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Galectin-3 (Gal3) is a β-galactoside binding lectin that mediates many physiological functions, including the binding of cells to the extracellular matrix for which the glycoprotein α

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic conditions, protein and lipid compositions, membrane curvature) and the different partners associated with specific membrane reshaping processes, we undertake in vitro bottom-up approaches. In recent years, there has been keen interest in revealing the role of septin proteins associated with major diseases. Septins are essential and ubiquitous cytoskeletal proteins that interact with the plasma membrane. They are implicated in cell division, cell motility, neuro-morphogenesis, and spermiogenesis, among other functions. It is, therefore, important to understand how septins interact and organize at membranes to subsequently induce membrane deformations and how they can be sensitive to specific membrane curvatures. This article aims to decipher the interplay between the ultra-structure of septins at a molecular level and the membrane remodeling occurring at a micron scale. To this end, budding yeast, and mammalian septin complexes were recombinantly expressed and purified. A combination of in vitro assays was then used to analyze the self-assembly of septins at the membrane. Supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), and wavy substrates were used to study the interplay between septin self-assembly, membrane reshaping, and membrane curvature.

Transmembrane proteins (or integral membrane proteins) are synthesized in the endoplasmic reticulum where most of them are core glycosylated prior to folding and in some cases assembly into quaternary structures. Correctly glycosylated, folded, and assembled transmembrane proteins are then shuttled to the Golgi apparatus for additional posttranslational modifications such as complex-type glycosylations, sulfation or proteolytic clipping. At the plasma membrane, the glycosylated extracellular domains are key to communicate with the cellular environment for a variety of functions, such as binding to the extracellular matrix for cell adhesion and migration, to neighboring cells for cell-cell interaction, or to extracellular components for nutrient uptake and cell signaling. Intracellular domains are essential to mediate signaling cascades, or to connect to cytosolic adaptors for internalization and intracellular compartmentalization. Despite its importance for the understanding of molecular mechanisms of transmembrane protein function, the determination of their structures has remained a challenging task. In recent years, their reconstitution in lipid nanodiscs in combination with high resolution cryo-electron microscopy has provided novel avenues to render this process more accessible. Here, we describe detailed protocols for the solubilization of heavily glycosylated α

The fine regulation of actin polymerization is essential to control cell motility, architecture and to perform essential cellular functions. Formins are key regulators of actin filament assembly, known to processively elongate filament barbed ends and increase their polymerization rate. Based on indirect observations, different models have been proposed to describe the molecular mechanism governing the processive motion of formin FH2 domains at polymerizing barbed ends. Using electron microscopy, we directly identified two conformations of the mDia1 formin FH2 domains in interaction with the barbed ends of actin filaments. These conformations agree with the open and closed conformations of the “stair stepping” model proposed by Otomo and colleagues1. We observed the FH2 dimers to be in the open conformation for 79% of the data, interacting with the two terminal actin subunits of the barbed end, while they interact with three actin subunits in the closed conformation. Further, our data reveal that the open state encompasses a continuum of states where the orientation of the leading FH2 domain with respect to the filament long axis varies from 108 to 135 degrees. In addition, we identified FH2 domains encircling the core of actin filaments, providing structural information for mDia1 away from the barbed end. Based on these direct observations, we propose a model of formin in interaction with the growing filament end, as well as with the core of the filament.

SUMMARYMembrane contact sites (MCSs) between organelles are heterogeneous in shape, composition and dynamics. Despite this diversity, VAP proteins act as receptors for multiple FFAT motif-containing proteins and drive the formation of most MCSs involving the endoplasmic reticulum (ER). Although the VAP‒FFAT interaction is well characterized, no model explains how VAP adapts to its partners in various MCSs. We report here that VAP-A localization to different MCSs depends on its intrinsically disordered regions (IDRs). We show that VAP-A interaction with PTPIP51 and VPS13A at ER‒mitochondria MCS conditions mitochondria fusion by promoting lipid transfer and cardiolipin buildup. VAP-A also enables lipid exchange at ER‒Golgi MCS by interacting with OSBP and CERT. However, removing IDRs from VAP-A restricts its distribution and function to ER‒ mitochondria MCS, at the expense of ER‒Golgi MCS. Our data suggest that IDRs of VAP-A do not modulate its preference towards specific partners, but adjust its geometry to the constraints linked to different MCS organization and lifetime. Thus, VAP-A conformational flexibility mediated by its IDRs ensures membrane tethering plasticity and efficiency.

