular myopathies (CNM) are a group of severe muscle diseases characterized by muscle weakness and organelles mis-positioning in myofibers. The severe X-linked form, also called myotublar myopathy, is caused by loss-of-function mutations in the phosphoinositide phosphatase Myotubularin (MTM1), while two main autosomal forms are due to mutations in membrane remodeling proteins, Amphiphysin 2 (BIN1) and Dynamin 2 (DNM2). Albeit these proteins are linked to lipid metabolism and membrane trafficking, the underlying CNM pathomechanisms are still unclear. Moreover, there is a strong need for therapeutic solutions for the different CNM forms.

In order to decipher the CNM pathway and test rescuing strategies, we have created and validated corresponding faithful myopathic mouse models mutated in MTM1, BIN1 and DNM2, and modulated these genes in the different models. We found down-regulation of DNM2 level, either by genetic cross, antisense oligonucleotides, or AAV (adeno-associated virus) expressing shRNA, efficiently prevent or revert the muscle weakness and the intracellular disorganization observed in myopathic mice mutated in any of the 3 CNM genes. In addition, overexpression of BIN1 though genetic cross with a mouse overexpressing human BIN1 or through AAV transduction can also rescue the myopathy in the MTM1 CNM mouse model. Lifespan is greatly extended, muscle weakness ameliorated and the intracellular disorganization rescued. We found MTM1 controls cell adhesion and integrin localization in mammalian muscle. Alterations in this pathway in the MTM1 CNM mouse model correlated with defects in myofiber shape and size that are main CNM hallmarks. BIN1 overexpression rescued integrin and laminin alterations and restored myofiber integrity, supporting MTM1 and BIN1 functional link is necessary for focal adhesion in skeletal muscle.

Overall, these data highlight that MTM1, BIN1 and DNM2 are part of a common pathway regulating myofiber organization and function. BIN1 and DNM2 represent two novel therapeutic targets for different forms of myotubular and centronuclear myopathies.


54

Monitoring force generation of phagocytosing macrophages

Florence Niedergang

Mularski M.1, Le Clainche C.

2, Balland M.

3 and Niedergang F.

1

Institut Cochin (Inserm U1016, CNRS UMR8104, Université Paris Descartes), 75014 Paris, France

Phagocytosis is important for a variety of immune functions such as remodelling of tissues, disposal of dead cells and bacterial clearance. Actin polymerisation provides the force that drives the membrane deformation required to engulf particulate matter during phagocytosis. Key to dissecting the mechanism by which this occurs, is understanding how the complex mechanosensitive machinery of actin binding proteins sense force and stabilize actin anchoring during phagocytosis, from initial receptor binding, through to phagosome formation and closure. A novel experimental approach utilizing traction force microscopy is being used to observe phagocytosing macrophages on substrates of biologically relevant stiffness. Inhibition of key actin binding proteins will provide greater understanding of the role of these proteins in the related mechanosensitive processes of phagocytosis and adhesion.


55

Up-regulation of flotillins, new marker of metastatic development, deregulates endocytosis and vesicular trafficking to induce Epithelio-to-Mesenchymal Transition and cellular invasion

Cécile Gauthier-Rouviere

Gauthier-Rouvière C., Genest M., Planchon D., Comunale F and Bodin S. CRBM, CNRS UMR5237, 34293 Montpellier, France

Recently flotillin 1 and 2, two ubiquitous and conserved membrane proteins able to oligomerize to form membrane microdomains, were reported to be upregulated in a wide variety of invasive carcinomas and sarcomas. This overexpression is associated with a poor prognosis. Through various in vitro and in vivo approaches, we showed that in non tumoral epithelial cells, overexpression of flotillins is sufficient by itself to induce epithelio-to-mesenchymal transition (EMT), extracellular matrix degradation and cellular invasion. These observations illustrate the critical role of flotillin upregulation in the acquisition of invasive properties by cancer cells. Our current data suggest that when upregulated, flotillins are promoting membrane curvatures of the plasma membrane and formation of intracellular vesicles. The cellular distribution of upregulated flotillins is dramatically modified with a strong enrichment in vesicular compartments that we characterized as non degradative-endolysosomes. We propose that flotillin overexpression induces a trafficking pathway that we named UFIT-pathway (Upregulated flotillin Induced Trafficking pathway) and that promotes cellular invasion. The aim of our study is to identify: 1) the molecular cargoes affected by this trafficking pathway; 2) the molecular mechanisms involved in the induction of this pathway.

We used non tumoral mammary breast epithelial cells (MCF10A and NMuMg) having low flotillin expression level to generate stable cell lines (MFC10AF1F2 and NMuMgF1F2) expressing flotillins at levels similar to those observed in MDA-MB-231 invasive breast cancer cells. In addition, we stably downregulated flotillins in MDA-MB-231 cells. Using these cellular models and their parental counterparts, we performed several global comparative analyses (transcriptomic, proteomic, phosphokinase arrays). We demonstrated that the upregulation of flotillins modified the differentiation state of mammary epithelial cells by inducing an EMT perturbing cellular adhesion and promoting invasion. We further showed that flotillin-upregulation in the non tumoral mammary epithelial cells is sufficient to activate multiple oncogenic signaling pathways. We next identified protein cargoes of the UFIT pathway such as transmembrane signaling receptors whose endocytosis, trafficking and activity is modified by flotillin upregulation.

Our results suggest that the UFIT pathway generates flotillin-positive endolysosomes actin as a “si nalosome compartments” involved in the activation of si nalin pathways stimulating EMT and cellular invasion.


56

Single-molecule analysis of IL-2 receptors reveals their clustering during endocytosis in lymphocytes

Laura Salavessa

Salavessa L., Grassart A., Malardé V. and Sauvonnet N. Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, INSERM U1202, 28 rue du Docteur Roux, 75015 Paris, France

Receptor-mediated endocytosis is essential for eukaryotic cells to uptake specific cargos. Several endocytic pathways exist but the clathrin-mediated route remains the best characterized. Although different clathrin-independent endocytosis (CIE) pathways have been uncovered, most of their fundamental mechanisms are still elusive.

The interleukin-2 receptor (IL-2R) was one of the first physiological cargos described as internalized through CIE. These receptors are critical for tolerance and immunity and transduction of signaling is highly regulated by their endocytosis and degradation. Therefore, deregulation of IL-2R internalization can lead to immunodeficiency or autoimmune diseases. A distinctive feature of this mechanism of endocytosis is the absence of a coat protein, like clathrin or caveolin, driving the pit and vesicle formation, hence raising the question on how the CIE vesicle is initiated. Protein crowding or clustering is known to mediate forces that can induce membrane conformational changes, as membrane bending. Additionally, a mechanism for IL-2R clustering at the base of membrane protrusions, along with other factors involved in CIE, was previously suggested. This led to our current hypothesis that clustering of the receptor would initiate and stabilize the pit, that would then mature into a vesicle in a process involving cholesterol, dynamin, actin-linked proteins and membrane-bending proteins. To test this hypothesis, we are currently determining the stoichiometry of IL-2R during endocytosis.

We generated a CRISPR-edited T-cell line that endogenously expresses the gamma chain of the IL-2R (IL-2Rγ) tagged with GFP. Using TIRF microscopy coupled to a single-molecule calibration and endocytic tracking and analysis, we are able to infer on the number of IL-2Rγ molecules present at the plasma membrane and correlate different clustering combinations with the rate of receptor endocytosis.

Firstly, we identified three different populations of IL-2Rγ according to their lifetimes: a short-lived population, likely corresponding to newly secreted receptor; a long-lived population representing the actively endocytic receptor; and a static population. IL-2Rγ seems to be secreted to the plasma membrane as a pre-cluster of three molecules, which then undergoes addition of further receptor molecules creating an endocytic viable cluster comprising four to five molecules. Interestingly, the static population is characterized by a higher number of molecules, suggesting that larger complexes might render the receptor inefficient for endocytosis. Importantly, the presence of the ligand, IL-2, leads to an increase in the number of IL-2Rγ molecules in the cluster, a shorter lifetime of the endocytic population and a higher rate of receptor uptake, stressing the importance of clustering for CIE internalization.

Currently we are studying the role of some factors involved in CIE that might impact IL-2Rγ clustering and/or pit formation, as cholesterol, actin cytoskeleton and dynamin. Our preliminary data points to the impact of cholesterol depletion on cluster size, rate of uptake and IL-2 signaling, suggesting that larger IL-2R clusters highly signal while being inefficient for endocytosis.

Several signaling receptors, as G-protein-coupled and tyrosine kinase receptors, are internalized through this endocytic mechanism. Therefore, clustering of the IL-2R as an enhancer of pit formation might be a conserved feature, highlighting the relevance of cytokine receptor endocytosis.


57

Exchange dynamics of dynamin measured in living cells during endocytic vesicle formation

David Perrais

Claverie L., Rosendale M., Butler C., Sibarita JB., Choquet D. and Perrais D. Institut Interdisciplinaire de Neurosciences, CNRS et Université de Bordeaux, Bâtiment Broca Nouvelle Aquitaine, 146 rue Léo Saignat, 33076 Bordeaux, France

During clathrin mediated endocytosis (CME), the GTPase dynamin is recruited to the neck of nascent clathrin-coated vesicles (CCVs) where it oligomerizes into helical filaments. Conformational changes induced by the hydrolysis of GTP catalyze the scission of the vesicle neck. This process has been studied in great detail with in vitro reconstitution on membrane tubules but it needs to be established in living cells, where dynamin interactions with other proteins such as amphiphysin are critical. Live cell TIRF imaging with the ppH assay [1] allows the detection of CCV formation with high spatial (~100 nm) and temporal (2 s) resolutions. It has revealed that dynamin is recruited to maturing clathrin coated pits (CCPs) in two phases with a peak at the time of scission [2] but the parameters of its recruitment in living cells remain unclear.

To determine these parameters, we have performed live cell imaging of dynamin recruitment at collective and single molecule levels during acute perturbations of its function. First, we showed that Dyngo4a, a cell permeable blocker of dynamin GTPase activity, or GTPγS dialysed through a patch-clamp pipette, quickly blocked CME and led to the accumulation of dynamin-mCherry at CCPs. Partial block decreased the rate of dynamin recruitment before CCV formation, suggesting that GTPase activity regulates its recruitment at early stages of CCV formation. To avoid any effect of diffusion, we have performed photo-activation of DMNPE-GTPγS: the compound is inactive before UV illumination, but blocks endocytosis within 4 s while provoking dynamin accumulation on a longer timescale. We next investigated the parameters of dynamin recruitment to forming CCVs. FRAP analysis showed that dynamin exchange is fast and complete and was only moderately impaired by saturating concentrations of Dyngo4a, suggesting that dynamin exchanges with an extra-CCP pool at all times, including association in the oligomeric spiral around the tubular neck. To get better insight into the modes of dynamin recruitment at all stages of CCP maturation we conducted dual ppH/single protein tracking (sptPALM) imaging in cells expressing dynamin-mEOS3.2. Dynamin is recruited to the plasma membrane, diffuses outside of CCPs and is trapped at CCPs. The number of detected molecules increases as scission approaches but single molecules are equally immobilized at all stages of CCP maturation. We conclude that dynamin exchanges with an extra-CCP pool at all times: this would allow for its further recruitment by addition of new binding sites and its ability to narrow the vesicle neck after GTP hydrolysis.

References [1] Merrifield CJ, Perrais D, Zenisek D. Coupling between Clathrin-Coated-Pit Invagination, Cortactin Recruitment, and Membrane Scission Observed in Live Cells. Cell. 2005;121: 593–606. [2] Taylor MJ, Perrais D, Merrifield CJ. A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis. Schmid SL, editor. PLoS Biol. 2011;9: e1000604.

GEM PhD Thesis Prize Talk Thursday, April 4th

58

GEM PhD thesis prize talk

Dystrophin-membrane interaction: 3D structure of dystrophin fragments in the presence of phospholipids

Raphael Dos Santos Morais

Dos Santos Morais R.1,2,3

, Delalande O.1

, Pérez J.3

, Combet S.2

and Hubert J-F.1

1Institut de Génétique et Développement de Rennes, CNRS UMR 6290, Université de Rennes 1.

2Laboratoire Léon-Brillouin, UMR 12 CEA-CNRS, Université Paris-Saclay.

3SWING Beamline, Synchrotron SOLEIL.

Dystrophin is a peripheral membrane protein supporting the plasma membrane of muscle cells (Le Rumeur et al., Biochim. Biophys. Acta, 2010). Gene mutations lead either to a total deficit of dystrophin or to the presence of the protein in truncated forms and are responsible of Duchenne and Becker muscular dystrophies (DMD and BMD) respectively. Exhaustive knowledge of dystrophin/membrane interactions is essential to contribute to the design of gene therapy strategies devoted to DMD and BMD, one of them aiming to design a minimal functional mini-dystrophin. Most part of the dystrophin consists in a central domain, made of 24 spectrin-like repeats (R1 to R24), of which the 3D-structure is not accessible by classical high-resolution methods and thus alternative approaches were employed to obtain structural information (Molza et al., Faraday Discuss., 2014; Delalande et al., J. Biol. Chem., 2018).

Here, we focused on the three first spectrin-like repeats (R1-3) of dystrophin central domain, that are known to interact with membrane lipids (Legardinier et al., Biochim. Biophys. Acta, 2008, and J. Mol. Biol., 2009) and that are found in potential therapeutic mini-dystrophins (Le Guinier et al., Nat. Commun., 2017; Ramos et al., Mol Ther., 2019). First, protein/lipid interactions of R1-3 were characterized with zwitterionic or anionic phospholipid-based bicelles as a membrane mimic. We notably used Microscale thermophoresis (MST, NanoTemper Techonologies#) to highlight protein/lipid interactions and to determine a dissociation constant of the complexes. To our knowledge, it is the first time that bicelles are used in MST experiments and we do believe that this approach could be extended to others protein/lipid systems. Then, using small-angle neutron scattering (SANS), we specifically probed the solution structure of R1-3 either free or bound to zwitterionic as well as anionic contrast-matched bicelles (Dos Santos Morais et al., Langmuir, 2017). Our results highlight that when bound to zwitterionic bicelles, no significant conformational modifications of R1-3 are detected. On the other hand, when R1-3 is bound to anionic bicelles, SANS data demonstrate large modifications of its 3D-structure. The R1-3/anionic bicelle complex was further analyzed by classical coarse-grained molecular dynamic simulations (CG-MD) and interactive CG-MD (Delalande et al., J. Comput. Chem., 2009). The final models proposed for R1-3 bound to membrane lipids are totally in adequacy with the experimental SANS data. Moreover, we propose an accurate mapping of the protein/lipid interactions obtained by coupling click-chemistry with mass spectrometry, in line with the in silico data (Dos Santos Morais et al., Biophys. J., 2018). We hypothesize that the membrane anchoring that likely occurs during the contraction/elongation process of muscles could be ensured by the opening of the first spectrin-like repeat of R1-3. Understanding these structural changes may help for the design of rationalized efficient mini-dystrophins devoted to gene therapy. Last but not least, we expect that our approaches can be extended to other peripheral and even integral membrane proteins.

#This talk is sponsored by NanoTemper Techonologies (https://nanotempertech.com/)

59

SESSION 5

Biophysical Methods

Chairpersons : Nathalie Sauvonnet & Erick Dufourc

SESSION 5 | Biophysical Methods Friday, April 5th

61

Nanostructured materials interacting with synthetic and natural lipid mesophases: challenges and opportunities

Debora Berti

Department of Chemistry “U o Schiff”, University of Florence and CSGI, Via della Lastruccia 3, Sesto Fiorentino, 50019, Florence, Italy

In spite of the tremendous achievements in the design, synthesis and preparation of nanosystems for medicine, the implementation of nanomaterials into biomedical applications is still very limited. One of the main issues is a scarce knowledge of the interaction of nano-objects and biologically relevant interfaces, from complex biological fluids to biomembranes. In this contribution I will provide an overview on our results on the interaction of nanostructured materials on synthetic and natural biomembranes. Our approach entails the understanding of mechanistic details gathered in model lipid mesophases, either based on bilayered structures (liposomes, supported lipid bilayers and giant unilamellar vesicles) or cubic mesophases. Unravelling the nature of these interactions also provide the opportunity to design new physico-chemical methods to gather compositional,structural and dynamical information on real cell membranes. References M. Mendozza et al.. Journal of Colloid and Interface Science, 541, 329-338 (2019). T. Pfeiffer et al., The Journal of Physical Chemistry Letters. 10(2), 129-137 (2019). M. Mamusa et al., Langmuir 34(30), 8952-8961 (2018). C. Montis, et al., Advanced Biosystems 2, 1700200 (2018). C. Montis et al., Nanoscale 10(33), 15442-15446 (2018). M. Mendozza et al., Nanoscale 10(7), 3480-3488 (2018).


62

An extracellular sphingolipid-binding domain on synaptotagmin: biochemical characterization and biological function

Jose-Jorge Ramirez-Franco

Ramirez Franco J-J., Flores A., Desplantes R., Fantini J., Debreux K., Wernert F., Lévêque C. and El Far O. INSERM UMR_S 1072, 13015 Marseille, France

Synaptotagmin is a synaptic vesicle protein important for neurotransmission. Because of its sensitivity to calcium ions, it determines the synchronicity between calcium influx into synaptic terminals and neurotransmitter release. The luminal N-terminal domain of Synaptotagmin is transiently exposed to the neuronal surface upon exocytosis, where it encounters molecular partners. Consequently, its diffusion appears hindered by physical barriers as it directly interacts with cholesterol in lipid microdomains, and is confined with cone-shaped glycosphingolipids (gangliosides) present on the extracellular leaflet of plasma membranes. In nerve terminals, gangliosides and Synaptotagmin 1 /2 serve as co-receptors for botulinum neurotoxin type B (BoNT/B), a potent neurotoxin. After binding, the toxin uses the endocytic pathway to enter the nerve terminal and then exerts its toxic activity by cleaving VAMP2 and potently inhibiting neurotransmission.

Using techniques including SPR, Langmuir film balance, ELISA, circular dichroism (CD), and fluorescence spectroscopy we identified a direct interaction between the N-terminal juxta-membrane domain of SYT1/2 and gangliosides. Molecular dynamics simulations corroborated these results and highlighted a potential physiological importance.

We will discuss the consequences of perturbing this interaction on BoNT/B toxicity and its potential physiological relevance.


63

Endocytosis across scales

Armando Maestro

Maestro A., Cicuta P., Zaccai N., Kelly B. and Owen D. Institut Laue-Langevin,71 avenue des Martyrs, 38000 Grenoble, France

Clathrin-mediated endocytosis is a crucial cell biology process allowing internalization of many cell-surface proteins, and other cargo, in eukaryotes. Clathrin-coated vesicles (CCVs) are assembled with their cargo at the plasma membrane, then transport to the early endosome inside the cell. A CCV consists of a clathrin scaffold coating a lipid vesicle, in which the cargo is embedded, linked by adaptor proteins that are associated with effectors of CCV assembly, stability and disassembly. Owen’s team recently determined that a single adaptor protein AP2 is sufficient to initiate and drive clathrin-coated bud formation on appropriate membranes, enriched in PtdIns(4,5)P2 [1,2].

In vivo, AP2 interacts solely with one leaflet of the cellular membrane. Therefore, an alternative valid model system is to explore clathrin assembly on a flat lipid surface (in our case a Langmuir monolayer). This allows us to probe the system with a set of state-of-the-art characterization methods typical of soft matter and physical chemistry, including AFM, neutron reflectometry, interfacial tensiometry and rheology. We thus have been able to analyse the first stages of CCV assembly by using cargo embedded in a lipid monolayer [3]. We show here in particular the influence of AP2, and subsequently the clathrin scaffold, on the composition, structure and mechanics of the complex layer that self-assembles in stages.

References

[1] Miller SE, Owen DJ et al, (2011) Cell. 147, 1118-31. [2] Kelly BT and Owen DJ, Science. (2014) 345, 459-63. [3] Maestro A, Zaccai N, Kelly B, Owen D and Cicuta P, Submitted.


64

Bacteria internalization: how to discriminate adhesion from entry on line

Frank Lafont

CMPI Group Center for Infection and Immunity of Lille Institut Pasteur de Lille, 1 rue du Pr Calmette, 59021 Lille, France

Internalisation of pathogens as bacteria is the initial step of infection but the immediate early event is the adhesion of the bacterium on the cell surface. Neisseria meningitidis for instance uses adhesion and signaling membrane receptors.The question arises of how discriminating between signaling triggered at the adhesion step versus that activated during the entry per se. Using several pathogens as models (e.g. Yersiniae), we developed correlative microscopy and force spectroscopy tools and methods to tackle this question that we propose to discuss during the meeting.


65

SAXS studies of lipid oxidation in mimetic membranes

Francesco Spinozzi

Spinozzi F., De Rosa R. and Itri R. Department of Life and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy

Many lipid molecules that form biological membranes shows unsaturated groups and therefore are prone to oxidation [1]. For polyunsaturated lipid molecules this process occurs spontaneously. On the contrary, monounsaturated phospholipids, such as POPC and cholesterol, require a highly oxidizing component (such as a singlet oxygen) to to initiate the reaction [2]. Since the biological function of membranes is directly associated with their composition, any biophysical alteration generated by lipid oxidation can positively or negatively affect the functioning of the membrane. For example, oxidative stress has been successfully used for the treatment of skin diseases. On the other hand, it is well known that lipid oxidation may be related to serious diseases such as cancer, hypertension, diabetes, Parkinson's, Alzheimer's, Huntington's and Sclerosis [3].

We here present a synchrotron SAXS investigation of large unilamellar vesicles (LUVs) composed by POPC and two oxidized species, namely POPC-OOH, its hydro-peroxidized form, and PazePC, a lipid that has a carboxyl group at the end of its truncated sn-2 chain. The replacement of POPC by either POPC-OOH or PazePC, with oxidized lipid molar ratio x varying from 0.00 up to 1.00, permits to experimentally inspect changes in the membrane structural properties due to oxidation. SAXS data analysis has been performed within the SDP (Scattering Density Profile) methodolgy [4] by using a new representation of the volume fraction probability of each chemical group of the lipid based on the error function, named Modified Scattering Density Profile (MSDP) [5].

Results, in agreement with recent Molecular Dynamics Simulations outcome [6], indicate that 95% of the hydroperoxide group lies in the membrane polar moiety, near the carbonyl and phosphate groups, whereas just 5% of OOH group experiences the polar/apolar interface, for all values of x studied. In the case of PazePC up to x=0.33, a bimodal distribution of the carboxyl group in the interior and polar regions of the lipid membrane is obtained, probably due to a dynamic movement of the shortened alkyl chain towards the water interface.

