Taras Sych

Thomas Schubert

Romain Vauchelles

Josef Madl
email: josef.madl@universitaets-herzzentrum.de

Ramin Omidvar

Roland Thuenauer
email: roland.thuenauer@cssb-hamburg.de

Ludovic Richert

Yves Mély

Winfried Römer
email: winfried.roemer@bioss.uni-freiburg.de

Robert Murphy

Maokai Xu

Lukas J Patalag

Annika Ries

Katharina Kettelhoit

Yves Mely
email: yves.mely@unistra.fr

Claudia Steinem

Daniel B Werz

GUV-AP: multifunctional FIJI-based tool for quantitative image analysis of Giant Unilamellar Vesicles

Keywords: Shiga toxin, LecA, primary cilium, plasma membrane, membrane order, lipidmediated endocytosis, Pseudomonas aeruginosa, giant unilamellar vesicles

P. aeruginosa lectin PAO1L StxB B subunit of Shoga toxin from S. dysenteriae

GSL

Summary

Plasma membrane glycosphingolipids (GSLs) are important membrane components involved in cell adhesion, signaling and endocytosis. They consist of a hydrophobic ceramide backbone embedded in the lipid bilayer and a carbohydrate head group exposed to the extracellular environment. GSLs can be highjacked by viruses, bacteria and bacterial products (such as toxins) which recognize their oligosaccharides using special carbohydrate binding proteins (called "lectins"). Lectin-GSL interactions are exploited by pathogens for binding to the membrane surface which can further result in pathogen uptake by the host cell. For instance, the entry of the pathogenic bacterium Pseudomonas aeruginosa into a host cell strongly depends on the specific interactions of its surface lectin LecA with the plasma membrane GSL globotriaosylceramide (Gb3). It has recently been shown that LecA-Gb3 interactions can initiate the formation of a socalled "lipid zipper" which leads to the bacterial internalization.

In this work, we investigated the interaction of LecA (coupled to bacteria or purified) with Gb3. Moreover, we compared LecA to another well-known lectin which binds Gb3, the Bsubunit of Shiga toxin from Shigella dysenteriae (StxB). We have strong indications that, despite sharing the same receptor, the two lectins behave differently as they induce different signaling events and follow different intracellular trafficking pathways. We hypothesized that these differences can originate already at the plasma membrane and can be explained by the selective binding of LecA and StxB to Gb3 incorporated into membrane nanodomains of different composition and organization.

We reconstituted the binding of lectins to Gb3 in model membranes (giant unilamellar vesicles -GUVs) of different membrane composition and order. We developed a method for quantitative description of lectin binding to GUVs based on the analysis of fluorescence microscopy images. Using this method, we confirmed that StxB prefers more ordered lipid bilayers, whereas LecA is less selective and binds efficiently to membranes of low order as well. Moreover, we studied the lectin binding to synthetic Gb3 species with 24:0 and 24:1 fatty acyl chains as well as to the synthetic construct FSL-Gb3. Using these species, we elucidated the role of the Gb3 molecular structure in the binding of lectins. Remarkably, using the information obtained from our binding studies, we succeeded to recreate the segregation of LecA and StxB in GUVs.

In the second part of the project, we explored the impact of LecA and StxB on membrane organization. Here, we were particularly interested in the rearrangement of ordered nanodomains in the plasma membrane by both lectins. The visualization of such nanodomains in the native plasma membrane is an extremely challenging task, which is why we mimicked them with liquid-ordered (Lo) domains in phase separated supported lipid bilayers (SLBs). We functionalized such lipid bilayers with Gb3 receptor molecules and found that StxB shrinks existing Lo domains and induces the formation of novel Lo domains. This suggests that StxB clusters "stabilize" the existing Lo domains. Moreover, the efficient clustering of Gb3 receptors by StxB induces the formation of new Lo domains. LecA shows a completely opposite behavior. Applied to SLBs, LecA induces the dissolution of Lo domains, homogenizing the membrane. Finally, P. aeruginosa applied to SLBs was found to dissolve Lo domains as well. This dissolution induced by the bacterium was observed for the first time. In summary, we learned/understood/demonstrated/revealed that the LecA-Gb3 interaction is the key to the dissolution of Lo domains by P. aeruginosa.

Zusammenfassung

Als wichtige Komponenten der Plasmamembran sind Glykosphingolipide (GSLs) an Zelladhäsion, Signalprozessen und Endozytose beteiligt. Sie bestehen aus einem hydrophoben, in die Lipiddoppelschicht eingebetteten Ceramid-Rückgrat und einer Kohlenhydrat-Kopfgruppe, die zur extrazellulären Umgebung exponiert ist. GSLs können von Viren, Bakterien und bakteriellen Produkten (wie Toxinen) gekapert werden, die deren Oligosaccharide mit Hilfe spezieller zuckerbindender Proteine (genannt "Lektine") erkennen. Lektin-GSL-Interaktionen werden von Pathogenen zur Bindung an Membranoberflächen genutzt, was auch zur Aufnahme des Pathogens in die Zielzelle führen kann. Beispielsweise hängt der Eintritt des pathogenen Bakteriums Pseudomonas aeruginosa in eine Wirtszelle stark von der spezifischen Interaktion seines Oberflächenlektins LecA mit dem Plasmamembran-GSL Globotriaosylceramid (Gb3) ab. Es wurde kürzlich gezeigt, dass LecA-Gb3-Interaktionen die Bildung eines sogenannten "Lipid-Reißverschlusses" induzieren können, der wiederum die bakterielle Internalisierung ermöglicht.

In der vorliegenden Arbeit untersuchten wie die Interaktionen zwischen LecA (an Bakterien gekoppelt oder aufgereinigt) und Gb3. Darüber hinaus verglichen wir LecA mit einem weiteren bekannten Gb3-bindenden Lektin, der B-Untereinheit des Shiga-Toxins aus Shigella dysenteriae (StxB). Vorangegangene Untersuchungen deuteten darauf hin, dass sich die Lektine, obwohl sie den gleichen Rezeptor teilen, verschieden verhalten, da sich induzierte Signale und intrazellulärer Transport unterscheiden. Wir vermuteten, dass diese Differenzen bereits an der Plasmamembran entstehen und durch die selektive Bindung von LecA und StxB and Gb3 in verschiedenen Nanodomänen der Membran mit unterschiedlicher Zusammensetzung und Organisation erklärt werden können.

Wir rekonstituierten die

Résumé

Les glycosphingolipides membranaires plasmatiques (GSL) sont d'importants composants membranaires impliqués dans l'adhésion cellulaire, la signalisation et l'endocytose. Ils se composent d'un squelette en céramide hydrophobe noyé dans la bicouche lipidique et d'un groupe de têtes glucidiques exposées à l'environnement extracellulaire. Les GSL peuvent être détournées par des virus, des bactéries et des produits bactériens (comme les toxines) qui reconnaissent leurs oligosaccharides en utilisant des protéines spéciales de liaison aux glucides (appelées "lectines"). Les interactions Lectine-GSL sont exploitées par les pathogènes pour se lier à la surface de la membrane, ce qui peut entraîner l'absorption de pathogènes par la cellule hôte. Par exemple, l'entrée de la bactérie pathogène P. aeruginosa dans la cellule hôte dépend fortement des interactions spécifiques de sa lectine de surface LecA avec la membrane plasmatique GSL globotriaosylceramide (Gb3). Il a été récemment démontré que les interactions LecA-Gb3 peuvent initier la formation de ce que l'on appelle la "lipid zipper" qui conduit à l'internalisation bactérienne.

Dans ce travail, nous avons étudié l'interaction de LecA (couplé à des bactéries ou purifié) avec Gb3. De plus, nous avons utilisé pour la comparaison une autre lectine bien connue qui se lie au Gb3, la sous-unité B de la toxine Shiga de S. dysenteriae (StxB). Nous avons de fortes indications que malgré le partage du même récepteur, les deux lectines se comportent différemment car elles induisent des événements de signalisation différents et suivent des voies de trafic intracellulaire différentes. Nous avons émis l'hypothèse que ces différences peuvent déjà provenir de la membrane plasmatique et s'expliquer par la liaison sélective de LecA et StxB au Gb3 incorporés dans des nanodomaines membranaires de composition et d'organisation différentes.

Nous avons reconstitué la liaison des lectines au Gb3 dans des membranes modèles (vésicules unilamellaires géantes -GUVs) de composition et d'ordre différents. Nous avons développé une méthode de description quantitative de la liaison de la lectine aux GUVs basée sur l'analyse d'images microscopiques à fluorescence. En utilisant cette méthode, nous avons confirmé que le StxB préfère les bicouches lipidiques plus ordonnées, alors que le LecA est moins sélectif et se lie efficacement aux bicouches lipidiques d'ordre inférieur également. De plus, nous avons étudié la liaison de la lectine aux espèces synthétiques Gb3 à chaînes acyles grasses 24:0 et 24:1 ainsi qu'à la construction synthétique FSL-Gb3. En utilisant ces espèces, nous avons élucidé le rôle de la structure moléculaire du Gb3 dans la liaison des lectines. Notamment, en utilisant les informations obtenues à partir des études des interactions de LecA et StxB avec le Gb3, nous avons réussi à recréer la ségrégation de LecA et StxB dans les GUVs.

Dans la deuxième partie du projet, nous avons exploré l'impact du LecA et du StxB sur l'organisation de la membrane. Ici, nous nous sommes particulièrement intéressés au réarrangement des nanodomaines ordonnés dans la membrane plasmique par les deux lectines. La visualisation de ces nanodomaines dans la membrane plasmique est une tâche extrêmement difficile, c'est pourquoi nous les avons imités avec des domaines à ordre liquide (Lo) dans des bicouches lipidiques supportées et séparées en phase (SLBs). Nous avons fonctionnalisé ces bicouches lipidiques avec des molécules réceptrices de Gb3. Nous avons constaté que le StxB rétrécit les domaines Lo existants et induit la formation de nouveaux domaines Lo. Cela suggère que les clusters StxB "stabilisent" les domaines Lo existants. De plus, le regroupement efficace des récepteurs Gb3 par StxB induit la formation de nouveaux domaines Lo. LecA montre un comportement complètement opposé. Appliqué aux SLBs, LecA induit la dissolution des domaines Lo, homogénéisant la membrane. Enfin, P. aeruginosa appliqué à SLB dissout également les domaines Lo. Nous avons constaté que l'interaction LecA-Gb3 est le processus clé pour la dissolution des domaines Lo induits par P. aeruginosa. Cette dissolution des domaines Lo dans les SLBs induite par la bactérie a été observée pour la première fois.

I Introduction 1. Structure et fonctions de la membrane plasmique

La membrane plasmique cellulaire (figure 1) est un objet biologique fascinant. Il définit la barrière entre le cytosol et l'environnement extracellulaire, détermine la forme de la cellule et maintient son intégrité. La membrane plasmatique assure l'absorption et la libération de matériaux par la cellule, processus cruciaux pour le métabolisme cellulaire. La transduction du signal et la communication cellule-cellule sont effectuées par divers composants membranaires plasmatiques spécifiques. De plus, la membrane plasmique est un exemple exceptionnel d'auto-assemblage et d'auto-organisation. Il représente une barrière semi-perméable, basée sur une bicouche lipidique avec une grande variété de composants et des propriétés uniques. Micrographie EM de la membrane plasmique de Chara corallina [START_REF] Coster | The Physics of Cell Membranes[END_REF]. Image en microscopie confocale de la coupe transversale de cellules MDCK-II: membrane plasmique (myristoyl-LYN-GFP -verte); noyau (DAPI -bleu). Barre d'échelle -10µm. Image fournie par le Dr. Roland Thuenauer.

Auto-assemblage des lipides dans l'eau, bicouche lipidique

Les lipides sont parmi les éléments les plus importants de la vie. Ils contribuent au stockage de l'énergie, participent aux événements de signalisation et composent l'épine dorsale de la membrane plasmique. Les lipides membranaires sont des molécules amphiphiles, composées d'un groupe de tête hydrophile et d'une ou plusieurs chaînes acyle grasses hydrophobes. (Israelachvili, 2011;van Meer et al., 2008). Les stérols sont un sous-groupe de stéroïdes dont la structure centrale est constituée de quatre cycles fusionnés avec un groupe hydroxyle en position 3 du cycle A. Adapté de [START_REF] Fahy | A comprehensive classification system for lipids[END_REF][START_REF] Jäpelt | Vitamin D in plants: a review of occurrence, analysis, and biosynthesis[END_REF].

Les glycérophospholipides sont la classe de lipides membranaires la plus abondante (van Meer et al., 2008). Ils sont composés d'un acide glycérophosphorique, qui sert de squelette à deux chaînes acyle grasses (partie hydrophobe) et d'un groupe de tête hydrophile (figure 2). Les sphingolipides membranaires, une autre classe importante de lipides membranaires, sont organisés sur la base d'un squelette en céramide, auquel différents groupes de tête peuvent être attachés (figure 2). Les stérols membranaires, en particulier le cholestérol (figure 2), forment un groupe de petits lipides présents dans les membranes des cellules animales (Maxfield and Tabas, 2005).

Exposés à l'eau, les lipides (ou les molécules amphiphiles similaires en général) y compactent des fractions hydrophobes et mettent leurs groupes de tête hydrophiles en contact avec de l'eau en même temps (Israelachvili et al., 1977;Sych et al., 2018a). Cela conduit à l'assemblage du «noyau hydrophobe», isolé du milieu aqueux. Leur assemblage dépend de la géométrie lipidique, à savoir du volume de la partie non polaire V, de la longueur de la chaîne acyle grasse 1 et de la surface du groupe de tête polaire A. La combinaison de ces trois paramètres définit la géométrie de l'ensemble lipidique dans l'eau (Gennis, 2013;Israelachvili, 2011). Par exemple, le volume Vm d'une micelle sphérique composée de N molécules lipidiques peut être exprimé par:

𝑉 𝑚 = 4 3 𝜋𝑅 3 = 𝑁𝑉
où R est le rayon de la micelle. La surface Am d'une telle micelle est:

𝐴 𝑚 = 4𝜋𝑅 2 = 𝑁𝐴
Par conséquent, le rayon d'une telle micelle sphérique peut être exprimé par:

𝑅 = 3𝑉 𝑚 𝐴 𝑚
Pour une micelle sphérique, R≤l, donc:

𝑉 𝑚 𝐴 𝑚 𝑙 ≤ 1 3
Le rapport Vm / Aml est appelé paramètre de tassement critique et, numériquement, le type d'assemblage lipidique basé sur les paramètres géométriques des molécules lipidiques individuelles. La longueur 1 et le volume v de la partie lipidique hydrophobe peuvent être déterminés par la structure chimique de la chaîne acyle grasse, tandis que la surface spécifique du groupe de tête, a, dépend en outre du contenu ionique de la solution aqueuse (pH, pK) et la charge du groupe de tête (Israelachvili, 2011). Sur la base du paramètre d'emballage critique, les lipides membranaires peuvent être classés en trois groupes principaux (figure 3):

1) Les lipides en forme de cône inversés (v / la0 <1/2) s'assemblent en micelles avec une surface de courbure positive. Ce groupe comprend principalement des lipides à une seule chaîne acyle grasse, tels que les lysophospholipides. Pour être assemblés dans des micelles sphériques, le paramètre de compression critique des lipides doit être inférieur à 1/3, tandis que les lipides avec 1/3 <v / la0 <1/2 s'assemblent sous forme de micelles cylindriques [START_REF] Fan | Staying Out" Rather than "Cracking In[END_REF].

2) Les lipides en forme de cônes (v / la0> 1) s'auto-organisent en micelles hexagonales (phase HII) à courbure négative (en milieu acide). Ce groupe contient des lipides polyinsaturés qui préfèrent être incorporés dans des membranes à courbure négative (Jouhet, 2013). 3) Les lipides en forme de cylindre (1/2 <v / la0 ≤ 1) s'assemblent dans une bicouche lipidique (également appelée «phase lamellaire»). Ce groupe d'espèces lipidiques, représenté par des lipides à deux chaînes d'acyle gras de longueur et de degré de saturation différents, revêt une importance particulière du fait qu'il forme principalement la matrice lipidique de la membrane plasmique. Ils peuvent être assemblés dans les bicouches lipidiques souples de liposomes (1/2 <v / la0 <1) ainsi que dans les bicouches lipidiques planaires (v / la0 1) (Angelova and Dimitrov;[START_REF] Méléard | Giant Unilamellar Vesicle Electroformation[END_REF].

Figure 3 | Auto-assemblage des lipides dans l'eau. a) Les lipides en forme de cône inversé s'assemblent dans des micelles en contact avec de l'eau; b) Les lipides en forme de cônes s'assemblent en micelles inversées (phase HII) au contact de l'eau; c) Les lipides de forme cylindrique s'assemblent dans des bicouches lipidiques dans de l'eau. Révisé de (Sych et al., 2018a).

Les lipides membranaires peuvent exister dans divers états de phase lamellaires qui déterminent l'ordre membranaire et la diffusion translationnelle des lipides (Lee, 1977a(Lee, , 1977b)). Les bicouches lipidiques à un composant peuvent subir une transition de phase entre les phases solide et cristalline liquide (Aufderhorst-Roberts et al., 2017;Busto et al., 2014;van Meer et al., 2008). Une phase bicouche lipidique solide se caractérise par une faible mobilité et un compactage élevé de ses composants, tandis que la phase cristalline liquide, au contraire, présente un ordre faible et une mobilité de translation élevée. Une telle phase lipidique faiblement ordonnée et à forte mobilité peut également être appelée phase désordonnée des liquides (Ld). La température de transition de phase (également appelée température de fusion de chaîne) pour les bicouches lipidiques composées d'une seule espèce lipidique dépend de la structure moléculaire des molécules lipidiques, le rôle prédominant étant la longueur et le degré de saturation des chaînes acyle grasses, mais également de la structure du groupe de tête. Pour les phospholipides, cette température peut varier de 70 ° C pour 20: 4 PC (1,2-diarachidonoyl-sn-glycéro-3-phosphatidylcholine) avec des chaînes acyle polyinsaturées à 100 ° C pour DSPE (1,2-distéaroyl-sn-glycéro-3phosphatidyléthanolamine) avec de longues chaînes acyle saturées [START_REF] Silvius | Thermotropic Phase Transitions of Pure Lipids in Model Membranes and Their Modifications by Membrane Proteins[END_REF]. La structure du groupe de tête (figure 4) a également une influence sur la température de transition de phase principale. Par exemple, les lipides PS et PE ont des températures de transition plus élevées que les lipides PC et PG, même s'ils sont tous composés de chaînes acyle grasses identiques.

Figure 4 | Glycérophospholipides. Différents types de groupes de tête et de structures en chaîne acyle grasse des glycérophospholipides. Révisé de [START_REF] Aktas | Membrane lipids in Agrobacterium tumefaciens: biosynthetic pathways and importance for pathogenesis[END_REF].

Les sphingolipides à membrane, en particulier la sphingomyéline (SM), ont tendance à former des bicouches lipidiques en phase solide avec une température de transition de phase de 38 ° C (figure 5). L'introduction de stérols (cholestérol, par exemple) dans les bicouches lipidiques peut avoir un effet intéressant [START_REF] Gracià | Effect of cholesterol on the rigidity of saturated and unsaturated membranes: fluctuation and electrodeformation analysis of giant vesicles[END_REF]van Meer et al., 2008;Veatch and Keller, 2005). Dans les bicouches lipidiques riches en SM, le cholestérol agit comme un espaceur entre les molécules de SM étroitement emballées et améliore par conséquent la mobilité des lipides dans ces membranes. Comme l'ordre de la membrane reste élevé, ces membranes adoptent un nouvel état de phase lipidiquedit à commande liquide (Lo). Il est à noter qu'en raison de la liaison hydrogène unique entre le SM et le cholestérol, ces deux lipides forment des domaines Lo particulièrement stables. [START_REF] Quinn | The liquid-ordered phase in membranes[END_REF]. Il est intéressant de noter que l'introduction de cholestérol (cholestérol) dans les bicouches lipidiques de Ld constituées de phospholipides non saturés (par exemple le 1,2-dioléoyl-sn-glycéro-3-phosphocholine (DOPC)) améliore l'ordre de ces membranes et, par conséquent, le DOPC / chol. Les bicouches lipidiques présentent un ordre membranaire supérieur à celui de Ld. Des domaines membranaires ségrégés spatialement de différentes phases peuvent coexister dans une bicouche lipidique à plusieurs composants [START_REF] Almeida | A Simple Thermodynamic Model of the Liquid-Ordered State and the Interactions between Phospholipids and Cholesterol[END_REF]Bacia et al., 2005). Ce phénomène s'appelle la séparation de phase. Habituellement, il se produit dans les bicouches lipidiques contenant des lipides avec différentes températures de transition de phase principale. Par exemple, la bicouche lipidique à deux composants composée de DOPC (Tm = -17 ° C) et de sphingomyéline (Tm = 38 ° C) présente une séparation de phase aux températures situées entre les transitions de phase principales des deux lipides. (Leonenko et al., 2004). Dans ce cas, il contient des domaines en phase solide enrichis en domaines SM et en phase liquide (Ld) riches en DOPC. Une addition supplémentaire de cholestérol à ces bicouches lipidiques peut convertir les domaines de la phase solide riches en SM en Lo (van Meer et al., 2008). De plus, à des rapports molaires spécifiques SM / DOPC / chol, les trois phases peuvent coexister dans la bicouche lipidique. (Aufderhorst-Roberts et al., 2017). La séparation de phases est réversible et disparaît en augmentant la température de la bicouche lipidique.

Ce phénomène de séparation de phases a fait l'objet de nombreuses études au cours des trois dernières décennies, à l'aide de diverses techniques, telles que la calorimétrie par titrage isotherme. (Mabrey and Sturtevant, 1976), microscopie à fluorescence [START_REF] Almeida | A Simple Thermodynamic Model of the Liquid-Ordered State and the Interactions between Phospholipids and Cholesterol[END_REF]Carravilla et al., 2015), microscopie à force atomique (Mingeot-Leclercq et al., 2008;Yilmaz and Kobayashi, 2015) et simulations de dynamique moléculaire (Koldsø et al., 2014;[START_REF] Wang | DPPC-cholesterol phase diagram using coarse-grained Molecular Dynamics simulations[END_REF]. Les interactions lipides-lipides contrôlent la formation et la dynamique de tels domaines membranaires, qui revêtent une importance capitale dans les membranes hautement complexes à plusieurs composants des cellules vivantes.

Organisation lipidique d'ordre supérieur dans la membrane plasmique

Le premier modèle descriptif de la membrane cellulaire a été présenté par Singer et Nicolson en 1972(Nicolson, 2014;Singer and Nicolson, 1972). Ce modèle indique que tous les composants de la membrane sont intégrés à la matrice bicouche lipidique séparant l'intérieur de la cellule de l'environnement extracellulaire. Selon ce modèle, tous les composants de la membrane (lipides et protéines) sont répartis de manière homogène dans la matrice bicouche lipidique et peuvent diffuser librement dans le plan latéral de la membrane. Ce modèle a été appelé «modèle de mosaïque fluide», car il décrit la membrane plasmique comme un liquide bidimensionnel. Des études ultérieures de la membrane biologique utilisant divers détergents (par exemple Triton X 100) ont montré la séparation du matériau de la membrane en deux fractions principalement: une membrane résistante aux détergents (DRM) et une membrane soluble pour détergents (DSM). Il est intéressant de noter que la fraction DRM s'est révélée être enrichie en sphingolipides à longues chaînes d'acyle gras saturés et en cholestérol, alors que la fraction DSM était enrichie en phospholipides membranaires. Ces résultats suggèrent d'abord l'association du SM et du cholestérol dans la membrane plasmique (Fra et al., 1994;Mahfoud et al., 2009;Seddon et al., 2004). Des études ultérieures sur la composition des feuillets membranaires plasmatiques ont révélé la préférence des lipides SM et PC pour le feuillet membranaire plasmatique externe (extracellulaire), alors que la majorité des lipides chargés négativement (tels que PE et PS), ainsi que PI et PG étaient principalement prédominants. situé dans la notice interne (Devaux, 1991;Devaux and Morris, 2004). Des lipides glycosylés ont été trouvés dans la foliole externe de la membrane plasmique avec des fragments de sucre exposés à l'environnement extracellulaire. (Simons and Van Meer, 1988). Une telle localisation des espèces lipidiques est activement maintenue par la cellule afin de prendre en charge diverses fonctions cellulaires. Par exemple, les espèces de PI, situées dans la feuille de membrane interne, peuvent être phosphorylées trois fois pour former PIP, PIP2 et PIP3, qui revêtent une importance cruciale pour la transduction du signal cellulaire (Yoshida et al.). Le cholestérol est également représenté dans les deux feuillets membranaires (Rothman and Lenard, 1977). L'hétérogénéité de la membrane plasmique dans le plan latéral a été constatée dans des cellules épithéliales polarisées (Simons and Van Meer, 1988). Les membranes apicales et basolatérales des cellules épithéliales sont séparées par des jonctions serrées. La membrane apicale est enrichie en sphingolipides et en cholestérol, tandis que la membrane basolatérale -en phospholipides [START_REF] Gerl | Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane[END_REF]. Ces deux membranes présentent également un ordre lipidique différent, la membrane apicale étant plus ordonnée. [START_REF] Ikenouchi | Lipid Polarity Is Maintained in Absence of Tight Junctions[END_REF]. Des domaines membranaires latéraux plus petits ont été découverts pour la première fois sous forme de fosses membranaires plasmatiques d'un diamètre de 60 à 80 nm, appelées cavéoles. [START_REF] Barnett-Norris | Lipids, lipid rafts and caveolae: Their importance for GPCR signaling and their centrality to the endocannabinoid system[END_REF][START_REF] Parton | Caveolae as plasma membrane sensors, protectors and organizers[END_REF][START_REF] Parton | The multiple faces of caveolae[END_REF]. Ils sont stables dans le temps et enrichis en sphingolipides et en cholestérol, ce qui suggère un ordre membranaire élevé. Les cavéoles condensent également un grand nombre de composants de la membrane plasmique. Ils sont impliqués dans l'endocytose, la transcytose et la transduction du signal [START_REF] Collins | Structure-Based Reassessment of the Caveolin Signaling Model: Do Caveolae Regulate Signaling through Caveolin-Protein Interactions?[END_REF][START_REF] Pelkmans | Endocytosis Via Caveolae[END_REF][START_REF] Tuma | Transcytosis: Crossing Cellular Barriers[END_REF]. De plus, ils sont considérés comme les réservoirs de la courbure de la membrane plasmique qui aident une cellule à survivre au stress mécanique [START_REF] Sinha | Cells Respond to Mechanical Stress by Rapid Disassembly of Caveolae[END_REF].

Sur la base de ces résultats, Simons et Ikonen ont amélioré le modèle de membrane plasmique en 1997 (figure 6). Ils ont présenté les «radeaux lipidiques», qui correspondent à des plates-formes dynamiques, hautement ordonnées, très bien organisées, enrichies en sphingomyéline, en cholestérol et en protéines membranaires et en glycolipides (Sezgin et al., 2017;Simons and Ikonen, 1997). Contrairement aux cavéoles, les radeaux lipidiques plans sont très dynamiques, ils s'assemblent et se réorganisent très rapidement [START_REF] Fan | Formation and regulation of lipid microdomains in cell membranes: Theory, modeling, and speculation[END_REF]. La formation de radeaux lipidiques est une caractéristique unique de la feuille externe de la membrane plasmique. [START_REF] Van Meer | The different hues of lipid rafts[END_REF]. La notice membranaire cytosolique ne contient pas de radeaux lipidiques; cependant, on sait que les amas lipidiques PS et PI dans la foliole interne de la membrane s'interdigitent avec les radeaux lipidiques (Raghupathy et al., 2015). Le cholestérol est uniformément distribué entre les monocouches lipidiques. Adapté de (Sych et al., 2018a).

Les radeaux lipidiques peuvent inclure ou exclure des protéines membranaires (Simons and Gerl, 2010;Simons and Toomre, 2000). Ils fournissent la configuration nécessaire au bon fonctionnement des protéines membranaires, notamment dans la signalisation cellulaire. On sait que les protéines à ancrage glycosylphosphatidylinositol (GPI) et d'autres protéines liées à la membrane par des groupes cholestérol ou palmitoyle sont associées aux radeaux lipidiques (Simons and Ikonen, 1997). Les radeaux lipidiques hébergent les hélices a des protéines G, ce qui permet leur implication dans la signalisation par GPCR [START_REF] Barnett-Norris | Lipids, lipid rafts and caveolae: Their importance for GPCR signaling and their centrality to the endocannabinoid system[END_REF]. En outre, les radeaux lipidiques sont impliqués dans la signalisation du récepteur de l'antigène des lymphocytes T [START_REF] Owen | Quantitative imaging of membrane lipid order in cells and organisms[END_REF], ainsi que dans la signalisation de la tyrosine kinase (Simons and Toomre, 2000). Les radeaux lipidiques participent à l'endocytose, au trafic cellulaire et à l'exocytose. Un exemple typique est l'entrée et la libération cellulaires du virus de l'immunodéficience humaine (VIH). L'entrée du VIH dépend du récepteur CD4 de la glycoprotéine cellulaire, connu pour s'associer aux radeaux lipidiques [START_REF] Carter | HIV entry in macrophages is dependent on intact lipid rafts[END_REF]. Au cours de la libération du virus, la protéine Gag myristoylée du VIH s'accroche à la feuille de membrane plasmatique cytosolique de la cellule hôte et induit le regroupement de radeaux lipidiques dans la feuille de membrane extérieure, ce qui entraîne la formation de particules virales et la formation de bourgeons (Ono and Freed, 2001). En conséquence, l'enveloppe virale est enrichie en sphingomyéline et en cholestérol, qui sont principalement des composants associés aux radeaux lipidiques [START_REF] Sengupta | A lipid-based partitioning mechanism for selective incorporation of proteins into membranes of HIV particles[END_REF].

Composants membranaires glycosylés et radeaux lipidiques, Glycosphingolipides

La surface externe de la membrane plasmique est fonctionnalisée avec une grande variété de molécules différentes qui permettent à la cellule d'interagir avec son environnement. Les lipides et les protéines membranaires peuvent subir des modifications post-traductionnelles, telles que la glycosylation [START_REF] Ambrosi | Lectins: tools for the molecular understanding of the glycocode[END_REF]. De plus, les parties lipidiques ou protéiques de telles molécules glycosylées servent d'ancres bicouches lipidiques pour les glucides, exposés à l'environnement extracellulaire. En particulier, les glycosphingolipides (GSL) présents dans la foliole externe de la membrane [START_REF] Schnaar | Glycosphingolipids. In Essentials of Glycobiology, A[END_REF], incorporer dans la bicouche lipidique avec la fraction hydrophile de céramide. Les GSL sont définis par les structures des glucides attachés au squelette des céramides. Sur cette base, trois groupes de GSL peuvent être mis en évidence (figure 7):

1) Les gangliosides [START_REF] Svennerholm | The gangliosides[END_REF] sont des glycosphingolipides avec des groupes de sucre contenant de l'acide N-acétylneuraminique (NANA, également appelé acide sialique). Ils peuvent également être subdivisés en fonction du nombre (un, deux, trois ou quatre) de fractions acide sialique associées au glucide GSL. Les représentants typiques sont GM1, GD1, GT1, GQ1; 2) Les cerebrosides [START_REF] Tan | The cerebrosides[END_REF]) sont des glycosphingolipides avec un seul sucre fixé au squelette des céramides (également appelés monoglycosylcéramides). Comme ce sucre peut être du glucose ou du galactose, les deux groupes de cérébrosides sont les glucocérébrosides et les galactocérébrosides;

3) Les globosides [START_REF] Jones | Globoside as a Membrane Receptor: A Consideration of Oligosaccharide Communication with the Hydrophobic Domain[END_REF] sont des glycosphingolipides à sucres multiples liés au squelette des céramides. Les représentants typiques sont Gb3 et Gb4.