International audience; The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol (“reverse topology”), but they can also be directed toward the cytosol (“normal topology”). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA + ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B–IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B–IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B–IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9. Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9 thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled the first-time isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin/microtubule cytoskeleton.SummaryHuman septins in cells form hetero-octameric complexes containing the ubiquitously expressed SEPT9. Iv et al. describe the first-time isolation of recombinant human septin octamers with distinct SEPT9 isoforms. Reconstitution studies show that octamers with either a long or a short SEPT9 isoform form higher-order filament assemblies and directly bind and cross-link actin filaments.

Cryo-electron microscopy (cryo-EM) is a technique for imaging biological samples that plays a central role in structural biology, with high impact on research fields such as cell and developmental biology, bioinformatics, cell physics and applied mathematics. It allows the determination of structures of purified proteins within cells. This review describes the main recent advances in cryo-EM, illustrated by examples of proteins of biomedical interest, and the avenues for future development.La cryo-microscopie électronique révèle une nouvelle vision de la cellule et de ses composants.La cryo-microscopie électronique (cryo-EM) est une technique d’imagerie du vivant qui prend désormais une place prépondérante en biologie structurale, avec des retombées en biologie cellulaire et du développement, en bioinformatique, en biomédecine ou en physique de la cellule. Elle permet de déterminer des structures de protéines purifiées in vitro ou au sein des cellules. Cette revue décrit les principales avancées récentes de la cryo-EM, illustrées par des exemples d’élucidation de structures de protéines d’intérêt en biomédecine, et les pistes de développements futurs.

International audience; Cryo-electron microscopy (cryo-EM) is a technique for imaging biological samples that plays a central role in structural biology, with high impact on research fields such as cell and developmental biology, bioinformatics, cell physics and applied mathematics. It allows the determination of structures of purified proteins within cells. This review describes the main recent advances in cryo-EM, illustrated by examples of proteins of biomedical interest, and the avenues for future development.; La cryo-microscopie électronique (cryo-EM) est une technique d'imagerie du vivant qui prend une place prépondérante en biologie structurale avec des retombées en biologie cellulaire et du développement, en bioinformatique, en biomédecine ou en physique de la cellule. Elle permet de déterminer des structures de protéines purifiées in vitro ou au sein des cellules. Cette revue décrit les principales avancées récentes de la cryo-EM, illustrées par des exemples de protéines d'intérêt en biomédecine, et les pistes de développements futurs. Comment des journaux scientifiques anglo-saxons se sont-ils convertis à un vocabulaire révolutionnaire traditionnellement réservé à des expressions en langue française ? En décrivant la cryo-microscopie électronique (cryo-EM), comme dans les éditoriaux de Science en 2014 « The resolution revolution », et de Nature en 2015 « The revolution will not be crystallized », et 2020 « Revolutionary cryo-EM is taking over structural biology ». La cryo-EM s'est en réalité développée depuis les années 1970, la dernière décennie ayant connu une

A species-specific region, denoted SpG8-1b allowing hydroxycinnamic acids (HCAs) degradation is important for the transition between the two lifestyles (rhizospheric versus pathogenic) of the plant pathogen Agrobacterium fabrum. Indeed, HCAs can be either used as trophic resources and/or as induced-virulence molecules. The SpG8-1b region is regulated by two transcriptional regulators, namely, HcaR (Atu1422) and Atu1419. In contrast to HcaR, Atu1419 remains so far uncharacterized. The high-resolution crystal structures of two fortuitous citrate complexes, two DNA complexes and the apoform revealed that the tetrameric Atu1419 transcriptional regulator belongs to the VanR group of Pfam PF07729 subfamily of the large GntR superfamily. Until now, GntR regulators were described as dimers. Here, we showed that Atu1419 represses three genes of the HCAs catabolic pathway. We characterized both the effector and DNA binding sites and identified key nucleotides in the target palindrome. From promoter activity measurement using defective gene mutants, structural analysis and gel-shift assays, we propose N5,N10-methylenetetrahydrofolate as the effector molecule, which is not a direct product/substrate of the HCA degradation pathway. The Zn2+ ion present in the effector domain has both a structural and regulatory role. Overall, our work shed light on the allosteric mechanism of transcription employed by this GntR repressor.