References

[1] Mertins et al., Biophys. J. 106, 162 (2014). [2] Itri et al. Biophys. Rev. 6, 47 (2014). [3] Cortez et al., FEBS Letters 584, 625 (2010). [4] Kučrka et al., Biophys. J. 95, 2356 (2008). [5] De Rosa et al., BBA - Biomembranes 1860, 2299 (2018). [6] Garrec et al., J. Phys. Chem. Lett. 5, 1653 (2014).


66

Out-of-equilibrium active membranes: incorporation of bacteriorhodopsin in a floating lipid bilayer

Tetiana Mukhina

Mukhina T., Gerelli Y., Fragneto G. and Charitat T. Institut Laue-Langevin , 71 Avenue des Martyrs, F-38042 Grenoble, France.

Transmembrane proteins have a pivotal role in the large number of cellular processes and provide essential functions of the cell membranes. By itself, membranes exhibit thermal fluctuations, but membrane protein activity breaks the fluctuation-dissipation theorem leading to out-of-equilibrium fluctuations. Active fluctuations have been widely described theoretically [1], but there are only few techniques for their experimental study. The outstanding model system as fluid floating phospholipid bilayer [2] and original scattering techniques as off-specular reflectivity [3] gave us ability to study the fluctuations of a single floating bilayer near a substrate [4] as well as membrane-membrane interactions [5].

The main aim of this project is to investigate out-of-equilibrium fluctuations of phospholipid membranes induced by active transmembrane protein bacteriorhodopsins (BR). Model systems such as tethered phospholipid bilayer, solid-supported single and floating phospholipid bilayers, prepared by Langmuir-Blodgett/Langmuir-Schaefer techniques, are routinely used to mimic and study phospholipid membranes and their interactions. As a first step the detergent-mediated incorporation method [6] was adapted to perform the insertion of BR into the phospholipid bilayer at the interfaces, using such sugar- based detergents as DDM and DOTM.

We present the proof of the possibility of inserting BR in supported POPC phospholipid bilayers and in a double bilayer system consisting of DSPC (bottom, supporting bilayer) and DPPC (top, floating bilayer) as recently demonstrated by neutron and x-ray reflectometry, QCM-D, fluorescence microscopy and AFM. Newly developed sample environment – solid-liquid cells suitable for the NR, XRR synchrotron and inhouse measurements, with the ability of sample illumination during measurements and solution injections – will be demonstrated. A further step of this work will be to check the protein activity, which can be triggered by the absorption of the green light, in the supported bilayer systems by means of NR. The lateral features of the membrane embedded proteins system and then out-of-equilibrium fluctuations caused by the protein activation will be probed by synchrotron radiation off-specular measurements.

References

[1] J. Prost, J.-B. Manneville, and R. Bruinsma. EPJ B, 1:465–480 (1998). [2] Charitat T., Bellet-Amalric E., Fragneto G. and Graner F., EPJ B, 8, 583-593 (1999). [3] Malaquin L., Charitat T. and Daillant J., EPJ E, 31, 3, 285-301 (2010). [4] Daillant J., Bellet-Amalric E., Braslau A., Charitat T., Fragneto G., Graner F., Mora S., Rieutord F. Stidder B., PNAS, 102, 11639-11644 (2005). [5] Hemmerle A., Malaquin L., Charitat T., Lecuyer S., Fragneto G. and Daillant J., PNAS, 2012, 109, 19938.


67

Mechanobiology of endocytic vesicle formation analyzed by Sla2 force sensors

Marc Abella Guerra

Abella M., Andruck L., Malengo G. and Skruzny M. Max-Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Straße 10, 35043 Marburg, Germany Mechanical forces exerted by protein machines are indispensable for many vital cellular processes. One of the best-studied examples is membrane reshaping during clathrin-mediated endocytosis, a principal vesicle trafficking route responsible for nutrient uptake, signalling regulation, and plasma membrane homeostasis. During endocytosis, a small region of the plasma membrane reshapes from a flat sheet to a closed vesicle. This reshaping requires mechanical force, which is provided by specific endocytic proteins and by actin polymerization. Several theoretical models have been already proposed to describe force-dependent processes during endocytosis. They are often focused on endocytosis in yeast, where actin is essential for vesicle budding and where key protein players and membrane-shape profiles have been established. However, to mechanistically understand force-dependent endocytic vesicle formation, applied forces need to be analysed in the cellular context to report real force values applied on the native membrane by a synergy of involved endocytic proteins.

To achieve that, we used FRET (Förster Resonance Energy Transfer) tension sensors (Freikamp et al., 2016), which allow the measurement of pN forces in vivo, and inserted them into the endocytic protein Sla2. Sla2 is part of the essential Sla2-Ent1 (Hip1R-epsin in human) protein linker transmitting force of polymerizing actin cytoskeleton to the plasma membrane (Skruzny et al., 2012, 2015). We followed force transmitted over Sla2 protein in real time by measuring FRET changes of Sla2 force sensors during the progression of individual endocytic events. Using sensors sensitive to different force ranges we estimated the force applied on Sla2 to be higher than 9 pN.

Next, we have analysed contributions of important membrane remodelling factors (e.g. BAR-domain proteins, regulators of actin polymerization) to force-dependent steps of endocytosis. Finally, ongoing experiments aim to monitor endocytic force requirements under various environmental and genetic conditions changing plasma membrane properties and cellular turgor pressure.

We believe that our data will help to construct a comprehensive, experimentally-based biomechanical model of endocytic vesicle formation, which could be also valuable for other cellular membrane reshaping processes based on actin polymerization and membrane remodelling factors.


68

Molecular transport in membranes investigated by neutron scattering: lipid exchange and translocation

Lionel Porcar

Porcar L., Gerelli Y. and Perez-Salas U. Institut Laue Langevin, 38042 Grenoble, France The great variety of lipid molecules in the cell membrane suggests their complex and unique role in cell function. The cell has further established unique lipid composition in different membranes within the cell for directed functionality. In addition, in membranes like the plasma membrane (PM), there is an asymmetric distribution of lipids between the outer or exoplasmic and the inner or cytoplasmic leaflets and the physiological fate of cells depends on the strict maintenance of this asymmetry. However, the exact mechanisms and energetic toll by which these lipids arrive and particularly remain at their locations, such as in the PM, are not fully understood. Reliable values of passive lipid translocation rates are a necessary starting point for a detailed mechanistic understanding of the lipid distribution landscape in cellular membranes. However, obtaining these values has been hampered by artifacts emerging from different methodologies. As a result, the reported rates for similar lipids can vary by several orders of magnitude, from less than a second to hours. Work in the last few years by several groups including ours, have shown that only those techniques free of artifacts can potentially be used in the study of the transport of lipids across membranes. Combining time resolved small angle neutron scattering and neutron reflectivity, we show that it is possible to capture inter and intra vesicular exchange as well as lipid composition differences in the leaflets of a model bilayer with the sub-nanometer spatial resolution and for times scales as short as a few minutes.

By in situ monitoring the structure of a) a solid supported lipid bilayer exposed to a solution of isotopically labeled vesicles and b) a bulk mixture of hydrogenated and deuterated vesicles, we can provide new insight on the characteristics of inter- and intra-bilayer rearrangement processes. We will report on the rates and energetics of pure lipids as well as cholesterol transfer in different lipids environments.

We will also present our recent results on the production of asymmetric membranes without the requirement of molecules such as cyclodextrin to study the intra-membrane flip-flop of lipids and cholesterol across a single lipid bilayer using SANS, NMR, and fluorescence techniques.


69

A spectrum of light sheet instruments optimized for different imaging demands

Colin Monks

Intelligent Imaging Innovations, Inc. 3509 Ringsby Ct. , 80216 Denver, USA

3i designs and manufactures technologies for living cell, live cell, and intravital fluorescence microscopy including digital holography, spinning disk confocal, multi-photon and light sheet. Customers worldwide use 3i’s SlideBook microscopy software to mana e everythin from instrument control to image capture, processing and data analysis. In my presentation, I will discuss a variety of light sheet options and strategies for imaging different types of specimens and will review the photo physics that lets people evaluate one type of light sheet against another.


70

Linking architecture and function of organelle contact sites

Wanda Kukulski

Cell Biology Division, MRC Laboratory of Molecular Biology, Cambridge, UK

Cellular membranes generate boundaries that compartmentalize the cell and at the same time host a large diversity of functions. The diverse roles of cellular membranes are tightly coupled to their architecture. Despite their importance, we know little about how membrane shape, protein composition and supramolecular organization reciprocally impact each other. Our lab’s oal is to understand mechanisms by which membrane architecture contributes to cellular functions. In this talk, I will focus on membrane contact sites, where specific proteins tether organelles to each other. Such organelle contacts play roles in lipid distribution, Calcium transport and organelle biogenesis. We aim to understand how the architecture built from proteins and apposing membranes drives lipid transfer reactions. Our main tools are correlative microscopy methods. By localizing fluorescent signals in electron tomograms of resin-embedded cells with high precision, we link the presence or absence of key proteins to the 3D membrane ultrastructure. To identify and visualize proteins associated with cellular membranes, we combine cryo fluorescence microscopy with cryo electron tomography of cells thinned by cryo-focused ion beam milling. We complement these appraoches with biochemical and genetic perturbations and live cell imaging.

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SESSION 6

Structure of Exo-Endocytotic Membrane Systems :

From Isolated Molecules to Cells

Chairpersons : Olivier Seksek & Christophe Lamaze

SESSION 6 | Structure of Exo-Endocytotic Membrane Systems : Friday, April 5th From Isolated Molecules to Cells

73

From Complex Biomembrane Mimics to Live Cells: Lessons Learned from Scattering Techniques

Georg Pabst

Institute of Molecular Biosciences, University of Graz, Graz, Austria

Lipid-only mimics of biological membranes serve as valuable platforms for studying the functional role of membrane lipids under chemically well-defined conditions. Of recent, we have focused on complex mimics of mammalian and bacterial plasma membranes with either lateral or transbilayer inhomogeneities. In particular, we have developed protocols for fabricating and analyzing asymmetric lipid vesicles, which are sufficiently stable and which are amenable for biophysical studies using diverse techniques. These include small-angle X-ray and neutron scattering, and allow us to address lipid-mediated transbilayer coupling mechanisms, which is of potential significance for diverse physiological processes, including cellular signalling. Our studies suggest that lipid intrinsic curvature plays a pivotal role in this scenario. At the same time, we are also exploiting the potential of scattering techniques to study the structural integrity of live bacteria. Early experiments demonstrate that there is an experimental window to address the activity of antimicrobial peptides on the subsecond time scale, enabling us to couple insight from membrane mimics to events on the cellular level.


74

Allosteric regulation of small GTPases at the surface of membranes : a structural and biophysical perspective

Jacqueline Cherfils

Nawrotek A., Benabdi S., Niyomchon S., Kryszke MH., Ginestier C., Caneque T., Tepshi L., Mariani A., Charafe-Jauffret E., Rodriguew R., Zeghouf M. and Cherfils J. ENS Paris-Saclay, 61 avenue du Président Wilson, 94235 Cachan, France

Lipidated small GTPases regulate most aspects of cell logistics » including signalling, membrane traffic and cell shape and motility, and they are associated with myriad severe diseases. To function, they assemble multiprotein complexes at the surface of membranes to propagate actions in the cell, but an integrated understanding of their interactions with the lipid bilayer is still lacking. I will describe recent research in which we combined X-ray crystallography, SAXS, molecular dynamics, HDX-MS and fluorescence kinetics to understand the inner workings of small GTPases and their regulators on membranes, and how it led to new concepts in drug discovery.


75

The membrane organization of skeletal muscle-derived extracellular vesicles regulated by S-palmitoylation: A comprehensive biochemical and structural characterization

Mauro Manno

Raccosta S., Buffa V., Romancino D., Noto R., Martorana V., Bongiovanni A. and Manno M. CNR Institute of Biophysics, via Ugo La Malfa 153, I90146 Palermo, Italy

Several cell types have the capacity to secrete nanometer-sized extracellular vesicles (EVs) working as potent signal transducers and cell-cell communicators. Skeletal muscle (SkM) cells can release EVs positive to the protein Alix, a bona fide exosomal regulator which may undergo S-palmitoylation, a reversible lipid post-translational modification, involved in different biological processes.

Here, we have used an integrated biochemical-biophysical approach to determine whether S-palmitoylation contributes to the regulation of extracellular vesicle production in skeletal muscle cells.

We ascertained that Alix is S-palmitoylated and that this post-translational modification influences its subcellular localization and protein-protein interaction, particularly with the tetraspanin protein CD9.

Further, we evaluated the effect of palmitoylation inhibition on the size and heterogeneity of EV, by using Atomic Force Microscopy (AFM) and Dynamic Light Scattering (DLS). In particular, we focus on DLS experiments and on a dedicated analysis to cope with the heterogeneity of such complex samples.

Small-Angle X-ray Scattering (SAXS) and Small-Angle Neutron Scattering (SANS) experiments showed that the structural organization of the lipid bilayer of the small (nano-sized) extracellular vesicle membrane with altered palmitoylation is qualitatively different compared to mock control vesicles. The combination of SAXS and SANS measurements on the same sample was crucial to clearly identify the structural features of the lipid belayer in such a complex and heterogeneous system.

We propose that the role of S-palmitoylation in SkM-derived EV biogenesis is to regulate the proper function of Alix in facilitating the interactions among extracellular vesicle-specific regulators and maintains the proper EV membrane structural

organization.Beyond its biological relevance, our study also provides the means for a comprehensive structural characterization of EVs, which is expected to be crucial in the design of engineered vesicles to be employed in EV-based therapies.


76

Subnanometer structure of the membrane-assembled retromer coat by cryo-electron tomography

Oleksiy Kovtun

Kovtun O., Leneva N., Bykov YS., Ariotti N., Schaffer M., Engel BD., Owen DJ., Collins BM. and Briggs JAG. MRC, Laboratory of Molecular Biology , CB2 0QH Cambridge, UK

Protein coats are molecular machines that select cargo and drive formation of transport vesicles in intracellular trafficking pathways. Understanding the mechanism of coat action requires structural information about their membrane bound form. Recent improvements in cryo-electron tomography permitted visualisation of membrane-associated coats at high resolution despite their relative heterogeneity. Here we report the structure and arrangement of the BAR/retromer coat of endosomal transport tubules. The tubules were reconstituted in vitro and subjected to dose-symmetric tomogram collection followed by subtomogram averaging with ab initio modeling. The structure is different from previously proposed models and provides new insights into mechanisms of endosomal sorting. The membrane-associated BAR layer is stabilised by overlaying chains of arch-like retromer dimers. The local arrangement of the coat, which would be hidden when using a helical reconstruction approach due to deviations from helical symmetry, is resolved in this structure. We observe several arrangements of retromer arches arising from the redundancy of their docking sites on the BAR-lattice.


77

Systematic superresolution analysis of endocytosis reveals an actin nucleation nano-template that drives efficient vesicle

Jonas Ries

Mund M., vd Beek J., Deschamps J., Dmitrieff S., Monster J., Picco A., Nédélec F., Kaksonen M. and Ries J. EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany

Clathrin-mediated endocytosis is an essential cellular function of all eukaryotes. It relies on a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies that mediate vesicle formation. How so many proteins can be organized to produce endocytic vesicles with high precision and efficiency is not understood. To address this gap, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. This allowed us to visualize where individual proteins are localized within the machinery throughout the endocytic process.

By combining superresolution imaging, live-cell microscopy and Brownian dynamics simulations, we aim to identify the architectural features that allow the endocytic machinery to create vesicles with high efficiency and robustness. We found that actin filament nucleation is pre-patterned by a nucleation nanotemplate, which directly links molecular organization to the mechanics of endocytosis, and might represent a general design principle for directional force generation in other membrane remodeling processes such as during cell migration and division.

I will present first results on a dynamic reconstruction of the yeast endocytic machinery from thousands of images of fixed structures.

Reference Mund et al. “Systematic analysis of the molecular architecture of endocytosis reveals a nanoscale actin nucleation template that drives efficient vesicle formation,” Cell, (2018).


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Unraveling molecular arrangements and membrane morphogenesis through live and super-resolution microscopy with MemBright probes and ICY SODA plugin (Standard Object Distance Analaysis)

Lydia Danglot

Collot M., Lagache T., AshokkumarP., Mély Y., Olivo-Marin J-C., Klymchenko A., Galli T. and Danglot L. Institut de Psychiatrie et Neurosciences Paris, Inserm Umr_S 1266, 102 rue de la Santé, 75014 Paris, France

Existing plasma membrane probes are generally non-efficient due to their highly lipophilic nature. They generally require 1-5 mM concentration, which is ∼100-fold larger than that needed to achieve a probe/plasma membrane lipid ratio of 1/100. Their use in cell imaging is usually disappointing and almost inexistent in tissue imaging. Nowadays, red to near-infrared fluorescent probes are in high demand due to the widespread use of green and yellow fluorescent proteins and because they operate in a convenient spectral window for both in vitro and in vivo imaging. Up to now, membrane probes have shown very poor performance in this spectral region.

In this work, In collaboration with a team of chemists leader in the field of fluorescent molecular probes (Mayeul Collot within the team of A. Klymchenko), we developed a family of highly efficient red to near-infrared probes (MemBright) that turn-on their fluorescence on binding to the plasma membrane of the cells (Cell Chemical Biology 2019). MemBright surpass by far commonly used commercial probes due to their very bright, efficient and selective plasma membrane staining allowing their use at low concentration (as low as 20 nM) and long term imaging (up to 13 h).

The high brightness of MemBright probes allowed us to visualize small cell membrane protrusions in live cells and population of cells both in epithelial tissue (liver) and even in acute brain slices. Owing to unprecedented preferential staining of neurons, these probes provide with a new and fast way to visualize neurons in live brain slices as well as detection of motor neurons in tissue imaging. We successfully validated them in a variety of fluorescence microscopy techniques, such as laser scanning confocal, two-photon excitation imaging of cells and animal tissues as well as super-resolution imaging, which make them a universal toolbox for multicolor bio-membrane imaging. 3D super-resolution microscopy (STORM) with MemBright revealed fine structures of neurons (spines), which have not been accessible using previously developed fluorescent molecular probes. Since MemBright is compatible with cell fixation and permeabilization, it constitutes a unique tool that can be used in combination with immunofluorescence and open a wide avenue to multicolor imaging at the nanoscopic resolution. As a proof-of-principle, we showed here that it can be used even in multicolour STORM microscopy, providing a membrane reference for nanoscale mapping and we show that glutamate receptor aggregate at site of membrane contact between axon and dendrite.

To unravel fine distance and molecular arrangement we developed a new user friendly plugin called SODA for Statistical Object Distance Analysis in collaboration with the BioImage analysis unit in Pasteur Institute (Nature Comm. 2018). SODA uses micro- and nano-scopy to significantly improve standard colocalisation analysis and is freely available in ICY. Based on Ripley’s function our method considers both the geometry of the cell and the densities of molecules to provide colored maps of isolated and statistically coupled molecules.

We will show how SODA and memBright are versatile and effective tools to statistically map large data sets of multi-color molecular assemblies with high spatial resolution.


79

Targeting CCR5 trafficking to inhibit HIV-1 infection

Gaelle Boncompain

Boncompain G., Herit F., Tessier S., Lescure A., Del Nery E., Gestraud P., Staropoli I., Fukata Y., Fukata M., Brelot A., Niedergang F.

and Perez F.

1

Institut Curie - CNRS UMR144, 26 rue d'Ulm, 75248 Paris Cedex 05, France

Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. Importantly, they have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they dramatically decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery.


80

Structural Biology in situ: The Promise and Challenges of Cryo-Electron Tomography

Wolfgang Baumeister

Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany

Traditionally, structural biologists have approached cellular complexity in a reductionist manner by characterizing isolated and purified molecular components. This 'divide and conquer' approach has been highly successful. However, awareness has grown in recent years that only rarely can biological functions be attributed to individual macromolecules. Most cellular functions arise from their acting in concert. Hence there is a need for methods developments enabling studies performed in situ, i.e. in unperturbed cellular environments. Sensu stricto the term 'structural biology in situ' should apply only to a scenario in which the cellular environment is preserved in its entirety. Cryo electron tomography has unique potential to study the supramolecular architecture or 'molecular sociology' of cells. It combines the power of three-dimensional imaging with the best structural preservation that is physically possible.

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SESSION 7

Therapeutic delivery

Chairpersons : Marie-Pierre Rols & Claire Desnos

SESSION 7 | Therapeuthic Delivery Saturday, April 6th

83

Transfection by cell-penetrating peptides

Ülo Langel

Department of Neurochemistry, The Arrhenius Laboratories for Natural Sciences, Stockholm University, Svante Arrhenius väg 16B, SE-10691, Stockholm, Sweden

PepFect delivery technology for oligonucleotide transfection by transportan based cell-penetrating peptides in vitro and in vivo is described. Recent data on mechanisms and applications of PepFect strategies are summarized on the variety of different cargoes including plasmid, antisense and siRNA oligonucleotides. Nanomaterials, including graphene oxides (GOs), and cell penetrating peptides (CPPs) in combination are presented and are shown promising as non-viral vectors for gene delivery. Possible mechanisms of oligonucleotide delivery by PepFects is discussed and novel data on genome analysis involved in these mechanisms are discussed. References [1] Freimann,K. et al. (2016) J.Control.Release, 241, 135-143. [2] Cerrato,C.P. et al. (2017) Exp.Opinion Drug Del., 14(2), 245-255. [3] Dowaidar,M. et al. (2017) Scientific Reports. 7(1), 12635, 1-14 [4] Kurrikoff,K. et al. (2017) Sci.Rep. 7: 17056 [5] Alvarez,M.J. et al. (2018) Nature Genetics, 50 (7), 979-989


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Structural and functional investigations of histidine rich-peptides with potent cell penetrating, antimicrobial and lentiviral transduction activities

Morane Lointier

Lointier M., Marquette A., Wolf J., Juhl D. and Burkard B. Biophysique des Membranes et RMN - Institut de Chimie (UMR-7177), 1 rue Blaise Pascal, Université de Strasbourg, 67000 Strasbourg, France

The 26-residue LAH4 peptides were designed [1] using linear cationic antimicrobial peptides as a template (AMPs). Besides their antimicrobial action LAH4 sequences have also been shown to exhibit powerful gene [2] and siRNA transfection activities as well as help the cell penetration of proteins, peptides, nanodots, adeno associated - and lentiviruses [3]. To enhance this latter activity, derivates of LAH4 were designed and analysed [4]. From a series of derivatives some peptides exhibit distinct shifts in activities towards antimicrobial [5], transfection [6] or lentiviral transduction [7] despite their similarity in composition. The core of all peptides is made up from alanines and leucines with four histidines interspersed into this sequence to form amphipathic helices with different physico-chemical properties. Two lysines at each terminus make the peptides more soluble.