Les glycanes de surface cellulaire remplissent une grande variété de fonctions [START_REF] Varki | Biological roles of glycans[END_REF]. Entre autres, les glycanes de surface cellulaire participent à la communication cellule-cellule, à l'adhésion et à la formation du glycocalyx cellulaire [START_REF] Shurer | Physical Principles of Membrane Shape Regulation by the Glycocalyx[END_REF]. Ils participent également à la modulation de l'organisation de la membrane plasmique et de la communication entre feuillets. Ici, en particulier, les GSL à long squelette en céramide permettent une interdigitation efficace (Manna et al., 2017). Les glycanes médient la transduction du signal et l'initiation de la cascade de signaux sur la membrane plasmique. De plus, les glycanes sont utilisés par les virus et les bactéries afin de permettre une adhésion et une internalisation efficaces dans la cellule hôte.

Imagerie de la membrane plasmique par microscopie à fluorescence

En biologie cellulaire, la microscopie à fluorescence est une méthode de choix car elle offre la capacité unique de suivre des composants cellulaires spécifiquement marqués et de suivre visuellement leur comportement au cours de processus cellulaires complexes. En particulier, depuis la découverte de la protéine fluorescente verte (GFP), la microscopie à fluorescence est devenue un outil essentiel pour déchiffrer la localisation et le devenir de la protéine [START_REF] Tsien | THE GREEN FLUORESCENT PROTEIN[END_REF]. Les protéines fluorescentes peuvent être utilisées comme marqueurs de protéines en les fusionnant avec la protéine cible au niveau génétique. En outre, la microscopie à fluorescence offre l'opportunité de l'imagerie de cellules vivantes et constitue un outil puissant pour élucider les mécanismes moléculaires au niveau de la membrane plasmique cellulaire. (Kwiatek et al., 2014). La membrane plasmique peut être marquée avec des fluorophores organiques à base de lipides ou lipophiles (Klymchenko and Kreder, 2014). Cependant, ces marqueurs peuvent induire une cytotoxicité par le biais du stress photo-oxydant. En variante, les PF fluorescents peuvent être conjugués à des protéines transmembranaires ou à des protéines ancrées GPI. Cette approche est moins susceptible d'induire une phototoxicité. De plus, il est possible de marquer des composants de la membrane plasmatique (par exemple des espèces lipidiques distinctes ou des protéines membranaires) avec des fluorophores et de suivre leur localisation et leur trafic (Klymchenko and Kreder, 2014;[START_REF] Zhang | Choosing the right fluorophore for single-molecule fluorescence studies in a lipid environment[END_REF]. La multitude de techniques de microscopie à fluorescence utilisées de nos jours est impressionnante. Pour la visualisation de la membrane plasmique, les approches classiques de la microscopie confocale ou à champ large sont souvent associées à des techniques quantitatives (imagerie de durée de vie en fluorescence, microscopie à corrélation de fluorescence) ou à des techniques de super résolution afin d'améliorer la résolution spatio-temporelle ou d'extraire des informations supplémentaires sur la membrane locale. environnement (Kwiatek et al., 2014;Sezgin et al., 2017). Ici, je passerai brièvement en revue deux techniques de microscopie à fluorescence largement utilisées: la microscopie confocale à balayage laser (LSCM) et la microscopie à fluorescence à réflexion interne totale (TIRFM).

Fluorescence

La fluorescence est un processus d'émission de lumière par une substance initialement excitée par un rayonnement électromagnétique [START_REF] Lakowicz | Principles of fluorescence spectroscopy[END_REF]. Pour être transféré à l'état excité, un fluorophore doit absorber un quantum de lumière -un photon. L'excitation lumineuse induit la transition d'un électron de l'état fondamental à une orbitale supérieure, amenant la molécule à l'état excité. Par la suite, cette molécule peut revenir à l'état fondamental avec l'émission d'un photon. Habituellement, les processus de lumière d'absorption et d'émission sont affichés à l'aide du diagramme de Jablonski (figure 8A). La transition de la molécule a lieu de l'état 0-singulet (S0 -état fondamental) au premier état singulet (S1 -état excité). L'électron revient dans l'état fondamental avec l'émission d'un photon ou de manière non radiative. De plus, un passage intersystème à l'état triplet est possible. 9) [START_REF] Pawley | Handbook of Biological Confocal Microscopy[END_REF]. Cela permet non seulement d'enregistrer l'image d'un plan de l'échantillon, mais également d'obtenir l'image 3D de l'échantillon en l'imaginant image par image. La taille de l'échantillon pouvant être imagée est définie par la distance de travail de l'objectif. Cette technique est également largement utilisée pour l'imagerie de cellules vivantes. Les principales limitations de LSCM proviennent de la limite de diffraction qui définit la résolution optique. Habituellement, les microscopes confocaux sont capables d'imager des objets avec une limite de résolution de 200 nm dans le plan latéral et de 400 à 500 nm dans la direction z. Un autre inconvénient de la microscopie confocale est la vitesse d'acquisition, qui est relativement lente en raison de la nécessité de numériser -1 à 2 images par seconde pour une image de 1024 × 1024 pixels. Cette fonctionnalité a été partiellement surmontée par le développement et la mise en oeuvre de scanners plus rapides (par exemple, le «mode résonant»).

Microscope confocal à balayage laser

Microscopie à fluorescence à réflexion interne totale

La focalisation du laser sur le plan focal arrière de l'objectif permet l'éclairage de l'échantillon avec le faisceau de lumière laser parallèle. Cela permet une excitation uniforme d'une zone relativement grande à la fois et augmente considérablement la vitesse d'acquisition pour une seule image. La fluorescence est détectée à l'aide d'une caméra (CCD, CMOS, etc.) capable de réaliser un instantané de l'ensemble de la zone d'imagerie. Ce type de microscopie est appelé "champ large" [START_REF] Axelrod | Total internal reflection fluorescent microscopy[END_REF]) (figure 10). L'avantage évident est la vitesse d'acquisition des images. Cependant, une excitation de champ large produit une quantité considérable de fluorescence de fond. Afin d'éliminer le fond de fluorescence, le faisceau d'excitation laser est incliné. Le meilleur rapport signal / bruit de fond est obtenu par microscopie à fluorescence par réflexion interne totale (TIRFM). Ici, le laser est incliné par rapport à l'angle TIR au niveau de l'interface lamelle / échantillon [START_REF] Axelrod | Total Internal Reflection Fluorescence Microscopy in Cell Biology[END_REF]. Le laser ne peut plus pénétrer dans l'échantillon, mais l'onde évanescente, induite à l'interface verre de protection / échantillon, permet toujours une excitation efficace d'une couche mince de l'échantillon. La principale limite de cette technique est qu'elle ne peut exciter qu'une couche de l'échantillon d'une épaisseur de 100 nm, adjacente au verre de protection. Un bon compromis entre l'élimination du fond et l'épaisseur de la couche d'excitation peut être obtenu en utilisant un éclairage appelé HILO (Tokunaga et al., 2008) -éclairage lamellaire fortement incliné de l'échantillon avec le faisceau laser. Le champ large en mode EPI / TIRFM / HILO est particulièrement efficace pour l'imagerie de cellules vivantes car il permet une acquisition à haute vitesse (des dizaines de millisecondes par image). Le TIRFM est particulièrement utile pour l'imagerie de la membrane plasmique à la surface inférieure des cellules vivantes.

Reconstitution de l'organisation de la membrane plasmique dans un système synthétique

L'étude de l'organisation membranaire dans les membranes cellulaires natives est une tâche exigeante (Klymchenko and Kreder, 2014;Kwiatek et al., 2014). Dans les cellules vivantes, les radeaux lipidiques sont très mobiles et de petite taille (Sezgin, 2017). De plus, afin de réaliser tous les différents processus cellulaires, ils assemblent et dissocient en permanence [START_REF] Fan | Formation and regulation of lipid microdomains in cell membranes: Theory, modeling, and speculation[END_REF]. Malgré les progrès récents des techniques de microscopie à super-résolution avec une augmentation impressionnante de la résolution spatiale et temporelle, la visualisation directe des nanodomaines membranaires reste difficile. De plus, l'abondance des composants de la membrane plasmatique limite l'analyse aux lipides et protéines membranaires uniques dans des processus cellulaires spécifiques.

Membranes modèles

L'organisation membranaire est non seulement maintenue activement par la cellule, mais également par les interactions lipides-lipides et lipides-protéines dans la membrane plasmique (Sprong et al., 2001). Ces interactions peuvent être isolées de la machinerie cellulaire active par reconstitution synthétique en une bicouche lipidique multicomposante composée d'espèces lipidiques et / ou protéiques distinctes (Bacia et al., 2005;[START_REF] Lin | Preparation of artificial plasma membrane mimicking vesicles with lipid asymmetry[END_REF]. Une telle approche «ascendante» permet un contrôle précis du contenu et de la structure de la membrane artificielle obtenue. Les membranes artificielles peuvent imiter les caractéristiques spécifiques de la membrane plasmique. Ils sont extrêmement utiles pour caractériser l'organisation de la membrane [START_REF] Dietrich | Lipid Rafts Reconstituted in Model Membranes[END_REF], reconstitution de l'adhésion cellulaire [START_REF] Villringer | Lectin-mediated protocell crosslinking to mimic cell-cell junctions and adhesion[END_REF] and endocytose (Römer et al., 2007). De nombreux types de membranes modèles ont été développés au cours des quatre dernières décennies. Les vésicules unilamellaires géantes (VUV), qui sont largement utilisées pour la reconstitution synthétique du transport transmembranaire, sont particulièrement intéressantes [START_REF] Hermann | Automated analysis of giant unilamellar vesicles using circular Hough transformation[END_REF]. Alternativement, de grandes plaques planes de bicouche lipidique étalées sur un support solide (Windschiegl et al., 2009), par conséquent, les bicouches lipidiques supportées (SLB) sont utilisées pour étudier l'organisation de la membrane à grande échelle.

Vésicules unilamellaires géantes

Des sphères bicouches lipidiques avec deux surfaces hydrophiles et un noyau hydrophobe entre elles offrent la possibilité de compartimenter de petits volumes de solutions aqueuses et de les isoler de l'extérieur aqueux [START_REF] Yang | Selfassembly of size-controlled liposomes on DNA nanotemplates[END_REF]. De telles sphères (également appelées liposomes) sont naturellement utilisées par la cellule pour les moyens de transport (endocytose [START_REF] Pakkanen | From endosomes onwards: membranes, lysosomes and viral capsid interactions[END_REF], transcytose [START_REF] Thuenauer | Pathways of protein and lipid receptor-mediated transcytosis in drug delivery[END_REF], trafic intracellulaire (Sens et al., 2008), exocytose et communication cellulecellule [START_REF] Xu | A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion[END_REF]. En fait, le cytosol cellulaire est isolé du liquide extracellulaire selon un principe similaire: il est confiné par la barrière bicouche lipidique. Les liposomes peuvent être utilisés pour imiter une telle barrière. Cependant, les liposomes qui se forment spontanément en appliquant une solution aqueuse aux lipides ont normalement une taille beaucoup plus petite (0,1 à 1 µm de diamètre) que les cellules régulières (dizaines de micromètres) [START_REF] Antonietti | Vesicles and Liposomes: A Self-Assembly Principle Beyond Lipids[END_REF]. De plus, des vésicules dites multilamellaires se forment fréquemment. Pour obtenir des structures unilamellaires, les liposomes doivent être décomposés en vésicules unilamellaires par extrusion ou sonication. Angelova et Dimitrov ont mis au point un protocole permettant d'obtenir des liposomes unilamellaires à la taille d'une cellule à partir de films lipidiques étalés sur une surface en 1986 (Angelova and Dimitrov). La procédure s'appelle l'électroformation et fournit des bicouches lipidiques sphériques de plusieurs dizaines de centaines de µm de diamètre -vésicules unilamellaires géantes (GUV). Il est important de pouvoir contrôler la formation de GUV avec des espèces distinctes de lipides et de protéines [START_REF] Dezi | Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents[END_REF]Madl et al., 2017). Ainsi, divers processus cellulaires peuvent être reconstitués à l'aide de GUV. En particulier, les GUV sont des systèmes modèles parfaits pour les études de transport à travers la bicouche lipidique dans le contexte de la formation de pores (Gilbert et al., 2014), absorption et libération cellulaire [START_REF] Mellander | Two modes of exocytosis in an artificial cell[END_REF]Römer et al., 2007;[START_REF] Sasai | Artificial Exocytotic System That Secretes Intravesicular Contents upon Ca2+ Influx[END_REF]. Les GUV peuvent également être utilisés pour imiter l'adhésion cellulaire [START_REF] Tareste | SNAREpin/Munc18 promotes adhesion and fusion of large vesicles to giant membranes[END_REF] et fusion de vésicule [START_REF] Witkowska | A convenient protocol for generating giant unilamellar vesicles containing SNARE proteins using electroformation[END_REF]. De plus, des composants cytosoliques cellulaires peuvent être ajoutés à la lumière du GUV. Par exemple, un maillage d'actine recréé dans des GUV a permis d'étudier le rôle du cytosquelette dans la déformation de la membrane [START_REF] Vogel | Minimal systems to study membrane-cytoskeleton interactions[END_REF]. De plus, l'intérieur des GUV peut être fonctionnalisé avec une machinerie cellulaire reconstituée synthétiquement pour la réplication de l'ADN [START_REF] Xu | Synthetic Protocells to Mimic and Test Cell Function[END_REF]. Ces GUV à plusieurs composants peuvent être utilisés pour imiter une variété de processus cellulaires tels que la communication cellule-cellule [START_REF] Joesaar | DNA-based communication in populations of synthetic protocells[END_REF] et même la division cellulaire [START_REF] Xu | Synthetic Protocells to Mimic and Test Cell Function[END_REF]. Globalement, de tels systèmes peuvent être utilisés pour générer des protocellules et des prototissues synthétiques [START_REF] Gobbo | Programmed assembly of synthetic protocells into thermoresponsive prototissues[END_REF][START_REF] Omidvar Ramin | Glycan-decorated protocells: novel features for rebuilding cellular processes[END_REF][START_REF] Villringer | Lectin-mediated protocell crosslinking to mimic cell-cell junctions and adhesion[END_REF] afin de recréer les principes de base de la vie.

Les GUV marquées avec des marqueurs lipophiles fluorescents peuvent être visualisées avec les techniques de microscopie conventionnelles, alors que l'imagerie de vésicules plus petites (<200 nm de diamètre) est limitée par la limite de diffraction. [START_REF] Dietrich | Lipid Rafts Reconstituted in Model Membranes[END_REF].

Bicouches lipidiques supportées

Le dépôt d'une bicouche lipidique sur un support solide offre la possibilité d'étudier les interactions lipide-lipide à très grande échelle dans d'immenses plaques membranaires planaires. Des bicouches lipidiques supportées peuvent être obtenues par l'application de vésicules unilamellaires sur une surface hydrophile. L'adhésion de la vésicule au support augmente la tension superficielle dans la bicouche lipidique, ce qui entraîne l'explosion de la vésicule et l'étalement de la bicouche lipidique sur le substrat hydrophile [START_REF] Jing | Formation of supported lipid bilayers on silica: relation to lipid phase transition temperature and liposome size[END_REF]. Normalement, de petites vésicules unilamellaires fortement incurvées (de diamètre inférieur à 100 nm) sont utilisées pour le dépôt sur le support plan. Les principales limitations des bicouches lipidiques supportées (SLB) proviennent des interactions de la membrane avec le support. Le choix du support est particulièrement important dans la conception générale des expériences impliquant des SLB. Afin d'étudier les SLB par microscopie à fluorescence, les chercheurs utilisent classiquement un support de verre. Le verre est un matériau amorphe et la surface du verre nécessite un traitement supplémentaire (par exemple, nettoyage à l'acide ou nettoyage au plasma) avant l'application de liposomes [START_REF] Cremer | Formation and Spreading of Lipid Bilayers on Planar Glass Supports[END_REF]. De plus, le couplage de la feuille inférieure à la surface du verre est très fort. Il limite presque complètement la diffusion des lipides dans la foliole inférieure [START_REF] Hennesthal | Pore-Spanning Lipid Bilayers Visualized by Scanning Force Microscopy[END_REF]. Une autre solution consiste à utiliser une surface de mica comme support (Glazier and Salaita). Le mica est un minéral de silice naturelle avec une charge de surface et un clivage basal presque parfait. La surface hydrophile en mica permet une adhésion efficace des vésicules lipidiques. Les substrats de mica sont classiquement utilisés comme support bicouche lipidique pour la microscopie à force atomique. Les multicouches épaisses de mica ne sont pas transparentes, ce qui empêche leur application en microscopie à fluorescence pour de telles études SLB. Néanmoins, le clivage du mica en couches minces permet l'application de certains modes de microscopie à fluorescence (Madl et al., 2017). En outre, malgré le couplage de la foliole bicouche lipidique inférieure sur le support en mica, la mobilité lipidique au niveau de cette foliole inférieure est supérieure à celle sur le support en verre. [START_REF] Richter | Following the Formation of Supported Lipid Bilayers on Mica: A Study Combining AFM, QCM-D, and Ellipsometry[END_REF]. Les autres matériaux solides utilisés pour le support de la bicouche lipidique sont SiO2, Or, TiO2, PDMS, etc. [START_REF] Tero | Substrate Effects on the Formation Process, Structure and Physicochemical Properties of Supported Lipid Bilayers[END_REF]. Le support solide empêche l'insertion de protéines transmembranaires et, en outre, aucune étude de déformation membranaire ne peut être réalisée. Afin de remédier à ces inconvénients, les supports à polymère [START_REF] Kühner | Lipid mono-and bilayer supported on polymer films: composite polymer-lipid films on solid substrates[END_REF] ou bicouches lipidiques planes suspendues dans les pores (Römer and Steinem, 2004) peut être employé.

L'épaisseur de la bicouche lipidique étant d'environ 5 nm, il est possible de l'étudier avec une microscopie à excitation en couche mince, telle que TIRF ou HILO (Figure 12).

Etudes d'ordre membranaire utilisant des sondes membranaires sensibles à l'environnement

L'ordre des membranes est déterminé par l'alignement de la chaîne acyle grasse et le degré de tassement des lipides dans la bicouche lipidique [START_REF] Almeida | A Simple Thermodynamic Model of the Liquid-Ordered State and the Interactions between Phospholipids and Cholesterol[END_REF]. L'ordre des membranes est essentiel pour la caractérisation de l'organisation et de la composition des membranes locales. Les marqueurs membranaires lipophiles ou les lipides fluorescents (voir le chapitre précédent) indiquent les positions des domaines Ld et Lo dans les membranes modèles. Cependant, cette approche assez simple ne permet pas de résoudre le problème du degré de compression dans les phases Ld ou Lo. Modification de la composition de la chaîne acyle grasse de la sphingomyéline et des lipides de PC, variation du contenu en cholestérol et introduction de glycosphingolipides dans les membranes modèles peuvent modifier de manière significative l'ordre des membranes des phases Ld et Lo (Carravilla et al., 2015;[START_REF] Dewitt | Interaction of Cholesterol in Ternary Lipid Mixtures Investigated Using Single-Molecule Fluorescence[END_REF]. Afin de déchiffrer les modulations fines de l'ordre de la membrane, des techniques plus sensibles doivent être appliquées.

Le degré de tassement de la bicouche lipidique peut être détecté par son niveau d'hydratation [START_REF] Sanchez | Laurdan generalized polarization: from cuvette to microscope[END_REF]. Les molécules d'eau peuvent se localiser dans la bicouche lipidique au niveau des groupes de tête lipidiques. La quantité d'eau dans la bicouche lipidique est modulée par le tassement lipidique -des membranes plus fluides peuvent contenir de l'eau au niveau des groupes de tête polaires, alors que l'eau est presque complètement exclue des bicouches lipidiques rigides. L'eau est un solvant polaire et le noyau hydrophobe de la membrane est clairement un environnement non polaire. Les spectres de fluorescence de sondes insérées dans un environnement polaire présentent un décalage de leur fluorescence maximale vers de plus grandes longueurs d'onde [START_REF] Lakowicz | Principles of fluorescence spectroscopy[END_REF]. Ce «décalage vers le rouge» est dû à la relaxation du solvant, due à l'interaction des moments dipolaires du solvant avec le moment dipolaire du fluorophore à l'état excité. En conséquence, une nouvelle population de fluorophores à énergie réduite de l'état excité se forme et émet une lumière de plus grande longueur d'onde. Pour plusieurs molécules fluorescentes, le décalage vers le rouge induit par l'augmentation de la polarité du solvant est radical (Klymchenko and Kreder, 2014). Ces sondes sensibles à l'environnement peuvent être utilisées pour détecter la rigidité de l'environnement de la membrane.

Au cours des dernières décennies, de nombreuses sondes lipohiles sensibles à l'environnement ont été développées et appliquées pour la détection locale de l'ordre et de la rigidité des membranes dans les cellules et les membranes modèles. Dans mon projet, j'ai utilisé le dérivé Nile Red -NR12S (figure 13) développé par Kucherak et al. [START_REF] Kucherak | Switchable Nile Red-Based Probe for Cholesterol and Lipid Order at the Outer Leaflet of Biomembranes[END_REF]. Cette sonde a différents spectres de fluorescence dans les domaines de phase Ld (composé de DOPC pur) et Lo (composé de SM / chol 2/1). Le spectre de fluorescence de NR12S dans les membranes Ld pures est décalé vers le rouge (figure 13). Dans les membranes modèles à séparation de phase, le NR12S s'intègre avec succès dans les domaines de phase Lo et Ld. Les GUV et les SLB marqués par NR12S peuvent être imagés davantage dans deux canaux de couleur d'émission distincts. La sélection des canaux de couleur est basée sur les spectres de fluorescence du NR12Svert (525/50 nm) et rouge (700/75 nm). La microscopie confocale a révélé que, dans le canal de détection vert, les domaines Lo sont plus lumineux que les domaines Ld, tandis que dans le canal de détection rouge, les domaines Ld apparaissent plus lumineux. Sur la base de ces images bicolores, la polarisation générale (GP) de NR12S dans les membranes modèles peut être quantifiée pour chaque pixel de l'image confocale. Les valeurs de GP peuvent varier de -1 à 1. Elle est faible pour le NR12S incorporé dans des domaines de membrane moins ordonnés et augmente en même temps que l'ordre de la membrane. Cette approche peut être utilisée pour cartographier l'ordre des membranes des membranes plasmiques natives et des systèmes membranaires synthétiques. [START_REF] Kucherak | Switchable Nile Red-Based Probe for Cholesterol and Lipid Order at the Outer Leaflet of Biomembranes[END_REF]. Les bandes verte et rouge représentent les parties verte et rouge des spectres pris pour le calcul de la polarisation générale (GP); C) Formule pour le calcul du médecin généraliste [START_REF] Sanchez | Laurdan generalized polarization: from cuvette to microscope[END_REF]; D) Imagerie ratiométrique à deux couleurs de GUV et SLB à phases séparées utilisant la microscopie confocale. Les filtres passe-bande pour les canaux de couleur verte et rouge sont sélectionnés comme indiqué en B. L'image GP est reconstruite par application de la formule en C aux canaux de couleur pixel par pixel. Plus le GP est élevé, plus l'ordre des membranes est élevé. Selon la barre de calibration, la valeur GP en Lo est plus élevée. Barres d'échelle -10 µm.

La séparation de phase dans les membranes modèles a été largement étudiée à l'aide de sondes membranaires sensibles à l'environnement [START_REF] Almeida | Sphingomyelin/Phosphatidylcholine/Cholesterol Phase Diagram: Boundaries and Composition of Lipid Rafts[END_REF]Carravilla et al., 2015;[START_REF] Sanchez | Laurdan generalized polarization: from cuvette to microscope[END_REF]Veatch et Keller, 2005). Souvent, des domaines coexistants ordonnés et désordonnés dans des liquides dans des membranes modèles sont produits en utilisant des compositions ternaires de DOPC, de cholestérol et de SM. L'ordre des membranes dépend des rapports molaires entre les trois lipides. Il est clair que l'augmentation de la teneur en SM et en cholestérol chez les VUG non séparés en phase augmente l'ordre des membranes. En utilisant la sonde écologiquement sensible Laurdan, Carravilla et al. (Carravilla et al., 2015) ont mesuré l'ordre membranaire de différentes compositions ternaires et obtenu le diagramme de phase ternaire pour les GUV composées de DOPC, de SM (extrait d'oeufs) et de chol. En outre, ils ont mis en évidence la zone du diagramme de phase (figure 14), où la séparation de phase a lieu à température ambiante. De plus, l'ordre membranaire des domaines Lo et Ld dans des vésicules à phases séparées à différents rapports a été quantifié. Il est intéressant de noter que l'ordre des membranes des domaines Lo et Ld purs dans les GUV à phases séparées varie selon le rapport entre les lipides. Ces diagrammes de phase ternaires sont extrêmement utiles pour la préparation ultérieure de VUV homogènes ou à séparation de phase avec un ordre de membrane distinct des domaines de phases Ld et Lo. A) L'ordre des membranes dépend du rapport molaire entre les trois lipides. Le code de couleur illustre la valeur GP des VUC composées de différents rapports molaires DOPC, SM et chol. La zone grise décrit les rapports auxquels se produit la séparation de phase. L'ordre des membranes des domaines Lo et Ld dans les vésicules à phases séparées varie également; B) Ordre membranaire des domaines Lo dans des vésicules à phases séparées à différents rapports molaires de lipides; C) Ordre membranaire des domaines Ld dans des vésicules à phases séparées à différents rapports molaires de lipides; Révisé de (Carravilla et al., 2015).

Investigation of the cellular uptake using model membranes

Endocytosis is a mode of active cellular transport of extracellular material into the cell [START_REF] Doherty | Mechanisms of Endocytosis[END_REF]. It is involved in cell-cell exchange of substances and mediates the entry of various pathogens. Different cells possess different mechanisms of endocytosis. Phagocytosis [START_REF] Underhill | Information processing during phagocytosis[END_REF] is the uptake of large extracellular particles (cell debris, microorganisms, apoptotic cells etc.) for further decomposition and recycling [START_REF] Cox | A Rab11-containing rapidly recycling compartment in macrophages that promotes phagocytosis[END_REF]. Phagocytosis requires extensive actin rearrangement. Only some cells are equipped with this mechanism (macrophages, monocytes, etc.). Non-phagocytic cells can internalize extracellular material via clathrinmediated endocytosis [START_REF] Kaksonen | Mechanisms of clathrin-mediated endocytosis[END_REF] (clathrin assembly on the cytosolic side of the plasma membrane promotes the formation of the endocytic bud), caveolaemediated endocytosis (highly curved caveolae contain multiple cellular receptors that mediate uptake) or pinocytosis ("cell drinking" -the uptake of small volumes of extracellular fluid, which normally occurs at regions of the plasma membrane where a lot of membrane ruffles are located). The induction of endocytic pathways often requires the activation of distinct plasma membrane receptors (Eierhoff et al., 2012). Glycosylated lipids and proteins, embedded in the plasma membrane are particularly important and abundant class of membrane commponents.

Lectins and glycosphingolipids as the main players in the uptake initiation

Carbohydrate groups of glycosylated membrane components present an attractive target for extracellular pathogens (viruses, bacteria) and pathogenic products (such as toxins) for specific binding and the subsequent exploitation of the native cellular endocytic pathways (Aigal et al., 2015;Schubert and Römer, 2015). The recognition of the carbohydrates is granted by carbohydrate binding proteins (lectins), that are ubiquitously present in viruses and bacteria [START_REF] Ambrosi | Lectins: tools for the molecular understanding of the glycocode[END_REF][START_REF] Lis | Lectins: carbohydrate-specific proteins that mediate cellular recognition[END_REF]. Lectins highjack the glycosphingolipids and induce activation of cellular signaling pathways. Moreover, clustering of GSLs induced by lectins can lead to the induction of local asymmetric stress in the lipid bilayer that results in endocytic tubule nucleation (Ewers et al., 2010;Römer et al., 2007). Plasma membrane tubules grow longer and sequester lectins on the negatively bent surface. Finally, an endocytic bud is detached from the plasma membrane and follows intracellular transport pathways [START_REF] Duncan | Endocytosis and Intracellular Trafficking as Gateways for Nanomedicine Delivery: Opportunities and Challenges[END_REF].

Toxins/lectins act as clustering devices and generate membrane deformation

Over the past decade, clustering of glycosphingolipids was shown to be a decisive event in internalization of lectins (Aigal et al., 2015). Despite the obvious involvement of cellular cytosolic components, such as clathrin and actin, the plasma membrane deformation relies on asymmetric stress induced by the specific multivalent binding of lectins to the plasma membrane surface (Pezeshkian et al., 2016). For example, the galactose-specific B subunit of Shiga toxin (StxB, expressed by S. desynteriae or E. coli [START_REF] Sandvig | Shiga toxins[END_REF]) is known to follow the clathrin-dependent endocytic pathway (Johannes and Römer, 2010). However, it was shown by Römer et al. (2007) thatalternative uptake strategies take place when the cytosolic endocytic machinery is inhibited or absent (figure 15). StxB recognizes the 𝛼-1-4-galactose of the GSL globotriaosylceramide (Gb3) and induces membrane deformation. Remarkably, the uptake of StxB was reconstituted synthetically in GUVs. These model membranes were constructed using the minimal set of lipids for lipid bilayer formation -DOPC, chol and 5 mol % of Gb3 receptor. StxB bound to Gb3, formed aggregates and induced membrane tubular invaginations, evenin such a minimalistic system. As suggested by theoretical models, the formation of lectin aggregates on the membrane surface is necessary for the membrane deformation (Johannes et al., 2014;Pezeshkian et al., 2016). In order to compensate for the bending rigidity of the lipid bilayer, such aggregates must grow until the tubule is nucleated. The origin force for the lectin aggregation is yet unclear, however, Pezeshkian et al. proposed an interesting model based on their molecular dynamic simulation studies [START_REF] Pezeshkian | Mechanism of Shiga Toxin Clustering on Membranes[END_REF]. They suggest that the aggregation of lectins is driven by the fluctuation forces of the membrane, the analogue of the Casimir effect. At once, StxB can bind up to 15 Gb3 molecules. Such a multivalent interaction grants an efficient clustering of Gb3, which is necessary for the induction of asymmetric stress and tubule nucleation. The role of the specific binding sites in the uptake initialization was explored using site-directed mutagenesis. It was shown that the depletion of the binding sites 3 or 2 (figure 15) decreases the binding affinity of StxB to Gb3, whereas the deletion of the binding site 1 does not [START_REF] Soltyk | A Mutational Analysis of the Globotriaosylceramide-binding Sites of Verotoxin VT1[END_REF]. Remarkably, deletion of the binding site 1 prevents StxB from efficient clustering and tubule formation (Römer et al., 2007). This suggests a crucial importance of this particular binding site for Gb3 clustering by StxB. Similar studies were performed for the fucose-specific Ralstonia solanacerum lectin (RSL). This protein is a trimer with two binding sites per monomer (Arnaud et al., 2013). It is also known to induce membrane invaginations in GUVs spiked with fucosylated lipids.