International audience; Budding yeast septins are essential for cell division and polarity. Septins assemble as palindromic linear octameric complexes. The function and ultra-structural organization of septins are finely governed by their molecular polymorphism. In particular, in budding yeast, the end subunit can stand either as Shs1 or Cdc11. We have dissected, here, for the first time, the behavior of the Shs1 protomer bound to membranes at nanometer resolution, in complex with the other septins. Using electron microscopy, we have shown that on membranes, Shs1 protomers self-assemble into rings, bundles, filaments or two-dimensional gauzes. Using a set of specific mutants we have demonstrated a synergistic role of both nucleotides and lipids for the organization and oligomerization of budding yeast septins. Besides, cryo-electron tomography assays show that vesicles are deformed by the interaction between Shs1 oligomers and lipids. The Shs1-Shs1 interface is stabilized by the presence of phosphoinositides, allowing the visualization of micro-metric long filaments formed by Shs1 protomers. In addition, molecular modeling experiments have revealed a potential molecular mechanism regarding the selectivity of septin subunits for phosphoinositide lipids.

Endosomal sorting complexes required for transport-III (ESCRT-III) are thought to assemble in vivo inside membrane structures with a negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and conversely how ESCRT-III polymers shape membranes is still unclear. Here, we used human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron microscopy, cryo-electron tomography and high-speed AFM. We show that CHMP4B filaments bind preferentially to flat membranes or to membrane tubes with a positive mean curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes, the latter winding around the tubes. Although combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes, they also reshape large unilamellar vesicles into helical membrane tubes with a pipe surface shape. Sub-tomogram averaging reveals that the filaments assemble parallel to the tube axis with some local perpendicular connections, highlighting the particular mechanical stresses imposed by ESCRT-III to stabilize the corkscrew-like membrane architecture. Our results thus underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature of ESCRT-III required for all or selected cellular membrane remodeling processes.

Cell membrane deformations are crucial for proper cell function. Specialized protein assemblies initiate inward or outward membrane deformations that the cell uses respectively to uptake external substances or probe the environment. The assembly and dynamics of the actin cytoskeleton are involved in this process, although their detailed role remains controversial. We show here that a dynamic, branched actin network is sufficient to initiate both inward and outward membrane deformation. The polymerization of a dense actin network at the membrane of liposomes produces inward membrane bending at low tension, while outward deformations are robustly generated regardless of tension. Our results shed light on the mechanism cells use to internalize material, both in mammalian cells, where actin polymerization forces are required when membrane tension is increased, and in yeast, where those forces are necessary to overcome the opposing turgor pressure. By combining experimental observations with physical modelling, we propose a mechanism that explains how membrane tension and the architecture of the actin network regulate cell-like membrane deformations. The same type of polymer network deforms cell membranes inward, to absorb external material, and outward, to facilitate signal transmission. Experiments and theory show that these deformations are regulated by membrane tension and network mesh size.

Septins are cytoskeletal filaments that assemble at the inner face of the plasma membrane. They are localized at constriction sites and impact membrane remodeling. We report in vitro tools to examine how yeast septins behave on curved and deformable membranes. Septins reshape the membranes of Giant Unilamellar Vesicles with the formation of periodic spikes, while flattening smaller vesicles. We show that membrane deformations are associated to preferential arrangement of septin filaments on specific curvatures. When binding to bilayers supported on custom-designed periodic wavy patterns displaying positive and negative micrometric radii of curvatures, septin filaments remain straight and perpendicular to the curvature of the convex parts, while bending negatively to follow concave geometries. Based on these results, we propose a theoretical model that describes the deformations and micrometric curvature sensitivity observed in vitro. The model captures the reorganizations of septin filaments throughout cytokinesis in vivo, providing mechanistic insights into cell division., Septins are cytoskeletal filaments that localize at constriction sites and impact membrane remodeling. Here authors examine the curvature sensitivity of septins using bilayers on wavy patterns and derive a theoretical model that quantitatively describe the results.