Here, to better understand the mechanisms of action and the differences between sequences, different peptide of the LAH4 family have been prepared, studied and compared. Their structures in solution and in membrane environments were investigated using dynamic light scattering and circular dichroism. Additionally, we tested their activities using dye release experiments using different membrane models. Furthermore, the fiber formation of some peptide sequences, which has been shown important for lentiviral transduction, is investigated at high-resolution by state-of-the art solid-state NMR spectroscopy.

References [1] Bechinger B. Towards membrane protein design: pH-sensitive topology of histidine-containing polypeptides. J Mol Biol. 1996; 263 (5): 768-775. [2] Kichler A, Mason AJ, Bechinger B. Cationic amphipathic histidine-rich peptides for gene delivery. Biochim Biophys Acta - Biomembr. 2006. [3] Moulay G, Leborgne C, Mason AJ, Aisenbrey C, Kichler A, Bechinger B. Histidine-rich designer peptides of the LAH4 family promote cell delivery of a multitude of cargo. J Pept Sci. 2017. [4] Majdoul S, Seye AK, Kichler A, et al. Molecular determinants of vectofusin-1 and its derivatives for the enhancement of lentivirally mediated gene transfer into hematopoietic stem/progenitor cells. J Biol Chem. 2016; 291 (5): 2161–2169. [5] Danos O, Bechinger B, Kichler A, Leborgne C, Ma J. Histidine-rich amphipathic peptide antibiotics promote efficient delivery of DNA into mammalian cells. PNAS. 2003; 100 (4): 1564-1568. [6] Vermeer LS, Hamon L, Schirer A, et al. Acta Biomaterialia Vectofusin-1 , a potent peptidic enhancer of viral gene transfer forms pH-dependent a -helical nanofibrils , concentrating viral particles. Acta Biomater. 2017; 64: 259-268. [7] Vogt TCB, Bechinger B. The Interactions of Histidine-containing Amphipathic Helical Peptide Antibiotics with Lipid Bilayers. J Biol Chem. 1999; 274 (41):29115-29121.


85

Extracellular Vesicles as a novel strategy of cell-to-cell communication

Nunzio Iraci

University of Catania, Torre Biologica, Via Santa Sofia, 97, 95123 Catania, Italy

Extracellular vesicles (EVs) are membrane particles involved in the exchange of a broad range of bioactive molecules between cells and the microenvironment. While it has been shown that cells can traffic metabolic enzymes via EVs much remains to be elucidated with regard to their intrinsic metabolic activity. Accordingly, herein we assessed the ability of neural stem/progenitor cell (NSC)-derived EVs to consume and produce metabolites. Both our metabolomics and functional analyses revealed that EVs harbour L-asparaginase activity catalysed by the enzyme Asparaginase-like protein 1 (Asrgl1). Critically, we show that Asrgl1 activity is selective for asparagine and is devoid of glutaminase activity. We found that mouse and human NSC-derived EVs traffic ASRGL1. Our results demonstrate for the first time that NSC EVs function as independent, extracellular metabolic units able to modify the concentrations of critical nutrients, with the potential to affect the physiology of their microenvironment.


86

Biophysical studies on the interaction of antimicrobial peptides containing un-natural residues with bacterial model membranes

Rosario Oliva

Oliva R., Chino M., Petraccone L., Notomista E. and Del Vecchio P. Department of Chemical Sciences, University of Naples "Federico II", Via Cintia 4, 80126 Napoli, Italy.

The increasing spread of bacteria resistant to conventional antibiotics has become a worldwide emergence. In fact, due to massive and out-of-control use of these drugs in humans and animals, bacteria have developed a series of mechanisms which render antibiotics ineffective. Thus, there is an urgent need of a new class of antimicrobial agents. Antimicrobial peptides (AMPs) are an interesting class of small peptides which can overcome this problem. It is believed that, due their unique and non-specific action mechanisms which involve a direct destabilization and interaction with the lipid matrix of patho ens’ membranes, they could help in fighting infections caused by multidrug-resistant (MDR) bacteria. In this line of research, synthetic peptides as antimicrobial agents exhibiting both resistance to proteolysis and selective toxicity have attracted much attention. In this study, it is reported the calorimetric and spectroscopic characterization of the interaction of a synthetic AMP containing un-natural residues, named P9Nal(SS) with liposomes composed by DPPC/DPPG (8/2 mol/mol) mimicking the cytoplasmic bacterial membrane. In addition, to explore the effect of amino acid substitution on the membranotropic activity of the peptide, a comparison with two derived peptides, named P9Nal(SR) and P9Trp(SS), it is achieved. Each peptide includes three lysine residues, two aromatic residues (either tryptophan or 2-naphthyl-L-alanine), two cysteine derivatives (a thioether or a mixed disulfide) and N- and C-terminal 6-aminohexanoic acid residues. The results reveals important similarities and differences on the interaction with cytoplasmic bacterial model membrane between the two peptides and with the parent peptide P9Nal(SS). The three peptides act through the same general action mechanism which involves peptides absorption on membrane surface, conformational changes and formation of lipid domains. Upon reaching a threshold lipid-to-peptide ratio (L/P), the peptides insert in the bilayer at different levels perturbing, or not, the regular lipid packing. The collected data suggest that the penetration abilities follow the order: P9Nal(SS) >> P9Nal(SR) ≥ P9Trp(SS) which correlate with peptides’ hydrophobicity. It seems that the partition inside the membrane is not fundamental in destabilizing the membrane, since all the peptides have similar activities against Gram-negative bacteria. Rather, the formation of lipid domains is the key step in promoting membrane perturbation. Overall, these results could represent an important contribution in understanding the action mechanism of AMPs and for the development of new peptide-based drugs for biomedical application.


87

Membrane biophysics and biointerfaces in nanotherapeutics development

Marlene Lúcio

CF-UM-UP (Centre of Physics of University of Minho and Porto), Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal

Nanotechnology has been successfully applied to medicine and pharmaceutical development; however, many doubts persist regarding the efficacy and safety of nanotherapies (NTs). One of the concerns with NTs is that these innovative systems have a large surface and consequently are subject to greater interactions with their immediate environment in our body. In this context, one of the main challenges of nanomedicine for the next 15 years is the development of methods that allow, using simple mimetic models, such as membrane mimetic systems, to predict the biological behavior of NTs [1]. In vitro biophysical assays may be interesting solutions in response to this challenge, since they allow the understanding of NT properties at the molecular level. Furthermore, under controlled conditions, the mimetic models of biological interfaces can help to rationalize and predict NT behaviors and their interactions in vivo.

This communication aims to present some routines developed in our research group with a view to predicting aspects related to the physiological barriers presented to drugs that can condition the therapeutic efficacy of NT. The interactions of drugs with membrane model systems are used to determine their membrane/water partition coefficient, which is a predictor of drug affinity to the membranes as well as drug permeability and absorption, giving important information regarding the most appropriate nanocarrier for drug loading. Further information about the degree of penetration of the drugs in a lipid nanocarrier can be also gained through in depth-dependent fluorescence quenching experiments (steady-state and time-resolved). This will help understand if the drug release will be immediate or more controlled. In addition, it is possible to evaluate the influence of the drugs changing the cooperativity and the main phase transition temperature of the lipid assembled nanocarriers giving a prevision of NTs stability. The stealth properties of NTs can be further monitored by binding studies to human serum albumin based in fluorescence quenching of intrinsic protein fluorophores. Finally, the fusion of the NTs with a membrane endosomal model system allows prediction of cellular uptake studied by FRET, further complemented by bio tracking of the drug by fluorescence lifetime imaging microscopy.


88

Mechanism of cellular internalization of siRNA-loaded WRAP5 nanoparticles

Konate Karidia

Konate K., Dussot M., Aldrian G., Vivès E., Deshayes S. and Boisguerin P. CRBM, 1919 route de Mende, 34293 Montpellier, France Nowadays, cell-penetrating peptides (CPPs) have been widely developed for the delivery of therapeutics such as oligonucleotides (Boisguerin et al. 2015). Amphipathic CPPs through a noncovalent strategy constituted a very potent family of vectors for both in cellulo and in vivo delivery of siRNA (Konate et al. 2016, Aldrian et al. 2017). WRAP5 is an arginine and tryptophan rich amphipathic peptide especially designed for siRNA transfection (Konate et al. 2018). WRAP5 complexed with siRNA forms small nanoparticles (NPs) of 100 nm size able to enter cells and to induce an important knock-down (KD) of luciferase in a wide variety of cell lines such as HT29, Neuro2a or U87 cell lines.

Transfection efficiency of a CPP is often correlated to its mechanism of entry into cells. However elucidation of this mechanism remains a tricky subject because it is a multifactorial process which depends on physico-chemical characteristics of CPPs, nature of the molecule to transfect and also methodology used for its investigation.

In general, CPPs internalization mechanism occurs either through endocytosis pathway or by direct translocation trough membrane plasma (Takeuchi et al. 2016). Previous investigations on internalization kinetics of WRAP5:siRNA-Cy5 nanoparticles showed a very fast process (within 15 min) suggesting a direct translocation mechanism which is partially confirmed by leakage assays on liposomes reflecting the plasma membrane. Thereafter, chemical agents used to inhibit well-known endocytic pathways (macropinocytose, clathrin, caveole and raft dependent) revealed high luciferase KD level on U87 cell line as the non-treated ones. Results were confirmed by confocal microscopy showing no co-localization between internalized WRAP5:siRNA-Cy5 nanoparticles and specific endosomal pathway markers. We also checked the possible WRAP5:siRNA-Cy5 co-localization with early and late endosomes or lysosomes.

Because the use of chemical component could influence the mechanism of internalization of WRAP5:siRNA-Cy5, a specific dynamin triple knock-out cell line (no endocytosis) were used to confirm that WRAP5 internalization mechanism is independent of endocytic pathways.

In conclusion, the WRAP5:siRNA nanoparticles are mainly internalized in cells independently of any endocytosis mechanism. Because the siRNA is not entrapped in endosomes, it is rapidly accessible to perform its biological activity as therapeutic molecule.


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PIP2 contributions for internalization of the cell-penetrating peptide Penetratin. Cell-free reconstitution and model membranes

Leïla Bechtella

Bechtella L., Walrant A. and Sachon E. UPMC – LBM Tour 33-23, 4 place Jussieu, 75005 Paris, France

Cell-penetrating peptides (CPP) can cross cell membranes and deliver biologically active molecules into cells. Understanding their internalization mechanisms is of first interest for an appropriate use in the medical field or for biotechnological applications. Previous work showed that CPPs could remodel the actin cytoskeleton, interacted strongly with negatively charged lipids and PIP2 could play a role in Penetratin internalization. This PhD project aims to analyze the role of the negatively charged lipid PIP2, a known regulator of actin polymerization, as a binding partner of the CPP Penetratin and as a potential effector of Penetratin internalization.

Penetratin effect on thermotropic phase behavior of PIP2-containing model membranes was studied by Differential Scanning Calorimetry (DSC). It showed that Penetratin can interact with polar head groups, inserts in fatty acid chains and impacts the lipid bilayer fluidity of PIP2-containing liposomes. It indicated that presence of PIP2 in liposomes triggers Penetratin-lipid interaction.

Moreover, Penetratin binding affinity for PIP2-containing lipid vesicles was estimated using tryptophan fluorescence. It pointed out that Penetratin interacts with negatively charged lipids and has a higher affinity for PIP2 than for PS.

Upon irradiation, a Penetratin peptide functionalized with a benzophenone photoprobe can be covalently bound to lipid fatty acid chains in its immediate environment. This method allows us to identify Penetratin interaction partners using photo-crosslinking coupled to mass spectrometry (MS). Using this techniques, we studied the interactions of Penetratin with negatively charged lipids, focusing on PIP2-containing liposomes or even more complex lipid mixes. PIP2 was found to be a good interaction partner for Penetratin and was preferably labelled in liposomes containing PC, PS and PIP2.


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91

Poster Session I

Poster Session I Wednesday, April 3rd

93

P1 Lipid bilayer composition triggers membrane damage by Abeta1-42 as revealed by Atomic Force Microscopy Mehdi Azouz

Azouz M., Cullin C., Lecomte S. and Lafleur M. CBMN, 1 Allée Geoffroy Saint-Hilaire, 33600 Pessac, France

Alzheimer’s Disease (AD) is a devastating age-related pathology which constitutes one of the most prevalent medical concerns of the 21st century, with an increasing number of affected individuals following the rise of the world population life expectancy. If the mechanisms occurring behind the neurodegeneration process are not elucidated, the pathogenic agents involved have been identified. The amyloid peptide Abeta1-42, the main component of the senile plaques found in the patient’s brain as one of the hallmarks of the disease, is considered as a major culprit regarding the pathology. It is now admitted that neurotoxicity is provided by soluble oligomers constituting intermediate species formed during the auto-assembly process of Abeta. As many evidences have shown that membrane might play an important role in triggering the production of toxic species, membrane/peptide interactions are under intensive investigations. Especially, the nature of lipids such as cholesterol and ganglioside GM1 constituting the lipid bilayer play a crucial role in the promotion of these interactions. Liquid-liquid phase separation in the fashion of lipid rafts are also suspected to be involved. We hence aimed at revealing the effects of these lipids in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) supported lipid bilayers and two phase separated systems using Atomic Force Microscopy (AFM) with the Abeta 1-42 peptide and an oligomeric mutant (oG37C). Pure zwitterionic bilayers did not induce any interaction with Abeta1-42 while the mutant accumulated on the membrane, targeting disordered lipid areas. Effects on cholesterol-containing membranes showed detergent effects with both peptides as observed with GM1-containing bilayers. Eventually, 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol(DOPC/DPPC/chol) and DOPC/Brain Sphingomyelin/cholesterol ternary systems were studied to elucidate how the peptides behave regarding liquid-liquid phase separated systems. Abeta1-42 and the mutant exclusively aggregated on the liquid disordered phase in both cases. This work aims at clarifying our understanding of Abeta1-42/membrane interactions by providing a comprehensive insight on the membrane compositional factors that might trigger binding and potential toxicity.


94

P2 Impact of membrane Poly-Unsaturated-Fatty-Acids (PUFAs) on Dopamine D2 receptor signaling pathways Rim Baccouch

Baccouch R.1, De Smedt-Peyrusse V.

2, Jobin M-L.

2, Heuninck J.

2, Durroux T.

3, Trifilieff P.

4 and Alves I.

5

1. CBMN, CNRS UMR 5248, Univ. Bordeaux, Allée Geoffroy St Hilaire, 33600 Pessac, France 2. NutriNeuro, INRA UMR 1286, Univ. Bordeaux, 146 rue Léo Saignat, 33000 Bordeaux, France 3. CNRS UMR 5203, INSERM U1191, IGF, Univ. Montpellier 1 et 2, Montpellier, France

The dopamine D2 receptor (DR2) is a 7-transmembrane G-protein coupled receptor (GPCR) expressed in the central nervous system that is involved in the control of the reward system. The alteration of the signaling pathways downstream of this receptor is involved in several psychiatric disorders such as depression, bipolar disorder and schizophrenia. Therefore, the DR2 is considered as a major target of antipsychotics. It is believed that the activity of this GPCR is strongly affected by membrane lipids and in particular PUFAs. Indeed, the brain is highly enriched in PUFAs, which are known to impact the physic-chemical properties of membranes as well as the activity of GPCRs. Recent in vitro data obtained with cells expressing the DR2 that have been enriched in PUFAs indicates that n-3 and n-6 PUFAs enhance both agonist and antagonist affinity for DR2 which suggest that membrane PUFA content impacts the DR2 activity. Moreover, in vivo data showed that a diet deprived in PUFAs decreases motivation in mice, a major symptom of psychiatric disorders. The open question to unravel concerns the impact of PUFAs on the DR2 signaling cascades following ligand-receptor interaction. To decipher the molecular mechanisms of DR2 signaling and PUFAs impact, our approach is to use techniques based on resonance energy transfer (RET) which allows the monitoring of RD2 signaling cascades as cAMP response, G-protein interaction and receptor internalization.

Preliminary results suggest that indeed PUFAs impact goes beyond receptor ligand interaction and affect downstream signaling cascades as receptor internalization. Other experiments are envisaged in order to demonstrate the considerable role of PUFAs on RD2 signaling with the long-term goal of using PUFAs as adjuvants to enhance the therapeutic level of classical antipsychotics used nowadays.


95

P3 An intrinsically disordered region in OSBP acts as an entropic barrier to control protein dynamics and orientation at membrane contact sites Joëlle Bigay

Bigay J., Jamecna D., Polidori J., Mesmin B., Dezi M., Levy D. and Antonny B. IPMC-CNRS, UMR 7275, 660 route des Lucioles, 06560 Valbonne, France

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 tetherin as well as domains involved in lipid transfer. Here, we show that a lon ≈ 90 aa 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 to homotypically tether 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.


96

P4 Identification of new cellular partners of galectin 3, a cancer associated protein, involved in clathrin independent endocytosis Abdeldjalil Boucheham

Boucheham A., Bar S., Rinaldi B., Hammann P., Kuhn L., Chicher J., Rechreche H. and Friant S. GMGM UMR 7156, Institut Botanique, 28 rue Goethe, 67000 Strasbourg, France

Galectin-3 is a member of β-galactoside-binding proteins family. It is a multi-functional protein that regulates à plethora of functions, including apoptosis, metastasis, cell adhesion and migration, cell-cell interactions and membrane trafficking. Galectin-3 is mainly a cytosolic protein, but can easily traverse the intracellular and plasma membranes to translocate into the nucleus, mitochondria or get externalized by non-classical pathway. Ludger Johannes (Institut Curie, Paris) and colleagues showed that Galectin-3 elicits glycosphingolipid-dependent formation of clathrin-independent endocytic structures. Here, by studying the interactome of endogenous Galectin-3 by immunoprecipitation coupled to mass spectrophotometry analyses, we have found that Galectin-3 interacts with a G protein coupled receptor GPCR and a palmitoyl-transferase. Our interactomic approach was validated by the identification of the Galectin-3 binding protein in our samples. The study of these new galectin-3 interactors and their validation in normal and pathological (cancer) conditions will need further investigations to better understand the pathway of Galectin-3 dependent endocytosis.


97

P5 Involvement of the putative mechanosensitive ion channel CmpX in envelope homeostasis in P. aeruginosa. Emeline Bouffartigues

David A., Omnes J., Tortuel D., Maillot O., Taupin L., Tahiroui A., Azuama CO., Lagy C., Depayras S., Poc C., Heipieper H., Orange N., Feuilloley M., Cornelis P., Dufour A., Chevalier S. and Bouffartigues E. LMSM EA 4312 (Univ Rouen) , CSSN ,55 rue saint Germain, 27 000 Evreux, France

Mechanosensitive ion channels are ubiquitous proteins whose opening under the effect of membrane constraints allows the passage of ions or molecules according to the selectivity thereof. In E. coli the low conductance channel MscS is one of the most studied. It is involved in the regulation of the turgor pressure during a sudden lowering of the osmolarity of the medium and it is regulated by RpoS, the stationary phase sigma factor. In P. aeruginosa CmpX is homologous to MscS and its physiological function is unknown. Its gene is part of a transcriptional unit located upstream sigX and oprF. SigX is an extracytoplasmic function sigma factor (ECF) for envelope stress response that directly controls the expression of cmpX. OprF, the major outer membrane proteinis involved in envelope integrity and environment perception. In order to specify the role of CmpX, a deletion mutant was constructed in strain H103 and characterized at molecular (qRT-PCR) and phenotypic levels for (i) its resistance to thermal shocks, (ii) its membrane fluidity (by generalized polarization and fluorescence) and composition (FAME), (iii) its production of virulence factors. In stationary phase, we show that the absence of CmpX causes a decrease of the fluidity in deep regions of the membranes hydrophobic core, the activation of the envelope stress response and an alteration of the outer membrane protein profile as well as the vesicles structure. These membrane alterations are associated with a decrease in the production of rhamnolipids, a virulence factor under the control of the Rhl I / R communication system. This work suggests that CmpX plays a key role in envelope homeostasis and pathogenicity in P. aeruginosa under stress conditions such as the stationary phase.


98

P6 Annexin A6 localization in matrix vesicles and in proteoliposomes: function and membrane binding properties René Buchet

Veschi EA., Bolean M., Ciancaglini P., Strzelecka-Kiliszek A., Bandorowicz Pikula J., Pikula S., Millán JL., Bottini M., Mebarek S., Granjon T., Magne D. and Buchet R. Université Lyon 1, ICBMS UMR 5246 CNRS, 49 boulevard du 11 Novembre 1918, 69622 Villeurbanne, France

Matrix vesicles (MVs) are 100-300 nm diameter vesicles released from hypertrophic chondrocytes. MVs serve as nucleation site for the initial mineral deposition in growth cartilage, during endochondral ossification. Annexin A6 (AnxA6) is the largest member of annexin family of proteins present in MVs. Freeze-thaw, use of Triton X-100 and proteolysis combined with ultracentrifugation revealed by Western Blot that AnxA6 is present in the lumen of MV, on surface of internal leaflet of MVs, on the external leaflet of MVs membranes and is inserted in the hydrophobic bilayer membrane and co-localized with cholesterol (Chol).

Proteoliposome models using giant vesicles served to determine the mechanisms of AnxA6 binding to MVs and protein translocation from MV lumen to external leaflet of MV bilayer. Addition of 2 mM CaCl2 promoted the recruitment of AnxA6-Alexa546 on the surface of 80:20 by weight phosphatidylcholine (PC): phosphatidylserine (PS) liposome as probed by confocal microscopy. Moreover, AnxA6 was bound externally to asolectin liposomes in a calcium dependent manner, as shown by transmission electron microscopy. Differential scanning calorimetry (DSC) using unilamellar vesicles (~100-200 nm) confirmed the protein incorporation into the bilayers through the assessment of the changes in the thermodynamic parameters of the liposomes composed of PC and 9:1 PC:PS (molar ratio). AnxA6 in presence of 2mM CaCl2 decreased the values of phase transition enthalpy (ΔH) associated to either PC or PC:PS suggesting a translocation or interaction of AnxA6 into the lipid bilayer preventing some lipid molecules from contributing in the phase transition parameters. However, when Chol is present in the liposome composition (5:4:1 PC:Chol:PS, molar ratio), there is an increased phase transition cooperativity.