Studies employing the modification of the lectin valence via site-directed mutagenesis or (Arnaud et al., 2013) or production of neolectins with engineered binding sites based on RSL (Arnaud et al., 2014) showed very clearly the implication of the distinct binding sites in the tubule nucleation. Remarkably, the neolectins where the two closest binding sites were present, showed almost the same efficiency in tubule formation as its natural hexavalent analogue. Other divalent or even trivalent lectins displayed a significantly reduced tubule formation. All these studies show the particular importance of lectin geometry and valence for endocytic tubule formation.

Lectin-induced lipid sorting and membrane reorganization

Apart from membrane deformation, lectins are also known to induce lipid sorting in GSL-containing membranes (Windschiegl et al., 2009). Clearly, curvature-driven lipid sorting occurs during various cellular processes, including uptake and release (Callan- Jones et al., 2011). As indicated by Pinot et al. (2014) the polyunsaturated phospholipids are abundantly implicated in the local membrane tubule or bud formation. They can adapt their configuration (mainly via the cis-trans isomerization of the unsaturated fatty acyl chains) in order to fit the curved profile of the plasma membrane in endocytic or exocytic buds. Furthermore, based on their molecular geometry (cone, cylinder, inverted cone), lipids prefer surfaces of their native curvature (Roux et al., 2005). This effect was extensively studied in asymmetric model membranes; however, a significant curvature-driven sorting based solely on lipid geometry could not even be observed even in highly curved lipid bilayers (Callan- Jones et al., 2011). Nevertheless, this effect is possible in presence of membrane-bound proteins (e.g. lectins). Interesting studies of lipid sorting in model membranes were performed by pulling a tether from GUV membranes using optical tweezers and micropipette aspiration. With this technique, Sorre et al. (2009) demonstrated that curved and non-curved membranes differ in their lipid composition. Again, unsaturated lipids (DHPE-Texas Red) were found to associate with the highly curved membrane. Furthermore, Sorre et al. spiked the GUV with the ganglioside GM1 and applied the B subunit of Cholera toxin (CtxB) to the membrane. It was found that such CtxB-Gm1 interaction even amplified the sorting of unsaturated DHPE molecules to the highly curved tether. The composition of the lipid bilayer played a very important role in this phenomenon. Significant sorting could occur only in GUVs with a lipid composition in close the phase demixing point (e.g. DOPC/chol/SM -1/1/1). Lectin-induced lipid sorting in non-curved planar lipid bilayers has similar features. It was shown that applying CtxB or StxB to a lipid bilayer composed of a ternary mixture near the demixing point can induce phase separation (Safouane et al., 2010). Such an effect, of course, requires efficient binding of the lectins to their membrane receptors has toin the GUV. Subsequently, these lectins induce phase separation and localize at the newly formed Lo domains. The distinct lipid composition of the membrane tubules formed by lectins is still an open question (Safouane et al., 2010;Sorre et al., 2009). On one hand, highly curved membrane surfaces clearly attract unsaturated lipids, on the other hand lectins are bound to glycosphingolipids that associate with the saturated sphingolipids and cholesterol. All in all, the resulting composition of the endocytic tubule is determined by the interplay between the attraction of unsaturated lipids and the sequestering of lectins associated to saturated sphingolipids.

The role of GSL fatty acyl chain structure in lectin binding and internalization

The GSLs are usually categorized by its carbohydrate moiety. However, the ceramides of GSLs with identical sugars can contain fatty acids of various lengths and saturation degrees. For example, the Gb3 naturally occurs in various isoforms (figure 16). The ratio between the content of the isoforms depends on the cell from which the mixture was extracted [START_REF] Shin | A Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometric Assay for the Quantification of Fabry Disease Biomarker Globotriaosylceramide (GB3) in Fabry Model Mouse[END_REF].

Figure 16| Glycosphingolipid globotriaosylceramide. Gb3 is defined by its carbohydrates (galactosylα1-4galactosylβ1-4glucosyl). However, different Gb3 species (isoforms) can vary in their fatty acids of the ceramide backbone. Revised from [START_REF] Shin | A Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometric Assay for the Quantification of Fabry Disease Biomarker Globotriaosylceramide (GB3) in Fabry Model Mouse[END_REF].

The structure of the lipid anchor (as well as the local lipid bilayer composition) moderates the exposure of the sugar group to the extracellular space, which in turn can prevent glycan recognition by a lectinlectins if the distinct position of the sugar group is unfavorable [START_REF] Lingwood | Cholesterol modulates glycolipid conformation and receptor activity[END_REF]. Consequently, altering the fatty acyl chain can inhibit the binding, clustering and formation of tubular invaginations by lectins. Schütte et al. (2015) studied the Gb3 C24:1 OH species in the context of StxB uptake in giant unilamellar vesicles. They spiked GUVs with Gb3 molecules with either natural (Gb3-R) or unnatural (Gb3-S) configuration of the hydroxyl group. Both species showed similar high nanomolar affinity to StxB. However, StxB induced tubular invagination twice less efficient in GUVs spiked with Gb3-R in comparison to Gb3-S. Remarkably, such minor modification of the fatty acyl chain significantly altered the behavior of Gb3 in the lipid bilayer.

In their further work, Schütte et al. studied the organization of supported lipid bilayers that contained 5 mol % of Gb3 species with either a C24:0 or a C24:1 fatty acyl chains (Schütte et al., 2014). In phase separated (Lo and Ld) supported lipid bilayers, Gb3 with the C24:1 fatty acid induced the formation of a phase domain of intermediate order, which they called liquid-intermediate phase (Li). In SLB spiked with Gb3 C24:0 such an effect was not observed. StxB applied to the supported lipid bilayer, in both cases successfully recognized Lo phase domains, whereas Ld and Li domains were not recognized by the toxin. These domains may be either depleted of sufficient amounts of Gb3 molecules, or the Gb3 molecules incorporated into these domains cannot be recognized due to an altered exposure of the sugar groups to the aqueous solution.

In the context of intracellular trafficking of the cholera toxin from V. cholerae (Ctx), Chinnapen et al. (2012) studied the cellular sorting of isoforms of the gangliosides GM1. They found that during the uptake and trafficking of Ctx only the GM1 molecules with unsaturated fatty acyl chains (16:1) are successfully transported to the trans-Golgi network and endoplasmic reticulum, whereas the GM1 with saturated fatty acyl chains.

GSLs are important molecules for plasma membrane interleaflet communication and signaling (Manna et al., 2017). The binding of lectins to GSLs at the plasma membrane can induce a signaling cascade required for triggering a certain cellular process (e.g. lectin internalization). Interestingly, lectins specific to the same glycans can induce different signaling events. For example, the α-galactose specific lectin StxB from S. dysenteriae and LecA from P. aeruginosa target the same GSL globotriaoylceramide (Gb3), but they induce different signaling events [START_REF] Zheng | The Pseudomonas aeruginosa lectin LecA triggers host cell signalling by glycosphingolipid-dependent phosphorylation of the adaptor protein CrkII[END_REF]. These differences may originate from a selective recognition of the Gb3 trisaccharide exposed in functional membrane domains of distinct composition [START_REF] Lingwood | Cholesterol modulates glycolipid conformation and receptor activity[END_REF]. The collective behavior of membrane lipids alters the exposure of the carbohydrate headgroup to the extracellular space. In turn, StxB and LecA can have preferences for Gb3 receptor molecules with differently oriented the head groups of the Gb3 receptor molecules.

All these fascinating findings emphasize crucial importance of the fatty acyl chain of glycosphingolipids for lectin binding, induction of membrane reorganization and deformation and, subsequently, intracellular trafficking of lectins.

Lectin-GSL interactions in viral and bacterial uptake

Lectin-glycan interactions are abundantly exploited byhuman pathogens like viruses and bacteria (Eierhoff et al., 2012). Bacteria produce lectins in order to target host cell glycans for different purposes. Shiga (Johannes and Römer, 2010) and cholera toxins [START_REF] Haan | Cholera toxin: A paradigm for multi-functional engagement of cellular mechanisms (Review)[END_REF], introduced above, are secreted by their bacteria and exert their cellular toxicity after their uptake. Lectins can also assist bacteria in targeting cell surface glycans for the means of adhesion to the host cell (Cossart, 2004). Many noninvasive bacteria adhere to cell and form biofilms, which is oftenlectin-mediated [START_REF] Johansson | Inhibition and Dispersion of Pseudomonas aeruginosa Biofilms by Glycopeptide Dendrimers Targeting the Fucose-Specific Lectin LecB[END_REF][START_REF] Kadam | A Glycopeptide Dendrimer Inhibitor of the Galactose-Specific Lectin LecA and of Pseudomonas aeruginosa Biofilms[END_REF]. Furthermore, bacteria can stimulate a targeted cell to polymerize actin intracellularly and engulf the extracellular bacterium(Pizarro-Cerdá and [START_REF] Pizarro-Cerdá | Bacterial Adhesion and Entry into Host Cells[END_REF]. Unlike bacteria, viruses require the cellular machinery for proliferation (Eierhoff et al., 2012). Some viruses express lectins on their surfaces in order to recognize host cell surface glycans [START_REF] Carter | HIV entry in macrophages is dependent on intact lipid rafts[END_REF]Ewers et al., 2010;Rydell et al., 2013). The interaction facilitates the viral uptake and further trafficking to the correct cellular compartment, where the viral genetic material can be integrated into the cellular DNA.

Glycan-mediated uptake of viruses

Numerous viruses are functionalized withlectins in order to recognize cell surface glycans (Eierhoff et al., 2012). Enveloped viruses like HIV or herpes virus make use of their lectins in order to fuse with the plasma membrane of the host cell and deliver the content of the viral particle into the cytosol. Alternatively, enveloped viruses like Influenza A [START_REF] Ho | Influenza M2 Transmembrane Domain Senses Membrane Heterogeneity and Enhances Membrane Curvature[END_REF] bind to the plasma membrane and are internalized via cellular endocytic pathways. The viral capsid of some non-enveloped viruses is composed of lectins assembled in lattices. A very well-studied example is the SV40 polynoma virus. The viral capsid of SV40 mainly consists of 72 Vp1 proteins, arranged in icosahedral particle. Vp1 is a pentavalent lectin specific to the ganglioside GM1. Ewers et al. (2010) rebuilt the uptake of SV40 in GUVs spiked with GM1. They showed that the viral capsid-GM1 interaction alone is sufficient for membrane tubule induction. Moreover, the application of soluble Vp1 protein molecules to the same GUV membranes also induced tubular invaginations, albeit less quickly than the viral particles. SV40 required only a few seconds for membrane tubule formation whereas soluble lectins (CtxB, StxB and also Vp1) required few minutes to induce a tubule.

Recently, the Vp1 -GM1 interaction was characterized more precisely using molecular dynamic simulations [START_REF] Kociurzynski | Binding of SV40's Viral Capsid Protein VP1 to Its Glycosphingolipid Receptor GM1 Induces Negative Membrane Curvature: A Molecular Dynamics Study[END_REF]. Multivalent binding of Vp1 to GM1 induces local curvature of the surrounding membrane, which further leads to tubule nucleation. Although, Vp1 proteins induce membrane tubules either being soluble or arranged as a lattice, these two processes are different. The Vp1 proteins assemble in aggregates on the membrane surface (similarly to StxB) and induce asymmetric stress in the membrane, whereas the viral capsid "imprints" its own curvature into the plasma membrane via adhesion. The latter allows bending of the membrane with high tension that cannot be bent by multivalent lectins aggregation.

Glycan-mediated uptake of bacteria

Bacteria do not require the host cell machinery for replication. However, some bacteria (e.g. Listeria, Shigella, etc…) internalize into the intracellular environment, where they can stay protected from the immune system. Such pathogens can be taken up by phagocytic cells (macrophages, monocytes, dendritic cells etc.) together with extracellular fluid (pinocytosis). Furthermore, bacteria modify the intracellular environment in order to survive and proliferate (Eierhoff et al., 2012). The invasion of non-phagocytic cells by bacteria requires the pathogen to develop an additional machinery (Cossart, 2004). The two main mechanisms utilized by numerous bacteria for cell entry, are called "trigger" and "zipper" mechanisms. The trigger mechanism relies on type 3 secretion system (T3SS). T3SS forms a transient pore in the host cell membrane and is used by bacteria to inject their virulence factors directly into the host cell cytosol. The local plasma membrane environment is critical for the stability of thisa pore. It has been shown, that some bacteria that enter the host cell via "triggering" target specific membrane nanodomains, enriched in cholesterol and sphingolipids, for pore insertion. Virulence factors injected via T3SS, trigger an extensive actin polymerization within the host cell leading to bacterial engulfment. Typical representatives of bacteria that utilize the trigger mechanism are Salmonella enterica, Shigella flexneri, and Pseudomonas aeruginosa (Eierhoff et al., 2012;[START_REF] Pizarro-Cerdá | Bacterial Adhesion and Entry into Host Cells[END_REF]. As an alternative mechanism for bacterial internalization, the "zipper" mechanism requires a specific multivalent interaction of bacterial particles with cellular receptors. This interaction further activates cellular signaling cascades that induce the actin rearrangement for bacterial uptake. The modification of actin is, however, less severe in comparison to the "trigger" mechanism. Moreover, the interaction with multiple cellular receptors during the bacterial adhesion to the host cell seems to be the decisive event for the "zipper mechanism", as the wrapping of the bacteria with the host plasma membrane can also occur without actin polymerization. Typical representatives of bacteria that take advantage of the zipper mechanism are Listeria monocytogenes (targets the membrane glycoprotein E-cadherin), Yersinia pseudotuberculosis (targets the membrane protein β1 integrin), and Pseudomonas aeruginosa (targets the membrane glycolipid Gb3, integrins and asialo-gangliosides). Remarkably, their efficient adhesion to the target plasma membrane surface again relies on the local plasma membrane organization (Duncan et al., 2004;Zaas et al., 2005). Membrane domains enriched in sphingomyelin and cholesterol do not only enclose various plasma membrane receptors required for adhesion, but also serve as important platforms for the signal transduction required for actin polymerization (Eierhoff et al., 2014;Lafont and Van Der Goot, 2005).

The lectin LecA in the invasion of P. aeruginosa

Pseudomonas aeruginosa is an opportunistic, gram-negative, pathogenic bacterium [START_REF] Emam | Glycolipid-dependent, protease sensitive internalization of Pseudomonas aeruginosa into cultured human respiratory epithelial cells[END_REF]. It is responsible for a high number of hospital-acquired infections and poses a high threat particularly for patients with cystic fibrosis or with a compromised immune system. The difficulties in treating P. aeruginosa arise from its versatile defense machinery. Among others, forming biofilms is a very efficient mechanism to protect individual bacteria from antibiotics and the host immune system [START_REF] Johansson | Inhibition and Dispersion of Pseudomonas aeruginosa Biofilms by Glycopeptide Dendrimers Targeting the Fucose-Specific Lectin LecB[END_REF]. Bacterial biofilms form by adhesion of bacteria to the surface of the host cell and their neighboring bacteria. Furthermore, bacteria secret hydrolytic enzymes and toxins to promote infection. Various compounds of the bacterial outer membrane are involved in efficient adhesion. Particularly important are bacterial lectins that are utilized to specifically target the host's cellular glycans. Adhesins (Eierhoff et al., 2012), specific to asialo-GM1 and -GM2 are of major importance for the bacterial adhesion. The bacterium P. aeruginosa expresses the two lectins, LecA and LecB [START_REF] Chemani | Role of LecA and LecB Lectins in Pseudomonas aeruginosa-Induced Lung Injury and Effect of Carbohydrate Ligands[END_REF], that are particularly specific to α-galactose and l-fucose, respectively. Surprisingly, studies of bacterial infection of host cells that were depleted of glycosphingolipids (by blocking the biosynthesis of glucosylceramide) revealed that the glycosphingolipids are rather important for the bacterial internalization than for adhesion (Eierhoff et al., 2012;[START_REF] Emam | Glycolipid-dependent, protease sensitive internalization of Pseudomonas aeruginosa into cultured human respiratory epithelial cells[END_REF]. P. aeruginosa can invade a host cell following the "trigger" mechanism. Using the T3SS, the bacterium injects the effector proteins ExoT and ExoS, which promote actin rearrangement in the host cell, leading to the bacterial engulfment. Interestingly, Eierhoff et al. (2014) showed that P. aeruginosa is capable of invadinginvading host cells via the "zipper" mechanism as well. Here, however, the zipper is formed by multivalent lectin interactions with the host plasma membrane glycosphingolipid Gb3. Hence, this mechanism was coined "lipid zipper". Gb3 molecules are targeted by the P. aeruginosa lectin LecA that is enriched at the surface of the bacterium (figure 17 A). LecA (figure 17 B) is a homotetramer with four binding sites specific to α-galactosylated membrane components (e.g. Gb3 -figure 17 C). The two pairs of binding sites are oriented in opposite directions. Such an orientation of binding sites allows the protein to cross-link proximal lipid bilayers of GUVs functionalized with glycans [START_REF] Villringer | Lectin-mediated protocell crosslinking to mimic cell-cell junctions and adhesion[END_REF]. Presumably, in a similar way, LecA mediates the adhesion of the bacterial outer membrane to the host cell surface [START_REF] Novoa | A LecA Ligand Identified from a Galactoside-Conjugate Array Inhibits Host Cell Invasion by Pseudomonas aeruginosa[END_REF]. This multivalent adhesion is a key event in the initialization of the lipid zipper. Remarkably, the adhesion to model GUVs leads to membrane deformation and bacterial engulfment even in the complete absence of actin (figure 17D). Furthermore, during the uptake of P. aeruginosa into H1299 lung epithelial cells, a pronounced colocalization of actin filaments with the internalized bacteria was not observed (figure 17E). The deletion of LecA decreases the internalization of the bacteria in host cells or GUVs drastically (more than 60 % (Eierhoff et al., 2014)). Moreover, blocking of LecA with a di-antennary galactose reduced the internalization of P. aeruginosa by more than 80% [START_REF] Novoa | A LecA Ligand Identified from a Galactoside-Conjugate Array Inhibits Host Cell Invasion by Pseudomonas aeruginosa[END_REF]. These findings prove that the lectin-GSL interaction is a decisive event in the bacterial invasion. However, the role of specific plasma membrane nanodomains (e.g. lipid rafts) in bacterial adhesion and internalization has to be further explored. Recently, [START_REF] Song | Pseudomonas aeruginosa quorum-sensing metabolite induces host immune cell death through cell surface lipid domain dissolution[END_REF] demonstrated that the P. aeruginosa quorum sensing metabolites can induce a dissolution of ordered membrane domains. This effect was also reconstituted in model membranes -GUVs and SLBs. The role of the lectin-GSL ligation in fine-tuning membrane organization remains unclear. In particular, the implication of the multivalent LecA-Gb3 interaction requires further understanding. (Eierhoff et al., 2014). Actin colocalization with the newly formed invaginations induced by bacteria is negligible. This implies that the key process of bacterial uptake is the LecA-Gb3 interaction, whereas the involvement of actin is insignificant. Scale bars are 10 μm and 2.5 μm, respectively.

II Aims

LecA and StxB are both specific to the plasma membrane GSL globotriaosylceramide (Gb3). However, they are known to induce different signaling events [START_REF] Zheng | The Pseudomonas aeruginosa lectin LecA triggers host cell signalling by glycosphingolipid-dependent phosphorylation of the adaptor protein CrkII[END_REF] and follow different pathways of intracellular trafficking [START_REF] Müller | Gb3-binding lectins as potential carriers for transcellular drug delivery[END_REF]. From the evidences, provided by the studies, reviewed in the introduction, we can hypothesize that these differences arise from a combination of two factors: First, LecA and StxB exhibit distinct binding preferences for Gb3 receptor molecules embedded in differently composed and organized membrane nanodomains. Second, the impact on spatial plasma membrane organization of LecA and StxB varies. Both these factors may determine the signaling and intracellular trafficking of LecA and StxB. Nevertheless, the questions of binding selectivity of LecA and StxB and the membrane reorganization induced by LecA and StxB were not properly addressed yet.

In this work, we propose to test our hypotheses by synthetically reconstituting the lectin-Gb3 interactions in model membranes. Collective effects of membrane lipids control the recognition of receptors by lectins [START_REF] Lingwood | Cholesterol modulates glycolipid conformation and receptor activity[END_REF]. As a result, LecA and StxB may indeed target different membrane domains. The optimal strategy for investigating the binding preferences of lectins to membranes of different compositions is to employ synthetic membrane systems (e.g. GUVs). GUVs provide the ability to control the membrane composition and elucidate the role of specific components. Such cell-sized model membranes are frequently used in combination with fluorescence microscopy techniques for monitoring protein binding. However, usually fluorescence microscopy provides only qualitative information ("binding"/"no binding"). Methods for quantitative binding evaluation require further development. Hence, we aimed to develop a method for quantitative evaluation of lectin binding to GUVs based on fluorescence microscopy. Furthermore, in order to analyze sets of fluorescence microscopy images in a fast and unbiased manner, we aimed to develop software for fast automated image analysis.

Furthermore, we used GUVs with ternary mixtures of DOPC, cholesterol and SM. By varying the ratios between these three lipids we were able to control the membrane order of the lipid bilayer. Moreover, GUVs exhibiting phase separation into Lo and Ld phase domains were produced. Furthermore, to elucidate the role of the Gb3 ceramide structure in lectin-Gb3 binding, we functionalized GUVs with Gb3 species having distinct fatty acyl chains (provided by our collaborators, the group of Prof. Daniel Werz, TU Braunschweig). In combination with the previously developed approach for quantitative evaluation of lectin binding, we aimed to analyze the binding preferences of StxB and LecA to the Gb3 receptor species incorporated in GUVs of different lipid compositions.

In the last part of the thesis, we aimed to study the membrane reorganization induced by LecA and StxB. Rearrangement of plasma membrane nanodomains is necessary for various pathogens to promote infection. In particular, several bacteria utilize lectins to deform membranes and/or initiate downstream signaling events. In this context, we mimicked plasma membrane organization on the microscopic scale using phase-separated SLBs. We, then, applied lectins to such model membranes and visualized membrane rearrangements over time using fluorescence microscopy, with the aim to follow and characterize the impact of StxB and LecA on the membrane organization of phase-separated SLBs.

Remarkably, we demonstrated that StxB and LecA have an opposite impact on SLB organization. StxB induced the formation of new Lo domains, whereas LecA dissolved Lo domains and induced complete membrane mixing. We hypothesized that membrane reorganization can originate from the geometry and valence of lectins. In order to understand the role of the specific binding sites of LecA, we used the commercially available lectin RPL α-Gal. This protein exists as a homodimer in aqueous solution and resembles a dimeric version of LecA with two Gb3-binding sites in total. Applying this lectin to the SLB, we aimed to elucidate the impact of LecA valence and geometry on the membrane reorganization.

The LecA-Gb3 interaction is decisive for the P. aeruginosa adhesion to the host cell membrane. It was reported that P. aeruginosa induces rearrangements of the plasma membrane in order to facilitate the infection [START_REF] Kierbel | Pseudomonas aeruginosa exploits a PIP3-dependent pathway to transform apical into basolateral membrane[END_REF][START_REF] Song | Pseudomonas aeruginosa quorum-sensing metabolite induces host immune cell death through cell surface lipid domain dissolution[END_REF].

Here we aimed to investigate the role of LecA-Gb3 interaction in the membrane organization rearrangement induced by P. aeruginosa. We applied live P. aeruginosa to SLBs and followed the subsequent membrane reorganization. Remarkably, we found that LecA, coupled to the bacterial surface, in addition to allowing an efficient bacterial adhesion, also plays a key role in the Lo domain dissolution. In order to confirm the key role of LecA for the lipid bilayer rearrangement process, we also used a LecA-deficient mutant of P. aeruginosa (ΔLecA). In the absence of LecA, Lo domain dissolution did not occur.

III Results and discussion

1. Quantitative evaluation of lectin-Gb3 binding efficiency using confocal microscopy [START_REF] Sych | GUV-AP: multifunctional FIJI-based tool for quantitative image analysis of Giant Unilamellar Vesicles[END_REF], for complete article see Chapter VII

Model membranes are widely used to evaluate the binding of proteins to membranes of different compositions. In particular, GUVs are often employed to address the question of lectin binding to the glycan moieties of glycolipids or glycoproteins incorporated into the GUV membrane. The binding of numerous lectins has been investigated with GUVs spiked with glycosylated components (Arnaud et al., 2013;Chinnapen et al., 2012;Römer et al., 2007;[START_REF] Villringer | Lectin-mediated protocell crosslinking to mimic cell-cell junctions and adhesion[END_REF]. GUVs are considered an ideal system for studying the binding of StxB and LecA to lipid bilayers of different compositions. Nevertheless, till now, binding studies with GUVs have been limited to qualitative information (binding/no binding). In order to provide quantitative results, we developed an approach for quantitative analysis of the lectin-Gb3 binding on GUVs imaged with confocal microscopy [START_REF] Sych | GUV-AP: multifunctional FIJI-based tool for quantitative image analysis of Giant Unilamellar Vesicles[END_REF]. This approach enables comparative studies of lectin binding efficiencies to the GUVs of different lipid bilayer composition and organization. Furthermore, we developed a FIJI-based macro for fast and unbiased automated processing of GUV images [START_REF] Sych | GUV-AP: multifunctional FIJI-based tool for quantitative image analysis of Giant Unilamellar Vesicles[END_REF] We tested the macro and validated the quantification approach using StxB binding to homogeneous or phase-separated GUVs of different lipid compositions. As expected, StxB binding to GUVs was allowed by introducing Gb3 receptor molecules (extracted from porcine brain, also referred to as "wild type" mixture, from now on -Gb3-mix) to the bilayer. In phase-separated GUVs (figure 18A), the Gb3-mix can be recognized by StxB exclusively in liquid-ordered (Lo) domains. The localization of StxB on GUVs can also be represented by circular profiles of fluorescence intensity (figure 18 B). The profiles confirm that Lo domains, in which the intensity of Bodipy is low, host StxB (red), whereas the binding of StxB to Ld domains enriched in Bodipy is negligible. Furthermore, from this image we extracted the mean fluorescence intensity of the protein bound to the GUV (𝐵𝑜𝑢𝑛𝑑 𝑝𝑟𝑜𝑡𝑒𝑖𝑛). Additionally, we extracted the mean fluorescence intensity of the protein that is freely dissolved in the solution outside of the GUV (𝐹𝑟𝑒𝑒 𝑝𝑟𝑜𝑡𝑒𝑖𝑛). Based on these two values we evaluated the binding of the proteins to the GUV by the following expression: 𝜀 = 𝐵𝑜𝑢𝑛𝑑 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 -𝐹𝑟𝑒𝑒 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝐹𝑟𝑒𝑒 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 ; We named this value relative binding efficiency. It is "0" when no binding occurred. The positive values of the binding efficiency denote the extent of protein binding to the GUV. For example, in figure 18B, the circular profile of StxB shifts from 0 in the Ld domain (no binding) to 2.5 in the Lo domain. In fact, this value of 2.5 represents the fluorescence signal contrast of the bound protein over the free protein and, with proper calibration, can be converted into the actual number of protein molecules bound to the GUV [START_REF] Weinberger | Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes[END_REF]. This approach can be used to compare the binding efficiency of the same protein to GUVs of different compositions.

We validated this approach using homogeneous GUVs spiked with different amounts of Gb3 receptors. We prepared GUVs containing 0, 2.5, 5 and 10 mol % of Gb3, and recorded their images with confocal microscopy (figure 19 A-D). As expected, the binding efficiency increased together with the amount of Gb3 in the membrane (figure 19E). The membrane without Gb3 was not recognized by StxB and the binding efficiency to such GUV was calculated as zero.

Still, the method has a few limitations. Since it is based on the ratio between the bound protein and free protein in solution, the concentration of the protein in the solution must be kept the same for all experimental conditions used for comparison. The selection of an appropriate concentration is a very crucial task. If the concentration is selected too low, only a small amount of protein will be free, which will complicate the evaluation of the "free protein" value. On the other hand, a too high concentration of protein can create a problem with the binding detection, as the free protein in this case will produce a too high fluorescence signal. Furthermore, a quantitative comparison of different proteins is not possible. The method is not yet calibrated to be able to estimate the binding constants of the proteins for the membrane. Nevertheless, this method allows the comparison of lectin binding to GUVs of different lipid compositions. Moreover, protein binding to plasma membrane-derived liposomes (giant plasma membrane vesicles) or to spherical cells can also be examined. Moreover, one of the most important features of the macro is its capability to detect and separately process Lo and Ld domains in phase-separated vesicles.

Selective recognition of Gb3 species by StxB and LecA in model

membranes of different membrane order (Schubert T, Sych T et al., manuscript in preparation), for complete article see Chapter VII

LecA and StxB are both specific to the α-galactose residue of Gb3. However, we found that they segregate when applied simultaneously to the plasma membranes of live cells. They form spatially separated microdomains at the plasma membrane and inside the tubular invaginations of HeLa cells. Furthermore, in polarized epithelial cells (MDCK-II), LecA binds more efficiently to the basolateral (BL) membrane than to the apical (AP) whereas StxB prefers to bind the AP membrane. Remarkably, the primary cilium is targeted by StxB exclusively. Such selectivity may originate from the binding preferences of lectins to Gb3 incorporated in membrane domains of different composition and order. It is known that AP and BL membranes differ in lipid content and membrane order [START_REF] Gerl | Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane[END_REF][START_REF] Ikenouchi | Lipid Polarity Is Maintained in Absence of Tight Junctions[END_REF]. The AP membrane is more ordered and contains more sphingolipids, whereas the BL membrane is less ordered and enriched in phospholipids. Moreover, the structure of the Gb3 ceramide (namely its fatty acyl chain length and saturation degree) can alter the recognition of the carbohydrate moiety by lectins. In addition, the Gb3 species with different ceramides may sort selectively to AP or BL membranes which also can render the binding preferences of the lectins.

In this work, we reconstituted the binding of LecA and StxB to Gb3 in model membranes (GUVs) of different compositions. We used homogeneous and phase-separated GUVs spiked with Gb3 receptor molecules. Furthermore, we examined the influence of the Gb3 molecular structure on the binding preferences of StxB and LecA. The Gb3 species were synthesized and provided to us by our collaborators -the group of Prof. Daniel Werz, TU Braunschweig. We tested Gb3 species with lignoceric (Gb3-24:0) and nervonic (Gb3-24:1) fatty acyl chains. We also compared Gb3 species with distinct fatty acids to the Gb3-mix. Additionally, we used a synthetic function-spacer-lipid construct Gb3-FSL-DOPE (Gb3-FSL). This construct consists of the sugar moiety of Gb3 linked to the glycerophospholipid DOPE via a poly-ethylene glycol spacer (Blake et al., 2011). This construct is purely artificial; however, it gives an interesting insight into how the modification of the lipid backbone can alter the behavior of glycolipids in the lipid bilayer.