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We have investigated the most efficient way of preparing biocompatible gold nanorods (GNR) used as tool for cancer imaging and therapy. The surface of cetyltrimethylammonium bromide-stabilized gold nanorods (GNR-CTAB) was functionalized with various thio-polyethylene glycols of the general formula HS-PEG mX ( m = 356–10,000; X = −OMe, −NH 2 ). The influence of several parameters such as PEG chain length, reaction conditions and purification method on long-term stability, morphology and optical properties of the produced GNR-S-PEG mX was studied, demonstrating the existence of a threshold HS-PEG mX chain length (with molecular weight m ⩾ 2000) for efficient steric stabilization of GNR. Several purification techniques were compared: dialysis, centrifugation and a rarely used technique in this field, size exclusion chromatography. While a very weak efficiency of dialysis was evidenced, both centrifugation and size exclusion chromatography were found to provide pure GNRs, though the latter method yielded nanoparticles with a significantly higher stability. Finally, the long-term stability of the produced GNRs was investigated in various media: water, PBS buffer and serum.

We report on the self-assembly, in water and in bulk, of amphiphilic liquid crystal block copolymers consisting of a cholesterol-based smectic LC polymer block (PAChol) and poly(ethylene glycol) (PEG) block. Two series of block copolymers, PEG 45 - b -PAChol and PEG 114 - b -PAChol (45 and 114 are the degree of polymerization of PEG blocks) with different hydrophilic/hydrophobic weight ratios were synthesized and characterized in detail. Depending on the diblock composition, smectic polymer vesicles and/or nanofibers were formed by adding water into a dilute solution of copolymers in dioxane. If THF is used instead of dioxane as solvent, solid spherical aggregates were obtained upon water addition for PEG 45 - b -PAChol series, while macroscopic precipitation occurred for PEG 114 - b -PAChol series. The mesomorphic and microphase segregation structures of the block copolymers in bulk were studied by X-ray scattering, DSC and POM. The interdigital smectic A (SmA d ) phase with a lamellar period of 4.25 nm was detected in all block copolymers. For PEG 114 - b -PAChol 5 (PEG/PAChol weight ratio = 66/34) and PEG 114 - b -PAChol 12 (45/55), lamellar type of microphase segregation was observed.

Amphiphilic diblock copolymers, poly(ethylene glycol)-block-poly(acrylate), bearing truxillic acid derivatives at the junction point between the two blocks are described. The truxillic acid junction can be selectively cut by UV light, leading to a disassembly of the nano-objects made by self-assembly of the amphiphilic copolymers in water.

What could be the self-assembly behavior in aqueous solution for an amphiphilic block copolymer with a very flexible hydrophobic backbone possessing however a liquid crystalline (LC) order? To answer this question, five amphiphilic block copolymers were prepared by grafting, via a thiol−ene addition reaction, four different thiol-functionalized LC mesogens or LC-like side groups on two commercially available poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PB) polymers. These copolymers form monodisperse nanospheres in dilute deionized water with the aid of 1,4-dioxane as cosolvent (dioxane−H2O system), while in the THF−H2O system, some give heterogeneous polydisperse micelles and others form large hollow concentric spherical vesicles (“Russian dolls” arrangement). Smectic stripes inside the vesicles and the nanospheres are clearly visualized by cryo-TEM technique.

ABCG2 is a human membrane ATP-binding cassette half-transporter that hydrolyzes ATP to efflux a large number of chemotherapeutic agents. Several oligomeric states of ABCG2 from homodimers to dodecamers have been reported depending on the overexpression systems and/or the protocols used for purification. Here, we compared the oligomeric state of His6-ABCG2 expressed in Sf9 insect cells and in human Flp-In-293/ABCG2 cells after solubilization in mild detergents. His6-ABCG2 was purified through a new approach involving its specific recognition onto a functionalized lipid layer containing a Ni-NTA lipid. This approach allowed the purification of His-ABCG2 in presence of all solubilized membrane components that might be involved in the stabilisation of native oligomers and without requiring any additional washing or concentration passages. ABCG2 purified onto the NiNTA lipid surfaces were directly analyzed by electron microscopy and by biochemical assays. Altogether, our data are consistent with a tetrameric organization of ABCG2 when expressed in either heterologous Sf9 insect cells or in human homologous cells.