These findings substantiated possible mechanism of AnxA6 translocation within MVs. First, AnxA6 in the presence of calcium ions binds to PS, reflecting binding of AnxA6 on the inner surface of the leaflet of MV bilayer. Secondly, during apatite formation, local pH drops promoting AnxA6 insertion in the bilayer of MVs. Thirdly, AnxA6 binds to PC mimicking the attachment of AnxA6 with the external leaflet of MV bilayer. The proposed mechanisms are consistent with the distinct types of binding of AnxA6 with PS as well as with PC, in the presence or absence of Ca2+.


99

P7 Reconstituting in vitro the remodeling of membrane nanotubes by actin dynamics Clément Campillo

Allard A., Lamour G., Labdi S., Sykes C. and Campillo C. LAMBE, CNRS UMR8587, Université d'Evry Val d'Essonne, Boulevard François Mitterrand 91025 Evry Cedex, France

Inside living cells, the remodeling of membrane nanotubes by the dynamics of acto-myosin networks is crucial for processes such as intracellular traffic or endocytosis. However, the mechanisms by which acto-myosin dynamics affect nanotube morphology are largely unknown. How much radial and axial forces are generated on the tube by acto-myosin dynamics ? Can these forces lead to nanotube scission? How do they relate to the structure of the acto-myosin network ? To address these questions, we perform in vitro experiments to decipher the physics of nanotube remodeling in biochemically controlled assays recapitulating key aspects of cellular membranes and actin dynamics. We use two complementary techniques to form membrane nanotubes on which we reconstitute acto-myosin networks from purified proteins. By using optical tweezers, we will measure the forces implied in nanotube formation and maintenance in presence of acto-myosin. In parallel, we develop a novel assay to image supported nanotubes and acto-myosin networks polymerizing on such nanotubes at the nanometric scale by using Atomic Force Microscopy. By combining these two techniques, we investigate how the structure of the acto-myosin network at the nanometric scale dictates tube reshaping at the micrometric scale and how this explains the results obtained in cells.


100

P8 Endoplasmic reticulum - plasma membrane contacts sites are closely associated to docked secretory granules during regulated exocytosis in neuroendocrine cells Sylvette Chasserot-Golaz Gabel M., Delavoie F., Ledain F., Thahouly T., Royer C., Gasman S., Vitale N. and Chasserot-Golaz S. INCI, UPR3212, 8 allée du Général Rouvillois, 67000 Strasbourg, France

Contact sites between endoplasmic reticulum and the plasma membrane (ER-PM contacts) represent an exciting and emerging area of research regarding lipid distribution and the impact of plasma membrane organization on specific cellular functions such as endocytosis and/or exocytosis in specialized cell types such neurons. Our aim is to study the functional importance of ER-PM contacts during exocytosis in chromaffin cells.

Performing dual immunogold labelling of plasma membrane sheets observed with transmission electron microscope, we highlighted the presence of ER-PM contacts at the vicinity of secretory granules docked to plasma membrane. Moreover, we found also that the area of ER-PM contacts increases upon cell stimulation. These suggest that ER-PM contacts could play a role in calcium-regulated exocytosis. To go further we will explore if ER-PM contacts are involved in the intake of certain lipids such as PIP2 necessary for the docking and fusion of secretory granules or in the regulation of calcium concentrations at exocytotic sites in neuroendocrine cells.


101

P9 GLUT4 vesicle exocytosis in live mice: an intravital microscopy system to study actin/tropomyosin function in vivo Anita Chitsaz

Chitsaz A., J Kee AJ., Masedunskas A., Lucas CA., Heydecker M., Bryce NS., Gunning PW. and Hardeman EC. 725 UNSW village, gate2, High street, UNSW Kensington campus, 2052 Sydney, Australia

Insulin and exercise-stimulated glucose uptake in muscle tissue requires the movement of the glucose transporter GLUT4 from intracellular storage vesicles to the cell membrane via an exocytic pathway. Actin filaments located at the cell cortex, and in particular those associated with the tropomyosin Tpm3.1, regulate this fundamental process . Our understanding of GLUT4 trafficking comes almost exclusively from studies using cultured adipocytes and myoblasts that are morphologically quite distinct from adipocytes and muscle fibres in adult tissue. They lack the complex 3-dimensional tissue architecture or the cellular and systemic interactions of tissues in the living organism. We have developed an intracellular intravital microscopy approach to measure GLUT4 trafficking in vivo in skeletal muscle. One of the limitations to study GSV trafficking in vivo was that individual GSV fusion events could not be unambiguously detected due to the absence of a clear fluorescent signature. We have overcome this by using a dual colour GLUT4 probe (rGLUTpHluor) comprised of pHluorin inserted into the first exofacial loop of GLUT4 and tdTomato at the C-terminus. We electroporated mouse skeletal muscle in vivo with rGLUTpHluor and have been able to track GSV behaviour prior to fusion and record fusion events at the sarcolemma.

With this system, using low magnification confocal microscopy, we could demonstrate the increase in GLUT4 vesicle translocation to the sarcolemma with insulin stimulation and 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) stimulation that mimics exercise. Using our dual florescence tagged construct, for the first time we could distinguished between the vesicles that translocated from intracellular storage compartments to the cell membrane versus the vesicles that went through the complete fusion with the membrane. Our data suggests the vesicle fusion stimulation as the main effect of insulin and AICAR stimulation. To study the GLUT4 vesicles behaviour prior and during fusion events, high magnification intravital imaging has performed using Highly Inclined Laminated Optical sheet (HILO) microscope. This represents the first demonstration of sub-diffraction-sized vesicle fusion events in vivo. Fusion events’ analysis su gests a significantly longer dispersion time in basal and AICAR- stimulated events versus insulin-stimulated events. Since the basal and AICAR stimulated events have the endosomal origin and a bigger vesicular size than insulin stimulated events, the longer dispersion time can represent this size differences . This demonstrates the first distinction between different intracellular vesicular pools in vivo. Using our system and recruitment of cytochalasinD as well as anti-tropomyosin 1001 reagent, we could elucidate the role of the actin-tropomyosin cytoskeleton in GLUT4 exocytosis, in both insulin and AICAR stimulation pathways. We have shown that disruption of actin-tropomyosin cytoskeleton decrease the rate of GLUT4 fusion events significantly and the increase the dispersion time in vivo and under physiological conditions.


102

P10 LIMONADA : a database dedicated to the simulation of complex lipid membranes Jean-Marc Crowet

Crowet J-M., Buchoux S., Belloy N., Sarazin C., Lins L. and Dauchez M. Université de Reims Champagne-Ardenne, UMR CNRS 7369, UFR Sciences Exactes et Naturelles, Chemin des Rouliers, 51100 Reims, France

Biological membranes can be composed of hundreds of different lipids distributed heterogeneously between and within lipid leaflets. Their ratios are organism and organelle specific and several studies have yielded detailed membrane compositions of different cell types. Moreover, lipidomic studies have established that there could be over 100,000 different lipids. Besides, the conjunction of a greater computing power and methodological developments has led to an ever-increasing size and complexity of the lipid systems simulated by molecular dynamics (MD).

Tens of model membranes with several lipid species designed to represent specific biological membranes have already been simulated. Modeling studies start then to encompass the wide diversity of biological membranes. A model plasma membrane with 63 lipid types has notably been recently simulated by Ingolfson et al. in 2014. Determination of complex lipid compositions is not trivial and simulating these membranes to equilibration can be quite computationally-demanding while only being a prerequisite for further simulations. Moreover, there are several forcefields (FF) available for membrane MD simulations and one major issue is that the number of lipid topologies is strongly FF-dependent.

In this work, we present LIMONADA (Lipid Membrane Open Network And Database; https://www.limonadamd.eu/) which has been developed as an open database (https://github.com/limonadaMD/) allowing to handle the various aspects of lipid membrane simulation from a lipidomic background. LIMONADA presents published membrane patches with their simulation files and the cellular membrane it models in a simulation-ready fashion. Limonada uses the lipid classification established by the LipidMaps consortium and every lipid is unequivocally identified by a four-digit ID (i.e. compliant with e.g. pdb files). From this basis, registered users can add new membranes, lipids, topologies and/or forcefields to the database with the only limitation that each addition must be sustained by a reference paper and that the primary source of the files stored by Limonada are also provided.


103

P11 A novel fission protein in endo-lysosomal membrane trafficking Maria Giovanna De Leo

De Leo MG., Gopaldass N., Knott G., Moreau D. and Mayer A. UNIL, Faculty of Biology and Medicine, Department of Biochemistry, chemin des Boveresses 155 - CP 51 - CH-1066 Epalinges, Switzerland

Cellular membranes constantly change their shape and exchange material between them, to maintain cellular homeostasis by providing a compartmentalised environment for carrying out different functions by various proteins and lipids. Since proteins and lipids are not always produced at the site of their activity membrane flux involves the fission and fusion of transport carriers is required. The morphology of transport carriers operating in different membrane transport routes varies from the classical small vesicles, to larger tubular structures. Numerous components required in the formation of transport carriers have been identified, such as the retromer complex and sorting nexins, while the fission of these carriers from their donor compartments remains poorly understood.

We described a novel role WIPI 1 at the endosomal membranes, where it can act as fission factor. WIPI 1 belongs to a family of conserved proteins called PROPPINs characterized by the presence of two lipid interaction sites with a capacity to bind PI3P, PI(3,5)P2, and PI(5)P phosphoinositides, that are enriched on endosomes, lysosomes, and autophagosomes respectively. A relevant feature found in all PROPPINs is a hydrophobic loop, located between these two lipid-binding sites, containing a stretch of at least 18 amino acids with the potential to fold into an amphipathic α-helix. This property was shown in yeast to be required for vacuole fission in vivo.

We hence speculated that other members of the PROPPINs family should display membrane fission activity. Indeed, we elucidated the relevance of this loop for WIPI 1 activity and we found that WIPI 1 fission activity is strictly dependent on its ability to bind PI(3,5)P2. Moreover, we showed that WIPI 1 inactivation strongly impacts on the endo-lysosomal membrane trafficking.


104

P12 VDAC1-SOD1 G93A aggregation in an Amyotrophic Lateral Sclerosis model: a patented peptide as an interaction scavenger. Implications for the neurodegenerative disease’s treatment Vito De Pinto

Magrì A.1, Reina S.

2, Guarino F.

2, Messina A.

1 and De Pinto V

2.

1. Dept. Biological, Geological and Environmental Sciences, University of Catania, Catania, Italy. 2. Dept. Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy.

Mitochondrial dysfunction in the neuromuscular disorder Amyotrophic Lateral Sclerosis (ALS) is an early and key event of motor neurons’ de eneration and correlates with the accumulation of misfolded forms of the antioxidant enzyme Superoxide Dismutase 1 (SOD1) onto the cytosolic surface of mitochondria. The Voltage-Dependent Anion selective Channel (VDAC) isoform 1 is the main porin of Mitochondrial Outer Membrane (MOM) and acts as docking site for several SOD1 mutants [1]. In physiological condition, VDAC1 allows metabolic exchanges with cytosol and, by interacting with Hexokinase 1 (HK1) it represses apoptosis [2]. However, the interaction of SOD1 G93A mutant with VDAC1 affects channel conductance and likely alters apoptosis regulation. We noticed that the mitochondrial-bound HK1 fraction is reduced in spinal cord’s motor neurons compared to brain’s neurons, suggesting that VDAC1 is more incline to interact with SOD1 mutants.

In this work, we describe the competition between SOD1 G93A and HK1 for the same VDAC1 docking site. In addition, the expression of SOD1 G93A, but not WT, in the motor neuronal-like cells NSC34 dramatically decreases HK1 affinity for mitochondria [4]. Based on these data, a short peptide designed on HK1 N-terminal domain sequence (NHK1) was synthetized and tested [5]. We found that addition of NHK1 to purified VDAC1 or to mitochondria strongly prevents SOD1 G93A deposition. Furthermore, in NSC34 cells expressing SOD1 G93A, NHK1 significantly improves both mitochondrial dysfunction and cell viability [3].

Overall, our results indicate a clear involvement of HK1 in mitochondrial dysfunctions in SOD1-mediated ALS cases and support the suitability of NHK1 peptide as a promising therapeutic tool for ALS treatment.

References [1] Israelson et al. Neuron, 2010, 67, 575–587. [2] Shoshan-Barmatz et al. Mol. Aspects Med., 2013, 31, 227–285. [3] Magrì et al. Sci. Rep., 2016, 6, 34802. [4] Allen Institute for Brain Science (http://www.brain-map.org). [5] Italian Patent n. 102016000026259; International Patents: application PCT/IB2017/051460.


105

P13 Unravelling the spatiotemporal dynamic of hepatitis B virus entry Maïka Deffieu

Deffieu MS., Dorobantu CM., Lučansk V. and Gaudin R. IRIM,CNRS 9004, 1919 Route de Mende, 34090 Montpellier, France

Hepatitis B virus (HBV) is an enveloped virus, leading to chronic infections often associated with liver diseases such as cirrhosis or hepatocellular carcinoma. Despite the existence of a preventive vaccine, the prevalence of HBV is estimated at 250 million people still carrying the virus and unable to be cured.

HBV mainly targets hepatocytes. The sodium taurocholate co-transporting polypeptide (NTCP) was identified as a hepatocyte-specific plasma membrane receptor necessary for HBV entry. Since its discovery, promising antagonistic peptides were developed for therapeutic treatment. However, the molecular mechanism involved in successful endocytosis upon HBV-NTCP interaction is unclear.

In order to study the spatiotemporal dynamics of HBV internalization, we developed fluorescent tools to observe HBV entry by 3D live cell imaging. In one hand, we used HepG2 cells (hepatocarcinoma cell line), which do not express NTCP and become permissive to HBV once this receptor is exogenously expressed. Thus, we generated stable HepG2 cell lines expressing the NTCP receptor fused to two different fluorescent proteins (Ruby3 or mNeonGreen) and further characterized their permissiveness to HBV. In the other hand, the HBV genome contains overlapping sequences that are difficult to modify. Specific HepAD38 cells stably secreting HBV virions, were transduced to express a fluorescent HBV core protein that is integrated within the viral particle upon budding. Using these tools, we performed high-resolution 3D confocal imaging to visualize virus attachment to cells, lateral diffusion, internalization, and the dynamic HBV-NTCP interactions during these events.

Our work provides seminal characterization of the spatiotemporal dynamics of HBV entry into cells. This was achieved thanks to recent advances in the HBV field as well the generation of novel tools that will be useful to further decipher the molecular mechanisms leading to productive HBV infection.


106

P14 Patch-Clamp Electrophysiology and BioMembrane Force Probe to assess mechanisms of Cell Penetrating Peptide uptake into cells Nathalie Dhayer

Dhayer N., Sophie C., Laurent V., Alain J., Sandrine S. and Nicolas R. Pierre et Marie Curie University, Paris, France Cell membrane is required for integrity and survival of living cells but can be a barrier for intracellular delivery of therapeutic agents. To counter this, Cell Penetrating Peptides (CPPs) can be used : these peptides are able to cross cell membrane even linked to a cargo. By this way, it is known that the cargo – such as small molecules, RNAs, fluorophores or drugs – can be delivered into the cytosol. Nevertheless, the cargo can be damaged when addressed to lysosomes. Therefore, a perfect understanding of the CPP uptake mechanisms is required to improve its efficacy. For this purpose, patch-clamp electrophysiology technique can be used to measure transitory current across cell membrane. When certain peptides are added in extracellular medium, a brutal current drop is reported which results from pore formation in the cell membrane. We hope to determine whether transient pore formation is part of the CPP uptake mechanism.

To go further, Biomembrane Force Probe (BFP) is used to measure the force exerted by the CPP on the cell membrane. This technique was successfully coupled to the electrophysiology one. We can measure simultaneously current through membrane and CPP action on it. We hope that this experiment will give details on the level of insertion of the CPPs and the concomitant membrane destabilization. Both techniques should lead us to a better understanding of the CPP uptake mechanisms.


107

P15 MOSPD2, a new "VAP-protein", builds sites of membrane contact between the ER and several organelles

Thomas Di Mattia

Di Mattia T., Ikhlef S., Wendling C., Drin G., Tomasetto CL. and Alpy F. IGBMC, 67100 Illkirch, France

Inter-organelle membrane contact sites are subcellular structures favoring exchanges and communication inside the cell. Such micro-domains are built by molecular bridges creating a physical connection between two distinct organelles. The field of contact sites is now flourishing with discoveries of new tethering molecules. In that context, we identified by an unbiased proteomic approach a novel scaffold protein named MOtile SPerm Domain-containing protein 2 (MOSPD2). MOSPD2 is an endoplasmic reticulum (ER)-resident protein able to interact with several organelle-bound proteins possessing a small motif, named FFAT (two phenylalanines in an acidic tract). Consequently, we showed that MOSPD2 and its protein partners build contacts between the ER and either endosomes, mitochondria or Golgi. These findings highlight a new way for docking organelles on the ER.


108

P16 Syntenin-phosphatidic acid interaction supports ILV budding Antonio Luis Egea Jimenez

Egea Jimenez AL., Ghossoub R. and Zimmermann P. Centre de Recherche en Cancérologie de Marseille. CRCM - Inserm 1068 - CNRS 7258 27 bd Leï Roure, BP 30059,13273 Marseille, France

Extracellular vesicles and exosomes are emerging as important organelles supporting cell-cell communication. Because of their potential therapeutic significance, important efforts are being made towards characterizing the contents of these vesicles and the mechanisms that govern their biogenesis.

We previously demonstrated that the lipid modifying enzyme phospholipase D2 (PLD2) is involved in the intraluminal budding of endosomes to form multivesicular bodies (MVBs) that will liberate exosomes (Ghossoub et al., 2014). PLD2 hydrolyses phosphatidylcholine (PC) to phosphatidic acid (PA). It is known that PA is important for membrane fission and fusion events. This is due to its ability to induce a negative membrane curvature because of its “small head”. Yet, a role for PA in the formation of MVBs or exosomes has not been documented.

We found that syntenin, a protein controlling the formation of intraluminal vesicles (ILVs) and exosomal biogenesis (Baietti et al., 2012), can directly recognize PA in liposomes mimicking the complex lipid composition of late endosomes/exosomes. By mutational analysis, we identified the syntenin-PA binding site in its first PDZ domain. We observed that syntenin mutants deficient for PA-binding affects endosomal budding. We are currently testing to what extend syntenin-PA interaction supports the biogenesis of ILVs and exosomes.

So far, our study indicates that syntenin might act as a sensor of late endosomal PA and that this interaction is required for intraluminal budding. This highlights a potentially new function of PA and unknown mechanism triggering late endosomal trafficking and exosome biogenesis


109

P17 Interplay between phosphoinositides and F-BAR proteins during endocytosis Fatima El Alaoui

El Alaoui F.,Rathar R., Sanchez-Fuentes D., Charlot B., Favard C., Muriaux D., Carretero-Genevrier A. and Picas L. IRIM, 1919 route de Mende,34090 Montpellier, France

Phosphoinositides are essential lipids in multiple cellular processes including membrane trafficking, cell signaling or cell growth. Among the different endocytic pathways, clathrin-mediated endocytosis is the best characterized and a paradigm of a process spatiotemporally organized by phosphoinositides. Different endocytic proteins such as the F-BAR domain only protein 1 and 2 complex (FCHo1/2) or the adaptor protein 2 (AP2) are reported to initiate clathrin-mediated endocytosis. For instance the F-BAR domain of Syp1, the yeast homologue of FCHo1/2, was shown to induce PI(4,5)P2 clustering on minimal systems. This was also the case of other endocytic proteins such as Epsin and AP180. Finally, AP2 was shown to create a local pool of PI(4,5)P2 by stimulating the metabolic activity of dedicated kinases, although it requires PI(4,5)P2 for its localization on membranes. Whether these proteins might be the first to drive the nanoscale reorganization of phosphoinositides such as the PI(4,5)P2 or its substrate PI(4)P4 at the plasma membrane or if they might bind to pre-existin phosphoinositide’s pools durin endocytosis is not well understood.

Here we have investigated the role of membrane curvature and PI(4,5)P2 organization on the dynamics of full-length F-BAR proteins such as FCHo2, FBP17 and their functional domains on in vitro and “cell-free” systems. Our results su est that PI(4,5)P2 clustering on membranes is enhanced by membrane curvature and would require the F-BAR oligomerization and the interaction of FCHo2 with effector proteins.


110

P18 Biomembrane mechanics tested in a microfluidic configuration: towards on-chip micropipette Marianne Elias

Elias M., Mesnilgrente F., Laborde A., Berti D., Montis C., Caselli L. and Joseph P. LAAS-CNRS, 7 avenue du Colonel Roche, 31400 Toulouse, France Cell membranes are highly complex systems, responsible for key functions of cell life. Many interests have grown towards the interaction of cells with nanoparticles and the effect on their nanotoxicity, and for further development of nanomedicine. To simplify the cells composition, biomimetic membranes of many kinds and shapes were developed. Giant Unilamellar Vesicles (GUV) are among the closest to cells reality.

Micropipette is one of the first tools used to characterize cells or biomimetic membranes mechanics [1]: it consists in determining how GUV area increases while drawing it inside a micropipette by applying a pressure drop [2]. However, it is complicated to handle, can only be applied to one GUV at a time, and does not permit to change easily the solution in which the measurement is performed. Therefore, in the spirit of recently published works [3,4], we developed a microfluidic platform that is able to trap and study the deformation of the GUV while applying a pressure, which should permit to extract the membrane tension, bending modulus and stretching modulus.

The concept of the chip for quantitative measurements is the following: an inlet and outlet connected with a very long serpentine channel of 40µm height and width. Another channel of 10 µm height and width, which plays the role of the micropipette, is connected to the middle of the long channel to trap GUV bigger than 10 µm.

Microfabrication of such desi n is challen in because of “suspended” micropipette channel. We have tried several approaches, in the clean room of LAAS-CNRS. The most promising is based on the lamination of five layers of a Dry Film photoresist with photolithography steps for each layer, on top of a 170 µm thick glass substrate for imaging.

Preliminary results are obtained on simpler designs: a U trap placed in the middle of a channel. The pressure at which GUV escape the trap, as well as the relation between membrane tension and area increase, are different for different types of lipid membranes (gel/liquid phase). Membranes incubated with gold nanoparticles show higher release pressure with respect to non-incubated ones. As perspective, we target quantifying the elationship, and we also plan to characterize the permeability of the membrane using Fluorescent Correlation Spectroscopy under a confocal microscopy.