Initially, we used GUVs composed of a DOPC/chol/Gb3 mixture (65/30/5). We quantified the relative binding efficiency of either StxB or LecA to such GUVs. We compared the binding of LecA or StxB to different Gb3 species incorporated into DOPC/chol membranes. Though the concentration of Gb3 receptor molecules was always kept constant, the binding efficiencies of LecA and StxB were varying. Interestingly, the most efficient binding was observed with Gb3-FSL. This may be caused by the altered exposure of the Gb3-FSL carbohydrate due to the presence of the linker. Otherwise, for both LecA and StxB, the binding efficiencies to Gb3-mix, Gb3-24:0 and Gb3-24:1 in DOPC/chol GUVs were similar (figure 20). In homogeneous membranes a very important factor is neglected, namely the sorting of Gb3 receptor molecules. In the plasma membrane, GSLs are considered to associate with membrane nanodomains rich in sphingolipids and cholesterol (i.e. Lo-like domains). Although, we reconstituted Lo and Ld domains in separate GUVs, it is important to study the binding of lectins in phase-separated GUVs, where both Lo and Ld are present. We prepared phase-separated GUVs of a DOPC/SM/chol/Gb3 (37.5/37.5/20/5) composition. In such phase-separated liposomes, Ld domains are mostly composed of DOPC and Lo domains consist of a SM/chol mixture. First, we studied the lectins binding to the Gb3-mix (figure 20). Due to their association to SM and chol, the majority of the Gb3-mix molecules are known to incorporate into Lo domains. Hence, only Lo domains can be recognized by Gb3-binding lectins. Indeed, StxB-Gb3-mix binding, reconstituted in phase-separated GUVs, was exclusive to Lo domains, whereas the binding efficiency of StxB to the Ld membrane was zero. However, remarkably, LecA recognized some Gb3-mix receptor molecules in Ld domains with a binding efficiency between 1 and 2. This indicates that some Gb3 species can incorporate into Ld domains, but they cannot be recognized by StxB.

In contrast to the Gb3-mix, the artificial Gb3-FSL based on DOPE clearly demonstrated a preferential partition to Ld. Both LecA and StxB bound very efficiently to Ld domains spiked with Gb3-FSL. Nevertheless, some Gb3-FSL was present in Lo domains and recognized by both LecA and StxB.

Further, we compared the recognition of Gb3-24:0 and Gb3-24:1 species by LecA and StxB. The preferential partition of the Gb3 isoforms in phase-separated liposomes is unknown to date. Unfortunately, it is not possible to directly visualize the partition of such Gb3 species. Attempts were made to directly label the Gb3 molecules with a fluorophore (Patalag et al.), but the introduction of the fluorescent moiety clearly altered the incorporation preferences of Gb3.

Gb3-24:1 was recognized by both LecA and StxB in Ld and Lo domains. Hypothetically, Gb3-24:1 should preferentially incorporate into Ld domains due to its monounsaturated fatty acyl chain. However, binding of both lectins to the Lo domains was more efficient. This means that either the hypothesis of Gb3-24:1 preference to Ld is wrong or that Gb3-24:1 can be better recognized by lectins when incorporated iton Lo.

The Gb3-24:0 in phase-separated vesicles showed an even more interesting behavior. The binding of either LecA or StxB to Ld domains was negligible. Moreover, StxB could not recognize Gb3-24:0 in Lo domains, whereas LecA bound to Gb3-24:0 in Lo quite efficiently. Clearly, Lo domains contained Gb3-24:0 molecules. Whether Gb3-24:0 can incorporate into Ld or incorporates exclusively into Lo is yet unclear. Even more puzzling is the inability of StxB to recognize Gb3-24:0 in the Lo domains of phaseseparated GUVs. Previously, we showed that StxB binds to Gb3-24:0 incorporated in GUVs composed of DOPC/chol. Interestingly, according to Schütte et al. (Schütte et al., 2015) and our observations presented in the supplementary information, StxB can bind to Gb3-24:0 in Lo phase domains of SLBs. The recognition of the Gb3-24:0 in the phase separated SLBs can be explained by the impact of the solid support is drastic enough to enable StxB binding, whereas in absence of the substrate, in phase-separated GUVs, Lo domains cannot be recognized. This observation again stresses the importance of the interleaflet coupling in the lipid bilayer.

We aimed to resolve the binding preferences of StxB and LecA to the AP and BL membranes of the epithelial cells. As the AP membrane is more ordered and enriched in SM and chol, we mimicked it with the GUVs composed of SM/chol (2/1). The less ordered BL membrane enriched in phospholipids we mimicked with pure DOPC. In fact, here we mimic AP and BL membranes with Lo and Ld -phase domains respectively. First, we explored the binding of LecA and StxB to of Gb3-mix (5 mol %) added to such GUVs. Remarkably, StxB clearly preferred binding to Gb3-mix incorporated in membranes rich in SM and chol, whereas the binding efficiency of StxB to DOPC/Gb3mix vesicles is very low (figure 21). LecA is less selective and binds equally efficient to GUVs composed of DOPC/Gb3-mix and to GUVs composed of SM/chol/Gb3-mix. This effect is even more drastically pronounced when Gb3-mix is replaced by Gb3-24:0 : StxB did not bind at all to GUVs composed of DOPC/Gb3-24:0, whereas the binding of StxB to SM/chol/Gb3-24:0 was concluded. Again, LecA bound to both types of GUVs with equal efficiency (figure 21).

Clearly, the lectins have preferences to membrane domains of different order. StxB prefers highly ordered membrane domains (i.e. Lo). Cholesterol seems to be particularly important for the recognition of Gb3-24:0 by StxB, since StxB binds efficiently to the GUVs composed of DOPC/chol/Gb3-24:0. On the contrary, LecA is less selective and also binds efficiently to Gb3 species incorporated into less ordered membrane domains (i.e. Ld).

Thereafter, using our knowledge about the binding preferences of StxB and LecA, we succeeded to rebuild the spatial segregation of StxB and LecA on the membrane surface of synthetic GUVs. For that, we used phase-separated GUVs spiked with two Gb3 species: Gb3-24:0 and Gb3-FSL.

All in all, understanding the binding selectivity of StxB and LecA, we propose to use these two lectins in combination as a comparative plasma membrane stain to evaluate the plasma membrane order.

Impact of the binding of lectins to

Gb3 on the membrane organization in Supported lipid bilayers (Sych et al., manuscript in preparation), for complete article see Chapter VII

The direct visualization of the plasma membrane reorganization in live cells is a very challenging task. Even with the recent improvements in the spatio-temporal resolution of the fluorescence microscopy techniques, the imaging of membrane nanodomains in live cells is very complex. As the solution, the approach of reconstitution lipid nanodomains in model membranes has been successfully employed. It was applied to resolve the mechanisms of lipid and protein clustering in the lipid bilayer. In this work, we mimicked the plasma membrane organization with phase separated supported lipid bilayer (SLB).

Phase separation into liquid-disordered (Ld -composed of DOPC) and liquid-ordered (Lo, composed of SM and chol) resembles the heterogeneity of the plasma membrane on the microscopic scale. We spiked the lipid bilayer with 5 mol % of Gb3 receptor molecules and, furthermore, applying lectins to such lipid bilayer, followed the rearrangement of the lipid domains in the SLB.

We produced a SLB with DOPC/chol/SM/Gb3-mix (37.5/20/37.5/5) composition. The SLB exhibited phase separation at room temperature. We visualized the phase separation using the lipophilic membrane marker βBodipy -C12 -HPC (Bodipy) which localizes preferentially into Ld domains. Furthermore, we applied lectins and imaged the bilayer over time with wide field (HILO) fluorescence microscopy.

StxB, applied to the lipid bilayer, binds preferentially to the Lo domains (figure 21). This is perfectly in line with the observations on GUVs. However, during the first few minutes of incubation, StxB-rich clusters appear in the Ld domain (figure 21). Simultaneously, Bodipy-depleted microscopic membrane domains appear in Ld and co-localize with clusters of StxB. We suggest that these Bodipy-depleted domains are new Lo domains induced by efficient Gb3 clustering by StxB. Moreover, we suggest that together with Gb3, such clusters contain SM and chol (despite the phase separation, low concentrations of these lipids are still present in Ld-domains). The StxB-induced membrane reorganization was studied in the context of StxB uptake initialization. In particular, Windschiegl et al. (Windschiegl et al., 2009) characterized the SLB reorganization induced by StxB using atomic force microscopy. According to their findings, StxB binding to SLB was shown to lead to efficient StxB cluster formation in homogeneous and phase-separated SLBs. Also, Safouane et al. (Safouane et al., 2010) showed the ability of StxB to induce phase separation in originally non-phase separated GUVs composed of DOPC/SM/chol/Gb3. These findings are perfectly in line with our observations suggesting that StxB acts as an efficient clustering agent.

LecA, also specific to Gb3 has different geometry and valence in comparison to StxB. It has only 4 binding sites. Its two pairs of binding sites are arranged to point in opposite directions. This enables LecA to cross-link membranes [START_REF] Villringer | Lectin-mediated protocell crosslinking to mimic cell-cell junctions and adhesion[END_REF]. Moreover, LecA was found to highly accumulate at such interfaces. We applied 200 nM of LecA to phase-separated SLBs in order to study its impact on membrane organization. We used Bodipy-labeled SLBs of identical composition (DOPC/SM/chol/Gb3 -mix (37.5/20/37. Importantly, the fluorescence intensity in these newly formed clusters is much higher than the fluorescence intensity of LecA bound to Lo domains, suggesting a condensed enrichment of LecA. These LecA clusters co-localize with domains characterized by high intensity of Bodipy (figure 22a -purple arrowhead). These new domains form and grow simultaneously with the LecA clusters. We suggest that these new domains are composed of two or several lipid bilayers assembled in stacks with the LecA sandwiched in between the lipid bilayers. We named these structures -membrane multilayers. We suggest that LecA sequesters the lipids from the SLB in order to compose such multilayers. The formation of the multilayers depletes the SLB in lipid material and consequently the SLB breaks down and large membrane defects emerge (figure 22a -orange arrowhead). The area of the membrane defects increase over time and after 1 hour of incubation with LecA, the SLB is partially destroyed. To further characterize the membrane multilayers, we quantified the fluorescence intensity of the membrane marker Bodipy in the multilayers and compared it to the fluorescence intensity of the Bodipy in Ld domains. A two-fold increase of Bodipy fluorescence intensity would indicate the formation of a membrane stack consisting of two lipid bilayers. However, we found that the membrane multilayers appear only approximately 1.5 times brighter than unilamellar Ld domains (figure 22e), which suggests that membrane multilayers have a distinct lipid organization that differs from the lipid organization in Ld or in Lo. Hence the amount of Bodipy in membrane multilayers is different. This question can be resolved by exploring the membrane order of SLBs at different time points of LecA-induced membrane reorganization.

We examined the membrane order using the environmentally sensitive lipophilic probe Nile Red 12 S (NR12S) in combination with the confocal ratiometric imaging. We found that membrane order of multilayers is indeed higher in comparison to the Ld domains, but lower than in Lo domains. This indicates that membrane multilayers are formed out of already mixed membrane, suggesting the importance of the Lo domains dissolution for the membrane multilayer formation.

In order to further understand the impact of LecA on membrane organization, we investigated the membrane multilayer formation and Lo domains dissolution, separately. At first, we applied LecA to non-phase separated SLB composed of DOPC/chol/Gb3-mix (65/30/5). Interestingly, in such SLB, the membrane multilayer formation together with complete membrane disintegration occurred very fast. Only after 15 min of incubation with 200 nM of LecA, the SLB was completely destroyed.

Separation of Lo domains dissolution from membrane multilayer formation is a more complicated task. We used the engineered protein RPL αGal (RPL). The monomer of this protein is similar to the monomer of LecA. However, it is stable as a dimer in solution.

The application of this protein to SLB leads to a partial dissolution of the Lo domains without the formation of membrane multilayers. Based on these findings, we propose an interpretation for the roles of the different LecA binding sites in the lipid bilayer reorganization (Figure 23). For the dimeric LecA analogue, multilayers do not form whereas the Lo domains dissolution occurs. This suggests that two binding sites pointing in one direction (as depicted on figure 23a -1 and 2 or 3 and 4) are sufficient for Lo domains dissolution. In contrast, the complete tetrameric structure is required for the formation of membrane multilayers. The exact mechanism of phase mixing is not clear yet and may be resolved using SLBs of more specific composition (specific DOPC/SM/chol ratios and specific structures of Gb3 fatty acyl chains) combined with studies of line tension at the phase domains borders. 4. Impact of P. aeruginosa on membrane organization studied in Supported lipid bilayers (Sych et al., manuscript in preparation), for complete article see Chapter VII LecA is the surface protein of P. aeruginosa and is of crucial importance for bacterial adhesion and internalization. In this work, we decipher the role of LecA in the membrane reorganization induced by P. aeruginosa. We investigated the membrane reorganization induced by two bacterial strains -P. aeruginosa strain PAO1 (wild type) and the P. aeruginosa strain deficient in LecA (ΔLecA). We reconstituted bacteriamembrane interactions in phase separated SLB of the same lipid composition as in the previous chapter (DOPC/SM/chol/Gb3 -(37.5/20/37.5/5)). However, since both bacterial strains were tagged with GFP, we changed the membrane marker. Here, we used Texas Red -DHPE that associates to the Ld domains.

Applied to the SLB, the wild type bacteria attach to the lipid bilayer and form aggregates (figure 24a -white arrowheads). These aggregates grow over time and accumulate more bacteria. On the contrary, ∆LecA strain bacteria do not form aggregates and the attachment of bacteria to the SLB is significantly reduced. Moreover, the ∆LecA bacteria can detach from the SLB (figure 24b -cyan arrowhead). The same reduction in attachment and absence of aggregation were observed when we applied both bacteria strains to SLB without Gb3 receptor (DOPC/chol/SM (37.5/20/37.5)). This clearly suggests that P. aeruginosa relies on the specific interaction of its proteins with the Gb3 membrane receptor for efficient adhesion and aggregation. Moreover, LecA is important for P. aeruginosa adhesion and requires the Gb3 receptor in the membrane.

We also examined the preferential locations of the bacterial attachment. Furthermore, we detected a local increase of the membrane marker signal at the spots of bacterial attachment (figure 24a -white circle, 24e). We suggest that bacteria sequester lipids out of the SLB to wrap themselves in membrane. This process seems to be analogous to the "lipid zipper" formation (Eierhoff et al., 2014) where bacteria by wrapping themselves with the membrane initialize their internalization. Here, however, the internalization is restricted by the SLB substrate, hence bacteria accumulate lipids on their surface. Importantly, LecA-Gb3 interaction is again crucial for this process. ∆LecA bacteria do not accumulate lipid material on their surface over time (figure 24b white circle).

Most importantly, bacteria induce dissolution of the Lo domains. The Lo domains decrease in size over time and some of them disappear (figure 24a -blue and green arrowheads). In the absence of LecA-Gb3 interaction, the SLB organization remains stable. Previous studies with polarized epithelial cells by Kierbel et al [START_REF] Kierbel | Pseudomonas aeruginosa exploits a PIP3-dependent pathway to transform apical into basolateral membrane[END_REF] also showed an extensive membrane reorganization induced by P. aeruginosa. Furthermore, recent work of Song et al. [START_REF] Song | Pseudomonas aeruginosa quorum-sensing metabolite induces host immune cell death through cell surface lipid domain dissolution[END_REF] suggested plasma membrane domains dissolution mediated by bacterial quorum sensing metabolites.

In our work, we observed that the Lo domains dissolution coincides with the bacterial aggregation mediated by bacteria-Gb3 interaction in absence of the cellular machinery. Moreover, we highlighted the importance of the lectin LecA in the dissolution of the Lo domains induced by bacteria.

IV Conclusions

Human pathogens are equipped with several independent mechanisms that ensure the successful binding and internalization into the host cell. Predominantly, pathogens initiate the uptake at the plasma membrane by specific targeting of various plasma membrane components. Particularly important are plasma membrane GSLs that can be recognized by lectins present in pathogens. Lectins bind to GSLs and induce membrane reorganization in order to promote infection. In this work, we explored the impact of bacterial lectins LecA and StxB on the membrane organization.

At first, we investigated the binding selectivity of StxB and LecA. Both proteins share a common receptor -globotriaosylceramide (Gb3). However, LecA and StxB exhibit preferences to Gb3 incorporated in membrane domains of different composition and organization. In the first part of my PhD project, we studied the binding preferences of LecA and StxB to Gb3 in model membranes (GUVs).

We developed a method for quantitative characterization of lectin binding to GUVs based on confocal fluorescence microscopy. In order to evaluate the binding, we calculate the mean fluorescence intensity of the bound proteins at the GUV rim and normalize it to the mean fluorescence intensity of the soluble protein. This provides a numerical evaluation of the imaging contrast which in turn represents the amount of proteins bound to the GUV. We named such value relative binding efficiency. Furthermore, in order to process numerous images with the GUVs in a fast and unbiased manner, we developed a FIJI-based macro for automated image processing. This approach is valid for the comparison of the binding efficiencies of the protein to GUVs of different compositions.

This high throughput method offers fast and unbiased analysis of the lectin binding to GUVs. However, it has several limitations. First, it does not provide the information on the binding affinity of the protein to the membrane. Furthermore, it allows direct quantifying of the amount of protein which is bound to the GUV only with proper calibration. Second, the value of relative binding efficiency depends on the concentration of the protein in the solution; the careful selection of the proper concentration is the responsibility of the user. Third, for now, the developed macro is limited to the GUVs or other round-shaped objects. Despite these limitations, the method was very successfully applied to explore the binding preferences of lectins to Gb3 incorporated in membranes of different organization.

In the second part of the project, we used GUVs for monitoring the LecA and StxB binding to Gb3. We controlled the membrane order of such GUVs by adjusting their composition. Also, we produced "phase separated" GUVs that contain spatially segregated domains of distinct membrane order. Moreover, we explored the lectin binding preferences to several Gb3 species, namely Gb3-24:0, Gb3-24:1 and to the artificial construct Gb3-FSL-DOPE. We found that StxB clearly prefers Gb3 receptor molecules incorporated in liquid-ordered (Lo) domains composed of SM and chol. LecA demonstrated a less selective behavior. Though it binds preferentially to Lo domains, it also binds to less ordered domains (e.g. liquid-disordered, Ld). These binding preferences of StxB and LecA were especially pronounced for the Gb3 species with a 24:0 fatty acyl chain.

The synthetic membrane systems have proven to be a very useful tool for the characterization of lectin -Gb3 interactions. The investigation of the LecA and StxB binding to GUVs allows elucidating the influence of composition and organization of the lipid bilayer on the lectin binding. In particular, we determined that the content of cholesterol in the membrane can influence lectin binding drastically. Moreover, the length and the saturation degree of the Gb3 fatty acyl chain alters the recognition of the carbohydrate group by lectins. Most importantly, the lipid order of the membrane plays crucial role in the lectin recognition and binding.

Our studies of LecA and StxB binding to live cells also revealed differences in the lectin behavior. In polarized epithelial cells, StxB preferentially targets the more ordered apical membrane, whereas LecA binds more efficiently to the less-ordered basolateral membrane. Moreover, the primary cilium was exclusively targeted by StxB. Since the membrane order of the primary cilium is unknown yet, we suggest that it mainly harbors membrane ordered domains. Most importantly, we succeeded to reconstitute the spatial segregation and binding selectivity of LecA and StxB in phase-separated GUVs.

In the last part of the project we studied the membrane reorganization induced by both LecA and StxB. By binding to Gb3, lectins induce the rearrangement of membrane nanodomains. As the studies of such nanodomains in living membranes are a very complicated task, we reconstituted membrane domains on the microscopic scale using supported lipid bilayers (SLBs).

We prepared SLBs that exhibit phase separation into liquid-ordered (Lo) and liquiddisordered (Ld) domains. By application of lectins, we observed changes in the lateral organization of the Lo domains. We found, that StxB shrinks existing Lo domains, but also induces novel Lo domains. This confirms that StxB is an efficient clustering agent. On the contrary, LecA induces the complete dissolution of Lo domains. Moreover, LecA induces the formation of membrane multilayers with LecA clusters accumulated in between the lipid bilayers that assemble in stacks. Despite both of these processes occur simultaneously, we proved by a valence reduction of LecA that Lo domains dissolution can occur independently. Moreover, the analysis of membrane order in membrane multilayers showed that they are formed out of membrane of "intermediate order" between Lo and Ld which suggests that Lo dissolution is necessary for "multilayers" formation.

Furthermore, we studied Lo domains dissolution by bacterium P. aeruginosa that expresses LecA. We found that the bacteria bind to SLB and induce Lo domains dissolution. Moreover, bacteria accumulate lipids from the SLB on their surface. Presumably, this is the analogue of lipid zipper formation but without the possibility to bend the membrane. We confirmed the importance of LecA for bacterium adhesion to SLB by using a LecA deficient strain. The ΔLecA bacteria do not bind well to SLBs and do not induce Lo domain dissolution. Lipid accumulation at the bacterial surface is not possible in the absence of LecA as well.

Supported lipid bilayers are very practical for the characterization of the lipid-lipid interactions on the large scale. Furthermore, the membrane deformation is constrained by the support; hence it is possible to study the lipid sorting and protein clustering explicitly. SLBs are widely used for the synthetic mimicry of the plasma membrane nanodomains (so-called lipid rafts). However, in these systems such domains are much bigger in size and temporally stable. Despite this, such system provides very interesting insights on how lectins and bacteria behave at the membranes of different composition and organization.

The impact of bacteria on the organization of the ordered domains is a very important observation. In native plasma membranes, the nanodomains of high order (lipid rafts) are implicated in various modes of signaling and initiation of endocytosis. Bacteria and viruses target lipid rafts and they exploit the machinery present in lipid rafts in order to promote infection. We believe that the Lo-domains dissolution, described in this work, may be common for numerous bacteria as a vital element of the host-pathogen interaction. Moreover, the Lo-domains dissolution driven by the interaction of the bacterial lectin with the plasma membrane GSL is reported for the first time.

V Outlook

In this work, we demonstrated several features of the interactions of the bacterial lectins with the GSL Gb3, reconstituted in model membranes. Although, some mechanisms of the StxB and LecA interactions with the membrane are now resolved, several open questions require further attention.

The sorting of the Gb3 species to the membrane domains of different composition and order is still elusive. Yet, we can recognize the presence of the Gb3 molecules in the lipid bilayer exclusively using the Gb3-binding lectins. However, lectins may induce the redistribution of the Gb3 in the membrane, hence the elucidation of the initial partition of the Gb3 species using lectins is challenging. The alternative strategies for the direct visualization of Gb3 molecules in the membrane must be employed. Our collaborators at TU Braunschweig (the group of Prof. Daniel Werz) developed the Bodipy-labeled synthetic Gb3 species. The unique feature of these constructs is that the fluorophore is attached to the sugar moiety; hence the introduction of such fluorophore should not influence drastically the incorporation of Gb3 in the lipid bilayer. However, the fluorophore coupled to the oligosaccharide group might interfere with the lectin recognition of Gb3. We aim to test in future the partition of such labeled Gb3 molecules in phase-separated model membranes, as well as the possibility to employ this constructs for the experiments with Gb3-binding lectins.

The membrane reorganization induced by LecA requires further characterization. The mechanism of the dissolution of the Lo domains is not yet resolved. The membrane reorganization studies in the SLBs that contain only single Gb3 species can give an interesting insight on how the specific interaction of LecA and Gb3 induces such extensive collective phenomenon in the lipid bilayer. Moreover, it can resolve the implications of different Gb3 species in the Lo domain dissolution induced by LecA. Furthermore, application of the Gb3 species tagged with the fluorophore can provide the information on the redistribution of Gb3 during the Lo dissolution. Also, the application of the molecular dynamic simulation can reveal very interesting details of this process. The formation of the membrane multilayers can be further characterized with the Atomic Force Microscopy. This method can provide information on the position of LecA at the lipid bilayer and inside the membrane multilayers as well as on the exact number of lipid bilayers in the multilayer.

The studies of the membrane order modifications by bacteria have to be further expanded to the investigation of the plasma membrane order during the bacterial uptake in live cells. The membrane order studies using the environmentally sensitive membrane markers can be employed. Using this techniques, the membrane order of the tubules formed by the "lipid zipper" in GUVs and live cells can be addressed.

VII Publications and manuscripts

In the following, all publications and manuscripts that I contributed to in the context of this thesis are listed and enclosed Biophysical Journal,116(3), pp.294a-295a.

First author publications and manuscripts

Lipid self-assembly and lectin-induced reorganisation of the plasma membrane

Taras Sych 1,2,3,4 , Yves Mély 4 and Winfried Römer

Abstract

The plasma membrane represents an outstanding example of self-organisation in biology. It plays a vital role in protecting the integrity of the cell interior and regulates meticulously the import and export of diverse substances. Its major building blocks are proteins and lipids, which self-assemble to a fluid lipid bilayer driven mainly by hydrophobic forces. Even if the plasma membrane appearsglobally spoken -homogeneous at physiological temperatures, the existence of specialised nano-to micrometer-sized domains of raft-type character within cellular and synthetic membrane systems has been reported. It is hypothesized that these domains are the origin of a plethora of cellular processes, such as signalling or vesicular trafficking.

This review intends to highlight the driving forces of lipid self-assembly into a bilayer membrane and the formation of small, transient domains within the plasma membrane. The mechanisms of selfassembly depend on several factors, such as the lipid composition of the membrane and the geometry of lipids. Moreover, the dynamics and organisation of glycosphingolipids into nanometer-sized clusters will be discussed, also in the context of multivalent lectins, which cluster several glycosphingolipid receptor molecules and thus create an asymmetric stress between the two membrane leaflets, leading to tubular plasma membrane invaginations.

1) The plasma membrane

The plasma membrane physically separates the cytoplasm of living cells from the extracellular environment and hence maintains the physical integrity of the cell. It acts as a barrier, which is selectively permeable to ions and organic molecules, and regulates transport processes into and out of the cell (1). The plasma membrane is involved in a multitude of cellular processes, such as signalling and adhesion, amongst others (2,3). It also helps to hold the cytoskeleton in place to preserve the cell shape (4). With respect to all these diverse functions, it sounds amazing that the plasma membrane represents a lipid matrix with a thickness of only 4-6 nm, with embedded integral and peripheral proteins (5,6). The principles how membrane lipids self-assemble into cell membranes and how lipidlipid interactions lead to the formation of small, transient domains within the membrane will be outlined in the following.

2) Self-organisation of lipids in aqueous solutions

The plasma membrane consists of a complex mixture of components, among which lipids are the major building blocks. Plasma membranes contain a large number of different types of lipids that form a lipid bilayer with an inner (cytosolic) and an outer (extracellular) membrane leaflet (7). Lipids exhibit an impressive diversity of properties and can be classified by their geometry ( 8), as well as by the type of assembly they form in contact with water.

Since lipids contain a hydrophilic (polar head group) and a hydrophobic part (fatty acyl chain) they are considered as amphiphilic molecules. Exposed to water, lipids compact their hydrophobic parts in the energetically most favourable way, forming a hydrophobic core (9). This simple principle is underlying the self-organisation of lipids in an aqueous environment, e.g. in living cells as well as in artificial membrane systems.

As indicated by Israelachvili (10,11), the lipid behaviour in aqueous solution can be easily predicted by taking into consideration the geometrical parameters of its hydrophobic and hydrophilic parts.

These are v, the volume of the nonpolar part, l, the length of the nonpolar part and a 0 , the optimal surface area of the polar head group. The parameters v and l can be simply determined by the lipid size and shape (11,12). The determination of the parameter a 0 is less straightforward. The possible repulsive interactions between head groups and parameters of the aqueous environment (ionic strength, pH) have to be taken into account. This factor is particularly critical for charged lipids, e.g. phosphatidylserine (PS) or phosphatidylglycerol (PG), where the head groups have to be far enough from each other that Coulomb's repulsive forces are equilibrated by lipid-lipid interactions.

One of the potential assembly modes of lipid molecules in water is the formation of spherical micelles (Figure 1) (12). If a spherical micelle is built up of N lipid molecules, the total micelle surface area a M and the volume of the micelle V M can be determined as:

a M = Na 0 = 4πR m 2 V M = Nv = 4/3πR m 3
Hence, the radius of the micelle R m can be expressed as:

R = 3v/a 0
Since R m ≤ l for spherical micelles, the following equation accounts:

v/la 0 ≤ 1/3
The expression of v/la 0 is called the critical packing parameter.

The interconnection between these three parameters determines the geometry of the lipid molecule and its behaviour in water. Using the same logic, the value of the critical packing parameter for a lipid bilayer can be obtained.

Lipids can be classified by their shapes into three major groups (Figure 1):

A) Inverted cone-shaped lipids (v/la 0 < 1/2), which form micellar phases with positive curvature in water. This group comprises mainly lipids with a single fatty acyl chain, such as lysophospholipids. To assemble as spherical micelles, the critical packing parameter of lipids has to be below 1/3, whereas lipids with 1/3< v/la 0 < 1/2 assemble as cylindrical micelles. The relevance of this type of lipids in native cell membranes is most obvious for pore formation (13) or vesicle budding (14).

B) Cone-shaped lipids (v/la 0 > 1), which self-organise in so-called hexagonal micellar phases of negative curvature (in acidic environment). This group of lipids includes mainly unsaturated lipids, which participate in uptake processes where the cell membrane is bended inwards (15).

C) Cylinder-shaped lipids (1/2 < v/la 0 ≤ 1), which assemble into a lipid bilayer that forms a lamellar phase when assembled in stacks. These lipids that are characterized by two fatty acyl chains of different length and saturation degree represent probably the most important group of membrane lipids and form the fluid lipid matrix of the cell membrane. They can assemble into flexible lipid bilayers of liposomes (1/2 < v/la 0 < 1) as well as planar lipid bilayers (v/la 0 ≈ 1).

In addition, factors such as the ion composition of aqueous solutions, pH or temperature can alter the lipid geometric parameters, and as a result, the assembly of lipids (11). A lipid bilayer can undergo solid-liquid phase transition , also referred to as main phase transition or chain-melting transition (16,17). Depending on the lipid, the main phase transition temperature varies from -17 ºC for unsaturated lipids, like dioleoylphosphatidylcholine (DOPC), up to 37 ºC for sphingomyelin (SM) with long saturated acyl chains (18). Glycolipids with sugar moieties have even higher solid-liquid phase transition temperatures (19). Lipid phase transitions have been intensively studied for already more than 50 years (20)(21)(22) using a large variety of techniques, such as X-ray diffraction (23), calorimetry (22), atomic force microscopy (AFM) (24) or fluorescence techniques on different membrane model systems (25,26). The length and saturation degree of the fatty acyl chain determines not only the temperature of the solid-liquid phase transition but also the degree of packing in the liquid phase. Quantitatively, this degree of packing can be described using the so-called acyl chain order parameter. Lipids with a low value of order parameter form a bilayer phase of low order and high mobility, known as liquid-disordered (Ld) phase (7). Lipids that form Ld phases are mainly unsaturated phospholipids (Figure 2). In contrast, bilayers formed of long-chain saturated lipids, like sphingomyelin, adopt a solid-like phase of high order and low mobility. Addition of sterols, such as cholesterol, renders the solid-like membrane more mobile turning it into a liquid phase. This phase is called the liquid-ordered (Lo) phase as the order still remains high. Interestingly, addition of cholesterol enhances acyl chain order in Ld-membranes (7,27,28). A lipid bilayer can contain spatially separated domains of different lipid phases. In binary mixtures composed of lipid species of low melting temperature (e.g. DOPC) and of high melting temperature (e.g. sphingomyelin), the bilayer exhibits spatial phase separation into solid and liquid-disordered phase domains. The temperature is the crucial factor for this process; phase separation can occur only at temperatures in between the main phase transition temperatures of the two lipids. Moreover, the coexistence of all three -Ld, Lo and solid phase -domains is possible by the addition of cholesterol (29). Remarkable is the preference of cholesterol to associate with sphingolipids and partition in sphingomyelin-containing Lo phases (30)(31)(32). Furthermore, phase separation can be reversed by increasing the lipid entropy (e.g. by increasing the temperature).

3) Higher-order organisation of lipids within the plasma membrane

To describe the structure of biological membranes, Singer and Nicolson introduced the fluid mosaic model in 1972 (33,34). According to this model, the cell membrane is considered as a twodimensional liquid arranged as a lipid bilayer with homogeneously distributed proteins. Lipids in a lipid bilayer are in continuous motion. In addition to translational diffusion (microseconds) and rotational movement (picoseconds -nanoseconds), they can flip from one leaflet to the other (milliseconds -seconds), or exhibit undulation movements (milliseconds -seconds), which are induced by transmembrane proteins or cytoskeleton (35)(36)(37). Further studies of biological membranes based on the use of detergents revealed the separation of the so-called detergent-resistant membrane (DRM) fractions, enriched in particular with sphingomyelin and cholesterol, from detergent-soluble membrane (DSM) fractions, enriched with rather unsaturated membrane phospholipids (38). Based on these and other important findings, the "lipid raft" theory was introduced by Simons and Ikonen in 1997 (39). According to this theory, the membrane is no longer a homogeneous fluid, where all components are randomly distributed, but contains highly ordered lipid (micro-) domains stabilized by sphingomyelin and cholesterol (Figure 3A). Lipid rafts are also enriched with (transmembrane) proteins and glycolipids, and are thought to play a key role in cell signalling, uptake and release (40)(41)(42). Further studies of membrane asymmetry revealed even more complicated structures of raft and non-raft membranes (43). Sphingomyelin-enriched rafts form mainly in the extracellular membrane leaflet while the cytosolic leaflet contains in total less than 10 % of all membrane sphingomyelin molecules (44)(45)(46). Consequently, the cytosolic leaflet is less viscous and does not sustain the formation of lipid rafts (47). In general, the cytosolic leaflet is enriched with lipids, such as phosphatidylinositol (PI), phosphatidylethanolamine (PE) and phosphatidylserine (PS). Remarkably, lipid raft-embedded GPI-anchored proteins in the extracellular leaflet interdigitate with PS clusters in the cytosolic membrane leaflet (48). Saturated PC lipids are found in the extracellular membrane leaflet.

The lipid raft theory is in line with data obtained from viral membranes. It was found that the HIV envelope is enriched in host cell sphingolipids and cholesterol, the typical lipid raft components (49).

This suggested that the assembly of HIV Gag proteins at the cytosolic leaflet of the cell membrane induces the accumulation of lipid rafts on the extracellular leaflet, so that the HIV envelope consists mainly of raft-like membrane after virus release (50). This strengthens the significance of lipid rafts as important signalling platforms for diverse cellular processes (6,51).

The size and lifetime of lipid rafts are far below the spatial and temporal resolution of most conventional microscopy techniques, which makes a direct visualisation of these domains in cell membranes nearly impossible (52). Though impressive progress in fluorescence microscopy has been made with the development of advanced techniques that offer high temporal and spatial resolution (52)(53)(54)[START_REF] Ott | Visualization of live primary cilia dynamics using fluorescence microscopy[END_REF](56), membrane nano-domains can hardly be visualised. In synthetic membrane systems, lipids self-assemble into a lipid bilayer mainly in a symmetric manner. PC: phosphatidylcholine; PS: phosphatidylserine; PI: phosphatidylinositol; PE: phosphatidylethanolamine; SM: sphingomyelin.

Plasma membrane organisation can be also explored in less dynamic systems with compositions close to native ones. This type of membrane systems, e.g. giant plasma membrane vesicles (57), plasma membrane spheres, or plasma membrane sheets ( 58) can be extracted from living cells by cell membrane disruption.

For elucidating the role of distinct membrane components, synthetic membrane systems (Figure 3B), such as unilamellar vesicles (59,60), supported (61) or pore-suspending ( 62) lipid bilayers, are employed. The main advantage of synthetic membrane systems is the possibility to adjust the lipid and/or protein content in order to prepare membranes of different properties. These systems are widely used to study the organisation of lipid bilayer phases (e.g. Lo and Ld) (63), membrane pore formation (64) and uptake processes [START_REF] Schubert | How synthetic membrane systems contribute to the understanding of lipid-driven endocytosis[END_REF]. Reconstitution of Lo domains using lipids found in DRMs (e.g.

sphingomyelin and cholesterol, in distinct ratios) enables mimicking some properties of lipid rafts at the microscopic scale. Various cellular processes (e.g. transport across the plasma membrane or induction of signalling events) require local reorganisation of the plasma membrane. For instance, extracellular (e.g. pore forming proteins [START_REF] Phillips | Emerging roles for lipids in shaping membraneprotein function[END_REF][START_REF] Yilmaz | Visualization of lipid membrane reorganization induced by a pore-forming toxin using high-speed atomic force microscopy[END_REF][START_REF] Gilbert | Membrane pore formation at protein-lipid interfaces[END_REF] and cell penetrating peptides [START_REF] Sodt | Molecular modeling of lipid membrane curvature induction by a peptide: more than simply shape[END_REF][START_REF] Maniti | Basic cell penetrating peptides induce plasma membrane positive curvature, lipid domain separation and protein redistribution[END_REF]) and cytosolic (e.g. actin [START_REF] Römer | Actin dynamics drive membrane reorganization and scission in clathrin-independent endocytosis[END_REF][START_REF] Head | Interaction of membrane/lipid rafts with the cytoskeleton: impact on signaling and function: membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling[END_REF], clathrin [START_REF] Saleem | A balance between membrane elasticity and polymerization energy sets the shape of spherical clathrin coats[END_REF], BAR domains [START_REF] Itoh | Mechanisms of membrane deformation by lipid-binding domains[END_REF][START_REF] Johannes | Building endocytic pits without clathrin[END_REF]) components can alter the lipid distribution within the lipid bilayer.

In this review, we focus on the membrane reorganisation driven by specific interactions of carbohydrate binding proteins (so-called lectins) with glycosphingolipids, their host cell receptors.

4) Glycosphingolipids self-organise in small clusters

Glycosphingolipids (GSLs) play crucial roles in intercellular communication and uptake processes, amongst others [START_REF] Johannes | Glycolipids and Lectins in Endocytic Uptake Processes[END_REF]. The GSL content in cell membranes varies from 5% to more than 20% [START_REF] Varki | Essentials of Glycobiology, Cold Spring Harbour[END_REF].

GSLs are mainly found in the extracellular membrane leaflet -the hydrophobic ceramide backbone being embedded in the lipophilic part of the membrane and the carbohydrate moieties being exposed to the extracellular environment [START_REF] Manna | The challenges of understanding glycolipid functions: an open outlook based on molecular simulations[END_REF]. The majority of GSLs is reported to be enriched in lipid rafts (5,39,[START_REF] López | Martini force field parameters for glycolipids[END_REF][START_REF] Fra | Detergent-insoluble glycolipid microdomains in lymphocytes in the absence of caveolae[END_REF][START_REF] Hooper | Detergent-insoluble glycosphingolipid/cholesterol-rich membrane domains, lipid rafts and caveolae[END_REF], where they associate with sphingomyelin and cholesterol. In addition, AFM studies as well as MD simulations have revealed the capability of GSLs to form separate clusters in lipid raftlike membranes [START_REF] Shi | GM1 clustering inhibits cholera toxin binding in supported phospholipid membranes[END_REF][START_REF] Mondal | Molecular level investigation of organization in ternary lipid bilayer: a computational approach[END_REF][START_REF] Koldsø | Lipid clustering correlates with membrane curvature as revealed by molecular simulations of complex lipid bilayers[END_REF][START_REF] Solovyeva | Shiga toxin induces membrane reorganization and formation of long range lipid order[END_REF]. Variations in the length and saturation degree of the fatty acyl chain of the ceramide backbone affect the orientation and partitioning of GSLs in the lipid monolayer [START_REF] Chinnapen | Lipid sorting by ceramide structure from plasma membrane to ER for the cholera toxin receptor ganglioside GM1[END_REF][START_REF] Schütte | Influence of Gb 3 glycosphingolipids differing in their fatty acid chain on the phase behaviour of solid supported membranes: chemical syntheses and impact of Shiga toxin binding[END_REF], and hence the exposure of the carbohydrate group rendering it partially inaccessible for ligands [START_REF] Mahfoud | Fatty acid-dependent globotriaosyl ceramide receptor function in detergent resistant model membranes[END_REF][START_REF] Schütte | 2-Hydroxy fatty acid enantiomers of Gb 3 impact Shiga toxin binding and membrane organization[END_REF].

GSL assemblies can modify locally membrane order and curvature, which is of high significance for various uptake processes. The long ceramide backbones of GSLs are also important elements for transbilayer coupling and communication since they interdigitate the two membrane leaflets [START_REF] Manna | Long-chain GM1 gangliosides alter transmembrane domain registration through interdigitation[END_REF].

5) Lectin-induced glycosphingolipid clustering re-organises the plasma membrane

Besides their natural functions, glycosphingolipids are hijacked by various pathogens (e.g. bacteria and viruses) [START_REF] Rydell | Human GII. 4 norovirus VLP induces membrane invaginations on giant unilamellar vesicles containing secretor gene dependent α1, 2-fucosylated glycosphingolipids[END_REF][START_REF] Ewers | GM1 structure determines SV40-induced membrane invagination and infection[END_REF][START_REF] Eierhoff | A lipid zipper triggers bacterial invasion[END_REF] and pathogenic products (e.g. toxins, lectins) [START_REF] Römer | Shiga toxin induces tubular membrane invaginations for its uptake into cells[END_REF][START_REF] Zheng | The Pseudomonas aeruginosa lectin LecA triggers host cell signalling by glycosphingolipid-dependent phosphorylation of the adaptor protein CrkII[END_REF]. Lectins [START_REF] Sharon | Lectins: carbohydrate-specific reagents and biological recognition molecules[END_REF] can either be soluble, prearranged as a viral capsid or attached to the outer bacterial membrane. They bind to the carbohydrate moieties of glycosphingolipids [START_REF] Aigal | Plasma membrane reorganization: A glycolipid gateway for microbes[END_REF], either by targeting preformed glycosphingolipid domains or by inducing domains by local recruitment of receptor molecules underneath their oligomeric structures [START_REF] Römer | Shiga toxin induces tubular membrane invaginations for its uptake into cells[END_REF]. Thereby, asymmetric stress between the extracellular and cytosolic membrane leaflets can lead to inward bending of the cell membrane and growth of tubular plasma membrane invaginations [START_REF] Römer | Shiga toxin induces tubular membrane invaginations for its uptake into cells[END_REF].

a) Toxins/ lectins act as clustering devices and generate membrane curvature

Based on cellular as well as synthetic membrane systems, it has been demonstrated that oligomeric toxins/ lectins bind to and cluster host cell glycosphingolipids, which leads to the formation of tubular plasma membrane invaginations as endocytic transport intermediates. For instance, the homopentameric B-subunit of Shiga toxin (StxB) from Shigella dysenteriae recognizes specifically the carbohydrate moieties of the GSL globotriaosylceramide (Gb3) on the extracellular membrane leaflet.

Up to 15 Gb3 receptor molecules can interact with one toxin pentamer [START_REF] Johannes | Shiga toxins-from cell biology to biomedical applications[END_REF]. This multivalent binding leads to Gb3 clustering and membrane reorganisation [START_REF] Windschiegl | Lipid reorganization induced by Shiga toxin clustering on planar membranes[END_REF]. Thus, the entropy of the system decreases together with lipid mobility (100), while the membrane order increases locally [START_REF] Solovyeva | Shiga toxin induces membrane reorganization and formation of long range lipid order[END_REF]101). StxB-induced Gb3 clustering modifies the area per lipid in the outer membrane leaflet. This creates asymmetric stress in the bilayer, and consequently a membrane tubule nucleates (102). Such a mechanism is probably in common for the cellular uptake of many oligomeric lectins [START_REF] Römer | Shiga toxin induces tubular membrane invaginations for its uptake into cells[END_REF]103,104). Nevertheless, the mechanism of lectin clustering on the extracellular membrane surface remains debated. Different reports claim that this process is either driven by capillary effect (105), by entropy modification of the lipid bilayer hydrophobic bulk (102,106) or by local lectin-induced curvature (107). However, Pezeshkian et al. (108) suggested that neither of these factors is critical and that multi-molecular lectin clustering is more likely related to membrane fluctuation-induced forces. Multiple carbohydrate binding sites of lectins ensure their efficient binding to GSLs and clustering on the membrane surface to induce cellular signalling and uptake processes. In order to explore the impact of lectin valency and binding site geometry on the induction of cellular processes, distinct carbohydrate binding sites of lectins have been modified by site-directed mutagenesis [START_REF] Römer | Shiga toxin induces tubular membrane invaginations for its uptake into cells[END_REF][START_REF] Windschiegl | Lipid reorganization induced by Shiga toxin clustering on planar membranes[END_REF]103,109), and tailor-made neolectins have been designed (110). In comparison to the natural lectin RSL from Ralstonia solanacearum, which is trimeric and hexavalent, a divalent, monomeric neolectin, which is highly similar in structure, was sufficient to induce tubular membrane invaginations on giant unilamellar vesicles by binding to a fucosylated glycolipid receptor. Amazingly, only the divalent neolectin with two carbohydrate binding sites nearby was able to form membrane invaginations whereas divalent neolectins with more distant binding sites did not, even though their binding constants were quite similar (110).

b) Toxins/ lectins generate membrane curvature Membrane curvature changes are the response to external or internal perturbations of the membrane, such as osmotic effects (111), membrane fusion (112), vesicle budding (113) or endocytosis (113)(114)(115). For instance, lectin-GSL interactions induce spontaneous curvature leading to tubular plasma membrane invaginations, which are energetically favourable. The efficiency of curvature induction and tubule formation depends on membrane rigidity and composition. In case of membranes with low rigidity, small protein clusters can initiate tubule nucleation, while in the case of membranes with higher rigidity larger protein clusters are required to bend the membrane (116,117). Once initiated, the endocytic membrane tubule grows and sequesters lectin molecules on its negatively bended surface.

With increasing lectin concentrations inside the tubule, a highly curved bud forms (114) that finally undergoes scission. In living cells, bud formation and scission can be significantly facilitated by the assembly of clathrin, actin (115) or BAR proteins at the cytosolic leaflet of the membrane [START_REF] Johannes | Building endocytic pits without clathrin[END_REF]118). (124) highlighted the importance of polyunsaturated, cone-shaped phospholipids in those uptake processes. Such lipids can adapt their conformation to the membrane curvature and may support endocytosis [START_REF] Schubert | How synthetic membrane systems contribute to the understanding of lipid-driven endocytosis[END_REF]124). Besides, curvature-driven lipid sorting may trigger the redistribution of membrane lipids based on their molecular geometry. However, numerous studies (119,(125)(126)(127)(128)(129) demonstrated that such curvature-driven lipid sorting is effective only in presence of membrane-bound or transmembrane proteins, otherwise entropy homogenises the lipid distribution even in highly curved membranes. The lipid composition of plasma membrane tubules is still elusive. Pulling a membrane tether from giant unilamellar vesicles using a micropipette (119,120,130) showed that tethers are enriched with unsaturated lipids, strongly arguing for their implication in tubule formation (130). However, as GSL clusters are associated with sphingomyelin, lectin-induced tubules may also contain saturated sphingolipids (101,130). Hence, successful tubule formation should involve an interplay between GSL clustering and attraction of saturated lipids (Figure 4C).

7) Conclusions

The plasma membrane is an exciting example of self-organisation in nature. Although many discoveries have been made concerning lipid self-assembly into a bilayer and the organisation of distinct lipids into functional units within the plasma membrane, the exact membrane organisation and its dynamics are still ill-defined. Synthetic membrane models are helpful tools in mimicking, and hence understanding plasma membrane organisation and dynamics, but one should not forget that the plasma membrane is a highly complex, asymmetric bilayer system consisting of a huge variety of lipid species that fulfil specific functions. Further improvements in microscopy techniques, in particular in super resolution live cell microscopy, as well as in the development of novel lipid labelling and detection methods are required to better decipher the principles of dynamic membrane organisation.

Introduction

Giant Unilamellar Vesicles (GUVs) represent cell-sized (tens of micrometers in diameter) spherical lipid bilayers. In combination with fluorescence microscopy techniques, GUVs became a frequently used approach to study cellular processes such as membrane permeabilization [START_REF] Hermann | Automated analysis of giant unilamellar vesicles using circular Hough transformation[END_REF], adhesion [START_REF] Villringer | Lectin-mediated protocell crosslinking to mimic cell-cell junctions and adhesion[END_REF] and endocytosis (reviewed in (Schubert and Römer, 2015)). GUVs with a spatial phaseseparation in liquid disordered (Ld) and liquid ordered (Lo) domains are of particular interest to mimic the heterogeneity of the plasma membrane including lipid rafts (reviewed in (Sych et al., 2018)). Acquired fluorescence microscopy data requires processing of a large number of data sets, especially if a quantitative description is requested. As manual image analysis is biased and highly time consuming, the use of high throughput methods implemented in automated software can significantly speed up the analysis and reduce biases. Current automated software packages for GUV analysis based on 3-D micrographs [START_REF] Husen | Morphometric Image Analysis of Giant Vesicles: A New Tool for Quantitative Thermodynamics Studies of Phase Separation in Lipid Membranes[END_REF][START_REF] Sendra | 2D map projections for visualization and quantitative analysis of 3D fluorescence micrographs[END_REF] or GUV contour [START_REF] Hermann | Automated analysis of giant unilamellar vesicles using circular Hough transformation[END_REF] require commercial software licences, are not compatible with different microscope output formats, or lack effective high throughput analysis capabilities. In this application note, we introduce a novel, powerful FIJI-based macro for fast and versatile analysis of multiple GUVs per image.

Presented macro

We have developed a macro using the ImageJ (FIJI) environment [START_REF] Rueden | ImageJ2: ImageJ for the next generation of scientific image data[END_REF][START_REF] Schindelin | Fiji -an Open Source platform for biological image analysis[END_REF], an open source microscopy image processing software. It has multiple built-in functionalities and most importantly it is compatible with the majority of microscopy data output formats thanks to the BioFormats plug-in [START_REF] Linkert | Metadata matters: access to image data in the real world[END_REF]. Briefly, the macro detects circular particles (e.g. GUVs, supplementary figure 1) in the microscopy image and determines their centers and radii.

The algorithm is explained in detail and step by step in the Manual.

GUV processing, validation and application

When GUVs are detected, various processing approaches can be applied.

Here, we demonstrate a selection. We tested the performance of GUV-AP based on laser scanning confocal microscopy images of GUVs. These GUVs were composed of DOPC, sphingomyelin and cholesterol

T.Sych et al.

in different ratios and labeled by various fluorescent probes (supplementary information -materials and methods).

Characterization of fluorescent membrane probes

We show in figure 1 as well as in the supplementary figures S2 and S3 that our macro can be applied to decipher the fluorescence properties (intensity, lifetime) of a membrane dye incorporated in the lipid bilayer as well as its preference to distinct lipid phases and its orientation in the lipid bilayer (Klymchenko and Kreder, 2014). For instance, the lipophilic membrane probe ß-Bodipy FL C5-HPC (Bodipy) is known to have preferences for the Ld phase (Figure 1A), whereas it is almost completely excluded from the Lo phase. Furthermore, the GUV analysis macro can also be used to display the circular profile that visualizes the variations of fluorescence intensity along the GUV contour as a 2D plot (figure 1B).

Quantification of protein binding to GUVs

In order to highlight the performance of our macro for the observation and quantification of protein binding, we imaged phase-separated GUVs containing the neutral glycosphingolipid globotriaosylceramide (Gb3; also known as CD77 or Pk blood group antigen) (reviewed in (Johannes and Römer, 2010)). Gb3 is the receptor for the galactose-binding Bsubunit of Shiga toxin (StxB) from Shigella dysenteriae (Römer et al., 2007). In phase-separated GUVs (figure 1A), Cy5-labeled StxB preferentially binds to Lo domains. This binding can be more accurately displayed using the circular profiles of Bodipy (figure 1B -green) and StxB-Cy5 (figure 1B -red).

To quantitatively evaluate the protein binding, we calculated the relative binding efficiency ε as shown in Figure 1C. ⟨Bound protein⟩ is the mean fluorescence intensity signal from the protein located on the vesicle contour and ⟨Free protein⟩ is the mean fluorescence intensity signal from the free protein in solution, extracted from the area of the image, which surrounds the GUV (Figure 1C). In absence of protein binding, the ⟨Bound protein⟩ value is equal to the ⟨Free protein⟩ value, so that the relative binding efficiency is ≈ 0. If ε is above 0, binding occurs. This binding efficiency value depends on the number of protein molecules bound to the membrane. Moreover, as shown by Weinberger et al. [START_REF] Weinberger | Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes[END_REF] it is possible to link such fluorescence intensity quantifications to the actual number of protein molecules bound to the membrane using proper calibration. The robustness of the method was validated by analyzing StxB binding to GUVs differing in their Gb3 content. As expected, the binding efficiency of StxB to GUVs increased with the amount of Gb3 molecules in the membrane (supplementary figure S4).

Conclusion

We developed a script for the recognition and analysis of circular segments of GUVs in order to study the partition of membrane probes as well as protein binding to membranes. In addition, we proposed a quantitative analysis to compare the binding efficiency of proteins to membranes of different compositions and biophysical properties. Our script is not limited to the analysis of GUVs, but can also process other spherical membranes like native giant plasma membrane vesicles, or any circular object. The script can be combined with time-frame analysis to study dynamics of binding processes and, with a tracking algorithm, to monitor GUV motions.

Materials and methods

GUV preparation 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), brain sphingomyelin (SM) and cholesterol (chol) were purchased from Avanti Polar Lipids. Globotriaosylceramide (Gb3) isolated from porcine brain was from Matreya. ß-Bodipy FL C 5 -HPC and di-4-ANEPPDHQ were from ThermoFisher Scientific. F2N12S and F2N12SM were provided by Dr. Andrey Klymchenko. GUVs were prepared as described in (Madl et al., 2016). In brief, solutions of lipids were prepared in chloroform. For phase-separated GUV preparation, a DOPC/chol/BSM/Bodipy (40/15/40/5/0.5) mixture was used. Three types of homogeneous GUVs differing in Gb3 content (0, 2.5, 5 and 10 mol %) were prepared using DOPC/chol/Bodipy (65/30/0.5), DOPC/chol/Gb3/Bodipy (65/30/2.5/0.5), DOPC/chol/Gb3/Bodipy (65/30/5/0.5) and DOPC/chol/Gb3/Bodipy (65/30/10/0.5) mixtures, respectively. The lipid solutions in chloroform were spread on the conducting surfaces of two Indium tin oxide (ITO) slides, and then chloroform was removed by drying under vacuum for 1 h. After chloroform was completely removed, GUVs were prepared by electroformation in a 300 µM sucrose solution for 3 h.

Protein labeling

StxB (Sigma-Aldrich) was labeled with Cy5 mono-reactive dye from GE Healthcare Life Sciences according to the amine-reactive probe labeling protocol from ThermoFisher Scientific. Briefly, 1 µl of a 10 mg/ml solution of the amino-reactive probe in DMSO was added to 100 µl of a 1 mg/ml protein solution supplemented with 100 µM NaHCO 3 . The mixture was incubated for 1 h at room temperature with continuous shaking. The labeled protein was purified using Zeba Spin Desalating Columns, 0.5 ml, from ThermoFisher Scientific.

Microscopy techniques

The fluorescence intensity measurements of GUVs (Figure 1, Figures S1,S2,S4) were performed on a confocal fluorescence microscope (Nikon Eclipse Ti-E with A1R confocal laser scanner, 60x oil objective, N.A. 1.49). For fluorescence excitation, 405 nm (F2N12SM), 488 nm (ß-Bodipy FL C 5 -HPC and Di-4ANEPPDHQ) and 642 nm (StxB-Cy5) lasers were used. The emission of ß-Bodipy FL C 5 -HPC and F2N12SM, as well as the green part of the emission di-4ANEPPDHQ were recorded using a 525/50 BP filter. The emission of the red part of di-4ANEPPDHQ emission and the emission of StxB-Cy5 were detected using a 700/75 BP filter. Fluorescence lifetime imaging microscopy (FLIM -Figure S3) was performed using a home built two photon laser scanning setup based on an Olympus IX70 inverted microscope with an Olympus 60x water immersion objective (N.A. 1.2) [START_REF] Kilin | Fluorescence Lifetime Imaging of Membrane Lipid Order with a Ratiometric Fluorescent Probe[END_REF]. Two-photon excitation was provided by an Insight Deep see laser (Spectra Physics)). The typical excitation power was ~2.5 mW (λ = 830 nm) at the sample. Photons were detected using an avalanche photodiode (SPCM-AQR-14-FC; Perkin Elmer) coupled to an HQ 585/40 bandpass filter and a single-photon-counting TCSPC module (SPC830; Becker & Hickl). FLIM data were analyzed with a binning of one using the SPCImage V4.6 software (Becker & Hickl), which uses an iterative reconvolution method to recover the lifetimes from the fluorescence decays.

Introduction

The plasma membrane controls the movement of substances between the interior and the outside of a cell. It is based on a lipid bilayer matrix that consists of lipids with diverse head groups and fatty acyl chains. The different membrane lipids are inhomogenously distributed in the plasma membrane: First, the inner and the outer leaflets of the lipid bilayer are composed of different lipid classes and this asymmetry is actively maintained by the cell (1)(2)(3)(4). Second, the plasma membrane is also heterogeneous along each leaflet: lipid domains of specific composition are formed (5)(6)(7) that serve as platforms for various cellular processes such as endocytosis (8) and signal transduction (9)(10)(11) and manifest in locally altered membrane order (12)(13)(14).

Detecting and following lipid domains in cells has turned out to be a challenging endeavor (15,16). Direct fluorescent labeling of a lipid is casting doubt on its natural behavior and phase partitioning properties (17)(18)(19). As alternative, environmentally sensitive probes that intercalate in the lipid bilayer and change their fluorescence spectra in dependence of the membrane order have been extensively used (19). However, such probes often show cytotoxic effects, can require technically advanced equipment, and can cause side effects by altering membrane order (20). An alternative approach is based on carbohydratebinding proteins, so called lectins, which specifically recognize the carbohydratemoieties of glycosphingolipids (21). For example, the B-subunit of cholera toxin (CtxB) targets the ganglioside monosialotetrahexosylganglioside (GM1). It has become a frequently used marker for highly ordered membrane domains termed as 'lipid rafts' (22)(23)(24)(25).

Here, we report that the lectins LecA from Pseudomonas aeruginosa (26)(27)(28) and the B-subunit of Shiga toxin (StxB) from Shigella dysenteriae (29)(30)(31), which recognize the same glycosphingolipid, the globotriaosylceramide (Gb3, also referred to as P K blood group antigen and CD77 (32)), segregate into different domains at the plasma membrane of cells. Furthermore, we were able to successfully rebuild LecA and StxB spatial segregation in synthetic lipid bilayer systems -giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs).

Such artificially prepared membranes with controlled lipid composition are extensively employed for studying lipid organization and function (14,33,34).

They allow to choose conditions to achieve lateral lipid segregation (5,35,36), resulting in the formation of liquid disordered (ld) and liquid ordered (lo) phases, which resembles lateral cellular plasma membrane heterogeneities.

Experiments with GUVs and SLBs enabled us to identify the interplay between the molecular structure of Gb3 (i.e. which Gb3 species it is) and the membrane environment, which both determine Gb3 membrane embedding and head group orientation (37), as critical parameters causing differential Gb3 recognition by LecA and StxB. In particular, we revealed that StxB has a preference for more ordered domains in comparison to LecA.

Based on these results we suggest that comparing the staining patterns of LecA and StxB yields a novel approach for evaluating membrane order in cells. To demonstrate the feasibility of this approach, we used epithelial cells. The apical plasma membranes of epithelial cells are known to be more ordered than basolateral plasma membranes (2,38). As expected from this order difference, StxB showed a stronger preference for the apical plasma membrane in comparison to LecA. In addition, we found that StxB, but not LecA, was able to recognize the primary cilium. This surprising finding suggests that the membrane of the primary cilium contains a lipid domain that has a higher order than the surrounding apical plasma membrane.

Results

LecA and StxB recognize different lipid domains on cells

The two lectins LecA and StxB share the same receptor, the globoside Gb3 (30,39). Upon application of LecA and StxB to either the apical or basolateral side of polarized Madin-Darby canine kidney (MDCK) strain II cells stably expressing Gb3 synthase ( 9), both lectins were found at higher concentration at apical cell membranes than at basolateral cell membranes (Fig. 1A). However, StxB showed a significantly stronger preference for the apical plasma membrane than LecA did (apical to basolateral signal intensity ratio for StxB 6.96 ± 1.04 versus 2.70 ± 0.47 for LecA). Furthermore, we found that StxB was able to bind the primary cilium (Fig. 1B), which confirms that Gb3 lipids are also found in this cellular organelle that shares a continuous lipid bilayer with the apical plasma membrane. Strikingly however, no LecA binding was detected at the cilium, even at relatively high LecA concentrations (Fig. 1B). Since the cilium represents only a tiny fraction of the area of the apical plasma membrane, StxB binding to the primary cilium cannot fully explain the higher apical to basolateral binding ratio for StxB in comparison to LecA. But measuring the total amount of bound lectin at the apical and basolateral plasma membrane provides only a global view and neglects any potential segregation of the two lectins at the plasma membrane on a smaller scale. Indeed, a closer look on Fig. 1B shows that at the (non-ciliary) apical plasma membrane LecA and StxB displayed only partially overlapping binding patterns. In agreement, previous studies found a partial segregation of the two lectins during early stages of their endocytosis (9), thus suggesting that the segregation between LecA and StxB occurs already at the plasma membrane.