References [1] Bassereau, P.; Sorre, B.; Lévy, A. Bendin Lipid Membranes: Experiments after W. Helfrich’s Model. Advances in Colloid and Interface Science 2014, 208, 47–57. [2] Henriksen, J. R.; Ipsen, J. H. Measurement of Membrane Elasticity by Micro-Pipette Aspiration. Eur. Phys. J. E 2004, 14 (2), 149–167. [3] Guo, Q.; Park, S.; Ma, H. Microfluidic Micropipette Aspiration for Measuring the Deformability of Single Cells. Lab Chip 2012, 12 (15), 2687–2695. [4] Lee, L. M.; Lee, J. W.; Chase, D.; Gebrezgiabhier, D.; Liu, A. P. Development of an Advanced Microfluidic Micropipette Aspiration Device for Single Cell Mechanics Studies. Biomicrofluidics 2016, 10 (5), 054105.


111

P19 Quantifying HIV-1 Gag plasma membrane binding in cells using spot variation FCS diffusion laws Cyril Favard

Noero J., Muriaux D., Berry H. and Favard C. IRIM, UMR 9004 CNRS, 1919 route de Mende, 34000 Montpellier, France

Many transient processes observed in cells arises from binding of cytosolic proteins to membranes. Monitoring and quantifying this process still remains a challenge in living cells. Fluorescence correlation spectroscopy (FCS) has provided a powerful way to investigate molecular motions in living cells. During this last decade, the establishment of FCS diffusion laws has revisited the non Brownian motions of molecules in membranes and characterized the nature of these deviations. Here, using numerical simulations, calculated FCS diffusion laws are established in the case of a membrane binding process. These diffusion laws exhibit non linearity and allow the determination of the apparent binding constant. Experimentally obtained HIV-1 Gag diffusion laws in cells, confirm this non linearity and estimate that, in average, roughly 30% of the total cellular protein is bound to the plasma membrane of cells.


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P20 Structural Characterization of Biomembranes: Moving Towards Complexity Giovanna Fragneto

Fragneto G., Delhom R., Joly L. and Scoppola E. Institut Laue-Langevin, 71 Avenue des Martyrs, 38042 Grenoble Cedex 9, France The structural characterizaton of lipid bilayers presents fundamental interest both in physics, for the study of thin fluctuating soft layers, and in biology, for the understanding of the function of biological membranes. This represents still a challenge: performing measurements on few nanometer thick, soft, visco-elastic and dynamic systems is close to the limits of the available tools and methods.

Neutron scattering techniques are rapidly developing for these studies. Since many biological processes occur at interfaces, the possibility of using neutron reflection to study structural and kinetic aspects of model as well as real biological systems is of considerable interest [1].

The most effective use of neutron reflection involves extensive deuterium substitution and this is becoming more and more an available option in biological systems in general and lipid bilayers in particular [2]. The use of deuterated lipid extracts presents relevant differences both with the hydrogenated counterpart and with synthetic systems [2-4].

The poster will review some progress made in the last few years by using neutron scattering at the ILL in the structural characterisation of biomembrane systems, efforts to build and characterize more and more complex systems [1-4], and will provide perspectives for future developments.

References [1] G. Fragneto, et al., Curr. Op. Coll. Int. Sci.e 2018 [2] A. de Ghellinck, et al., PlosOne 2014 [3] A. de Ghellinck et al., BBA-Biomembranes 2015 [4] A. Luchini et al., Coll. Surf. B 2018


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P21 Role of non-vesicular secretion in neuronal development Alessandra Gallo

Gallo A., Giordano F., Vannier C. and Galli T. Institut de Psychiatrie et Neurosciences Paris, Inserm Umr_S 1266, 102 rue de la Santé, 75014 Paris, France

The growth of axons and dendrites during neuronal development requires a massive increase of surface area via the insertion of new proteins and lipids. This event occurs through the fusion of secretory vesicles with the plasma membrane (PM), a general process of the secretory pathway. Recently, non-vesicular transfer of lipids at contact sites between the endoplasmic reticulum (ER) and the plasma membrane was shown to contribute to membrane expansion. Members of the ER-integral membrane protein Extended-Synaptotagmin (E-Syt) family have been identified as Ca2+-dependent lipid-transfer proteins (LTP) at ER-PM contact sites, and shown to transfer glycerophospholipids via their SMP domains.

My host laboratory has found that a novel ER-PM SNARE complex, composed of the ER-resident Sec22b and the neuronal plasmalemmal Syntaxin-1 (Stx1), is involved in neurite growth despite being unable to mediate membrane fusion (Petkovic et al., 2014, NCB). However, how this complex participates to neurite extension remains to be elucidated. In yeast, Sec22 interacts with LTPs of the OSH family, enriched at the ER-PM contact sites, supporting a role for Sec22-mediated ER-PM junctions in non-vesicular lipid transport between these bilayers.

Based on these observations, our starting hypothesis is that E-Syts-mediated non-vesicular lipid transfer at Sec22b-populated ER-PM contact sites, may cooperate with the conventional secretory pathway in the process of neurite outgrowth. The goal of my PhD project was to explore this hypothesis with two specific questions: 1. What are the partners of Sec22b complexes which might be involved in this novel mechanism for membrane expansion? 2. What is the mechanism by which the non-fusogenic SNARE Sec22b/Stx1 complex acts in neuronal development?

My results showed that Sec22b interacts with E-Syt2 and Stx1 in PC12 cells and with E-Syt2, E-Syt3 and Stx3 in HeLa cells. Sec22b-ESyt2 interaction appeared to depend on the presence of the Longin amino-terminal domain of Sec22b. Overexpression of E-Syt2 seems to stabilize Sec22b-Stx3 association, whereas silencing of E-Syt2 had the opposite effect. Overexpression of E-Syt2 full length, but not the forms lacking the SMP or transmembrane domains, increased membrane area as seen by the formation of filopodia particularly in the growing axon. Interestingly, treating neurons with the botulinum neurotoxin C1, that cleaves Stx1 and prevents Sec22b-Stx1 association, reverts the E-Syt2-induced phenotype of growth. These major findings support the idea that Sec22b/Stx1-containing contact sites may contribute to membrane expansion via the action of phospholipid transfer proteins such as E-Syts.


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P22 Unravelling the spatiotemporal dynamics of virus-receptor interactions during HCV entry Raphaël Gaudin

Clement C., Dorobantu C., Baumert T., Favard C., Ronde P., Mély Y., Lucansky V. and Gaudin R. IRIM , 1919 route de Mende, 34293 Montpellier, France Entry of enveloped viruses is a process that commonly includes attachment of the viral particle to the cell surface, interaction with cellular receptors, internalization into vesicular compartments and endosome-to-cytosol release of the viral genome. While molecular partners involved in these processes are relatively well-described, there is a lack of knowledge regarding our understanding of the spatiotemporal recruitment of the viral entry factors involved.

Occludin (OCLN) is a major HCV entry factor, which is part of the structural protein complex composing the tight junctions (TJs) at hepatocytic cell-cell contacts. Yet, how HCV particles meet with their receptor at the cell surface remains unclear. Indeed, whether particles slide at the surface of the cell toward the TJs or in contrast, whether “out-of-the-TJ” OCLN diffuses toward immobile particles remain controversial. Here, we characterized the spatiotemporal dynamics of HCV-OCLN interactions using 3D live cell imaging. To this end, the genome of human hepatocarcinoma-derived cells (Huh7.5.1) was edited using Crispr/Cas9 to insert a sequence coding for a fluorescent protein up/downstream of the receptor’s enes to obtain cells expressin endo enous levels of fluorescently labeled viral receptors. The permissiveness of the EGFP-OCLN+/+ edited cell line was characterized and the impact of the tag on OCLN function and distribution was assessed. In parallel, we produced a fluorescently labelled replication-competent HCV virus and performed live cell imaging of single viral particles infecting gene-edited cells. Image analyses revealed that endogenous OCLN is recruited outside TJs to interact with incoming HCV particles. Strikingly, we found that the amount of OCLN beneath HCV particles correlated with a change of the diffusive pattern of the virus. Further work is ongoing to statistically characterize the behaviour of these events and to identify the underlying mechanisms involved. Our work shed light on an important virus subversion strategy in which TJ-associated proteins are relocated by a mechanism that remains to be determined.


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P23 Translocation of cell penetrating peptides (CPPs) through droplet interface bilayers Pauline Gehan

Gehan P., Rodriguez N. and Cribier S. Pierre et Marie Curie University, Paris, France Cell Penetrating Peptides (CPPs) are small polycationic peptides able to cross the cell membrane even if they are attached to a cargo. They are interesting candidates as efficient vectors to deliver drugs directly into the cytoplasm. It has been shown that this crossing can occur through endocytosis pathway or direct translocation, however the mechanism of the latter remains unknown.

In our lab, we focused on the molecular description of translocation mechanisms and the determination of the peptide’s partners for this translocation. In this work, we focused on the role of lipids in the interaction and internalization of a well-known CPP: penetratin. For this purpose, we used a model lipid bilayer formed at the interface of two adhering aqueous droplets in oil, each covered with a lipid monolayer (figures a and b). By introducing the fluorescently labelled CPP into one of the droplet, we can monitor its translocation through the bilayer. Moreover, this system allows us to form asymmetric bilayers, as in cell membranes, and thus provides a versatile model for studying the effects of lipid composition on CPP translocation and obtaining insights on the mechanism involved.

Thus, we have showed that anionic lipids are required on both leaflets and that the nature of the head roup on the proximal leaflet (or “outer leaflet”) has a crucial importance to induce translocation of the studied CPP. To pursue this study, we are currently developing a microfluidic device (figure c) to form and trap the two-droplets population. This system, under development, will allow us to screen a lot of parameters and to evaluate the role of lipids in CPP internalization mechanisms. We also aim to introduce electrodes into droplets (figure d) in order to detect possible modifications of the bilayer in presence of the CPP and study the effect of the membrane potential.

These tools, under development, will provide more insights into the mechanisms of translocation of cell penetrating peptides.


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P24 Tetraspanin-6 controls the turnover of syntenin and syndecan and inhibits the release of CD63 positive exosomes Rania Ghossoub

Ghossoub R., Lembo F., Egea-Jimenez AL., Leblanc R., Rubinstein E., David G. and Zimmermann P. CRCM, 27 Boulevard Lei Roure, 13009 Marseille, France

Extracellular vesicles (EVs), including exosomes, transport complex information (lipids, proteins, nucleic acids) and contribute to the regulation of multiple physio-pathological processes. Tetraspanins are four membrane span protein scaffolds enriched in EVs [1]. EV subpopulations with different functional activities can be differentiated based on the presence of specific tetraspanins like CD9, CD63 and CD81 [2]. Yet, other membrane scaffolds like syndecan heparan sulfate proteoglycans and their PDZ adaptor protein syntenin can control up to 50% of the release of exosomes [3,4].

Here we aimed to gain insight in the molecular mechanisms governing EV diversity and to start clarifying the relationships between tetraspanins, syndecans and PDZ scaffolds. We therefore tested for the effect of TSPAN6, a poorly characterized tetraspanin interacting with PDZ scaffolds and in particular syntenin, on the EV repertoire of MCF-7 cells. We found that TSPAN6 restricts the formation of SDC-syntenin-CD63 exosomes. Moreover TSPAN6 addresses syntenin to degradation in a PDZ-independent but syndecan-dependent manner. Finally, TSPN6 controls syndecan trafficking and processing.

Taken together these data indicate that TSPAN6 impacts on EV diversity by controlling the trafficking and processing of syndecans and syntenin and point to the importance of membrane scaffold networking for the formation of extracellular organelles. References [1] Andreu Z, Yáñez-Mó M. Tetraspanins in extracellular vesicle formation and function. Front Immunol. 2014 Sep 16;5:442. [2 Tkach M, Théry C. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci U S A. 2016 Feb 23;113(8):E968-77. [3] Baietti MF, Zhang Z, Mortier E, Melchior A, Degeest G, Geeraerts A, Ivarsson Y, Depoortere F, Coomans C, Vermeiren E, Zimmermann P, David G. Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat Cell Biol. 2012 Jun 3;14(7):677-85. [4] Imjeti NS, Menck K, Egea-Jimenez AL, Lecointre C, Lembo F, Bouguenina H, Badache A, Ghossoub R, David G, Roche S, Zimmermann P. Syntenin mediates SRC function in exosomal cell-to-cell communication. Proc Natl Acad Sci U S A. 2017 Nov 21;114(47):12495-12500.


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P25 Thermotropic and mechanical properties of lipid mixtures: the role of Dimethyl Sulfoxide Beatrice Gironi

Gironi B.1, Paolantoni M.

2, Morresi A.

1 and Sassi P.

1,2

1. Dipartimento di Chimica, Biologia e Biotecnologie, University of Perugia, Via Elce di Sotto 8, 06123 Perugia, Italy 2.Centro di Eccellenza sui Materiali Innovativi Nanostrutturati (CEMIN), University of Perugia, Via Elce di Sotto 8, 06123 Perugia, Italy

The importance of studying model lipid bilayers comes from the increasing interest in mimicking the biological membrane and understanding its structural, mechanical and thermotropic properties. It is known that the presence of saturated, unsaturated phospholipids and cholesterol (Chol) reproduces segregated Chol-rich domains inside the bilayer, “lipid rafts”, also present in biolo ical membranes [1]. These domains are known to be involved in a lot of biological functions, solubilize most of the membrane proteins and it is important to improve the knowledge of their structural properties. In our study we prepared multi lamellar liposomes (MLV) increasing the complexity of their lipid composition. We prepared simple lipid mixture (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Sphingomyelin (SM) both with and without cholesterol) and complex binary and ternary mixtures (POPC/SM and POPC/SM/ Chol). All the systems analysed were suspended in water and water/Dimethyl Sulfoxide (DMSO) solutions. The presence of DMSO in the solvating medium was used to examine its effect on the phase behaviour and structural properties of complex lipid membranes. This basic study is also important to explain the strong cryoprotective properties of DMSO [2]. To this extent we used a multitechnique approach (FTIR spectroscopy, fluorescence microscopy, calorimetry, Flicker spectroscopy…) to monitor the different states of the membranes in a wide temperature range (-60 ~ +70 °C). We also prepared the lipid vesicles by inserting a vibrational probe, Hexacarbonyl tungsten, in the hydrophobic part of the bilayer: this allowed to study how the free volume inside the membrane organises in different conditions [3], and this is related to the structural and permeability properties of lipid bilayers. References [1] Nicolini, C.; Kraineva, J.; Khurana, M.; Periasamy, N.; Funari, S. S.; Winter, R. Temperature and Pressure Effects on Structural and Conformational Properties of POPC/SM/Cholesterol Model Raft Mixtures-a FT-IR, SAXS, DSC, PPC and Laurdan Fluorescence Spectroscopy Study. Biochim. Biophys. Acta - Biomembr. 2006, 1758 (2), 248–258. [2] Ricci, M.; Oliva, R.; Del Vecchio, P.; Paolantoni, M.; Morresi, A.; Sassi, P. DMSO-Induced Perturbation of Thermotropic Properties of Cholesterol-Containing DPPC Liposomes. Biochim. Biophys. Acta - Biomembr. 2016, 1858 (12), 3024–3031. [3] Möller, M. N.; Li, Q.; Chinnaraj, M.; Cheung, H. C.; Lancaster, J. R.; Denicola, A. Solubility and Diffusion of Oxygen in Phospholipid Membranes. Biochim. Biophys. Acta - Biomembr. 2016, 1858 (11), 2923–2930.


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P26 Synthesis and Aggregation Behavior of a Series of Single-Chain Alkyl-Branched Bolalipids: Effect of Lateral Chain Length Kai Gruhle

Gruhle K., Müller S., Meister A. and Drescher S. Institute of Pharmacy, Biophysical Pharmacy, Martin Luther University (MLU) Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, Halle (Saale) 06120, Germany

Purpose: Bipolar phospholipids (bolalipids), which consist of two hydrophilic headgroups connected by a long alkyl chain, are important for the outstanding stability of Archaea against harsh living conditions such as low pH values or high temperatures. Consequently, natural occurring bolalipids as well as their synthetic derivatives are attractive candidates for the stabilization of liposomal drug delivery systems. Since standardized bolalipids are difficult to isolate from natural membranes, considerable efforts have been devoted to the synthesis of novel bipolar lipids. In our group, we simplified the chemical structure of bolalipids while maintaining their membrane-stabilizing properties. The first representative of a simplified archaeal model lipid was PC-C32-PC. The mixing behavior of this model lipid with classical phospholipids, e.g. DPPC, is limited due to packing problems caused by the larger space requirement of the PC headgroup of PC-C32-PC com-pared to the small cross-sectional area of its alkyl chain. To overcome this restriction, struc-tural modifications of the bolalipid structure are necessary. Hence, we established a synthetic route to single-chain alkyl-branched bolalipids, where the additional alkyl chains should compensate the packing problems.

Synthesis: Starting from a retrosynthetic view, the corresponding 1,ω-diols has to be pre-pared at first. Therefore, we used a copper-catalyzed GRIGNARD bis-coupling reaction of a middle part (1,ω-dibromide) and two identical side parts. We used δ-alkyl lactones as start-ing material for the synthesis of the side parts. Since the availability of δ-alkyl lactones is limited with respect to different lengths of the side chain, a new route for the preparation of 1-bromoketones based on a GRIGNARD coupling reaction to 5-bromovaleric acid chloride has been established. A series of different 1,1’-alkyl branched diols were prepared. In the last step, the corresponding bis(phosphocholines) (PC-C32(1,32R)-PC) were synthesized by a phosphorylation and quarternisation reaction of the corresponding diols. Results and Conclusions: The temperature-dependent aggregation behavior of the novel bolalipids in aqueous suspension was investigated using differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). In the DSC thermograms, we found a strong shift in Tm values comparing PC-C32(1,32C3)-PC and PC-C32(1,32C6)-PC. Further-more, we were able to show that the different lateral chain lengths also have a strong influ-ence on the aggregation behavior of the bolalipids. For example, PC-C32(1,32C3)-PC self-assembled into sheet-like aggregates, whereas PC-C32(1,32C6)-PC and PC-C32(1,32C9)-PC tended to form nanofibers.


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P27 Understanding mechanisms of transport of two urea derivatives through Urea Transporter (UT-b) Marc Guéroult

Guéroult M., Azouzi S., Mouro-Chanteloup I. and Etchebest C . UFR Sciences Exactes et Naturelles, Université de Reims Champagne Ardenne, Moulin de la Housse , 51687 Reims Cedex 2, France Urea is a small organic molecule acting in diverse physiological process such as water reabsorption in kidney or nitrogen sources. The importance of such molecule needed a better understanding of passing though the plasmic membrane and specifically in red blood cell (RBC). Urea transporter are a family of transmembrane protein UT-B, which contains ten transmembrane helices with intracellular N-terminus and C-terminus, carries the Kidd (Jk) blood group antigens. 2 small molecules derivative from urea, dimethylurea and methylurea, are specifics inhibitors of urea passive diffusion through UT-B, with different behaviors since the methylurea is competitive inhibitor, while the dimethylurea is a suicide inhibitor. To understand the difference of behavior between these two inhibitors and the mechanism associate potential mean force (PMF) were carrying out. All simulations allow us to characterize two specific orientations of the solutes, all along the UT-b pore. The 2 most important energetical barrier identified corresponding to these specific orientations and the entrance and exit funnel of the UT-b pore. The extremities of UT-b pore are characterized by a particular amino-acids motif, where the most conserved residues are aromatic or apolar. Energetical barrier are also associated desolvation of solute, which was monitorated by hydrogen bonds evaluations along the UT-b pore.

The comparison of the different PMFs identified some important difference between the methylurea PMF and dimethylurea PMF mostly located in Sm region, which is known to stabilize and fix urea molecule. The transport of solute through the UT-b pore are driven mostly by desolvation force and specific orientation of solute induce by hydrophobic and apolar residue defining a particular motif.


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Poster Session II


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P28 Tunnel vision: Understanding the cell biology and biophysics of tunneling nanotubes Michael Henderson

Henderson M., Descroix S., Bassereau P. and Zurzolo C. Unité Trafic Membranaire et Pathogenèse, Institut Pasteur, 28 rue du Dr. Roux, 75015 Paris, France Novel cell protrusions known as tunneling nanotubes (TNTs) have been found to mediate cytoplasmic continuity between remote cells for purposes of cell-to-cell communication during development and disease transmission, suggesting that TNTs are a ubiquitous structure in both normal physiological and pathological contexts. However, the underlying mechanistic details behind TNT formation and fusion of the nanotube with a target cell are unknown. This new research project combines interdisciplinary expertises between Instituts Pasteur and Curie and aims to understand how protein-specific interactions between actin and the membrane are involved in forming TNTs, which leads to mechanical and dynamical differences that distinguish TNTs from other membrane protrusions such as filopodia. Through the development of novel microfluidic and substrate-patterned devices, directed TNT growth between positioned cell pairs will be controlled such that the involvement of actin, actin-regulating proteins (such as the actin-bundling protein Eps8), and actin-membrane connectors (such as inverted BAR domain proteins which can deform membranes and stabilize negative curvatures inherent to nanotubes) can be ascertained in real time and correlated to mechanical measurements made on TNTs. The new devices developed will also uniquely allow sequential sample loading to create heterotypic cell-cell or cell-liposome arrangements to establish the roles that actin organization and specific membrane compositions have in facilitating membrane fusion upon TNT contact with a target cell. By deciphering how TNTs are molecularly and mechanistically differentiated from filopodia, directed treatments can be created that specifically halt TNT formation in aggressive and treatment-resistant pathologies.


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P29 Characterization of PI4P-dependent trafficking of ATG9 vesicles during autophagosome formation Javier Hervas

Hervas JH., Judith D. and Tooze SA.

Autophagy is an evolutionarily conserved cytoplasmic degradation process for macromolecules and organelles, and is vitally important for cell and tissue homeostasis. At autophagy induction, e.g. by starvation, a crescent-shaped double membrane called the isolation membrane, or phagophore, forms de novo and sequesters cargo before closing on itself, forming the autophagosome. This then fuses with lysosomes to degrade its cargo.