To independently confirm these findings, we used HeLa cells, which endogenously express Gb3 (41). We applied energy-depletion (8) in order to inhibit energy-dependent scission of endocytic buds. This results in the formation of fairly stable tubular invaginations at the plasma membrane (8). The super-resolution microscopy technique TIRF-SIM revealed that LecA and StxB clearly segregate into different domains at the plasma membrane (Fig. 1C).

These findings demonstrate that -despite sharing the same receptor -LecA and StxB show different binding patterns on cells.

LecA and StxB also segregate on GUVs containing a mixture of Gb3 species

In order to identify the mechanism causing LecA and StxB spatial segregation, we applied model systems based on artificial lipid bilayers. To mimic the behavior and embedding of Gb3 in the cellular plasma membrane, we chose GUVs as model system. We produced GUVs containing 5 mol-% of a natural mixture of Gb3 species extracted from porcine brain (Gb3-mix) and applied both lectins (Fig. 2). Under certain conditions, StxB can induce tubular invaginations on GUVs (8,42). Hence, to rule out secondary effects, we imaged at time points before the lectins were able to induce tubules. With these settings LecA and StxB showed spatially separated binding patterns on the surface of phases-separated GUVs (Fig. 2A). In these experiment we included the ld phase marker β-BODIPY-FL-C5-HPC (17). This revealed that both lectins preferred the lo phase, but LecA and StxB only partially overlapped within the lo phase. In contrast, in non-phaseseparated GUVs no segregation between LecA and StxB was observed (Fig. 2B).

To rule out unspecific effects, we carried out several control experiments. First, we established that GUVs lacking Gb3 were not able to bind LecA or StxB (Fig. S1). Second, only negligible spectral crosstalk occurred between the membrane marker channel (β-BODIPY-FL-C5-HPC) and the channels used for the fluorescent labels of the lectins (Cy3 and Cy5) (Fig. S2).

These results demonstrate that the different binding patterns between LecA and StxB, which were observed in cells, could be successfully rebuilt in a model system of controlled composition based on GUVs. In such synthetic systems, in absence of the active cellular machinery, only the lectin-Gb3 interaction and the membrane environment can drive the spatial segregation between LecA and StxB .

Analysis of LecA and StxB preference for Gb3 species and membrane order

Based on previously reported observations with a specific class of Gb3-binding lectins, pentameric vero toxins (43)(44)(45)(46)(47)(48), we hypothesized that LecA and StxB prefer to recognize differently exposed carbohydrate head groups of Gb3. The exposure of the head group of a glycolipid is predominantly determined by the way it is embedded in the lipid bilayer (47,49). The embedding itself is influenced by (i) the length and saturation degree of its fatty acyl chain and (ii) the constitution of the surrounding lipid environment. To test how the structure of the fatty acyl chains influences LecA and StxB binding, we produced GUVs containing only Gb3 molecules with defined fatty acyl chains (Gb3-C24:0, Gb3-C24:1, and Gb3-FSL) and Gb3-mix as a control. Hereby, Gb3-FSL is a synthetic lipid that contains the Gb3 head group linked via a spacer (O(CH2)3NH) to a dioleoylphosphatidylethanolamine (DOPE) backbone (50). Furthermore, to investigate how the lipid environment influences LecA and StxB binding, we prepared non-phase-separated and phase-separated GUVs. To measure the binding of the two lectins in a quantitative manner, we used the recently developed FIJI macro GUV-AP that determines protein binding efficiency to GUVs and enables analysis of large datasets (51). For quantifying the binding efficiency, we determined the ratio between the signal of the lectin at the rim of the GUV and the free lectin in the solution (51). Such ratio represents quantitatively the contrast between the bound and free lectin. If the lectin cannot bind to the GUV, the contrast and thus the binding efficiency equals to 0. Furthermore, we included a feature in the macro to discriminate protein binding to ld and lo phases in phase-separated GUVs.

In non-phase-separated GUVs containing Gb3-mix, separately applied LecA and StxB were able to bind homogenously (Fig. 3A) and with comparable binding efficiency (Figs. 3E-F and Tables S1-S2). Introducing phase-separation to GUVs containing Gb3-mix showed that both, LecA and StxB, preferentially bound lo phases (Fig. 3B) as it has already been shown before (Fig. 2A). Interestingly, LecA recognized Gb3-mix in the lo phase much better than StxB.

In non-phase-separated GUVs containing Gb3-C24:0, LecA and StxB were able to bind with similar efficiency (Fig. 3C). But in phase-separated GUVs containing Gb3-C24:0, clear differences became apparent: Only LecA detectably bound to lo phases, whereas StxB hardly bound lo phases (Figs. 3D-F). In GUVs containing Gb3-C24:1 both lectins showed similar binding patterns (Fig. S3A andFigs. 3E-F).

In general, for Gb3-mix and all single Gb3 species tested so far, both lectins preferred the lo phase over the ld phase in phase-separated GUVs. However, Gb3-FSL demonstrated the opposite behavior: In phase-separated GUVs containing Gb3-FSL, LecA and StxB bound better to the ld phase (Fig. S3B and Figs. 3E-F). In addition, whereas LecA displayed higher binding efficiencies than StxB in non-phase-separated GUVs containing Gb3-C24:0 and Gb3-C24:1, the opposite was true for non-phase-separated GUVs containing Gb3-FSL (Fig. S3B and Figs. 3E-F).

These results demonstrate that by altering the incorporation of Gb3 in the lipid bilayer, different binding behavior of LecA versus StxB can be achieved.

The most obvious difference was seen for Gb3-C24:0, where LecA was able to recognize lo phase-localized Gb3, but StxB hardly bound. This difference could be caused by the lipid order of the lo phase domains that is dependent on cholesterol and sphingomyelin (5,35,52). Thus, to test the impact of cholesterol and sphingomyelin directly, we prepared 'pure phase' GUVs that did not contain cholesterol and sphingomyelin and approximate ld phases ('DOPC' in Fig. 4) and GUVs that did contain cholesterol and sphingomyelin approximating lo phases ('Chol + SM' in Fig. 4). Chol + SM-GUVs bound both lectins significantly better than DOPC-GUVs (Figs. 4A-B and4E-F), but the relative differences between Chol + SM-GUVs and DOPC-GUVs were more pronounced for StxB. This effect was enhanced for GUVs containing only Gb3-C24:0. In this case, LecA equally bound to DOPC-GUVs and Chol + SM-GUVs, but StxB was only able to recognize Chol + SM-GUVs (Figs. 4C-F and Tables S3-S4).

Taken together, using GUVs as model system enabled us to reveal that the two parameters 'Gb3 fatty acyl chain structure' and 'lipid environment' can cause conditions leading to preferential binding of either LecA or StxB. Both parameters influence Gb3 embedding in the lipid bilayer, which determines the orientation of the Gb3 head group (43,47,49). The head group conformation is known to affect the binding efficiency of lectins to glycosphingolipids (37). Our analysis demonstrated that both parameters have to be considered to explain the different binding behavior of LecA and StxB.

Redesign of LecA and StxB segregation using a minimal system with pure Gb3 species If the hypothesis is correct that the combination of Gb3 fatty acyl chain structure and membrane environment differentially determines the binding preferences of LecA and StxB, it should be possible to rebuild LecA and StxB segregation on the same GUV using minimal set of distinct Gb3 species. Since we observed the starkest differences between StxB and LecA for Gb3-C24:0 and Gb3-FSL, we chose to test LecA and StxB segregation in phase-separated GUVs containing 5 mol-% of each of these two Gb3 species. Indeed, this was sufficient to achieve different, but partially overlapping, staining patterns of LecA and StxB (Fig. 5) that resembled the patterns observed on cells (Figs. 1B-C) and on phaseseparated GUVs containing Gb3 mix (Fig. 2A). In particular, in GUVs containing only Gb3-C24:0 and Gb3-FSL, StxB was only able to bind the ld phase, whereas LecA showed a preference for the lo phase.

Moreover, we investigated whether such segregation can also be seen in phaseseparated SLBs containing 5 mol-% of Gb3-C24:0 and 5 mol-% of Gb3-FSL (Fig. S4A). Single microscopic images of flat SLBs provide a larger overview along the lateral direction of the lipid bilayer than images of GUVs. Therefore, in the images of SLBs multiple StxB clusters with distinct binding efficiencies for StxB within the ld phase became apparent. LecA bound in a pattern inverse to the StxB binding pattern and preferentially recognized lo phases. To gain more insights into LecA and StxB binding behavior on SLBs, we carried out experiments with SLBs containing only single Gb3 species. In SLBs containing Gb3-FSL, LecA formed clusters in the ld phase (Fig. S4B) and StxB homogeneously bound ld phases in SLBs containing Gb3-FSL (Fig. S4C). SLBs containing only Gb3-C24:0 showed preferential binding of LecA to lo phases (Fig. S4D). Surprisingly, in these SLBs StxB also bound lo phases (Fig. S4E) as also previously reported (53). However, this is in contrast to the results obtained with phase-separated GUVs containing Gb3-C24:0, where StxB was not able to bind significantly (Fig. 3D). This observations suggest that an additional parameter, such as altered inter-leaflet coupling in SLBs caused by the interaction of one leaflet with the underlying mica substrate, results in changes in Gb3 head group exposure that can be sensed by StxB.

Taken together, our results demonstrate that a minimal system containing only two selected Gb3 species is sufficient to replicate different binding patterns of LecA and StxB.

Discussion

The concept of LecA and StxB as sensors for membrane environment LecA and StxB bind both to the glycosphingolipid Gb3 (30,39). However, we found that LecA and StxB spatially segregated at the plasma membrane of cells.

By using GUVs, which combine the advantages of (i) being artificial membrane systems with well-defined lipid composition and phases with (ii) mimicking the incorporation of Gb3 in cellular plasma membranes, we were able to redesign the segregation between LecA and StxB. This proves that the different binding behavior of LecA and StxB is based on the direct interaction between the lectins and Gb3. Moreover, we identified two interdependent parameters that control the efficiency of LecA and StxB binding to Gb3: the structure of the fatty acyl chain of Gb3 (i.e. Gb3 species) and the order of the membrane environment. Both parameters determine how a single Gb3 molecule is embedded in the lipid bilayer, and hence, how the Gb3 head group is oriented. Thus, in accordance with previous studies (5,37,47), we propose that the conformation of the carbohydrate head group of Gb3 is the critical factor that alters binding efficiencies for LecA and StxB. Importantly, we demonstrated here that the specific binding preferences of LecA and StxB are different. This enables utilizing the comparison of staining patterns of the two lectins as readout for the local membrane environment.

The primary cilium harbors a unique lipid domain

The formation of distinct LecA and StxB domains on cells occurred on various scales. Even complete cellular organelles, like the primary cilium, were not recognized by LecA, while they were bound by StxB. The primary cilium membrane and the apical plasma membrane are continuous and there are no apparent obstacles that could directly block lipid movement along the outer plasma membrane leaflet (54). Nevertheless, our surprising finding suggests that the primary cilium membrane contains a distinct lipid domain of higher order that does not extend to the surrounding apical plasma membrane. Support for this hypothesis can be found in literature. Primary cilia were found to be cholesterol-rich [START_REF] Ott | Visualization of live primary cilia dynamics using fluorescence microscopy[END_REF] (56). The gangliosides GM1 and GM3 (57), and the Forssman glycolipid (58), which are all considered as lipid raft markers, localize to the primary cilium of mammalian cells. Furthermore, Trypanosoma brucei flagellar membranes, which resemble primary cilia, are enriched in lipid raft components (59).

Influence of membrane order on LecA and StxB binding

It is well established that the lipid composition and overall membrane order differ between the apical and basolateral plasma membrane of epithelial cells (2,38,60), with the apical plasma membrane displaying a higher order and containing more cholesterol and sphingolipids relative to the basolateral plasma membrane (61). LecA and StxB were also able to recognize these differences, with StxB showing a significantly stronger binding preference for the apical plasma membrane than LecA. This fits to the observations from our studies with artificial membranes, which revealed that, overall, LecA more tolerantly recognized different Gb3 species and membrane environments than StxB. By using GUVs, we were able to demonstrate in Fig. 4 that the apical preference of StxB can be explained by higher membrane order induced by cholesterol and sphingomyelin: For GUVs containing Gb3-mix, both LecA and StxB preferred the more ordered GUVs, but the preference was more pronounced for StxB, as it was for the apical to basolateral binding ratio on cells (Fig 1A). When using GUVs containing only the single Gb3 species Gb3-C24:0, the effect became drastically enhanced. LecA demonstrated equal binding to GUVs with and without cholesterol and sphingomyelin, whereas StxB only bound to GUVs with cholesterol or sphingomyelin.

Interplay between Gb3 species and membrane environment

Not only the total binding efficiencies of LecA and StxB at the apical and basolateral plasma membrane were different, but also on smaller scales distinct binding differences were seen: At the plasma membrane of energy depleted cells, LecA and StxB segregated into only partially overlapping nano-domains (Fig. 1C). This suggests that LecA and StxB segregation promptly develop after binding and most likely continue during endocytosis (9), which provides an explanation for the different intracellular trafficking of LecA and StxB we reported before (9). Importantly, we were able to rebuild such a domain separation between LecA and StxB in GUVs containing Gb3-mix and also in GUVs containing only the two Gb3 species Gb3-C24:0 and Gb3-FSL.

The reasons why Gb3-C24:0 and Gb3-FSL are minimally sufficient for reconstituting LecA and StxB segregation can be deduced from our experiments with single Gb3 species and illustrate how the interplay between Gb3 species and membrane environment governs different LecA and StxB binding: In phaseseparated GUVs containing Gb3-FSL, both lectins preferred the ld phase over the lo phase. This was in stark contrast to the results obtained with phase-separated GUVs containing Gb3-mix and single non-FSL Gb3 species in which both lectins preferred the lo phase over the ld phase. Most likely, this is caused by a preferential integration of Gb3-FSL into the ld phase, since lectins bound to Gb3-FSL always co-localized with the ld marker β-BODIPY-FL-C5-HPC (17) (Figure S3B). As already described above, our GUV experiments with Gb3-C24:0 illustrate that for StxB Gb3 is best exposed in lo environments and the membrane composition alone is able to block or mask the binding of StxB.

The results from SLBs are in line with findings from GUVs in case of LecA, but for StxB divergent outcomes were observed: In phase-separated GUVs StxB hardly bound lo and ld phases, while on phase-separated SLBs binding of StxB to lo and ld phases occurred. This demonstrates that the system has a high sensitivity:

Additional parameters that lead to subtle changes in the lipid bilayer and hence cause different Gb3 head group exposure, such as altered inter-leaflet coupling induced by the substrate of SLBs, can significantly shift the binding ability of StxB.

Conclusion and outlook

Taken together, our data revealed that the two Gb3-binding lectins LecA and StxB segregate into different domains at the plasma membrane of cells. We were able to reconstitute these effects in synthetic systems demonstrating that the driving force for segregation is controlled by parameters that determine the conformation of the Gb3 head group. These parameters, Gb3 fatty acyl chain structure and membrane environment, are interdependent, and enable LecA and StxB to act as sensors for membrane order on native cells with StxB indicating more ordered membranes. Thus, comparing LecA with StxB staining patterns could be used in future as a sensitive read out for determining membrane order during cellular processes.

Materials and Methods

Cell lines and cell culture

Madin-Darby canine kidney (MDCK) strain II cells and HeLa cells were maintained at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% (for MDCK) and 10% (for HeLa) fetal calf serum. MDCK cells stably expressing Gb3 were established by stable overexpression of Gb3 synthase as described earlier (9,42). For generating polarized monolayers, MDCK cells were confluently seeded to transwell filters (Corning, 0.4 µm pore size, polycarbonate membrane) and cultured for 4 d.

Lipids and preparation of lipid mixtures

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (Chol) and sphingomyelin (SM) were purchased from Avanti Polar Lipids, whereas Gb3-FSL was purchased from Sigma Aldrich. Gb3-mix was from Matreya, consisting of a mixture of Gb3 species extracted from porcine brain. The Gb3-species Gb3-C24:0 and Gb3-C24:1 were synthesized as described earlier (53). The fluorescent lipid 2- (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY-FL-C5-HPC) was from Invitrogen. All lipids were dissolved in chloroform, only Gb3-mix and Gb3species were dissolved in a 2:1 chloroform/methanol mixture. Dissolved lipids were stored with an argon layer on top at -20°C. Lipid mixtures were prepared with a concentration of 0.5 mg of total lipid mass per 1 mL organic solvent or aqueous environment or buffers. The lipid ratios of each composition were calculated in molar percentages.

Preparation of GUVs

GUVs were produced utilizing electro-swelling (62). Briefly, lipid mixtures were spread on each side of an indium-tin oxidized (ITO) glass slide (Sigma Aldrich) with a Hamilton syringe (Hamilton) and evaporated overnight. Electro-swelling was done in phosphate buffered saline (PBS, Carl Roth) supplemented with 1 mg/mL sucrose (Carl Roth) for 3 h at room temperature for non-phase- Chol + SM GUVs (red) containing 5 mol-% of either Gb3-mix, or Gb3-C24:0. For each condition, the middle horizontal line represents the median, the boxes the 25 th to 75 th percentiles, and the whiskers the min and max values. In Table S3 the descriptive statistics of the data is summarized, and Table S4 contains the complete data from the significance analysis.

Introduction

Host cell invasion is a strategy exploited by many bacteria to escape the host's immune defenses and to fulfill nutritive and metabolic requirements 1-3 . Commonly, invasion strongly relies on tight host-pathogen interactions, and the interplay of bacterial factors and the host cell machinery 4 . The bacterial uptake is mostly induced at the cell surface by binding to a wide range of plasma membrane molecules. These initial interactions can trigger diverse signaling events leading to actin polymerization, which is described as a major driving force for bacterial engulfment. Recently, we have introduced the concept of the "lipid zipper", a membrane invagination process that also leads to wrapping of the plasma membrane around the bacterium, but in a fully different way 1 . We have reported that the interaction of the bacterial surface lectin LecA 6,7 with the host cell glycosphingolipid Gb3 was fully sufficient for membrane bending and engulfment of the bacterium P. aeruginosa in the absence of actin polymerization. Remarkably, reconstitution of bacterium-Gb3 interactions in synthetic membrane systems (e.g. giant unilamellar vesicles) as well as theoretical modeling contributed largely to the understanding of the mechanism of LecA-triggered lipid zippering. We found that interfering with LecA-Gb3 interactions by LecA deletion or blocking, and Gb3 depletion led to a significant (more than 60%) reduction in invasiveness of P. aeruginosa 1 . Based on the crucial role of LecA-Gb3 interactions for bacterial uptake, divalent LecA binders have been designed, which reduced cellular invasiveness of P. aeruginosa more than 80% when applied in a concentration of 50 nM 8 .

Glycosphingolipids 9 , such as Gb3, are enriched in so-called lipid rafts, which play key roles as signaling platforms for the internalization of many bacteria 10,11 . Lipid rafts are highly ordered, dynamic membrane domains, mainly composed of sphingomyelin, cholesterol 12-14 and a large variety of plasma membrane lipids and proteins important for cell signaling and internalization.

For bacterial entry, the impact of the tetravalent, galactose-binding lectin LecA on plasma membrane organization and dynamics, in particular on lipid rafts, is elusive.

In this work, we mimicked the cell membrane organization by using phase-separated supported lipid bilayers (SLBs) as minimal synthetic membrane system. SLBs were prepared using a ternary mixture of the phospholipid DOPC, sphingomyelin (SM) and cholesterol (chol), supplemented with the glycosphingolipid Gb3. They exhibited phase-separation at room temperature and formed two distinct phases -liquiddisordered (Ld, mainly composed of DOPC) and liquid-ordered (Lo, mainly composed of SM/chol). The Lo domains in such SLBs can be considered as a minimalistic model of lipid rafts at the microscopic scale.

Furthermore, we explored the impact of P. aeruginosa and especially of its lectin LecA on the lipid organization of phase-separated SLBs. We found that the P. aeruginosa strain PAO1 adheres to SLBs, forms aggregates and dissolves Lo domains. We demonstrated that LecA-Gb3 interactions are the driving forces for these processes by using wild-type and LecA-deficient strains. Moreover, the dissolution of Lo domains was also induced by purified, tetravalent LecA and an engineered divalent LecA variant. The dissolution mechanism was investigated in more detail to decipher the crucial role of protein structure and valence for this process.

Results

Aggregation of P. aeruginosa at the membrane and partial dissolution of ordered domains.

Planar SLBs with lateral phase separation, which represent a widely used synthetic membrane system to mimic cellular lipid rafts, were prepared on mica surface using a lipid composition of DOPC, chol, SM and the glycosphingolipid Gb3 (37.5/20/37.5/5).

To monitor the membrane organization, the lipophilic probe Texas Red-DHPE was used. This probe preferentially associates to the less tightly packed Ld domains, whereas the more tightly packed Lo domains appear dark (figure 1a).

We applied 250 µL of a suspension of the P. aeruginosa wild type strain PAO1 1a and corresponding quantifications in figure S3a). After As the PAO1 wild type strain, the PAO1-ΔLecA strain preferentially targets the Lo/Ld domain boundaries (59% ± 6%). Furthermore, 44% ± 6 % of LecA-deficient bacteria adhere to Ld domains, and Lo domains are rather neglected (5% ± 2%) (figure 1b and corresponding quantifications in figure S3a). In addition, there was no formation of bacterial aggregates (figure S3c), indicating thatthe bacterial adhesion to and the aggregation at the membrane is mediated by LecA-Gb3 interactions. Importantly, the PAO1-ΔLecA strain does not induce the dissolution of Lo domains (figure 1b). The total area of Lo domains remains largely constant over the incubation time (figure 1c)

and the individual areas of Lo domains do not decrease (figure 1f). Furthermore, we did not observe the accumulation of membrane lipids around LecA-deficient bacteria (figure 1b -white circle and figure 1e).

As control experiments, we applied the P. aeruginosa wild type and ΔLecA strains to SLBs devoid of Gb3 consisting of DOPC, chol and SM (37.5/20/37.5). In comparison to Gb3-containg SLBs, the adhesion of both bacterial strains to Gb3-negative SLBs was strongly reduced (5.5 fold for PAO1 wt and 60 fold for PAO1 ΔLecA -figure S4).

All these findings confirm the key importance of the LecA-Gb3 interactions not only for the adhesion of P. aeruginosa to the membrane, but also for the dissolution of Lo domains. A more detailed description of the dynamics of Lo domain dissolution reveals a simultaneous reduction of the number of Lo domains (figure 2c) and the area of individual Lo domains (figure 2d). This is in line with our previous observations of altered membrane organization induced by LecA-expressing P. aeruginosa.

Roughly 5 min after application of LecA, more intense LecA clusters emerge (figure 2a). These clusters grow over time to micrometer size. Interestingly, this LecA clustering coincides with the appearance of areas characterized by enhanced Bodipy fluorescence intensity (figure 2a). As published recently 15 , LecA has the ability to crosslink opposing lipid membranes upon which it strongly accumulates at membrane interfaces. Accordingly we presume that these novel areas represent membrane-LecA-membrane sandwiches, i.e a double or even multilayer of bilayer membrane crosslinked by LecA. Furthermore, after 15 min, large defects in the SLB start to appear (figure 2a and supplementary movie 3). The total area of the membrane defects increases over time (figure 2b, figure S5) with the increasing area of membrane multilayers (figure 2b). However, the membrane multilayer formation precedes the membrane destruction (green curve in figure 2b). Hence, we suggest that the formation of multilayers depletes the initial SLB in lipids, which results in extensive membrane defects. Later on, we will use a dimeric LecA variant to elucidate the dynamics of Lo domain dissolution in absence of multilayer formation. LecA drastically alters membrane order.

Lo domains enriched in SM and chol exhibit a highly-ordered lipid organization, whereas the surrounding membrane environment is low-ordered (also called disordered). However, after LecA-induced Lo domain dissolution, SM and chol are still present in the membrane; they are redistributed and very likely modify the membrane order. Various techniques provide the possibility to sense the local membrane order of the lipid bilayer 35,36 . In this work, we employed fluorescencebased ratiometric imaging using the environmental sensitive membrane marker Nile Red 12 S (NR12S) 37 . This probe is sensitive to the membrane hydration and exhibits a drastic red-shift in polar environments (i.e. in Ld domains). The general polarization (GP) value of NR12S represents the local membrane order. The GP value can vary from -1 to 1. Low values correspond to low membrane order whereas higher GP values indicate a higher membrane order. NR12S was added to phase-separated SLBs (DOPC/chol/SM/Gb3 (37.5/20/37.5/5)). We reconstructed the GP image to map the membrane order of the SLB (figure 3a).

In absence of LecA, the GP image displays a clear distinction between Lo domains (green, with a GP value of approximately -0. 3a -gray circles). We excluded this multilayer from our quantifications. As a result, the membrane order of multilayers is higher (figure 3c) than the order of Ld domains and lower than the order of Lo domains of the original lipid bilayer. This intermediate membrane order is the consequence of the lipid redistribution in the membranes caused by the dissolution of Lo domains. We suggest that Lo dissolution is a prerequisite for multilayer formation. For a better understanding of the dynamics of these processes, it would be advantageous to analyze both separately from each other (figure 2b and figure S6b). To focus on multilayer formation, we used homogeneous SLBs consisting of DOPC/chol/Gb3 (65/30/5), where classical phaseseparation does not exist, and accordingly Lo domains cannot be dissolved. In this system, LecA (200 nM) induces multilayer formation and membrane disintegration (figure S7). Moreover, in this case (i.e. in the absence of phase separation) the membrane disintegration occurs very fast; the membrane is completely destroyed within 15 min after LecA application.

To study the dissolution of Lo domains separately, membrane multilayer formation has to be constrained. For that, the engineered homodimeric LecA variant RPL α-Gal (RPL) was used.

Dimeric LecA variant dissolves Lo domains without forming membrane multilayers. The amino acid sequence of RPL monomer is similar to the LecA monomer but the N-terminus of the protein is modified with a 6xHis-tag. This minor modification guarantees the stability of the protein as a dimer in aqueous solution and does not affect its specificity to the 𝛼-galactose moiety of glycoconjugates (e.g. Membrane multilayer formation was not observed in these experiments. Clearly, the membrane dissolution process is not a collective effect induced by multilayer formation, but rather an independent process.

Discussion

Multiple works suggested the crucial implication of the lipid rafts in the P. aeruginosa infection. Lipid rafts harbor big variety of components that can be targeted by P. aeruginosa in order to promote infection. Song et al. 20 demonstrated the ability of quorum-sensing metabolites, secreted by P. aeruginosa to induce the apoptosis of the immune cells via the dissolution of plasma membrane ordered nanodomains.

Lipid rafts are also highly involved in the bacterial internalization. In particular, such important lipid raft-associated plasma membrane components as ceramides 21,22 and cholesterol 23 are known to play crucial role in the P. aeruginosa uptake in the host cell. In polarized epithelial cells, the bacterial entry from the apical site requires the enrichment of PIP3 24 at the cytosolic plasma membrane leaflet and PIP3 is also raftassociated membrane component 25 . The cholesterol rich membrane domains are required for the type 3 secretion system of the P. aeruginosa to initiate the trigger uptake mechanism 26 . Also, the glycosphingolipids, enriched in lipid rafts, such as asialo-gangliosides GM1 and GM2 or globoside Gb3 are the membrane receptors that mediate the bacterial adhesion to the host cell membrane 1,27 . Moreover, the interaction of the P. aeruginosa with glycosphigolipid Gb3 can initiate the bacterial invasion through the lipid zipper 1 . The interaction of the P. aeruginosa with the Gb3 is granted by its surface lectin LecA. This galactose specific lectin mediates the P. aeruginosa adhesion and bacterial impact on the lipid rafts.

In this work, we demonstrated that P. aeruginosa can drastically alter the lipid organization in the model membranes. Namely, P. aeruginosa dissolves the ordered membrane domains. We observed this process in the SLBs -model membranes on the solid substrate. The Lo domains in such SLBs mimic the nanodomains (lipid rafts)

of the plasma membranes of living cells at the microscopic scale. Clearly, the native membrane nanodomains are smaller in size and highly dynamic, whereas the Lo domains in SLBs are large and stable over time (figure S8). Nevertheless, the principles that underlie the constitution of such ordered nanodomains can be elucidated from the SLBs. Moreover, the reorganization of the Lo domains in SLBs induced by various factors can be visualized and analyzed.

We applied bacteria to the SLBs and observed its binding and clustering (figure 1).

The interactions of the bacteria with the SLBs induced the decrease of the areas of the Lo domains, several Lo domains completely disappeared. We suggest that the rupture of membrane order is required for the P. aeruginosa to promote infection. We demonstrated, that the specific LecA-Gb3 interaction is responsible for the Lo domains dissolution in the SLB induced by P. aeruginosa. LecA-deficient strain showed the reduced binding to the SLB and also it was not able to rearrange the Lo domains. The similar situation was observed when both wild type and LecA-deficient strains were applied to the SLBs without Gb3.

Interestingly, we observed the accumulation of the SLB membrane at the bacterial surface. This process seems to be the analogue of the bacterial wrapping with the membrane via "lipid zipper". Clearly, this "wrapping" cannot induce the bacterial internalization as the membrane bending is constrained by SLB substrate. Hence, bacteria, wrapped in membrane remain at the support. Also, in absence of the LecA-Gb3 interaction, the bacterial "wrapping" with membrane was not observed.

The role of LecA and Gb3 for plasma membrane reorganization during P. aeruginosa adhesion was validated by the studies of the impact on the organization of SLBs induced by soluble purified LecA (recombined in E. coli). Interestingly, LecA prefers to bind to Lo domains, whereas bacteria prefer Lo/Ld boundaries. Furthermore, LecA induces Lo domains dissolution. Simultaneously, LecA forms micrometer-sized clusters at the SLB. These clusters sequester the lipids from the initial lipid bilayer in order to form membrane multilayers. This effect induces depletion of the SLB in lipids and as a result, it becomes disintegrated.

The impact of purified LecA is not a common feature for the Gb3-specific lectins. The multilayers with LecA sandwiched in between the membrane layers. Indeed, as we also observed for GUV membranes 15 , the preferential location for LecA is in the membrane interfaces.

In order to resolve the composition of the membrane multilayers, we compared the membrane order in multilayers to the membrane order in Ld and Lo. We found that the multilayers exhibit the intermediate membrane order in between the Ld and Lo domains. This suggests that membrane multilayers are composed out of the mixed content of former Ld and Lo domains. Hence, we assume that the Lo domains dissolution is required for the membrane multilayers formation.

Interestingly, for the membrane dissolution the whole tetravalent form of LecA is not necessarily required. Divalent LecA variant (α-galactose specific RPL α Gal) induces Lo domains dissolution, but the membrane multilayers are not formed. Hence, clearly, the complete tetrameric form is required for the multilayer formation.

Multiple bacteria exploit cellular machinery through interaction with lipid rafts in order to promote infection 10,11,31,32 . In this work, on the example of P. aeruginosa, we demonstrated that bacteria are capable of the induction of the extensive reaarangement of the membrane ordered domains: Lipid raft-like domains, reconstituted in synthetic SLBs were targeted by bacteria and dissolved. We suggest that the rearrangement of the ordered domains in the membrane might be required by bacteria for successful adhesion and/or internalization into the host cell. We also highlight the crucial importance of the host cell glycosphingolipids and bacterial lectins in this process. HILO -illumination microscopy. SLBs are often imaged using TIRF microscopy 33 .

Methods

However, in our case, the specific design of the SLB support doesn't allow to achieve the total internal reflection of the laser beam at the mica/SLB interface. Therefore, we used HILO -highly inclined thin layer illumination below the angle of total internal reflection 34 . This method provides a high signal to background ratio decreasing the illumination of the non-bound lectins in the solution.

  Bindung der Lektine an Gb3 in Modellmembranen (gigantische, unilamellare Vesikel -GUVs) unterschiedlicher Komposition und Ordnung. Wir entwickelten eine Methode zur quantitativen Beschreibung der Lektinbindung and GUVs basierend auf der Analyse von fluoreszenzmikroskopischen Aufnahmen. Mit dieser Methode bestätigten wir, dass StxB stärker geordnete Lipiddoppelschichten bevorzugt, während LecA weniger selektiv ist und Membranen geringer Ordnung ebenfalls effizient bindet. Des Weiteren studierten wir die Lektinbindung an synthetische Gb3-Spezies mit 24:0-und 24:1-Fettsäureketten sowie an das synthetische Konstrukt FSL-Gb3. Mit diesen Spezies klärten wir die Rolle der molekularen Struktur des Gb3 in der Bindung der Lektine auf. Es gelang uns auf Grundlage unserer Bindestudien die Segregation von LecA und StxB in GUVs zu nachzubilden. Im zweiten Teil des Projektes erkundeten wir die Auswirkung von LecA und StxB auf die Membranorganisation. Hierbei waren wir besonders an der Umgestaltung geordneter Nanodomänen in der Plasmamembran durch die Lektine interessiert. Die Visualisierung solcher Nanodomänen in der nativen Plasmamembran ist eine äußerst herausfordernde Aufgabe, weshalb wir sie mit flüssig-geordneten (liquid-ordered, Lo-) Domänen in phasenseparierten gestützten Lipiddoppelschichten (supported lipid bilayers, SLBs) nachahmten. Wir funktionalisierten diese Lipidschichten mit Gb3-Rezeptormolekülen und fanden heraus, dass StxB vorliegende Lo-Domänen schrumpfen lässt und die Bildung neuer Lo-Domänen induziert. Das weist darauf hin, dass StxB effizient Gb3 bündelt und den Rezeptor in Lo-Domänen stabilisiert. LecA zeigt ein völlig gegensätzliches Verhalten. Auf SLBs angewendet induziert LecA die Auflösung von Lo-Domänen und homogenisiert die Membran. Abschließend beobachteten wir, dass P. aeruginosa ebenfalls die Auflösung von Lo-Domänen in SLBs bewirkt. Diese durch das Bakterium induzierte Auflösung wird mit der vorliegenden Arbeit zum ersten Mal beschrieben. Zusammengefasst haben wir gelernt/verstanden/demonstriert/aufgedeckt, dass die LecA-Gb3-Interaktion den Schlüssel zur Auflösung von Lo-Domänen durch P. aeruginosa darstellt.

Figure 1 |

 1 Figure 1 | Membrane plasmique. Schéma de la membrane plasmique (Encyclopedia Britannica).Micrographie EM de la membrane plasmique de Chara corallina[START_REF] Coster | The Physics of Cell Membranes[END_REF]. Image en microscopie confocale de la coupe transversale de cellules MDCK-II: membrane plasmique (myristoyl-LYN-GFP -verte); noyau (DAPI -bleu). Barre d'échelle -10µm. Image fournie par le Dr. Roland Thuenauer.

Figure 2 |

 2 Figure 2 | Structures chimiques des espèces lipidiques membranaires. Les glycérophospholipides sont constitués d'un groupe de tête à base de phosphate et d'une ou plusieurs chaînes acyle grasses. Les sphingolipides sont basés sur un squelette en céramide (sphingosine + acide gras).Les stérols sont un sous-groupe de stéroïdes dont la structure centrale est constituée de quatre cycles fusionnés avec un groupe hydroxyle en position 3 du cycle A. Adapté de[START_REF] Fahy | A comprehensive classification system for lipids[END_REF][START_REF] Jäpelt | Vitamin D in plants: a review of occurrence, analysis, and biosynthesis[END_REF].

Figure 5 |

 5 Figure 5 | Les phases bicouches lipidiques. Dans un environnement aqueux, les bicouches lipidiques peuvent former des phases de tassement et de mobilité lipidique différents. a) phase solide (ici constituée de SM); b) Phases fluides: ordonnées liquides (ici formées de SM et de chol) et désordonnées liquides (ici formées de DOPC). Révisé de(Sych et al., 2018a).

Figure 6 |

 6 Figure 6 | Nanodomaines de la membrane plasmique. La membrane plasmique native contient des nanodomaines lipidiques compactés et hautement ordonnés appelés «radeaux lipidiques». Des radeaux lipidiques se forment dans la feuille externe de la membrane plasmique et sont enrichis en sphingomyéline et en cholestérol. Les radeaux lipidiques s'associent aux glycosphingolipides, aux protéines transmembranaires et ancrées au GPI. La foliole cytosolique de la membrane plasmatique est enrichie en PI, PG, PS et PE, tandis que la foliole externe contient la majorité des lipides SM et PC. Le cholestérol est uniformément distribué entre les monocouches lipidiques. Adapté de(Sych et al., 2018a).

Figure 7 |

 7 Figure 7 | Glycosphingolipides. Structure de trois classes principales de glycosphingolipides. Les structures chimiques sont adaptées de Avanti Polar Lipids.

Figure 8 |

 8 Figure 8 | Fluorescence. A) Diagramme de Jablonski (Lakowicz, 2006). Le fluorophore absorbe une quantité de lumière qui entraîne le transfert d'un électron de l'état fondamental (SO) à l'état excité (S1 -flèche bleue ou S2 -flèche violette). Les sous-états vibratoires des états électroniques sont indiqués par des lignes grises. Le processus d'absorption peut transférer un électron à plusieurs états vibratoires de l'état excité. La conversion interne non radiative amène l'électron à l'état vibratoire le plus faible de l'état excité S1. Par la suite, la fluorescence est la transition de l'électron à l'état fondamental SO par l'émission d'un photon. L'électron peut être transféré dans différents états vibratoires de l'état fondamental. Alternativement, un électron peut subir un croisement intersystème vers l'état de triplet excité (T1). B) Spectres d'excitation et d'émission de fluorescence du fluorophore organique Bodipy (Thermo Fisher Scientific). Les propriétés des fluorophores définissent leurs spectres d'absorption et d'émission. Le spectre d'absorption définit la longueur d'onde de la lumière à utiliser pour exciter le fluorophore. Les spectres d'absorption et d'émission (figure 8B) des fluorophores dépendent souvent des propriétés de l'environnement (polarité du solvant, viscosité, pH, etc.) et peuvent donc être utilisés en tant que capteurs environnementaux. De plus, les phénomènes de fluorescence sont largement utilisés en microscopie pour la localisation des fluorophores.

Figure 9 |

 9 Figure 9 | Microscopie confocale à balayage laser (LSCM). A) Schéma d'une configuration de microscopie confocale inversée. La lumière d'excitation laser est focalisée sur le plan focal de l'échantillon. La réflexion laser est coupée avec un miroir dichroïque. La lumière de fluorescence est transmise à travers un filtre d'émission approprié et détectée à l'aide d'un photomultiplicateur. Le balayage permet l'acquisition d'une image pixel par pixel; B) Image de microscopie confocale de la coupe transversale de cellules MDCK-II. La membrane plasmatique (myristoyl-LYN-GFP -verte); noyau (DAPI -bleu). Image fournie par le Dr Roland Thuenauer.

Figure 10 |

 10 Figure 10 | Microscopie à excitation large champ. A) Schéma d'une configuration de microscopie inversée à champ large. La lumière d'excitation laser est focalisée sur le plan focal arrière de l'objectif. L'échantillon est illuminé comme illustré en B. La réflexion du laser est coupée avec un miroir dichroïque. La lumière de fluorescence est transmise à travers un filtre d'émission approprié et détectée à l'aide d'une caméra (dans ce cas, CCD). B) Trois modes d'éclairage de l'échantillon: EPI, HILO et TIRF. C) Image TIRF des membranes plasmiques inférieures de cellules vivantes MDCK-II marquées avec myristoyl-LYN-GFP. Barre d'échelle -10 µm. Image fournie par Marco Frensch.

Figure 11 |

 11 Figure 11 | Vésicules unilamellaires géantes. A) Schéma d'une GUV à séparation de phase. B) Image confocale de la section transversale équatoriale d'un GUV homogène composé de DOPC / chol (70/30) marqué avec HPC-Bodipy. C) Image confocale de la coupe transversale équatoriale d'un GUV à séparation de phase composé de DOPC / chol / SM (2/1/2) marqué avec HPC-Bodipy. HPC-Bodipy incorpore de préférence le domaine Ld plus fluide (composé principalement de DOPC), tandis que le domaine Lo (composé principalement de SM et de cholestérol) apparaît sombre. Barres d'échelle -10 µm. Régime révisé du(Sych et al., 2018a).

Figure 12 |

 12 Figure 12 | Bicouches lipidiques soutenues. A) Schéma d'un SLB à séparation de phase. B) Image à grand champ (HILO) d'un SLB homogène composé de DOPC / chol (70/30) marqué avec HPC-Bodipy. C) Image à large champ (HILO) d'un SLB à séparation de phases composé de DOPC / chol / SM (2/1/2) marqué avec HPC-Bodipy. Parce que HPC-Bodipy préfère incorporer dans le domaine plus fluide de Ld (composé principalement de DOPC), le domaine Lo (composé principalement de SM et de cholestérol) apparaît sombre. Barres d'échelle -1o µm. Régime révisé du(Sych et al., 2018a).

Figure 13 |

 13 Figure 13 | Études d'ordre membranaire utilisant NR12S A) Structure de la sonde NR12S (Kucherak et al., 2010); B) Spectres de fluorescence de NR12S incorporés dans des bicouches lipidiques de Ld (DOPC) et de Lo (SM / chol -2/1) purs (Kucherak et al., 2010). Les bandes verte et rouge représentent les parties verte et rouge des spectres pris pour le calcul de la polarisation générale (GP); C) Formule pour le calcul du médecin généraliste (Sanchez et al., 2007); D) Imagerie ratiométrique à deux couleurs de GUV et SLB à phases séparées utilisant la microscopie confocale. Les filtres passe-bande pour les canaux de couleur verte et rouge sont sélectionnés comme indiqué en B. L'image GP est reconstruite par application de la formule en C aux canaux de couleur pixel par pixel. Plus le GP est élevé, plus l'ordre des membranes est élevé. Selon la barre de calibration, la valeur GP en Lo est plus élevée. Barres d'échelle -10 µm.

Figure 14 |

 14 Figure 14 | Diagramme de phase ternaire obtenu par des études d'ordre membranaire avec Laurdan.A) L'ordre des membranes dépend du rapport molaire entre les trois lipides. Le code de couleur illustre la valeur GP des VUC composées de différents rapports molaires DOPC, SM et chol. La zone grise décrit les rapports auxquels se produit la séparation de phase. L'ordre des membranes des domaines Lo et Ld dans les vésicules à phases séparées varie également; B) Ordre membranaire des domaines Lo dans des vésicules à phases séparées à différents rapports molaires de lipides; C) Ordre membranaire des domaines Ld dans des vésicules à phases séparées à différents rapports molaires de lipides; Révisé de(Carravilla et al., 2015).

Figure 15|

 15| Figure 15| Shiga toxin induces tubular invaginations in GUVs A) The ribbon diagram of pentameric Shiga toxin B subunit (StxB); B) The ribbon diagram of StxB monomer with indicated binding sites; C) StxB induces tubular invaginations in GUVs composed of DOPC/chol/Gb3 (65/30/5) labeled with HPC-Bodipy. StxB clusters on the outer leaflet of the GUV membrane and induces tubules. Tubules are enriched in StxB. Scale bar -10 µm.

Figure 17|

 17| Figure 17|The uptake of P. aeruginosa via a lipid zipper. A) GFP-expressing P. aeruginosa (green) after surface staining of its lectin LecA with an anti-LecA antibody (Alexa 647, red); Scale bar -2 µm. B) Ribbon diagram of LecA; C) Glycosphingolipid Gb3 -the cellular receptor targeted by LecA; D) The LecA-Gb3 interaction is sufficient for the lipid zipper formation and initiation of bacterial (GFP, green) internalization into GUVs composed of DOPC/chol/Gb3 (65/30/5) labeled with TexasRed (red). LecA deletion (D -bottom panel) inhibits the binding and internalization of the bacteria into GUVs(Eierhoff et al., 2014); Scale bars -5 µm. E) Bacteria induce membrane invaginations in H1299 lung epithelia cells by a lipid zipper(Eierhoff et al., 2014). Actin colocalization with the newly formed invaginations induced by bacteria is negligible. This implies that the key process of bacterial uptake is the LecA-Gb3 interaction, whereas the involvement of actin is insignificant. Scale bars are 10 μm and 2.5 μm, respectively.

Figure 18 |

 18 Figure 18 | StxB binding to GUVs. (A) StxB-Cy5 (red channel) binds to the Lo domains of a GUV composed of DOPC/chol/SM and the glycosphingolipid Gb3 (40/15/40/5). The Lo domains are not visible since the membrane probe Bodipy (green channel) preferentially localizes to Ld domains; (B) Circular profiles of the membrane marker Bodipy (green curve) and StxB-Cy5 (red curve). The units of the StxB-Cy5 circular profile (right Y axis) are quantified as shown in (C). (C) Calculation of the relative binding efficiency.

Figure 19 |

 19 Figure 19 | StxB binding to GUVs containing different molar percentages ofGb3-mix. A-D) LSCM images of GUVs (in green) containing 0% (A), 2.5% (B), 5% (C) and 10% (D) of Gb3, which have been incubated with 100 nM of StxB-Cy5 (red). StxB-Cy5 intensities were all scaled equally so that the free protein signals are visible in all images. E) Binding efficiency 𝜀 values for StxB. Error bars represent standard deviation. Number of replicas for 0%, 2.5%, 5% and 10% Gb3-containing GUVs was16, 12, 17 and 10, respectively.

Figure 20 |

 20 Figure 20 | The influence of Gb3 species and phase separation on LecA and StxB binding efficiencies. GUVs doped with 0.5 mol% of the membrane marker β-BODIPY-FL-C5-HPC (green) and 5 mol% Gb3-mix (A and B) or 5 mol% Gb3-C24:0 (C and D) were incubated with LecA-Cy5 (200 nM, orange) or StxB-Cy5 (200 nM, blue). Non-phase-separated GUVs (A and C) contained DOPC/cholesterol/Gb3 in the ratio 64.5/30/5 mol%, whereas phase-separated GUVs (B and D) consisted of DOPC/cholesterol/sphingomyelin/Gb3 in the ratio 42.5/14.5/42.5/5 mol%. The images A -D show equatorial sections through representative GUVs. Scale bars correspond to 5 μm.(E -F) Quantitative analysis of the binding efficiencies of LecA (E) and StxB (F) to non-phase-separated GUVs (red) or the Ld (green) or Lo (blue) phase of phase-separated GUVs containing 5 mol% of either Gb3-mix (mix), Gb3-C24:0 (C24:0), Gb3-C24:1 (C24:1), or Gb3-FSL (FSL). For each condition, the middle horizontal line represents the median, the boxes the 25th to 75th percentiles, and the whiskers the min and max values.

Figure 21 |

 21 Figure 21 | The interplay between Gb3 species and cholesterol content influences LecA and StxB binding efficiencies. GUVs doped with 0.5 mol% of the membrane marker β-BODIPY-FL-C5-HPC (green) and 5 mol% Gb3-mix (A and B) or 5 mol% Gb3-C24:0 (C and D) were incubated with LecA-Al647 (200 nM, orange) or StxB-Cy5 (200 nM, blue). GUVs approximating liquiddisordered membranes (DOPC, A and C) contained DOPC/Gb3 in the ratio 94.5/5 mol%, and GUVs resembling liquid-ordered membranes (Chol + SM, B and D) consisted of sphingomyelin/cholesterol/Gb3 in the ratio 64.5/30/5 mol%. The images A -D show equatorial sections through representative GUVs. Scale bars correspond to 5 μm. (E -F) Quantitative analysis of the binding efficiencies of LecA (E) and StxB (F) to DOPC GUVs (green) and Chol + SM GUVs (red) containing 5 mol% of either Gb3-mix, or Gb3-C24:0.

Figure

  Figure 21| StxB-induced SLB reorganization. The SLB was composed of DOPC/chol/SM/Gb3 (37.5/20/37.5/5). The SLB was labeled by Bodipy-HPC that preferentially incorporates in Ld domains. StxB-Cy5 (200 nM) was applied. StxB binds almost exclusively to Lo domains. Moreover, it induces a formation of new Lo domains (red and yellow circles -Merged) by efficient clustering of the Gb3 molecules incorporated in the Ld phase. Scale bars -10 and 5 µm respectively.

  5/5)) to the one used in the experiment with StxB. At the earlier time points, LecA behaved similarly to StxB, as it bound preferentially to Lo domains enriched in Gb3 (figure 22a -black arrowhead). However, over time, no formation of new microscopic Lo domains was observed. On the contrary, Lo domains were decreasing in size and completely disappear (figure 22a -blue arrowhead). The total area of the Lo domains (figure 22b -blue curve) as well as the number of Lo domains and the size of the individual Lo domains decreased over time (figure 22c, d). Remarkably, after 20 min of incubation with LecA, almost all Lo domains are dissolved. In parallel, LecA forms large microscopic clusters (figure 22a -white arrowhead).

Figure 22 |

 22 Figure 22 | SLB reorganization induced by purified LecA. SLB is labeled by 𝛽 Bodipy-C12-FL-HPC that localizes in Ld domains (cyan). LecA ia labeled by Cy5 (red). Images are acquired with wide field microscopy (HILO). a) Time series of LecA interaction with phase-separated SLB (DOPC/chol/SM/Gb3-mix (37.5/20/37.5/5)). LecA induces Lo domains dissolution (blue arrowhead) and multilayer formation (purple arrowhead). Membrane multilayers co-localize with the bright LecA clusters (white arrow). At later time points, large membrane defects appear (orange arrowhead) b) The total area of the Lo domains, membrane defects and membrane multilayers changes over time. c) The total number of Lo domains decreases over time; d) The sizes of the individual Lo domains decrease over time; e) The mean fluorescence intensity signal in membrane multilayers is approximately 1.5 times higher than the intensity of the original, single bilayer membrane. Scale bars are 10 and 5 µm, respectively.

Figure 23|

 23| Figure 23| Schematic representation of the membrane reorganization induced by LecA. a) Ribbon diagram of LecA tetramer. Gb3-specific binding pockets are marked with numbers 1-4; b) LecA binding to a phase separated lipid bilayer; c) Lo domains dissolution; d) Membrane multilayer formation. LecA accumulates at the lipid bilayer interfaces.

Figure 1 :

 1 Figure 1: Self-assembly of lipids in contact with water. Lipids are characterised by their critical packing parameter v/la 0 . A) Inverted cone-shaped lipids form micelles (micellar phase) of positive curvature. B) Cone-shaped lipids assemble into different structures of hexagonal (H II ) phase. C) Cylinder-shaped lipids form a lamellar phase.

Figure 2 :

 2 Figure 2: Lipid bilayer phases. Depending on the lipid composition, temperature and order parameters, the lamellar phase can adopt solid or fluid phases. A) Lipid bilayer of solid phase composed of sphingomyelin. B) Lipid bilayer of fluid phases can exist as liquid-ordered (composed of sphingomyelin and cholesterol) or liquid-disordered (composed of DOPC).

Figure 3 :

 3 Figure 3: From lipid rafts in native plasma membranes to liquid-ordered phases in synthetic membrane systems. A) Composition of raft and non-raft types of native plasma membranes. The membrane is asymmetrical -the extracellular membrane leaflet is enriched with SM and saturated PC lipids, while PS, PI and PE lipids associate mainly to the cytosolic leaflet. B) Synthetic membrane systems composed of liquid membrane domains to mimic native membrane phases. The liquid-ordered domain mimics a lipid raft membrane and the liquid-disordered domain mimics a non-raft membrane.

6 )

 6 Lectin-and curvature-driven lipid sorting as membrane reorganisation process Lipid sorting was reported for multicomponent lipid mixtures(119), where an interplay between membrane entropic forces and lipid-lipid interactions takes place. In such systems, like ternary mixtures close to phase separation (119,120), lipid sorting can be triggered by a change in temperature or lipid composition. When an oligomeric lectin binds to GSLs, it acts like a clustering device (Figure4A, B) that can activate lipid sorting and induce phase separation in otherwise homogeneously mixed membranes(101,(120)(121)(122)(123). Local asymmetric stress imposed by lectin-GSL interactions, and subsequent membrane deformation can change locally the lipid organisation of the bilayer.Pinot et al.

Figure 4 :

 4 Figure 4: Lectin-and curvature-driven lipid sorting. A) Lectins recognise GSL receptors and bind to the membrane surface. B) Lectins crosslink the GSL-enriched clusters. These clusters induce an asymmetric stress in the membrane, which leads to tubule nucleation and plasma membrane invagination. C) Lectin-induced plasma membrane tubules are enriched with sphingolipids due to their association with GSL clusters.

Figure 1 :

 1 Figure 1: GUV processing. A) StxB-Cy5 (red channel) binds to the Lo domain of a GUV composed of DOPC/chol/SM and the glycosphingolipid Gb3 (40/15/40/5). The Lo domains are not visible since the membrane probe Bodipy (green channel) preferentially localizes to Ld domains. B) Circular profiles of the membrane marker Bodipy (green curve) and StxB-Cy5 (red curve). The units of StxB-Cy5 circular profile (right Y axis) are quantified as shown in (C). C) Calculation of the relative binding efficiency.

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Segregation between LecA and StxB can be reconstituted in GUVs containing a natural mixture of Gb3 species GUVs containing 5 mol-% Gb3-mix were incubated with LecA-Al647 (200 nM, orange) and StxB-Cy3 (600 nM in A and 200 nM in B, blue) (A and B). The images show equatorial sections through representative GUVs with a diagram displaying the corresponding circular intensity profiles underneath. Phase-separated GUV (A) contained DOPC/sphingomyelin/cholesterol/ Gb3-mix in the ratio 42.5/14,5/42.5/5 mol-%, whereas non-phase-separated GUV (B) contained DOPC/cholesterol/Gb3-mix in the ratio 64.5/30/5 mol-%. GUVs in A and B were additionally spiked with 0.5 mol-% of the membrane marker β-BODIPY-FL-C5-HPC (green). Scale bars correspond to 5 μm.

Figure 3 :

 3 Figure 3: Investigation how Gb3 species and phase separation influence LecA and StxB binding efficiencies GUVs doped with 0.5 mol-% of the membrane marker β-BODIPY-FL-C5-HPC (green) and 5 mol-% Gb3-mix (A and B) or 5 mol-% Gb3-C24:0 (C and D) were incubated with LecA-Cy5 (200 nM, orange) or StxB-Cy5 (200 nM, blue). Nonphase-separated GUVs (A and C) contained DOPC/cholesterol/Gb3 in the ratio 64.5/30/5 mol-%, whereas phase-separated GUVs (B and D) consisted of DOPC/cholesterol/sphingomyelin/Gb3 in the ratio 42.5/14.5/42.5/5 mol-%. The images A -D show equatorial sections through representative GUVs. Scale bars correspond to 5 μm. (E -F) Quantitative analysis of the binding efficiencies of LecA (E) and StxB (F) to non-phase-separated GUVs (red) or the ld (green) or lo (blue) phase of phase-separated GUVs containing 5 mol-% of either Gb3-mix (mix), Gb3-C24:0 (C24:0), Gb3-C24:1 (C24:1), or Gb3-FSL (FSL). For each condition, the middle horizontal line represents the median, the boxes the 25 th to

Figure 4 :

 4 Figure 4: The interplay between Gb3 species and cholesterol content influences LecA and StxB binding efficiencies GUVs doped with 0.5 mol-% of the membrane marker β-BODIPY-FL-C5-HPC (green) and 5 mol-% Gb3-mix (A and B) or 5 mol-% Gb3-C24:0 (C and D) were incubated with LecA-Al647 (200 nM, orange) or StxB-Cy5 (200 nM, blue). GUVs approximating liquid-disordered membranes (DOPC, A and C) contained DOPC/Gb3 in the ratio 94.5/5 mol-%, and GUVs resembling liquid-ordered membranes (Chol + SM, B and D) consisted of sphingomyelin/cholesterol/Gb3 in the ratio 64.5/30/5 mol-%. The images A -D show equatorial sections through representative GUVs. Scale bars correspond to 5 μm. (E -F) Quantitative analysis

Figure 5 :

 5 Figure 5: Reconstituting LecA and StxB segregation in minimal systems (A) Phase-separated GUVs composed of DOPC/cholesterol/sphingomyelin/ β-BODIPY-FL-C5-HPC (green)/Gb3-FSL/Gb3-C24:0 in the ratio 40/14.5/40/0.5/5/5 mol-%) were incubated with LecA-Al647 (200 nM, orange) and StxB-Cy3 (600 nM, blue). Equatorial sections through a representative GUV are shown and the corresponding circular intensity profiles are displayed underneath. Scale bar corresponds to 5 μm.

  about 10 min of incubation, bacteria start to form aggregates with newly attaching bacteria, which grow in size over time (figure1a and S3b).Simultaneously with the formation of bacterial aggregates, SLBs get drastically reorganized (figure1a): Over time, the shape and size of Lo domains change: The total area of Lo domains (figure1c) as well as the areas of individual Lo domains (figure1d) decrease. Remarkably, several Lo domains disappear completely (figure1a, blue and green arrowhead). In parallel to the dissolution of Lo domains, the fluorescence intensity of the membrane marker Texas Red-DHPE increases around bacteria, indicating a local enrichment of lipid membranes. One selected event describing the subsequent adhesion of two bacteria (figure1a) was quantitatively analyzed (figure1e). At this point, we speculate that P. aeruginosa tries to cover its surface by recruiting lipid membrane from the SLB. By this means, bacteria seem to have an effect on the collective organization of the membrane since Lo domains that have not encountered any bacterium in close proximity, also dissolve (figure1a, blue arrowhead).AsEierhoff et al. highlighted the crucial role of the interaction of the P. aeruginosa lectin LecA with the host cell glycosphingolipid Gb3 for host cell invasion 1 , we wanted to elucidate the impact of LecA on membrane organization. Therefore, we studied the effects of the LecA-deficient PAO1 strain (PAO1-ΔLecA) on membrane organization of SLBs (figure1band supplementary movie 2). In the absence of specific LecA-Gb3 interactions, the number of PAO1-ΔLecA adhering to a Gb3-containing lipid bilayer was reduced (approx. 4 fold -figureS3) and detachment of bacteria away from the lipid bilayer was recorded (figure1b, cyan arrowhead).

Figure 1 |

 1 Figure1| SLB reorganization induced by P. aeruginosa. Bacteria expressing GFP (green) were applied to phase-separated SLBs composed of DOPC/chol/SM/Gb3(37.5/20/37.5/5) and labeled by Texas Red-DHPE (red). a) At the earlier time points bacteria prefer to attach to the Lo/Ld domain boundaries (purple arrowhead).Furthermore, the P. aeruginosa wild type strain forms bacterial clusters (white arrow). Bacteria accumulate membrane from the SLB at the spots of bacterial attachment (white circle). P. aeruginosa dissolves Lo domains (blue and green arrowheads). b) P. aeruginosa ∆LecA strain contact the lipid bilayer but do not remain stably attached to its surface (cyan arrowhead). Moreover, P. aeruginosa ∆LecA do not induce the disappearance of Lo domains. Bacteria do not accumulate membrane from SLB. c) In SLBs, P. aeruginosa wild type strain induces the decrease of the total area of Lo domains over time (red curve) whereas P. aeruginosa ∆LecA do not (black curve). d) P. aeruginosa wild type strain decreases the size of the individual Lo domains over time. e) P. aeruginosa wild type (red curve) accumulates membrane from the SLB on the bacterial surface. The membrane marker intensity at the spot where bacterium is attached (a -white circle), increases over time. Two successive events of bacterial docking to the SLB are visible, indicating that two bacteria accumulate membrane material one after the other. In contrast, P. aeruginosa ∆LecA do not accumulate membrane material (black curve, b -white circle). f) P. aeruginosa ∆LecA do not modify the size of the individual Lo domains. The scale bars are 10 and 5 µm, respectively. The complete time sequence for a and b are available online as the supplementary movies 1 and 2.

Figure 2 |

 2 Figure 2 | SLB reorganization induced by purified LecA. SLB is labeled by 𝛽 Bodipy-C 12 -FL-HPC that localizes to Ld domains (cyan). LecA is labeled with Cy5 (red). Images are acquired with wide field microscopy (HILO). a) Time series of LecA interaction with phase-separated SLB (DOPC/chol/SM/Gb3 (37.5/20/37.5/5)). LecA induces Lo domains dissolution (blue arrowhead). Simultaneously, LecA clusters at the SLB (white arrowhead). LecA clusters induce the formation of membrane multilayers (purple arrowhead). At the later time points, large membrane defects appear (arrowhead) b) The total area of the Lo domains, membrane defects and membrane multilayers changes over time. c) The total number of Lo domains decreases over time; d) The sizes of the individual Lo domains decrease over time; e) The mean fluorescence intensity signal in membrane multilayers is approximately 1.5 times higher than the intensity of the original, single bilayer membrane. Scale bars are 10 and 5 µm, respectively. The later timepoints are presented in figure S5. The complete time sequence is available online as the supplementary movie 3.

Figure 3|

 3| Figure 3| Membrane order modifications of SLB induced by LecA. The two-color imaging was performed using confocal microscopy. The time sequence in two color channels is presented in figure S6. a) GP images of the SLB before and at several time points after LecA application. The GP values are according to the color code on the right panel. Phase dissolution occurs in the same way as in figure 2 (red arrowhead). The membrane multilayers are highlighted by circles. b) GP histograms of the SLB before (black curve) and 20 min (red curve) and 40 min (blue curve) after LecA application. c) Membrane order of the Lo domains, Ld domains and membrane multilayers. Scale bar 10 µm.

  Gb3). The crystal structure of this lectin is not resolved yet, but, presumably, the geometry of the dimeric form is arranged as presented in figureS1. Applied to phaseseparated SLBs, RPL (200 nM) recognizes preferentially Lo domains (figure4a) and the SLB reorganization is triggered immediately after RPL addition. Lo domains fuse and decrease in size over the first 15 min (figure4a-c). At this point, the re-shaping of Lo domains stops. Nevertheless, the fluorescence intensity ratio between Ld and Lo domains (I Ld /I Lo ) indicates a two-fold drop over the next 45 min (figure4 a, d, e). This can be explained by asymmetric Lo domain dissolution. We hypothesize that in the upper membrane leaflet, which is directly exposed to RPL, Lo domains dissolve, whereas in the lower leaflet, which is in direct contact with the mica substrate, the dissolution of Lo domains is constrained.