Phosphoinositides play a crucial role in cell signalling, and mainly in highly dynamic processes involving membrane trafficking as is the case of autophagy. In this sense, PI3P has been shown to be a key signalling lipid for autophagy initiation in the last years. In addition, PI4P, which is present in large amounts in the Golgi, has been related to autophagosomelysosome fusion events. Interestingly, ARFIP2 protein has been described to bind PI4P in the Golgi membranes , and it is essential for the movement of ATG9 vesicles, the only autophagy related transmembrane protein described so far. As well as ARFIP2, PI4P metabolizing enzymes (PI4KIIIβ, PI4K2A and SACML1) have been described to be present in these ATG9 membranes and might play a role in the initial steps of phagophore formation. Particularly, knockdown of PI4KIIIβ reduces the autophagy flux and alters ATG9 trafficking, which indicates a function in phagophore formation. Thus, in this project we will develop an optogenetics approach to recruit specific PI4P kinases or phosphatases to the phagophore membrane in order to control the production and removal of PI4P or ARFIP2, and consequently study the effects of PI4P on protein recruitment during autophagosome expansion.

Poster Session II Thursday, April 4th

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P30

The role of specific lysosomal lipid - bis(monoaclyglycero)phosphate (BMP) - on membrane features and its association in acid environment Rosangela Itri

Tsubone T., Ramos AP. and Itri R. Department of Applied Physics, Institute of Physics, University of São Paulo, SP, Brazil The autophagic/lysosomal system includes a variety of vesicular compartments that undergo dynamic fusion events [1]. It because during autophagy, cytoplasmic materials are sequestered by the autophagosome and transported to the lysosome for digestion. The specific stages of autophagy are induction, formation of the isolation membrane (phagophore), formation and maturation of the autophagosome and, finally, fusion with a late endosome or lysosome [2]. However, the characteristics and factors modulating these autophagosome– lysosome fusion remain, for the most part, unknown. Koga and co-workers suggested that changes in the lipid composition may inhibit autophagic vesicular fusion [1]. However, the complexity of vesicular fusion events that take place in autophagy, makes it difficult to study in intact cells. To gain insights on the properties that govern membrane lysosomal fusion events, we have investigated the biophysical properties of mimetic membranes that contains bis(monoaclyglycero)phosphate (BMP) - a hallmark phospholipid of lysosomal membranes [3,4] - and their consequences on membrane fusion.

Considering that BMP has a unique structural characteristic, here we have used DOPG (that is a structural isomer of BMP) for comparison purpose. Interestingly, our results have shown that membrane fusion is significantly influenced by pH, unlikely the data from DOPG vesicles. The explanation for this phenomena can be related to properties of BMP on packing membranes differently at low pH. Langmuir isotherms of BMP in pH 4.5 indicates that the surface pressure rises more slowly than in BMP pH 7.4 isotherm suggesting that the presence of protons on the head group disturbs lipid film packing and reduce the surface area available for each molecule due to their higher compaction. In fact, at pH 4, BMP exhibits lower surface charge density than DOPG, indicating the electrostatic repulsion decreasing of the charged head groups of BMP.

Acknowledgements. FAPESP for financial support with scholarship grant (2016/23071-9) and CNPq research grant to RI.

References [1] H. Koga, S. Kaushik, A.M. Cuervo, Altered lipid content inhibits autophagic vesicular fusion, FASEB J. 24 (2010) 3052–3065. [2] S. Nakamura, T. Yoshimori, New insights into autophagosome–lysosome fusion, J. Cell Sci. 130 (2017) 1209–1216. [3] T. Kobayashi, E. Stang, K.S. Fang, P. De Moerloose, R.G. Parton, J. Gruenberg, A lipid associated with the antiphospholipid syndrome regulates endosome structure and function, Nature. 392 (1998) 193–197. [4] T. Kobayashi, M.H. Beuchat, J. Chevallier, A. Makino, N. Mayran, J.M. Escola, C. Lebrand, P. Cosson, T. Kobayashi, J. Gruenberg, Separation and characterization of late endosomal membrane domains, J. Biol. Chem. 277 (2002) 32157–32164.


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P31 Investigation of lipid dynamics in the presence of antimicrobial peptides PGLa and magainin 2 Maria Kardash

Biophysique des Membranes et RMN - Institut de Chimie (UMR-7177), 1 rue Blaise Pascal, Université de Strasbourg, 67000 Strasbourg, France

PGLa and magainin 2 are antimicrobial peptides (AMPs) extracted from the skin of African clawed frog Xenopus laevis. Antimicrobial peptides can interact with bacterial membrane, destroying it and causing cell death. This ability makes them an excellent alternative to antibiotics as bacteria can not develop resistance to peptides action. That is why the mechanism of AMPs interaction with biomembranes is so interesting.

Here, PGLa and magainin 2 influence on dynamics of phosphate lipid groups in model membranes was investigated. Pulsed phosphorus-31 nuclear magnetic resonance was used to determine longitudinal relaxation time, T1, and transverse relaxation time, T2, in the temperature range 270-320 °K. It is known that PGLa and magainin 2 has synergistic effect - enhancement of antimicrobial activity in the mixture comparing to the individual components of corresponding amounts. Hence, model membranes of different lipid compositions with addition PGLa, magainin 2 or their mixtures were investigated.

Three membrane compositions were studied: mixtures POPE/POPG, POPC/POPG and DMPC/DMPG. Amounts of peptides were 0.5 – 1mol%. Temperature dependencies of relaxation times T1 and T2 were obtained. For T1, addition of peptides (alone or in the mixture) didn’t influence si nificantly on the relaxation time. However, a shift of extremums in the melting point to the area of lower temperature - about 5 °K – was observed. There was no remarkable difference for the mixture of peptides comparing with individual peptides.

In T2 measurements the biggest influence on relaxation time was for DMPC/DMPG lipid membranes. Here, the big extremum in the transition point with addition of peptides was replaced by slow changing, and for the mixture of peptides there was almost not extremum point. We see here a significant influence of peptides on lipid dynamics, and the biggest effect is observed for the mixture that could be the consequence of their synergistic activity. Probably, peptides “soften” the membrane, so instead of el phase transited into the liquid it becomes liquid at the lower temperature. Mentioned above the effect of shifting of the extremum in the meltin point is also can be the consequence of “softenin ” the membrane, though not so much as it is observed for DMPC/DMPG lipid mixture.


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P32 Functional nanostructure of NhaA protein in tethered lipid bilayer membranes Sebastian Köhler

Köhler S., Fragneto G., Maccarini M., Alcaraz JP. and Martin DK. ILL, 71 Avenue des Martyrs, 38042 Grenoble, France For studies of active membrane proteins it is necessary to embed them into an environment which is as close to nature as possible to retain their function, while at the same time keeping the system as simple as possible to allow for an experimental characterization and to be able to identify factors which influence the system. Tethered lipid bilayers (tBLMs) represent an experimentally accessible and stable model for biological membranes. Due to their connection to a planar surface they can form a more natural environment for membrane protein incorporation than the widely used solid supported bilayers that is stable for months. Using a combination of neutron reflectometry (NR) and electrochemical impedance spectroscopy (EIS) we characterize the incorporation of antiporter membrane proteins into these tBLMs.

A membrane protein’s function stron ly depends on the structure of the lipid bilayer that forms its environment. We are interested in which structural factors of the surrounding membrane influence the incorporation and subsequently the activity of an antiporter protein. Using NR we determine the nanostructure of the membrane/protein system[1] while EIS can be used to obtain functional information about the system. It provides electrophysiological information of the bilayer system like ion permeability and can show whether the incorporated antiporter protein is active or not, as this is related to an ion flow across the membrane. A combination of these two methods can thus give both structural and functional information, making it a useful tool for the complete characterization of a protein-membrane system [2]. With this methodology we want to relate structural properties of the bilayer, which are influenced by e.g. different lipid composition and tether molecule architecture, to the incorporated proteins’ function to understand the mechanisms behind these dependencies.

Here we present a study on the incorporation of the sodium-proton antiporter A (NhaA) into PEG-tethered lipid membranes on gold surfaces. We show the nanostructural characterization of highly covering tBLMs of different lipid composition with large fractions of incorporated functional membrane protein and how EIS can be used to investigate its activity. References [1] M. Maccarini, E. Watkins, B. Stidder, JP Alcaraz, B. Cornell, D. Martin, Eur. Phys. J. E, 2016, 39, 123. [2] M. Maccarini, L. Gayet, J.-P. Alcaraz, L. Liguori, B. Stidder, E. Watkins, J.-L. Lenormand, D. Martin, Langmuir, 2017, 33, 9988-9996.


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P33 BAD-LAMP influence the spatio-teporal regulation of type I interferon production in human plasmactoid dendritic cell Dario Leone

Centre d'Immunologie Marseille-Luminy, 171 Avenue de Luminy, 13009 Marseille, France

The endolysosome pathway constitute an heterogenous and highly dynamic system in which endocytic recycling pathway as well as endogenous phagosome and recycling endosome convert. Moreover, the trafficking and localization within specific endolysosome subpopulation, characterized by distinct membrane protein, was showed to be crucial for the activation of spatio-temporally regulated signaling pathway. In particular, the regulation of type I interferon (IFN-I) secretion by an highly specialized subtype of innate immune cells termed plasmacyotid dendritic cell (pDC), was showed to be dependent on its localization into endosome expressing VAMP3 and LAMP-2 but not LAMP-1.

PDC respond to pathogenic nucleid acid (NA) secreting large quantity of type I interferon (IFN-I), a potent signaling molecules able to interfere with the cellular homeostasis by inducing an alert status in the acceptor cells. Together with B-cell, PDC are characterised by the unique expression of endosomal resident toll like receptor (TLR) -7 and -9, two sensors able to recognize NA and initiate a tightly spatio-temporal regulated signaling cascade that culminate into the production of a large but transient amount of IFN-I followed by a second wave of secretion of an anti-inflammatory cytokine named; tumor necrosis factor alfa (TNF-a). The uncontrolled production of IFN-I or TNF-a by pDC is associated respectively with autoimmune disease like Systemic Lupus Erythematosus (SLE) as well as Psoriasis and aggressive ovarian and breast cancer, consequently, the production of type both cytokines must be tightly regulated by several and partially overlapping secure mechanism that depend also on adapotor proteins able to regulate the intracellular trafficking and the transit of these signaling pathway complex.

In this context, our group have identified a novel protein called the Brain And Dendritic cell Lysosomal Associated Membrane Protein (BAD-LAMP or LAMP-5) that is uniquely expressed in terminally differentiated immature human plasmacytoid dendritic cells (pDC) and cortical neurons which is able to modify the localization and therefore the production of IFN-I and TNF-a. In details, BAD-LAMP localise into the endoplasmatic reticulum golgi intermediate compartment (ERGIC) where it interact with UNC93B1, a multispanning chaperon that sequester TLR7 and TLR9 in steady-state. Shortly after stimulation with the TLR9 agonist CpG A (1 to 3 hours), BAD-LAMP, UNC93B1 TLR9 together with the adaptor protein MyD88, traffic into specific endosome characterized by the expression VAMP-3 and LAMP-2 promoting the activation of a specific transcription factor (IRF7) and the subsequent IFN-I production. At later time points (3 to 6 hours) the same complex translocate into late endosomes/lysosomes that are LAMP-1+ resulting in the inhibition of IFN-I and the production of TNF-a.

The involvement of BAD-LAMP in controlling TLR9 trafficking was confirmed by its overexpression that results in increased secretion of TNF-a and a strong reduction of IFN-I, suggesting a role in promoting the trafficking of the TLR9 and MyD88 complex into LAMP-1+ lysosome. (Combes A. et al Nature Communications 2017 Oct 13;8(1):913).


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P34 Identification and characterization of lipids organizing membrane nanodomains competent for IFN-γR activation Changting Li

Li C., Christophe Lamaze C. and Blouin C.

Institut Curie, PSL Research University, CNRS UMR3666, INSERM U1143, F-75005,Paris, France

Lipids can regulate protein structure and function throught various types of protein-lipid interactions. As amphipathic molecules, lipids have polar head groups and nonpolar side-chains which can both interact with proteins on specific domains. In comparison with proteins that interact with lipid head groups, the study of the interaction of lipids within the hydrophobic bilayer with transmembrane domain of membrane receptors is more challenging. The IFN-γ receptor (IFN-γR) is the prototype of cytokine receptors that activate the JAK/STAT pathway, a major signaling axis in innate and adaptive immunity. A recent study from my host laboratory (Blouin et al., Cell, 2016) demonstrates that IFN-γR complex dynamically partitions into sphingolipid/cholesterol-dependent nanodomains. This study further established that this lipid environment is necessary for the IFN-γR complex conformational changes required for the activation of the associated JAK kinases after IFN-γ stimulation. Biochemical studies demonstrated the direct association of a sphingosine-derivative on IFNγR1 transmembrane domain (Contreras et al., Nature 2012). The aim of our project is to determine how plasma membrane lipids regulate IFNγR assembly and function. To do this, we will identify the membrane lipids that specifically interact with the transmembrane domain of each subunit IFNγR1 and IFNγR2 with native lipidomic analysis by mass spectrometry. Therefore, the proper isolation and surface immunoprecipitation of the receptor subunits is a key step for the identification of associated membrane lipids. Our first results show efficient immunoprecipitation of endogenous IFNγR1 and IFNγR2 at the plasma membrane. We will also use the reconstitution of IFNγR transmembrane domains in lipid bicelles, like we have done for the T Cell Receptor partner, CD3ζTMCD. This will allow us to study lipid-induced conformational changes and identify lipid binding sites on IFNγR subunits by NMR.


130

P35 An electrostatic switching mechanism controls the PS/PI4P exchange activity of Osh6p Nicolas-Frédéric Lipp

Lipp N-F., Gautier G., Magdeleine M., Renard M., Albanese A., Čopič A. and Drin G. IPMC, 660 route des lucioles, 06560 Valbonne, France

In eukaryotic cells, phosphatidylserine (PS) is made in the endoplasmic reticulum (ER), yet it is enriched in the plasma membrane (PM), where it provides negative charges and is critical for recruiting various signaling proteins. In yeast Osh6p and its close homologue Osh7p contribute to this accumulation by transporting PS from the ER to the PM via PS/phosphatidylinositol 4-phosphate (PI4P) exchange cycles. It is unclear how, at each cycle, these proteins escape from the large electrostatic attraction of the PM to move back to the ER. We addressed this issue with Osh6p combining cellular observations, in vitro assays and simulations. We show that this protein dissociates from anionic membranes, like the PM, once it traps one of its lipid ligand. The N-terminal lid of Osh6p, when it folds to close the binding pocket with the lipid inside, changes the electrostatic surface of the protein, thereby reducing its binding to anionic membranes. Owing to this electrostatic switching mechanism, Osh6p keeps a fast exchange activity between weakly and highly anionic membranes, i.e in a model system mimicking the ER/PM interface. Attenuation of the electronegativity of the lid impairs transport in vitro and in yeast. This study demonstrates how a lipid transfer protein self-limits its residency time on membranes to be efficient.


131

P36 Differential identity of Filopodia and Tunneling Nanotube revealed by the opposite functions of actin regulatory complexes and by ultra-structural analysis Nina Ljubojevic

Ljubojevic N., Bhat S., Cordero Cervantes D., Delage E. and Zurzolo C. Unité Trafic Membranaire et Pathogenèse, Institut Pasteur, 28 rue du Dr. Roux, 75015 Paris, France Tunneling Nanotubes (TNTs) are filopodia-like protrusions that play a pivotal role in long-range intercellular communication. Different pathogens can use these structures to propagate between cells. TNTs are also implicated in cancer and neurodegenerative diseases, making them promising therapeutic targets. Understanding the mechanism of their formation, and their relation with filopodia is of fundamental importance to uncover their physiological function, particularly since filopodia are not able to mediate the transfer of cargo between distant cells. Here we studied different regulatory complexes of actin, which play a role in the formation of both of these structures. We previously demonstrated that the filopodia-promoting CDC42/IRSp53/VASP network negatively regulates TNT formation and impairs TNT-mediated intercellular vesicle transfer. Conversely, elevation of Eps8, an actin regulatory protein that inhibits the extension of filopodia in neurons, increases TNT formation. Notably, Eps8-mediated TNT induction requires Eps8 bundling but not its capping activity. Thus, despite their similarities, filopodia and TNTs form through distinct molecular mechanisms. Our current results using Arp2/3 and formin inhibitors, as well as different Rho GTPases, further suggest that a switch in the molecular composition in common actin regulatory complexes is critical in driving the formation of either type of membrane protrusion. Furthermore, other actin-binding proteins known to positively regulate filopodia formation such as N-WASP, WAVE-2 and cofilin all showed no impact on TNT formation, thus confirming the hypothesis that TNTs and filopodia are regulated in opposite manners. Therefore, we are currently elucidating key actin regulators that lead to actin filament formation in tunneling nanotubes and directly observing their impact on actin dynamics in these structures.


132

P37 How septins sense and reshape membrane at micrometric scale Stéphanie Mangenot

Beber A., Taveneau C., Nania M., Bassereau P., Levy D., Cabral JT., Isambert H., Bertin A. and Mangenot S. Physico Chimie Curie, UMR 168, 11 Rue Pierre et Marie Curie, 75005 Paris, France

Septins are cytoskeletal proteins able to form supramolecular structures such as filaments, networks and rings. They are bound to the inner plasma membrane at specific location including the separation between the mother and daughter cell during cytokinesis and the basis of cilial cells. Septins are essential for cytokinesis, participate in the formation of diffusion barrier and might be involved in membrane deformation and rigidity. Using simplified biomimetic systems, we asked if septins were sensible to curvature, given their preference to be located at places of high curvature. To mimic specific curvatures and geometries observed in vivo, we have used PDMS substrates covered with a supported lipid bilayer. “Wavy” PDMS patterned substrates display both positive (“bumps”) and ne ative (“valleys”) curvatures. To our surprise, we have seen, usin Scannin electron Microscopy, that Septin filaments have a preference for negative micrometric size curvatures. On positively curved eometries (bumps), septins spontaneously ali n alon the “bumps” and thus orient towards null curvature.

This curvature preference is closely related to the ability of septins to reshape and deform membranes. When interactin with iant unilamellar vesicles, septins induce μm scale deformations with the formation of regular rigid spikes at the surface of the liposomes. We propose a theoretical model to take into account these observations and could be relevant to describe the organization of septins during cytokinesis in vivo.


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P38 Regulation of ER-Endosome contact formation Arthur Martinet

IGBMC, 67100 Illkirch, France

Eukaryotic cells are compartmentalized in organelles – subcellular structures delimited by membranes - having specialized functions allowing the division of labor in the cell. Organelles collaborate with one another and thus generate an interdependent network. Inter-organelle membrane contact sites (MCSs) are recognized as a mode of inter-organelle collaboration. MCSs are characterized by a close proximity of heterologous membranes from two different organelles. Most MCSs involve the endoplasmic reticulum (ER), which creates contacts with organelles such as mitochondria, endosomes, the Golgi apparatus, and the plasma membrane. MCSs are involved in several critical cellular functions such as calcium exchange and lipid homeostasis, and are also involved in the regulation of signaling pathways. At the molecular level, MCS are formed by protein-protein and/or protein-membrane interactions at the interface between the two organelles involved. At the ER level, VAP (Vesicle-Associated Membrane Protein Associated Proteins) and MOSPD2 (Motile sperm domain-containing protein 2) are responsible for the formation of many MCSs. Indeed, VAP and MOSPD2 proteins contain a MSP (Major Sperm Protein) domain facing the cytoplasm which acts as a molecular hook interacting with small protein motifs named FFAT (two phenylalanines in an acidic tract). FFATs are found on a variety of proteins attached onto other organelles, and their interaction with VAPs/MOSPD2 proteins allows the physical association between the ER and these organelles. MCSs are highly dynamic structures. In this work, we investigated the molecular mechanisms allowing the regulation of MCS formation.


134

P39 Molecular mechanisms of leptin transport across the tanycytes of median eminence Marion Millet

Millet M., Duquenne M., Haeberlé A., Prévot V., Ory S. and Gasman S. Institut des Neurosciences Cellulaires et Intégratives (INCI), CNRS, 8 allée Rouvillois, 67000 Strasbourg, France

Leptin is a hormone secreted by adipose tissue that acts in the central nervous system to regulate appetite. This dialogue between the periphery and the brain is essential to maintain energy homeostasis but is impaired in obese people. We have recently hypothesised that this so-called leptin-resistance, could be the consequence of a defective leptin transport into the brain.

The median eminence is an hypothalamic structure containing specialized ependymoglial cells called tanycytes. These cells line the floor of the third ventricle and contact the fenestrated vessels in the median eminence. It has been proposed that tanycytes act as « gatekeepers » by regulating the access of blood-borne signals to the hypothalamus, and are involved in leptin exocytosis into the cerebrospinal fluid from where leptin-sensitive regions can be reach. However, the cellular and molecular mechanisms controlling leptin endocytosis, its transcytosis as well as its release from apical site of the tanycytes remain unknown. Using fluorescence and electron microscopy on primary cultures of rat tanycytes, we are deciphering the endocytic route taken by leptin as well as the molecular mechanisms controlling leptin exocytosis. Altogether, these data constitute a promising start toward the understanding of the leptin journey in tanycytes.


135

P40 Mixing Behavior of Bilayer-Forming Phosphatidylcholines with Single-Chain Alkyl-Branched Bolalipids

Sindy Müller

Müller S., Gruhle K., Meister A. and Drescher S. Institute of Pharmacy, Biophysical Pharmacy, Martin Luther University (MLU) Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, Halle (Saale) 06120, Germany

Purpose: Liposomes have become the most widely used drug delivery system. However, despite numerous advantages of liposomes in drug encapsulation, no oral liposomal formulation has been approved so far. The reason therefore can be found in the instability of conventional phospholipids in the human gastrointestinal tract against, e.g., bile salts and low pH values. The aim of our research is to develop an oral liposomal formulation that is stabilized by so-called bolalipids. These bolalipids are artificial analogues of archaeal membrane lipids, which make it possible for the archaea to live under harsh conditions such as high temperatures and low pH values. Since both the extraction and the total synthesis of these bolalipids are very expensive and time-consuming, we synthesize lipids that are simplified in their chemical structure maintaining the stabilizing properties. Methods: The miscibility of our bolalipids with classical bilayer-forming phospholipids was investigated using DSC and TEM. By means of DLS, we determined the particle size, particle size distribution, and storage stability of liposomes prepared from lipid mixtures of bolalipids and phospholipids. To further study the shape of the modified vesicles, we recorded electron micrographs of vitrified specimens and replica of freeze-fractured samples. In addition, we investigated the permeability of bolalipid-containing membranes using a dithionite fluorescence assay. Finally, we started in vitro experiments and incubated the bolalipid-liposomes in artificial digestion media and examined changes in particle size and the release of an encapsulated, water-soluble fluorescent dye.