Figure 4|

 4| Figure 4| SLB reorganization induced by the dimeric LecA variant RPL 𝛼Gal. SLB is labeled by 𝛽 Bodipy-C 12 -FL-HPC that localizes to Ld domains (cyan). RPL is labeled by Cy5 (red). Images are acquired with wide field microscopy (HILO). a) The time series of the RPL interaction with phase separated SLB (DOPC/chol/SM/Gb3 (37.5/20/37.5/5)). RPL binds preferentially to the Lo domains. RPL induces fusion and a decrease in size of the Lo domains (purple arrowhead). b) The total area and the number of Lo domains decreases over time; c) The sizes of the individual Lo domains slightly decrease over time; d,e) The contrast of the fluorescence signal between the Ld and Lo domains at different time points. d) The Bodipy fluorescence intensity profile along the white line displayed in (a) at different time points. e) The intensity contrast (ratio between the fluorescence intensity signals of Bodipy in Ld and Lo domains) value at different time points for all Lo domains. After 1 hour, the contrast dropped to about half of the initial value. The scale bars are 10 and 5 µm respectively. The complete time sequence is available online as the supplementary movie 4.

  reorganization of membrane supplemented by Gb3 was formerly studied in context of cellular uptake of the B subunit of Shiga toxin from S. dysenteriae. The studies on synthetic membrane systems (GUVs 28 and SLBs 29 ) showed the specific preference of StxB to the Gb3 incorporated in Lo domains as well as efficient clustering of the Gb3 molecules induced by the multivalent binding of the StxB. Moreover, StxB shrinks Lo domains and induces the formation of novel Lo domains in supported lipid bilayers (figure S9, also demonstrated by Windschiegl et al. 29 ). Efficient Gb3 clustering by StxB is granted by the number, position and orientation of binding pockets (all 15 of them are pointing in one direction). Unlike StxB, LecA contains only 4 Gb3-specific binding sites and, in addition, the distance between them is larger than in case of StxB (ref). Despite, as well as StxB, LecA binds initially also to Lo domains, we did not observe new Lo domains formation as in case of StxB. On the contrary, LecA induces the dissolution of the Lo domains homogenizing the membrane. Moreover, LecA -Gb3 interaction is strong enough to detach the membrane from the solid support and to either wrap it around bacterium or to form

  dioleoyl-sn-glycero-3-phosphocholine(DOPC), sphingomyelin and cholesterol) were from Avanti Polar Lipids. Gb3 "wild type" (ceramide trihexosides) was from Matreya. The lipophilic membrane probe β-BODIPY C12-HPC(2)(3)(4),4a-Diaza-s-Indacene-3-Dodecanoyl)-1-Hexadecanoyl-sn-Glycero-3-Phosphocholine) and DHPE-TexasRed were from ThermoFisher Scientific. The environmental sensitive membrane marker NR12S was provided by Dr. Andrey Klymchenko (University of Strasbourg). Shiga toxin B subunit was from Sigma Aldrich. Recombinant LecA was produced from Escherichia coli following previously published procedures7 . Recombinant RPL 𝛼Gal was from Glycoselect. The marker for lectin labeling (Cy-5 monoreactive NHS esther) was from GE Healthcare. PBS (-/-) buffer was from Gibco.Bacterial culture. GFP-tagged P. aeruginosa PAO1 wild type and PAO1 ΔLecA were constructed according to the previously published procedure 1 .Lectin labeling. Lectins were labeled with Cy5 mono-reactive dye from GE Healthcare Life Sciences according to the amine-reactive probe labeling protocol from ThermoFisher Scientific. Briefly, 1 µl of a 10 mg/ml solution of the amino-reactive probe in DMSO was added to 100 µl of a 1 mg/ml protein solution supplemented with 100 µM NaHCO3. The mixture was incubated for 1 h at room temperature with continuous shaking. The labeled protein was purified using Zeba Spin Desalating Columns, 0.5 ml, from ThermoFisher Scientific.Solid supported lipid bilayer preparation. The lipid mixtures of DOPC/chol/SM/Gb3 with the molar ratio of 37.5/20/37.5/5 were prepared in chloroform. SLBs were labeled adding 0.1 mol % of Bodipy or TexasRed to the lipid mixture in chloroform. Chloroform was further removed by evaporation first under nitrogen atmosphere and under vacuum(10)(11)(12)(13)(14)(15). Lipids were resuspended in ultrapure water at 55 ºC with extensive vortexing in order to form large multilamellar vesicles. Furthermore, the vesicle suspension was extruded using the LiposoFast-Basic extruder (Avestin) with 50 nm pore size filter (55 ºC, 50 passages). The thin freshly cleaved layer of mica (PLANO) was attached to the microscopy glass coverslip (Menzel Gläser) using UV-curing glue (Norland optical adhesive). The extruded small unilamellar vesicles (SUV) suspension (50 𝜇𝑙) was applied to the mica together with 150 microliters of high (10 mM) CaCl 2 solution in pure water. The coverslips were incubated 30 min at 55 ºC in order to form the non-phase separated lipid bilayer. Thereafter, SLBs were washed with warm (55 ºC) PBS (-/-) to remove the lipids not attached to mica. Finally, SLBs were cooled down very slowly (1 -1.5 hours) to room temperature in order to induce the lipid phase separation on microscopic scale. For membrane order studies, lipid bilayers were prepared unlabeled and then NR12S (2 𝜇𝑙 of 0.1 mM NR12S in DMSO) was added to the SLBs when the bilayer formation has already been completed. After 1-2 min of incubation, the lipid bilayer was washed mildly with PBS. Each individual SLBcontaining well plate was further mounted on the microscopy holding dish for further imaging. Bacteria or lectins were further applied to the assembled well-plate.The imaging of the LecA at the surface of bacteria with Structured IlluminationMicroscopy. An overnight culture of P. aeruginosa strains PAO1-GFP wild type and PAO1-GFP ΔLecA were centrifuged at 6000 rpm and resuspended in 500 µl of PBS (-/-) . 1:1000 dilution of the bacterial stock was pelleted and used for the staining procedure. The bacteria were fixed with 4% PFA at RT for 20 min. After fixation, both strains were spun down and excess PFA was removed by NH4Cl quenching. After blocking with 3% BSA, bacteria were stained with anti-LecA antibody 1:200 for 45 min. This was followed by 3X washes with PBS (-/-). Bacteria were then incubated with secondary antibody (1:300) for 30 min. The stained bacteria were washed three times with PBS and the pellet was mixed with mowiol, spotted on a cover slip and mounted onto a glass slide. Imaging was performed with an N-SIM microscope system (Eclipse Ti-E with 100x oil objective, N.A.1.49) with ORCA-Flash 4.0 sCMOS camera (Hamamatsu Photonics K.K.). The images were acquired and reconstructed using the reconstruction slice system from NIS-Elements software (Nikon).Imaging of SLB reorganization induced by bacteria with the confocal microscopy. Overnight cultures of bacteria were pelleted and resuspended in PBS (-/-). Bacteria were applied to SLBs at room temperature and examined on the inverted confocal fluorescence microscope. Of note, due to the safety reasons (the imaging chamber must be tightly sealed before it can be mounted on the microscope), it was possible to start the imaging of live bacteria applied to SLBs only starting from the time point 5 min after bacterial application. The two color imaging was performed on a confocal fluorescence microscope (Nikon Eclipse Ti-E with A1R confocal laser scanner, 60x oil objective, N.A.1.49). For imaging of GFP-tagged bacteria, 488 nm laser for excitation and the emission filter 525/50 BP were used. Simultaneously, the SLB was imaged using 561 nm laser excitation and 585/40 BP emission filter.

  HILO-illumination imaging was performed on a (Nikon Eclipse Ti-E with 100x oil objective, N.A.1.49 and iXon 897 EMCCD camera (Andor Technology)). The 488 nm and 647 nm lasers were used to excite Bodipy and Cy-5 respectfully. After lectin application several areas of each SLB was imaged with exposure time of30 -50ms and interval of 20 seconds between frames.Time lapse data analysis. The resulting time-frame images of the SLBs were analyzed using the FIJI-based home-made macro. First the bleaching and laserprofile corrections were applied. Then, the total area of the membrane defects, Lo domains, Ld membrane and membrane multilayers for each frame was calculated. In addition, the sizes of the individual Lo domains were extracted.Confocal ratiometric microscopy. The two color ratiometric imaging was performed on a confocal fluorescence microscope (Nikon Eclipse Ti-E with A1R confocal laser scanner, 60x oil objective, N.A.1.49). For fluorescence excitation, 488 nm laser was used. The emission of NR12S was simultaneously recorded in two color channelsgreen (using 525/50 BP filter) and far red (700/75 BP filter). The two-color-channel raw image was analyzed using home-made FIJI-based macro in order to produce the general polarization (GP) value of NR12S spectrum for each pixel of the image using the formula:𝐺𝑃 = 𝐼 )*++, -𝐼 *+. 𝐼 )*++, + 𝐼 *+.where 𝐼 )*++, is the gray value of the pixel in green color channel and 𝐼 *+. is the gray value of the pixel in red color channel. The resulting GP image contained GP values for each pixel of the image. The GP histogram was extracted from the resulting GP image.Statistical data analysis and representation. Statistical data is visualized using box-whiskers plots, where the middle horizontal line represents the median, the boxes the 25th to 75th percentiles, and the whiskers -the standard deviation. For the plots in figureS1also the outliers are indicated with dots. The statistical significance was analyzed with oneway ANOVA. The p-values are indicated at the plots.

Figure S5|

 S5| Figure S5| Membrane defects induced by LecA. a) Membrane reorganization induced by LecA. The images correspond to the same experiments as in Figure 2, but at longer time points. Membrane defects grow over time and the SLB becomes partially disintegrated (orange arrow). The fluorescent LecA clusters accumulated in membrane multilayers fade over time. Most likely, it is due to the bleaching of the Cy 5 label. Scale bars -10 and 5 µm respectively.

Figure S7 |

 S7 Figure S7 | LecA-induced membrane reorganization of homogeneous SLB. The SLB was prepared from a non-phase separating lipid mixture (DOPC/chol/Gb3 (65/30/5). Membrane marker -HPC-Bodipy). Since such lipid bilayers do not exhibit phase separation, the membrane fluorescence signal (cyan) appears homogeneous. Application of LecA labeled with Cy5 (red) induces membrane multilayers formation (magenta arrows) and fast membrane disintegration (membrane defect is highlighted with red arrow). Scale bars -10 and 5 µm respectively.

Figure S8|

 S8| Figure S8| Negative control. Phase separated SLBs labeled by DHPE-Texas Red (a) and HPC-Bodipy (b) were imaged during one hour using confocal (a) and widefield (b labeling ). The Lo domains and their lateral distribution are stable during one hour of acquisition for both types of labeling and acquisition. A membrane defect (HPC-Bodipy -magenta arrow) that formed during SLB preparation is stable and neither expands or shrinks during the acquisition. Scale bars -10 and 5 µm respectively.

Figure

  Figure S9| StxB-induced SLB reorganization. The SLB was composed of DOPC/chol/SM/Gb3 (37.5/20/37.5/5). The SLB was labeled by Bodipy-HPC that preferentially incorporates in Ld domains. StxB-Cy5 (200 nM) was applied. StxB binds almost exclusively to Lo domains. Moreover, it induces a formation of new Lo domains (red and yellow circles -Merged) by efficient clustering of the Gb3 molecules incorporated in the Ld phase. Scale bars -10 and 5 µm respectively.

  Les lasers fournissent une émission lumineuse intense d'une bande spectrale très mince. Par conséquent, ils sont largement utilisés comme sources d'excitation pour la microscopie à fluorescence. La lumière laser peut être focalisée en un point distinct de l'échantillon avec l'objectif. De plus, la fluorescence induite par la lumière laser peut être collectée et analysée. En analysant l'échantillon point par point, vous pouvez obtenir une image de la tranche de l'échantillon située dans le plan focal de l'objectif, pixel par pixel. Le problème commun de la microscopie est d'établir un rapport signal sur bruit élevé. Une quantité considérable d'arrière-plan provient de la fluorescence en dehors du plan focal de l'objectif. Afin de surmonter ce problème, le signal provenant

d'au-delà du plan focal est éliminé par l'introduction d'un trou d'épingle dans le système de microscopie. Cette technique de microscopie s'appelle Microscopie confocale à balayage laser (LSCM, figure

  Remarkably, bacteria exhibited a clear preference to the border of the Ld and Lo domains in phase separated SLB (figure 24a -purple arrowheads). Up to 60 % of the bacteria at the early time points were located at the Lo/Ld borders. Moreover, Ld domains were also attractive spots (approx.. 35 %) for bacterial adhesion, whereas Lo domains remained almost completely ignored by bacteria. Taking into account that the Gb3 is also present in Lo, we suggest that despite bacteria rely on a specific interaction of LecA to Gb3 for successful adhesion; additional mechanisms may amplify the Gb3 recognition incorporated in a more favorable environment (e.g. Ld domains, Lo/Ld boundaries). Importantly, at later time-points, bacteria prefer to join existing bacterial aggregates instead of adhesion to SLB area not yet occupied by bacteria. Bacteria labeled by GFP (green) are applied to phase separated SLBs composed of DOPC/chol/SM/Gb3(37,5/20/37,5/5) and labeled by DHPE-TexasRed (red). Ld domains are enriched in DHPE-Texas Red, whereas the tightly packed Lo domains are depleted of the membrane marker and thus appear dark. Images are acquired with a laser scanning confocal microscope. a) The wild-type strain P. aeruginosa attaches to the SLB and forms bacterial clusters (white arrow). At the earlier time points, bacteria preferentially attach to the Lo/Ld domain boundaries (purple arrowhead). Furthermore, bacteria pull lipids out of the SLB to cover their surface with sequestered material (white circle). Additionally, P. aeruginosa alter the lipid organization of SLBs and dissolve the Lo domains (blue and green arrowheads). b) P. aeruginosa ∆LecA strain bacteria contact the lipid bilayer but do not remain stably attached to its surface (cyan arrowhead). Moreover, P. aeruginosa ∆LecA bacteria do not dissolve the Lo domains. c) In SLBs, P. aeruginosa wt induces the decrease of the total area of Lo domains over time (red curve) whereas P. aeruginosa ∆LecA does not. d) P. aeruginosa decreases the size of the individual Lo domains over time. e) P. aeruginosa wt (red curve) accumulates lipids from the SLB on the bacterial surface. The membrane marker intensity at the spot where bacterium is docked (a -white circle), increases over time. Two successive events of bacterial docking to the SLB are visible, indicating that two bacteria pull-out membrane material one after the other. In contrast, P. aeruginosa ∆LecA does not accumulate membrane material (black curve). The membrane marker intensity at the spot where bacterium is docked (b -white circle) does not increase over time. f) P. aeruginosa ∆LecA does not modify the size of the individual Lo domains. The scale bars are 10 and 5 µm, respectively.

Figure 24 | SLB reorganization induced by P. aeruginosa.

The bacterium Pseudomonas aeruginosa perishes membrane organization by its lectin LecA

	Other publications without context to this thesis
	Sych, T., Mély, Y. and Römer, W., 2018. Lipid self-assembly and lectin-induced Villringer, S., Madl, J., Sych, T., Manner, C., Imberty, A. and Römer, W., 2018. Lectin-reorganization of the plasma membrane. Philosophical Transactions of the Royal mediated protocell crosslinking to mimic cell-cell junctions and adhesion. Society B: Biological Sciences, 373(1747), p.20170117. Scientific reports, 8(1), p.1932.
	Zamotaiev, O.M., Shvadchak, V., Sych, T.P., Melnychuk, N.A., Yushchenko, D., Mely, Y. and Contribution: I contributed to the writing of the review and prepared the figures. Pivovarenko, V.G., 2016. Environment-sensitive quinolone demonstrating long-The accepted manuscript of the article is enclosed with permission of the publisher lived fluorescence and unusually slow excited-state intramolecular proton
	Royal Society. transfer kinetics. Methods and applications in fluorescence, 4(3), p.034004.
	Co -author manuscript
	Schubert, T., Sych, T., Madl, J., Xu M., Brandel A., Omidvar R., Patalag L., Ries A., Kattelhoit
	K., Mely Y., Steinem C., Werz D., Thuenauer, R. and Römer, W. Differential recognition
	of lipid domains by two Gb3-binding lectins [Manuscript in preparation]
	The manuscript is enclosed

Sych, T.,

Schubert, T., Vauchelles, R., Madl, J., Omidvar, R., Thuenauer, R., Richert, L., Mély, Y. and Römer, W., 2018

. GUV-AP: multifunctional FIJI-based tool for quantitative image analysis of Giant Unilamellar Vesicles. Bioinformatics.

Contribution: I wrote the macro code, performed all experiments, prepared figures and wrote the manuscript.

The accepted manuscript of the article is enclosed with permission of the publisher Oxford Academic. Sych, T., Schubert, T., Aigal. S., Thuenauer, R., Brandel, A., Omidvar, R.,

Eierhoff T., Richert, L., Mély, Y, Madl, J., and Römer, W. [Manuscript in preparation]

Contribution: I performed all experiments, prepared figures and wrote the manuscript.

The manuscript is enclosed.

Contribution: I performed some experiments for the figures 2 and 4, I analyzed data and contributed to the writing of the manuscript.

Baaken, G., Zaitseva, E., Petersen, S.,

Sych, T., Stefan, K. and Behrends, J.C., 2019. Array

of Freestanding Planar Lipid Bilayers for Parallel Optical and Electrical Recordings.

Table S1 .

 S1 Macro execution time. The macro was run on the Mac OS X El Capitan version 10.11.6, processor 2.9 GHz Intel Core i5, memory 8 GB 1867 MHz DDR3.

	Type of GUVs	Batch mode	Channels	Circularity check	GUV stitching	Number of images processed	Number of GUVs processed	Total execution time	Time per image	Time per GUV
	phase-	no	2	no	yes	25	108	13 min 46 s	33 s	7.7 s
	separated	yes	-	-	-	-	-	-	-	-
		no	2	yes	no	80	90	10 min 55 s	8.2 s	7.3 s
	homogeneous
		yes	2	yes	no	80	90	7 min 58 s	6 s	5.3 s

  2) and Ld domains (blue, with a GP value of around -0.7). Here, the GP histogram shows two distinct populations that correspond to Lo and Ld domains (figure 3b -black curve). After the application of 200 nM of LecA, Lo domain dissolution, formation of multilayers and membrane disintegration were observed (figure 3a and S6), as described above. In brief, Lo domains shrink, rearrange and disappear (figure 3a -red arrowhead). Since the total area of Lo domains is reduced at 20 min of LecA incubation (figure S6), the Lo population of the GP histogram is reduced accordingly, whereas the Ld population is

	membrane.
	Concurrent with Lo domain dissolution, membrane multilayers grow (figure 3a -all
	circles). Using raw confocal images (figure S6) we located multilayers for further
	quantification of membrane order (figure 3a -40 min, all circles). Interestingly, we
	observed that the upper bilayer can grow on the top of an existing Lo domain,
	apparently blocking its dissolution (figure

increased (figure 3b -red curve). At 40 min, the Lo domains are almost completely dissolved (figure

3a

). Here, the GP histogram shows only one distinct population (figure 3b -blue curve). This population is still located in the GP value range of lowordered membranes (i.e. Ld domains), but exhibits a slight shift towards higher GP values. This indicates the redistribution of SM and cholesterol in this novel "mixed"

Acknowledgements

This work was the biggest project I performed in my life so far. I would not be able to accomplish my aims without the help and support of multiple people who made a significant impact on my professional and private life over last years. Here, I am happy to acknowledge all of them.

Acknowledgements

This work was supported by the Excellence Initiative of the German Research Foundation [EXC 294] and the German Research Foundation grant [IRTG 1642 -Soft Matter Science: Concepts for the design of functional materials]. TS acknowledges the support by the Franco-German University (PhDtrack program "Polymer Sciences" and the program "Cotutelle de thèse") and the Collège doctoral européen (Programme doctoral international). We are grateful to Toshihide Kobayashi for proofreading of the manuscript.

Acknowledgements Funding: This work has been supported by the German Research Foundation (EXC 294, RO 4341/2-1, RO 4341/3-1 and IRTG 1642). Taras Sych acknowledges the support by the Franco-German University (programs "Polymer Sciences" and "Cotutelle de thèse") and the Collège Doctoral Européen (PDI). We are grateful to Andrey Klymchenko for providing the lipophilic membrane probes. Conflict of Interest: The authors declare no conflict of interest. Acknowledgements This work was supported by the German Research Foundation [EXC 294, RO 4341/2-1, RO 4341/3-1, IRTG 1642, RTG 2202, SFB 803], the Ministry of Science, Research and the Arts of Baden-Württemberg [Az: 33-7532.20] and a starting grant from the European Research Council [Programme "Ideas," ERC-2011-StG 282105]. Ta.S. acknowledges support by the Franco-German University (programs 'Polymer Sciences' and 'Cotutelle de thèse') and the Collège Doctoral Européen (PDI). Acknowledgments This study was supported by the German Research Foundation (DFG) under Germany's Excellence Strategy (CIBSS -EXC-2189 -Project ID 390939984, BIOSS -EXC 294), by DFG grants RO 4341/2-1, RO 4341/3-1, RTG 2202 and IRTG 1642, by the Ministry of Science, Research and the Arts of Baden-Württemberg (Az: 33-7532.20), by the German Federal Ministry of Education and Research (BMBF) in the framework of the EU ERASynBio project SynGlycTis, and by a starting grant from the European Research Council (Programme "Ideas," ERC-2011-StG 282105). T.S. acknowledges support by the Franco-German University (programs 'Polymer Sciences' and 'Cotutelle de thèse') and the Collège Doctoral Européen (PDI). We are grateful to Andrey Klymchenko for providing the lipophilic membrane probes. S.A. acknowledges support from the International Max Planck Research School for Molecular and Cellular Biology.

The ImageJ distribution package FIJI is freely available online: https://imagej.net/Fiji. The macro file GUV-AP.ijm is available at https://github.com/AG-Roemer/

Abbreviations

Lectins:

Supplementary figures

GUV-AP: multifunctional FIJI-based tool for quantitative image analysis of Giant Unilamellar Vesicles

Taras Sych 1,2, § , Thomas Schubert 1, § , Romain Vauchelles 2 , Josef Madl 1 , Ramin Omidvar 1 , Roland Thuenauer 1 , Ludovic Richert 2 , Yves Mély 2,* and Winfried Römer [START_REF] Kreder | Blue fluorogenic probes for cell plasma membranes fill the gap in multicolour imaging[END_REF]. The yellow dot and the dashed arrow respectively indicate the starting point and the direction used to extract the circular profile given in (B). This circular profile shows that the F2N12SM intensity is high in Ld phase and negligible in Lo phase. C) and D) show the polarization effect [START_REF] Haluska | Combining Fluorescence Lifetime and Polarization Microscopy to Discriminate Phase Separated Domains in Giant Unilamellar Vesicles[END_REF], which results in the variation of the fluorescence intensity of the probe in the bilayer along the GUV contour. I max is the fluorescence intensity where the molecules of the probe have parallel orientation to the excitation laser polarization. At I min probes are oriented perpendicular to the laser polarization. This effect can be used to determine the orientation of the probe in the bilayer; C) GUV composed of SM/chol (70/30) labeled by F2N12SM. The fluorescence intensity of F2N12SM varies along the GUV contour, as a result of the orientation of the dye in the membrane. D) Circular profile of the GUV in (C). [START_REF] Kilin | Fluorescence Lifetime Imaging of Membrane Lipid Order with a Ratiometric Fluorescent Probe[END_REF]. The colors of the pixels represent the lifetime values according to the color scale on the X-axis. The yellow dot and the dashed arrow, respectively, indicate the starting point and the direction of the circular profile extraction. B) Circular profile of GUVs presented in A. The variation of the apparent lifetime in the GUV composed of DOPC/chol is due to the polarization effect [START_REF] Haluska | Combining Fluorescence Lifetime and Polarization Microscopy to Discriminate Phase Separated Domains in Giant Unilamellar Vesicles[END_REF].

Abstract

The two lectins LecA from Pseudomonas aeruginosa and the B-subunit of Shiga toxin from Shigella dysenteriae (StxB) share the glycosphingolipid globotriaosylceramide (Gb3) as receptor. Counterintuitively, we found that LecA and StxB segregated into different domains after recognizing Gb3 at the plasma membrane of cells. Based on this observation, we proposed the hypothesis that how Gb3 is embedded in the lipid bilayer and thus how the carbohydrate head group of Gb3 is oriented, differentially influences Gb3 binding by LecA and StxB.

To test this hypothesis, we reconstituted lectin-Gb3 interaction using giant unilamellar vesicles and supported lipid bilayers. Also in these model systems we were able to rebuild LecA and StxB segregation. Furthermore, the structure of the fatty acyl chain of Gb3 and the local membrane environment, which both control Gb3 membrane embedding, distinctively modulated Gb3 recognition by LecA and StxB. Specifically, StxB preferred more ordered membranes in comparison to LecA.

Based on these findings, we propose comparing staining patterns of LecA and StxB as an alternative method to assess membrane order in cells. To verify our novel approach, we re-established that the apical plasma membrane of epithelial cells is more ordered than the basolateral plasma membrane. Additionally, we found that StxB was able to recognize Gb3 at the primary cilium and at the surrounding periciliary membrane, whereas LecA was only able to bind periciliary Gb3. This suggests that the membrane of the primary cilium is of higher order than the surrounding periciliary membrane.

Significance statement

Lipid order crucially mediates the function of plasma membrane components.

Sensing membrane order in cells is challenging and currently available ordersensitive fluorescent probes can cause side effects by their incorporation in the lipid bilayer. Here, we introduce the use of the lectins, LecA and StxB, which both recognize the glycosphingolipid Gb3, as alternative. The binding of the two lectins to Gb3 varied differentially in dependence of the lipid environment. In particular, StxB showed a much stronger preference for more ordered domains separated GUVs or for 3 h at 55°C for phase-separated GUVs. The imaging buffer for GUVs was adjusted to the same osmolarity as the PBS/sucrose solution.

Preparation of SLBs

Supported lipid membranes were prepared using vesicle fusion (63). Briefly, lipid films were resuspended in low-Ca buffer (20 mM TRIS/HCl, 100 mM NaCl, and 1 mM CaCl2, pH 7.5) at 55°C for 35 min. Small vesicles were created using an extruder with a 50 nm cut-off polycarbonate membrane (Avestin) and directly transferred to the sample chamber. The sample chamber was home-built using mica, either grade V1 or V3 (Plano GmbH), which was fixed with optical adhesive (NOA 61, Norland Products) to a glass bottom dish (35 mm, #1.5 glass, World Precision Instruments). To induce vesicle fusion, a high-Ca buffer (20 mM TRIS/HCl, 100 mM NaCl, and 10 mM CaCl2, pH 7.5) was added to increase the CaCl2 concentration and bilayers were heated to 55°C for 1 h, followed by gentle cooling to room temperature. To remove non-fused vesicles and other debris, samples were rinsed with low-Ca buffer.

Lectin production and labeling

Recombinant LecA was produced from Escherichia coli following previously published procedures (39). The StxB used was the B-subunit of Shiga toxin 1 produced in Escherichia coli (Sigma Aldrich). Lectins were dissolved at 1 mg/mL in PBS (for StxB) or in PBS supplemented with 100 mg/mL MgCl2 and 100 mg/mL CaCl2 (for LecA), sterile filtered, and stored at 4°C prior usage.

For fluorescent labeling, NHS-ester conjugated Alexa488 (Thermo Fisher), Cy3, and Cy5 (GE Healthcare Life Science) was used. Dyes were dissolved at 1 mg/mL in water-free DMSO (Carl Roth), aliquoted, and stored at -20°C before usage. To set up the labeling reaction, 200 µL of lectin (1 mg/mL) was supplemented with 25 µL of a 1 M NaHCO3 (pH 9) solution. The molar ratio between dye and lectin was set to 5:1. The labeling mix was incubated at room temperature for 50 min and uncoupled dyes separated using Zeba Spin desalting columns (7k MWCO, 0.5 mL, Thermo Fischer). Labeled lectins were aliquoted into working stocks and stored at -80°C until usage.

Microscopy

MDCK cells grown on transwell filters were fixed with 4% formaldehyde and mounted as described before (9).

GUVs and MDCK cell samples were imaged using a laser scanning confocal microscope system from Nikon (A1R), equipped with a 60x oil immersion objective with a numerical aperture (NA) of 1.49, and laser lines at 488 nm, 561 nm, and 647 nm. Image acquisition and processing was done using the software NIS-Elements (built version 4.5, Nikon).

SLBs were imaged with a Nikon Eclipse Ti-E inverted microscope, equipped with a TIRF-illuminator, a 100x oil immersion objective (NA 1.49) and an EM-CCD camera (Andor Ixon DU-879). Images were acquired using a highly inclined laser beam (HILO-configuration) (64). Image acquisition and processing was done using the software NIS-Elements (built version 4.5, Nikon). TIRF-SIM imaging was done using a Nikon-N-SIM microscope equipped with laser lines at 488 nm, 561 nm, and 647 nm, and a CFI apochromatic TIRF 100x oil objective (NA 1.49).

Image analysis

Quantification of lectin binding to MDCK cells was done by measuring fluorescent intensities in single cells that were not surrounded by labeled cells using a custom-written Matlab program as described before (40).

Quantification of lectin binding efficiencies to GUVs was done using the ImageJmacro GUV-AP (51). Furthermore, we included a feature in the macro to enable the independent assessment of binding to ld and lo phases in phase-separated GUVs. To this end we modified the macro with a procedure that recognizes and

Supplementary Materials

The bacterium Pseudomonas aeruginosa perishes membrane organization by its lectin LecA

Taras Sych 1,2,3,4 , Thomas Schubert 1,2,3 , Sahaja Aigal 1,2,5 , Roland

Résumé en anglais

The interaction of plasma membrane glycosphingolipids with the carbohydrate binding proteins (lectins) is of vital importance for the infection of the host cell by various viruses and bacteria. In this work, we explored the interaction of the lectins LecA from the bacterium P. aeruginosa and B subunit of Shiga toxin (StxB) from S. dysenteriae with its plasma membrane receptor globotriaosylceramide (Gb3). Moreover, we studied the interaction of the complete bacterium P. aeruginosa with Gb3. In order to decipher the lectin-Gb3 interaction in absence of other cellular components we employed the artificial membrane systems -Giant unilamellar vesicles (GUVs) and Supported lipid bilayers (SLBs). We observed the lectin binding using different modes of fluorescence microscopy (confocal, TIRF, etc…). We examine the binding of both lectins to the membrane domains of different ordere and composition. We found that StxB prefers more ordered membrane domains whereas LecA is less preferential. Moreover, both lectins induce the reorganization of the membrane domains: StxB stabilizes ordered domains, shrinks them and induces the formation of the novel ordered domains.

On the other hand LecA, as well as the bacterium P. aeruginosa induce the dissolution of the ordered domains. We believe, these membrane reorganization processes are crucial for the bacterial infection.

Keywords: Plasma membrane, Bacterial invasion, Lectins, Glycosphingolipids