Results: The mixing behavior of the three new alkyl-branched bolalipids PC-C32(1,32C3)-PC, PC-C32(1,32C6)-PC, and PC-C32(1,32C9)-PC with DPPC and DOPC in different molar ratios was studied. We were able to show, that PC-C32(1,32C6)-PC and PC-C32(1,32C9)-PC are miscible with the unsaturated DOPC and that the amount of bolalipid, which can be incorporated into a phospholipid bilayer, increased with increasing length of the lateral alkyl chains of the bolalipid. Thus, we were able to produce liposomes based on DOPC that contained our novel bolalipids. These modified liposomes showed a rough surface in EM images and were stable in size over at least 21 days of storage. We found that bolalipid-containing membranes are more permeable to smaller ions than the bilayers of conventional phospholipid liposomes presumably due to clustering of both lipid components. First in vitro experiments using artificial digestion media indicated that bolalipid-containing liposomes were stable in artificial gastric juice and were superior in this media to conventional liposomes regarding the release of a water-soluble fluorescent dye. Nevertheless, in artificial intestinal media containing bile salts, bolalipid-liposomes were solubilized.

Conclusions: We proved that the bolalipids PC-C32(1,32C6)-PC and PC-C32(1,32C9)-PC were miscible with DOPC. Hence, we were able to produce DOPC based liposomes containing these bolalipids, which is - to our knowledge - the first time for artificial single-chain bolalipids. In addition, a comprehensive physicochemical characterization of these modified liposomes was carried out. Initial in vitro experiments suggested increased stability of the bolalipid-containing liposomes in simulated gastric fluid compared to conventional vesicles.


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P41 HIV-1 Gag restricts specifically PI(4,5)P2 and cholesterol mobility creating a nanodomain platform for virus assembly: frommembrane model to host CD4 T cells. Delphine Muriaux

Favard C., Chojnacki J., Mak J., Eggeling C and Muriaux D. IRIM, 1919 route de Mende, 34170 Montpellier, France

The retroviral Gag protein targets the plasma membrane of infected cells for viral particle formation and release. The MA domain of Gag is myristoylated for membrane anchoring but also contains a highly basic region that recognizes acidic phospholipids. Gag targets lipid molecules at the inner leaflet of the plasma membrane including phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) and cholesterol. In this study, we address the question whether HIV-1 Gag is able to trap PI(4,5)P2 and/or other lipids during HIV-1 assembly in the host living CD4+ T cells. Here, we determine lipid dynamics within and away from HIV-1 assembly sites using super-resolution STED microscopy that is coupled with scanning Fluorescence Correlation Spectroscopy (sSTED-FCS). Analysis of HIV-1 infected CD4+ T lymphocytes revealed that, upon virus assembly, HIV-1 is able to specifically trap PI(4,5)P2, and cholesterol, but not phosphatidylethanolamine (PE) or sphingomyelin (SPM) at the cellular membrane. Furthermore, analysing CD4+ T cells expressing only HIV-1 Gag protein shows that Gag is the main driving force to restrict mobility of PI(4,5)P2 and cholesterol at the plasma membrane. Our data provide first direct evidence showing that HIV-1 Gag creates its own specific lipid environment for virus assembly, by selectively recruiting lipids to create a PI(4,5)P2/cholesterol enriched nanodomains favouring assembly, and that HIV-1 does not assemble on pre-existing lipid domains.


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P42 Exo-endocytosis coupling is calcium independent at small central synapses Marta Orlando Orlando M., Rosenmund C. and Herman M. Charité – Universitätsmedizin Berlin, Charitéplatz 1,10117 Berlin, Germany

Successful synaptic transmission requires release of neurotransmitter by synaptic vesicle exocytosis in the presynaptic terminal. The retrieval of synaptic vesicles from the plasma membrane by endocytosis is crucial for maintaining synaptic structure and efficient neurotransmission. Sites of synaptic vesicle fusion must be cleared rapidly; therefore, the coupling of exo- and endocytosis is crucial. Despite many years of research, the exact molecular and biophysical requirements for the coupling of exo- and endocytosis are still debated. The role of calcium in exo- and endocytosis coupling has remained particularly enigmatic. Because calcium influx is necessary to trigger synaptic vesicle fusion, it is difficult to deconvolve calcium’s role in initiatin the membrane retrieval after exocytosis as well. Therefore, to study the role of calcium in initiating endocytosis, we used a hypertonic sucrose solution to evoke calcium-independent neurotransmitter release from synapses in cultured hippocampal neurons. This is a well-characterized and established method determined by electrophysiology to induce exocytosis of the readily-releasable pool of vesicles in a calcium-independent manner.

We evoked synaptic vesicle exocytosis using hypertonic sucrose solutions and investigated membrane retrieval using high pressure freezing and transmission electron microscopy analysis of ultrastructure. We observed that sucrose induced formation of membrane invaginations, or endocytic pits, which were completely absent in synapses incapable of undergoing exocytosis. Even in the complete absence of calcium, obtained by incubating neurons in the calcium chelator BAPTA-AM, we could show that hypertonic sucrose still induced pit formation. This is, to our knowledge, the first proof that synaptic vesicle endocytosis can be initiated at presynaptic terminals in the absence of calcium influx. Further, we show that sucrose-induced initiation of endocytosis is clathrin-independent but requires actin polymerization. Our findings point to a central role of actin acting on membrane tension for an efficient coupling of exocytosis to endocytosis. Furthermore, we investigated whether calcium is necessary for the kinetics of endocytosis by genetically labeling a synaptic vesicle protein.

Preliminary evidence obtained by monitoring the endocytosis and reacidification of the pH sensitive variant of GFP, pHlourin, tagged to synaptophysin with live-cell imaging suggests that protein recycling occurs in the absence of calcium influx and has similar kinetics to the endocytosis of synaptic vesicle proteins after calcium-mediated fusion.

Our data clarify the controversial role of calcium in the initiation of endocytosis at presynaptic terminals. We suggest that membrane tension, and not calcium influx, is the key driving force for the coupling of exo- and endocytosis.


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P43 Phospholipid scramblase 1 (PLSCR1) regulates synaptic vesicle recycling Stéphane Ory

Caputo M., Estay-Ahumada C., Caquineau C., Toth P., Gasman S. and Ory S. Institut des Neurosciences Cellulaires et Intégratives (INCI), CNRS, 8 allée Rouvillois, 67000 Strasbourg, France

Neurotransmitter release occurs through a Ca2+-dependent exocytic process requiring the fusion between synaptic vesicle and plasma membranes. To maintain organelle homeostasis, exocytosis must be followed by compensatory endocytosis, a process that intends to preserve the plasma membrane integrity and to allow secretory vesicle recycling.

We have previously demonstrated in neuroendocrine cells that large dense core vesicle exocytosis leads to the disruption of plasma membrane asymmetry due to the Phospholipid Scramblase-1 (PLSCR-1) -dependent redistribution of phosphatidylserine (PS) from the inner to the outer membrane leaflet. We showed that, fommowing exocytosis, secretory granule membrane recapture was impaired in chromaffin cells of PLSCR1 knock-out mice. This demonstrated that PLSCR1-dependent lipid rearrangement was critical for compensatory endocytosis in chromaffin cells. It is currently unknown whether such phospholipid redistribution occurs in neurons and if PLSCR1 is involved in synaptic vesicle recycling.

Using FM1-43 dye uptake assay in neurons cultured from PLSCR1 KO mice, we showed that compensatory endocytosis was significantly decreased compared to wild type cells. In addition, monitoring the exocytosis/endocytosis cycle in live cells by expressing the fluorescent reporter consisting of the synaptophysin coupled to pHluorin (Syn-pH), we observed that the dynamic of compensatory endocytosis in PLSCR1 KO neurons is much slower compared to wild type neurons. The molecular mechanisms involved are currently under investigation but these data suggest that PLSCR1 and lipid scrambling may be a key component of the molecular machinery regulating synaptic vesicle recycling.


139

P44 Understanding the regulation of endocytosis and trafficking of an anthrax toxin receptor, CMG2 Numa Piot

Piot N., Abrami L. and van der Goot FG. École Polytechnique Fédérale de Lausanne (EPFL) SV GHI VDG, AI 3147 (Bâtiment AI), Station 19, CH-1015 Lausanne, Suisse

Capillary Morphogenesis Gene 2 (CMG2) is a transmembrane receptor at the plasma membrane known for more than a decade to be one of the two cellular receptors for anthrax toxin. Shortly later this discovery, Hyaline Fibromatosis Syndrome (HFS), a very rare and disfiguring disease, was shown to be induced by different mutations in CMG2. For a long time, CMG2 physiological functions remained unknown until two years ago when CMG2 was shown to be the receptor responsible for collagen VI endocytosis and degradation. CMG2-mediated collagen VI internalization is likely to use the same endocytic pathway as anthrax toxin, which hijacked CMG2 endocytosis to intoxicate cells. This work aim to increase the understanding of how CMG2 endocytosis and trafficking though the endocytic pathway are regulated which is essential to understand the molecular mechanisms behind both HFS and cell intoxication by anthrax toxin.

Endocytosis and trafficking are very tightly regulated processes through recruitment of adaptor proteins as well as post-translational modifications involving the action of many interacting proteins. In this context, we aim to identify these CMG2 interacting partners using mass spectrometry and APEX2-mediated biotinylation of neighboring proteins. We performed a first screen in presence and in absence of CMG2 ligand that identified a set of proteins related to actin cytoskeleton dynamics and endocytosis mainly, which is consistent with CMG2 function and localization.

In a second time, we used an assay monitoring anthrax toxin endocytosis using CMG2 endocytic pathway, which allowed us to validate the role of CMG2 interacting partners in this process. Two E3 ubiquitin ligases, Cbl and RNF41 as well as a kinase and a deubiquitinase, MARK2 and USP8 respectively, were previously identified as potentially involved in CMG2- mediated anthrax toxin entry. Using the anthrax toxin entry assay, we were able to show that Cbl plays a significant role in anthrax toxin endocytosis and RNF41 and MARK2 showed a tendency to be involved in this process, while USP8 showed no direct role in CMG2 endocytosis. Further experiments will need to be done to determine how and at which step of the endocytic pathway these interacting proteins act on CMG2.


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P45 Curvature dependence of SNARE TMD mediated membrane fusion Matthias Pöhnl

Pöhnl M. and Böckmann R. Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Staudtstr. 5, Geb. B1, 91058 Erlangen, Germany Membrane fusion is a key event in a wide range of biological processes like exocytosis, viral infection, fertilization and intracellular trafficking. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are thought to play a major part throughout all steps of the fusion process, e.g. docking the membranes, bringing them in close proximity by overcoming repulsive forces between the opposing membranes, initializing hydrophobic contacts, and in lowering the energy cost of highly curved membrane areas in fusion pore opening [1]. However, the microscopic details of the individual fusion steps and especially the role of the specific domains of the SNARE proteins remain elusive.

Substituting the SNARE transmembrane domain (TMD) with lipid anchors or mutating its primary structure was shown to alter the vesicle fusion process by altering the initiation and pore opening [2]. Molecular dynamics (MD) simulations of flat membranes were used to obtain a molecular description of these effects by investigating TMD-lipid interactions [2], [3] and TMD oligomerization [4]. The TMD regulates lipid mobility, and hence lipid protrusion events leading to first hydrophobic contacts between the merging membranes [3]. Furthermore the specific TMD primary structure regulates the oligomerization propensity, which is key for efficient membrane fusion.

However, these studies neglect effects like changed lipid protrusion probabilities and high energy costs caused by the curvatures involved in membrane fusion. Therefore, we extend previous work on the properties of SNARE TMDs and SNARE TMD mutants using coarse-grained MD simulations allowing for strongly curved membrane geometries. In a first step we investigate the curvature dependence of the localization of TMDs and their oligomers.

References [1] Jahn R. and Fasshauer D., Nature, 2012 [2] Dhara M., Yarzagaray A., Makke M., Schindeldecker B., Schwarz Y., Shaaban A., et al., eLife, 2016 [3] Han J., Pluhackova K., Bruns D., Böckmann R., BBA, 2016 [4] Han J., Pluhackova K., Wassenaar T., Böckmann R., Biophys. J., 2015


141

P46 Probing synaptotagmin oligomerization by Atomic Force Microscopy Lorena Redondo-Morata

Krishnakumar S., Janel S., Lafont F. and Redondo-Morata L. INSERM U1019 – Institut Pasteur de Lille, 1 rue du Professeur Calmette, 59021 Lille Cedex, France Atomic Force Microscopy (AFM)-based studies constitute today a fairly established methodology to observe the structure of biomolecules and to measure their mechanical properties. However, biomolecules are dynamic in nature; hence, to understand how they work we need to increase the spatiotemporal resolution of conventional AFM. In the last decade, High-Speed AFM (HS-AFM) was developed and successfully applied to several cellular machineries, either cytoplasmic or bound to membranes. The molecular movies obtained by this method provide insights otherwise not accessible by other means to date.

Recently, we have succeeded to visualize by these means the molecular mechanism of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) assembly formation. We observed the formation of spiraling filaments on lipid membranes and estimated that the accumulated elastic energy is sufficient to drive membrane deformation. In our most recent work, we studied the molecular role of Vps4, an ATPase that it is known to drive the disassembly of persisting filaments of ESCRT-III, revealing a dynamic ESCRT subunit exchange.

With a similar methodological approach, we are now studying the oligomerization of synaptotagmin 1 (Syt1). During calcium-regulated exocytosis, the constitutive fusion machinery is ‘clamped’ in a partially assembled state until synchronously released by calcium cations. The protein machinery involved in this process is known, but the supra-molecular architecture and underlying mechanisms are unclear. Recently, James Rothman group in Yale has demonstrated that Syt1 molecules readily form Ca2+-sensitive ring-like oligomers on lipid monolayers. This prompted a hypothesis that such Ca2+-dependent Syt1 oligomerization at the interface of the docked synaptic vesicle may play a critical role in regulation of neurotransmitter release. We employ High-Speed AFM and super-resolution optical microscopy approaches correlatively to solve the structures of the synaptic vesicle-plasma membrane junction in fully reconstituted systems. We aim to visualize the dynamic assembly and disassembly of the different oligomeric structures which serves to tether the synaptic vesicle to the plasma membrane. HS-AFM will provide sub-second temporal resolution and nanometer spatial resolution of label-free complexes assembly in the lipid membrane and to map the nanomechanical properties of the overall structures on the membrane. Our goal is to provide further information to validate the so-called ring hypothesis, as well as structural and mechanical insights.


142

P47 Endophilin-A3 controls a clathrin-independent endocytic route distinct from Endophilin-A2 Henri-François Renard

Renard H-F., Tyckaert F., Lo Giudice C., Hirsch T., Shafaq-Zadah M., Valades Cruz CA., Wunder C., Johannes L., Van der Bruggen P., Alsteens D. and Morsomme P. Croix du Sud 4-5 bte L7.07.07 (Carnoy B.384), 1348 Louvain-La-Neuve, Belgium While several clathrin-independent endocytic processes have been described so far, they often remain poorly characterized mechanistically. Lately, key elements of these machineries have been identified in the BAR domain protein family, which are specialized in membrane curvature induction and recognition. In particular, proteins from the Endophilin-A (EndoA) subfamily were shown to control the clathrin-independent uptake of various cargoes, such as β-adrenergic receptors, IL-2 receptor or bacterial toxins. Owing to their high sequence similarity, the three endophilin isoforms A1, A2 and A3 are mostly considered as redundant. In our lab, we showed that they actually do not colocalize and control the uptake of different subsets of cargoes in distinct endocytic carriers. Using proteomics and cell biology approaches, we identified ALCAM/CD166 as a new clathrin-independent cargo. We could demonstrate that EndoA3 isoform – but not A2 – specifically and functionally associates with ALCAM/CD166 early endocytic carriers. We also provided evidences that the uptake of ALCAM/CD166 and the recruitment of EndoA3 to endocytic sites at plasma membrane are driven by extracellular Galectin-8. Taken together, our data demonstrate the existence of a previously unnoticed clathrin-independent endocytic route controlled by Galectin-8 and EndoA3, essential for the turnover of cell surface proteins such as CD166. As CD166 is present at the surface of various cell types, including cancer cells, our findings might contribute to better understand its role in cell adhesion and migration.


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P48

MYO1C facilitates arrival at the Golgi apparatus through stabilization of branched actin Kristine Schauer

Capmany A., Yoshimura A., Kerdous R., Lescure A., Del Nery E., Coudrier E., Goud B. and Schauer K. Institut Curie, 12 rue Lhomond, 75005 Paris, France We aim at the identification of myosin motor proteins that control trafficking at the Golgi apparatus. In addition to the known Golgi-associated myosins MYO6, MYO18A and MYH9 (myosin IIA), we identify MYO1C as a novel player at the Golgi. We demonstrate that depletion of MYO1C induces Golgi apparatus fragmentation and decompaction. MYO1C accumulates at dynamic structures around the Golgi apparatus that colocalize with Golgi-associated actin dots. Interestingly, MYO1C depletion leads to loss of cellular F-actin, and Golgi apparatus decompaction is also observed after the inhibition or loss of the Arp2/3 complex. We show that the functional consequences of MYO1C depletion is a delay in the arrival of incoming transport carriers, both from the anterograde and retrograde routes. We propose that MYO1C stabilizes branched actin at the Golgi apparatus that facilitates the arrival of incoming transport at the Golgi.


144

P49 The role of VAPA-mediated membrane contact sites during collective cell migration Hugo Siegfried

Siegfried H., Le Borgne R., Heuzé M. and Verbavatz J-M. Institut Jacques Monod, CNRS UMR7592, 15 rue Hélène Brion, 75013 Paris, France

Recent studies have described the existence of distinct domains of membrane tethering, called membrane contact sites (MCS), between the endoplasmic reticulum (ER) and other organelles. MCS serve as platforms for lipid exchange between the two organelles and calcium fluxes. Despite the growing knowledge on molecular composition of MCS, their roles in physio-pathological processes are barely studied. Cell migration, in particular, highly depends on regulation of phosphoinositide (PI) pool at the plasma membrane (PM) giving rise to signalling pathways that mediate cytoskeleton reshaping.

Here, we study the involvement of an ER-resident protein that tethers ER-MCS, called VAPA (Vamp-Associated Protein A), during collective cell migration. VAPA and its homologs have been shown to modulate PI(4)P levels in different membrane compartments in yeast and mammalian cells, and to regulate actin nucleation on endosomes (Stefan et al, 2011; Dong et al, 2016). Moreover, ORP3, one of the VAPA partners at ER-PM contact sites, regulates cell adhesion and spreading (Lehto et al, 2007; Weber-Boyvat et al, 2015), indicating that ER-PM contact sites are required for cell motility processes.

We show that VAPA KD Caco2 cells lose their directionality in in vitro collective migration assays, affecting the capacity of monolayers to colonize space. Interestingly, VAPA KD single cells exhibit increased spreading on fibronectin and disorganized actin cytoskeleton.

Our results show that VAPA is essential during cell migration, probably through the regulation of lipid exchange. Even if the molecular mechanisms are still unknown, this preliminary study opens the doors to many perspectives about the role of MCS in cell migration.


145

P50 Membrane remodeling function of the retromer at “non-endosomal” organelles Julia Sirés Campos

Sires Campos J., Ripoll L., Marks MS., Raposo G., Houdusse A. and Delevoye C. Institut Curie, 12 Rue Lhomond, 75005 France, Paris

The sorting and transport of proteins are essential for the correct function of cellular organelles and therefore, organism development and physiology. Within the endolysosomal system, several protein complexes coordinate various tubulo/vesicular trafficking events: from the building of transport intermediates to the packaging of cargos and their transport to destination. Across all eukaryotes, one of these vital endosomal sorting machinery is the retromer complex. Consisting in a cargo selection complex (CSC; VPS35–VPS29–VPS26 trimer) and a membrane bending complex (MBC; dimer of sorting nexins), the retromer sorts transmembrane cargo proteins and recruits accessory molecules to the endosomal membrane, therefore helping the generation of tubulovesicular carriers.

While many researches have probed the role of retromer during trafficking from endosomes, much less is known regarding its functions at other organelles. We thus investigated the role of the retromer in a newly recycling pathway originating at the melanosome, the melanin pigment containing lysosome-related organelles of skin epidermal melanocytes and eye pigment cells. We recently defined that this melanosomal export pathway corresponded to tubular transport intermediates, which are severed and released through the coordinated action of the Myosin VI actin-based motor and the branched-actin polymerizing machinery. We hereby demonstrate that the retromer complex cooperates to export melanosome cargoes, suggesting for the first time that the retromer functions together with an actin-based motor. By combining super resolution fluorescence microscopy, electron microscopy and biochemical analyses, we found that the CSC and MBC sub-complexes were both implicated in the pigmentation process via the formation of recycling tubules at melanosomes. Depletion of one or other complex gives rise to: a) cargo accumulation at melanosomes; b) increased melanin production; and c) defects in tubule release from melanosomes.

These findings show that the retromer complex functions in parallel at multiple transport pathways originating from different organelles. By controlling together with actin and associated motors cargo sorting and export at melanosomes, the retromer coordinates the biogenesis and severing of tubular transport intermediates necessary for organellar and cellular functions. How such a single complex might achieve similar functions at distinct intracellular locations is currently under investigation.


146

P51 Visualisation of endocytosis and recycling of β-adrenergic receptors in neuronal dendrites Silvia Sposini

Sposini S. and Perrais D. IINS, 146 rue Leo Saignat, 33000 Bordeaux, France Endocytic trafficking and sorting of G-protein coupled receptors (GPCRs) are key determinants of cellular responses, controlling both the temporal and spatial parameters of cellular signalling. A growing body of evidence is pointing to the fact that distinct GPCRs are internalized through various mechanisms (Clathrin Mediated Endocytosis, Fast Endophilin Mediated Endoctytosis) and are differently organized into the endosomal pathway (classic Early Endosome, Very Early Endosome). This is particularly relevant for β1-adrenergic receptor (B1AR) and β2-adrenergic receptor (B2AR), which, despite their similarities, including activation by the same ligands, do not use the same cellular machinery for their trafficking in immortalised cell lines [1,2]. Understanding how such distinct trafficking behaviour is achieved and orchestrated is especially intriguing for those tissues where B1AR and B2AR are endogenously co-expressed. One such region is the hippocampus, a part of the brain essential for many forms of learning and for which synaptic plasticity in neurons is highly modulated by noradrenergic receptors. Considering that a detailed description of GPCR traffickin in neurons still needs to be mechanistically dissected, we sou ht to use β-adrenergic receptors as prototype GPCRs to have a better understanding on the molecular mechanisms, as well as the physiological role, of receptor trafficking in neurons.

To achieve our goal, we developed high spatial-temporal resolution microscopy methods to visualise internalisation, intracellular trafficking and recycling of these receptors in living neurons. Specifically, we have monitored the endo- and exo-cytosis of receptors tagged with pH sensitive probes using Total Internal Reflection fluorescence microscopy (TIR-FM) with the use of the ppH protocol [3]. To track individual receptors during their intracellular trafficking, we have used new generation quantum dots in combination with super resolution microscopy. With this tools, we will generate maps of B1AR and B2AR trafficking pathways in hippocampal neurons, confirming/rejecting the role of previously identified adaptor proteins (APPL1, arrestins, clathrin, endophilin, etc) in neurons. Overall these approaches will decipher the spatio-temporal organisation of the trafficking and sorting mechanisms of signalling receptors; importantly, these can be applied to other GPCRs and have a broader interest and application to other physiological contexts. References [1] Boucrot E, Ferreira APA, Almeida-Souza L, Debard S, Vallis Y, Howard G, et al. Endophilin marks and controls a clathrin-independent endocytic pathway. Nature. 2015 517:460-5. [2] Sposini S, Jean-Alphonse FG, Ayoub MA, Oqua A, West C, Lavery S, et al. Integration of GPCR Signaling and Sorting from Very Early Endosomes via Opposing APPL1 Mechanisms. Cell Rep. 2017;21: 2855–2867. [3] Rosendale M, Jullié D, Choquet D, Perrais D. Spatial and Temporal Regulation of Receptor Endocytosis in Neuronal Dendrites Revealed by Imaging of Single Vesicle Formation. Cell Rep. 2017;18: 1840–1847.


147

P52 Deformation and fission of symmetric and asymmetric model membranes containing polyunsaturated phospholipids Marion Tiberti

Tiberti ML., Manni M., Gautier R. and Antonny B. IPMC, 660 routes des lucioles, 06560 Sophia-Antipolis, France Phospholipid membranes form cellular barriers but need to be flexible enough to divide by fission. Phospholipids generally contain a saturated acyl chain at position sn1 whereas the sn2-acyl chain is saturated, monounsaturated or polyunsaturated. To obtain details at the molecular-level, we used coarse-grained and all atom molecular dynamics simulations. The development and application of several descriptors reveal that the vertical movements of the acyl chains along the membrane normal increase with polyunsaturation and are responsible for the increasing flexibility of the membranes. This effect was directly observed in membrane tether simulations in which a force is applied to a flat lipid bilayer, which leads to the formation of a tubule, which eventually brakes by fission. In agreement with the experiments, the simulations indicate that phospholipids with one docosahexaenoic acid chain (DHA; omega-3) make membranes more deformable than phospholipids containing less unsaturated acyl chains, including arachidonic acid, an omega-6 acyl chain. Experimentally, we also observe that phospholipids with two polyunsaturated acyl chains make membranes even more prone to vesiculation than phospholipids with one saturated and one polyunsaturated acyl chain. However, this effect correlates with a dramatic increase in membrane permeability to solutes. Thus, asymmetric sn1-saturated-sn2-polyunsaturated phospholipids, which are abundant in the brain and notably in synaptic vesicles, provide a trade-off between efficient membrane vesiculation and low membrane permeability. However, each leaflet of biological membranes has not the same lipid composition. In particular, polyunsaturated lipids are mainly present on the internal leaflet. To mimic more precisely the biological membranes, we have built new membrane systems with asymmetric lipid composition. We observe that it is easier to pull on the side of the polyunsaturated lipids than on the other side: the generated tube is longer and the radius is finer when the membrane is pulled on the polyunsaturated lipid side. We propose a model in which the polyunsaturated acyl chain adapts its conformation to the convex membrane leaflet undergoing deformation.


148

P53 Molecular mechanisms of the selection of a specific host lipid environment by the HIV-1 Gag protein Nario Tomishige

Tomishige N., Didier P., Pollet B., Sako Y., Mely Y. and Kobayashi T. LBP, Université de Strasbourg, Faculté de Pharmacie, 74 route du Rhin, CS 60024 , F-67401 Illkirch Cedex, France

Human immunodeficiency virus type 1 (HIV-1) obtains its lipid envelope by budding through the plasma membrane of infected host cells. Various studies have shown that the HIV-1 membrane differs from the producer cell plasma membrane, being enriched with specific lipids such as sphingomyelin (SM). This suggests that viruses bud from existing or induced membrane subdomains. HIV-1 Gag protein is the major structural component that functions to orchestrate HIV-1 assembly at the plasma membrane. Expression of Gag is sufficient to promote the formation of virus-like particles carrying a lipidic envelope derived from the host cell membrane. The binding of Gag to the plasma membrane is dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). In the plasma membrane of mammalian cells, lipids are asymmetrically distributed i.e. PI(4,5)P2 is in the inner leaflet whereas SM forms characteristic domains in the outer leaflet. One major question of the assembly of HIV-1 is how the binding of Gag to inner leaflet PI(4,5)P2 recruits outer leaflet SM-rich lipid domains.

We have developed original proteins/peptides that bind specific lipids, as well as small fluorescent molecules to monitor the physical properties of biomembranes. In combination with SM-specific probe, non-toxic lysenin (NT-Lys) and different optical microscopy techniques, we examined the interaction of Gag and SM on the plasma membrane in Gag-transfected HeLa cells. Super resolution PALM/STORM microscopy showed interbilayer co-localization of expressed Gag-mEos2 and Alexa Fluor647-NT-Lys. Furthermore, Gag-induced restriction of the mobility of SM was revealed by fluorescence recovery after photobleaching (FRAP) experiment of EGFP-NT-Lys. Possible interaction model of Gag and SM will be discussed.


149

P54 Endophilin-A3 controls a clathrin-independent endocytic route distinct from Endophilin-A2 François Tyckaert

Renard H-F., Tyckaert F., Lo Giudice C., Hirsch T., Shafaq-Zadah M., Valades Cruz CA., Wunder C., Johannes L., Van der Bruggen P., Alsteens D. and Morsomme P. Croix du Sud 4-5 bte L7.07.07 (Carnoy B.384), 1348 Louvain-La-Neuve, Belgium While several clathrin-independent endocytic processes have been described so far, they often remain poorly characterized mechanistically. Lately, key elements of these machineries have been identified in the BAR domain protein family, which are specialized in membrane curvature induction and recognition. In particular, proteins from the Endophilin-A (EndoA) subfamily were shown to control the clathrin-independent uptake of various cargoes, such as β-adrenergic receptors, IL-2 receptor or bacterial toxins. Owing to their high sequence similarity, the three endophilin isoforms A1, A2 and A3 are mostly considered as redundant. In our lab, we showed that they actually do not colocalize and control the uptake of different subsets of cargoes in distinct endocytic carriers. Using proteomics and cell biology approaches, we identified ALCAM/CD166 as a new clathrin-independent cargo. We could demonstrate that EndoA3 isoform – but not A2 – specifically and functionally associates with ALCAM/CD166 early endocytic carriers. We also provided evidences that the uptake of ALCAM/CD166 and the recruitment of EndoA3 to endocytic sites at plasma membrane are driven by extracellular Galectin-8. Taken together, our data demonstrate the existence of a previously unnoticed clathrin-independent endocytic route controlled by Galectin-8 and EndoA3, essential for the turnover of cell surface proteins such as CD166. As CD166 is present at the surface of various cell types, including cancer cells, our findings might contribute to better understand its role in cell adhesion and migration.


150

P55 Dynamics of VAPA-mediated membrane contact sites during cell division Jean-Marc Verbavatz

Sampietro M., Walch L., Pellier E., Siegfried H., Heuzé M., Jackson C. and Verbavatz JM. Institut Jacques Monod, 15, rue Hélène Brion, 75013 Paris, France

The hallmark of eukaryotic cells is their internal membrane organization. The membrane of each organelle has a specific lipid composition, an important determinant of organelle identity and function. Failure to maintain the lipid composition of membranes can lead to disease. The organization of cellular membrane systems, and their communication via vesicular trafficking, have been well-studied. Recently, a new form of transport has been uncovered: the exchange of lipids and ions at regions of close contact between membrane organelles. Membrane contact sites (MCS) are regions of close (< 30 nm) apposition without fusion, between the ER and other membrane organelles (plasma membrane, mitochondria, Golgi, endosomes...). The discovery of proteins functioning specifically at contact sites has revealed that MCS result from the assembly of molecular complexes, which tether the membranes of distinct compartments. One of the first MCS proteins identified was VAMP-associated protein (VAP). VAP is an ER membrane protein, which interacts through an FFAT motif, with several lipid transfer proteins (LTPs), such as OSBP and OSBP-related LTPs (ORPs). LTPs contribute to maintaining the lipid identity of organelles, by transferring lipids without membrane fusion at MCS, often against their concentration gradient, using the phosphatidylinositide (PI) gradient between membranes as the driving force. LTPs rely on VAP in the ER and a second targeting domain for their localization at MCS. It is unknown whether ER-MCS undergo remodeling and/or play any role in cellular processes such as cell division in mammalian cells, which involves a dramatic remodeling of cell membranes and membrane compartments. Plasma-membrane PIs, which are both binding partners and transported substrates of several LTPs, play multiple roles during cell division, such as cell rounding, spindle orientation and cytokinesis. Vertebrates have 2 VAP genes; VAPA and VAPB. The VAPA protein itself has 2 isoforms. We have evidence that VAPA-mediated MCS are regulated during cell division: the protein expression of one VAPA isoform is altered, and its interaction with ORP3, one of the OSBP-related protein, is dramatically increased. In order to investigate the molecular basis of VAPA-mediated MCS functions and their regulation during cell division, we have generated cells expressing fluorescently labeled VAPA isoforms and mutants. These cells will be used to study the dynamics and function of VAPA-mediated MCS in mammalian cells during cell division.


151

P56 High content analysis of α-synuclein pools in primary cultures of wild-type mouse cortical neurons challenged with recombinant α-synuclein assemblies Federica Zinghirino

De Giorgi F.1, 2, 3

, Zinghirino F.6, Laferrière F.

1,2, Faggiani E.

1,2, Benjmin Dehay B.

1, 2, Dovero S.

1, 2, Bouter A.

4,5,

Bezard E.1, 2

and Ichas F.1, 2, 3

1 Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, 33076 Bordeaux, France 2 CNRS, Institut des Maladies Neurodégénératives, UMR 5293, 33076 Bordeaux, France 3 INSERM, LNEC, U1084, 86073 Poitiers, France 4 CNRS, UMR5248, CBMN, Pessac, France 5 Université de Bordeaux, UMR5248, CBMN, Pessac, France; 6 Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy.

We developed a High-Content Screening (HCS) assay for detecting and profiling

candidates (i.e. genes or drugs) capable of interfering with both the onset and the pro ression of α-synucleinopathy. Our assay system consisted in wild type mouse cortical neurons challenged with exogenous α-synuclein assemblies in a standardized 96-well culture format. We exposed neurons to low concentrations (10 nM) of recombinant α-synuclein preformed fibrils (PFFs) and observed the early formation of scattered hybrid α-synuclein speckles at the level of the target neurons. These consisted in the apposition of exogenous human α-synuclein clumps with endogenous synuclein counterparts on the processes of most neurons. Later on, extensive phosphorylation of endo enous α-synuclein took place in a fraction of the neurons, showin this time a “whole cell” pattern instead of a dotted one. Hybrid speckles and extensive α-synuclein phosphorylation seem to proceed in parallel rather than in a sequential & causal manner. Bi exo enous α-synuclein assemblies (>100 nm) are not taken up by neurons but steadily dock to their plasma membrane and cause a local redistribution of presynaptic proteins and components. Smaller assemblies are readily taken up and trigger the extensive phosphorylation and aggregation of endogenous α-synuclein in certain neurons. The possible crosstalk between these two distinct phenomena is being explored.


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153

List of participants

Abbandonato Gerardo University of Milan

Milano, Italy [email protected]

Abella Guerra Marc

Max-Planck Institute for Terrestrial Microbiology

Marburg,Germany [email protected]

Advedissian Tamara Institut Pasteur Paris, France [email protected]

Albrecht Camille

Université de Reims Champagne-Ardenne

Reims, France [email protected]

Alves Isabel CBMN Pessac, France [email protected]

Antonny Bruno IPMC Valbonne, France [email protected]

Ayvazyan Naira Orbeli Institute of Physiology

Yerevan, Armenia [email protected]

Azouz Mehdi CBMN Pessac, France [email protected]

Baccouch Rim CBMN Pessac, France [email protected]

Basserau Patricia Institut Curie Paris, France [email protected].

Baumeister Wolfgang Max Planck Institute of Biochemistry

Martinsried, Germany

[email protected]

Bechinger Burkhard Institut de Chimie

Strasbourg, France [email protected]

Bechtella Leïla UPMC Paris, France [email protected]

Berti Debora University of Florence, Italy

Florence, Italy [email protected]

Bigay Joëlle IPMC Valbonne,France [email protected]

Blanchoin Laurent BIG Grenoble, France [email protected]

Boncompain Gaelle Institut Curie Paris, France [email protected]

Boucheham Abdeldjalil GMGM Strasbourg, France [email protected]

Bouffartigues Emeline LMSM Evreux, France [email protected]

Buchet René ICBMS Villeurbanne, France

[email protected]

Campillo Clément LAMBE Paris, France [email protected]

Chasserot-Golaz Sylvette INCI Strasbourg, France [email protected]

Cherfils Jacqueline ENS Paris-Saclay

Cachan, France [email protected]

Chitsaz Anita UNSW Sydney, Australia [email protected]

Čopič Alenka IJM Paris, France [email protected]

Coudrier Évelyne Institut Curie Paris, France [email protected]

Crowet Jean-Marc

Université de Reims Champagne-Ardenne


D'Ambrosio Juan Martin IJM Paris, France [email protected]


154

Danglot Lydia IPNP Paris, France [email protected]

De Leo Maria Giovanna UNIL Lausanne, Switzerland

[email protected]

De Matteis Antonella University of Naples, TIGEM

Naples, Italy [email protected]

De Pinto Vito University of Catania

Catania, Italy [email protected]

Deffieu Maïka IRIM Montpellier, France [email protected]

Delevoye Cédric Institut Curie Paris, France [email protected]

D'Errico Gerardino University of Naples

Napoli, Italy [email protected]

Descamps Estelle The Francis Crick Institute

London, France [email protected]

Desnos Claire CNRS UMR 8003

Paris, France [email protected]

Destainville Nicolas IRSAMC Toulouse, France [email protected]

Dhayer Nathalie ENS Bagneux, France [email protected]

Di Mattia Thomas IGBMC Strasbourg, France [email protected]

Dos Santos Morais

Raphael IGDR Rennes, France [email protected]

Drin Guillaume IPMC Valbonne, France [email protected]

Dufourc Erick CBMN Pessac-Bordeaux, France

[email protected]

Egea Jimenez Antonio Luis CRCM Marseille, France [email protected]

El Alaoui Fatima IRIM Montpellier, France [email protected]

El Far Oussama INSERM UMR_S 1072

Marseille, France [email protected]

Elias Marianne LAAS-CNRS Toulouse, France [email protected]

Favard Cyril IRIM Montpellier, France [email protected]

Fragneto Giovanna Institut Laue-Langevin

Grenoble, France [email protected]

Gagnoux Laurent IBV Nice, France [email protected]

Gallo Alessandra IPNP Paris, France [email protected]

Gasman Stéphane INCI-CNRS Strasbourg, France gasman

Gatti Evelina CIM -CNRS Marseille, France [email protected]

Gaudin Raphaël IRIM Montpellier, France [email protected]

Gauthier-Rouviere

Cécile CRBM Montpellier, France [email protected]

Gehan Pauline UPMC Paris, France [email protected]

Geli Maria-Isabel CSIC Barcelona, Spain

Ghossoub Rania CRCM Marseille, France [email protected]

Gironi Beatrice University of Perugia

Perugia, Italy [email protected]

Gruhle Kai MLU Halle (saale), Germany

[email protected]

Guéroult Marc

Université de Reims Champagne Ardenne



155

Henderson Michael Institut Pasteur Paris, France [email protected]

Herman Melissa Charité Berlin, Germany [email protected]

Hervas Javier The Francis Crick Institute

London, UK [email protected]

Inamdar Kaushik IRIM Montpellier, France [email protected]

Iraci Nunzio University of Catania


Itri Rosangela University of São Paulo

São Paulo, Brazil [email protected]

Jani Riddhi Institut Curie Paris, France [email protected]

Johannes Ludger Institut Curie Paris, France [email protected]

Joseph Pierre LAAS-CNRS Toulouse, France [email protected]

Kardash Maria Institut de Chimie

Strasbourg, France [email protected]

Karidia Konate CRBM Montpellier, France [email protected]

Köhler Sebastian Institut Laue-Langevin


Kovtun Oleksiy MRC-LMB Cambridge, UK [email protected]

Kukulski Wanda MRC -LMB Cambridge, UK [email protected]

Lafont Frank Institut Pasteur Lille, France [email protected]

Lamaze Christophe Institut Curie Paris, France [email protected]

Langel Ülo Stockholm University

Stockholm, Sweden [email protected]

Laporte Jocelyn IGBMC Illkirch, France [email protected]

Lavieu Grégory Institut Curie Paris, France [email protected]

Lebreton Stéphanie Institut Pasteur Paris, France [email protected]

Leone Dario CIML Marseille, France [email protected]

Lévêque Christian UNIS Marseille, France [email protected]

Li Changting Institut Curie Paris, France [email protected]

Lipp Nicolas-Frédéric IPMC Valbonne, France [email protected]

Lizarrondo Javier EMBL Hamburg, France [email protected]

Ljubojevic Nina Institut Pasteur Paris, France [email protected]

Lointier Morane Institut de Chimie

Strasbourg,France [email protected]

Lúcio Marlene CF-UM-UP Porto,Portugal [email protected]

Maestro Armando Institut Laue-Langevin

Grenoble,France [email protected]

Mangenot Stéphanie Institut Curie Paris,France [email protected]

Manno Mauro CNR Palermo,Italy [email protected]

Martel Anne Institut Laue Langevin

Grenoble,France [email protected]

Martinet Arthur IGBMC Strasbourg,France [email protected]

Mayer Andreas UNIL Lausanne, Switzerland

[email protected]

https://www.google.com/search?client=firefox-b-d&q=University+of+Catania&ludocid=17396269503809958265&sa=X&ved=2ahUKEwjG4e_3jf_gAhVEzoUKHWQXCfIQ8G0oADAUegQIBRAC

https://www.google.com/search?client=firefox-b-d&q=University+of+Catania&ludocid=17396269503809958265&sa=X&ved=2ahUKEwjG4e_3jf_gAhVEzoUKHWQXCfIQ8G0oADAUegQIBRAC


156

Millet Marion INCI Strasbourg, France [email protected]

Miserey-Lenkeï Stéphanie Institut Curie Paris, France [email protected]

Monks Colin Intelligent-Imaging

Denver, USA [email protected]

Montero Maité CURIB Mont-Saint-Aignan, France

[email protected]

Monticelli Luca MMSB Lyon, France [email protected]

Morsomme Pierre LIBST Louvain-la-Neuve, Belgium

[email protected]

Mukhina Tetiana Institut Laue Langevin


Müller Sindy MLU Halle, Germany [email protected]

Muriaux Delphine IRIM Montpellier, France [email protected]

Mus-veteau Isabelle IPMC Valbonne, France [email protected]

Niedergang Florence Institut Cochin Paris,France [email protected]

Oliva Rosario University of Naples "Federico II"

Acerra, Italy [email protected]

Orlando Marta Charité Berlin, Germany [email protected]

Ory Stéphane INCI Strasbourg,France [email protected]

Pabst Georg University of Graz

Graz, Austria georg.pabst(at)uni-graz.at

Perissinotto Fabio University of Trieste

Trieste, Italy [email protected]

Perrais David IIN Bordeaux, France [email protected]

Piantanida Luca INSERM TPR2 Marseille, France [email protected]

Picas Laura IRIM Montpellier, France [email protected]

Piot Numa EPFL Lausanne, Switzerland

[email protected]

Podkalicka Joanna Institut Curie Paris, France [email protected]

Pöhnl Matthias Friedrich–Alexander University

Erlangen, Germany [email protected]

Pokorny Laura UCL London, UK [email protected]

Porcar Lionel Institut Laue Langevin


Ramirez-Franco Jose Jorge UNIS Marseille, France [email protected]

Redondo-Morata Lorena Institut Pasteur Lille, France [email protected]

Renard Henri-François UCL Louvain-la-Neuve, Belgium

[email protected]

Ries Jonas EMBL Heidelberg, Germany

[email protected]

Risselada Herrer Jelger

Max-Planck-Institute for Biophysical Chemistry

Göttingen, Germany [email protected]

Rols Marie-Pierre IPBS Toulouse, France [email protected]

Rondelli Valeria University of Milan

Milan, Italy [email protected]



157

Roudaut Alban Covalab Villeurbanne, France

[email protected]

Royes mir Jorge ENS Paris, France [email protected]

Saint-Pol Julien BBB Laboratory Lens, France [email protected]

Salavessa Laura Institut Pasteur Paris, France [email protected]

Sauvonnet Nathalie Institut Pasteur Paris, France [email protected]

Schauer Kristine Institut Curie Paris, France [email protected]

Seksek Olivier IMNC Orsay, France [email protected]

Sens Pierre Institut Curie Paris, France [email protected]

Siegfried Hugo IJM Paris, France [email protected]

Sims Kacee Avanti Lipids Alabaster, USA [email protected]

Sirés Campos Julia Institut Curie Paris, France [email protected]

Spinozzi Francesco Polytechnic University of Marche

Ancona, Italy [email protected]

Sposini Silvia IINS Bordeaux, France [email protected]

Stella Lorenzo University of Rome “Tor Vergata”

Roma, Italy [email protected]

Tamm Lukas University of Virginia

Charlottesville, USA [email protected]

Tareste David IPNP Paris, France [email protected]

Tiberti Marion IPMC Valbonne, France [email protected]

Tomishige Nario LBP Illkirch-Graffenstaden, France

[email protected]

Tribet Christophe ENS Paris,France [email protected]

Tyckaert François UCL Louvain-la-Neuve, Belgium

[email protected]

Verbavatz Jean-Marc IJM Paris, France [email protected]

Zhang Wenxin The Francis Crick Institute

London, UK [email protected]

Zimmermann Pascale CRCM Marseille, France [email protected]

Zinghirino Federica University of Catania


Zobiack Nicole Intelligent-Imaging

Goettingen, Germany

[email protected]


158

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