Two families of amphiphilic diblock copolymers, in which the hydrophobic block was a cholesteryl-based smectic liquid-crystalline polymer and the hydrophilic block was either a neutral polymer with a lower critical solution temperature (LCST), poly(N,N-diethylacrylamide), or a copolymer containing acrylic acid moieties and poly(ethylene oxide) side chains, were successfully synthesized by reversible addition−fragmentation chain transfer (RAFT) polymerization. Nanoassemblies of these amphiphilic species in water or in buffer solution were prepared using the cosolvent method. The morphology of the nanoassemblies clearly depended on the weight fraction and the nature of the hydrophobic block. The amphiphilic liquid-crystal (LC) block copolymers with a hydrophobic/hydrophilic weight ratio of 74/26 or 65/35 formed long nanofibers, whereas the non-LC copolymers based on polystyrene of similar ratio formed vesicles or short cylindrical micelles. Such morphologies were assigned to the presence of a smectic order ...

International audience; Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV) within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms.

Myosin 1b is a single-headed membrane-associated motor that binds to actin filaments with a catch-bond behaviour in response to load. In vivo, myosin 1b is required to form membrane tubules at both endosomes and the trans-Golgi network. To establish the link between these two fundamental properties, here we investigate the capacity of myosin 1b to extract membrane tubes along bundled actin filaments in a minimal reconstituted system. We show that single-headed non-processive myosin 1b can extract membrane tubes at a biologically relevant low density. In contrast to kinesins we do not observe motor accumulation at the tip, suggesting that the underlying mechanism for tube formation is different. In our theoretical model, myosin 1b catch-bond properties facilitate tube extraction under conditions of increasing membrane tension by reducing the density of myo1b required to pull tubes.

Giant unilamellar vesicles (GUVs) are convenient biomimetic systems of the same size as cells that are increasingly used to quantitatively address biophysical and biochemical processes related to cell functions. However, current approaches to incorporate transmembrane proteins in the membrane of GUVs are limited by the amphiphilic nature or proteins. Here, we report a method to incorporate transmembrane proteins in GUVs, based on concepts developed for detergent-mediated reconstitution in large unilamellar vesicles. Reconstitution is performed either by direct incorporation from proteins purified in detergent micelles or by fusion of purified native vesicles or proteoliposomes in preformed GUVs. Lipid compositions of the membrane and the ionic, protein, or DNA compositions in the internal and external volumes of GUVs can be controlled. Using confocal microscopy and functional assays, we show that proteins are unidirectionally incorporated in the GUVs and keep their functionality. We have successfully tested our method with three types of transmembrane proteins. GUVs containing bacteriorhodopsin, a photoactivable proton pump, can generate large transmembrane pH and potential gradients that are light-switchable and stable for hours. GUVs with FhuA, a bacterial porin, were used to follow the DNA injection by T5 phage upon binding to its transmembrane receptor. GUVs incorporating BmrC/BmrD, a bacterial heterodimeric ATP-binding cassette efflux transporter, were used to demonstrate the protein-dependent translocation of drugs and their interactions with encapsulated DNA. Our method should thus apply to a wide variety of membrane or peripheral proteins for producing more complex biomimetic GUVs.

International audience; Thermoresponsive behavior of different kinds of polymersomes was studied using small angle neutron scattering (SANS), transmission electron microscopy (TEM), and proton nuclear magnetic resonance ((1)H NMR). The polymersomes were made of block copolymers containing a 2000 Da polyethylene glycol (PEG) as a hydrophilic block and either a liquidlike polymer (e.g., PBA: polybutylacrylate), a solidlike polymer (PS: polystyrene), or a liquid crystalline (LC) polymer as a hydrophobic block. Structural changes in polymersomes are driven in all cases by the critical dehydration temperature of PEG corona, which is closely related to the chemical structure and chain mobility of the hydrophobic block. No structural changes occur upon heating from 25 to 75 °C in the liquidlike polymersomes where the critical dehydration temperature of PEG should be higher than 75 °C. In contrast, glassy PEG-b-PS polymersomes and LC polymersomes show structural changes around 55 °C, which corresponds to the critical dehydration temperature of PEG in those block copolymers. Furthermore, the structural changes depend on the properties of the hydrophobic layer. Glassy PEG-b-PS polymersomes aggregate together above 55 °C, but the bilayer membrane is robust enough to remain intact. This aggregation is reversible, and rather separate polymersomes are recovered upon cooling. However, LC polymersomes display drastic and irreversible structural changes when heated above ∼55 °C. These changes are dependent on the LC structures of the hydrophobic layer. Nematic LC polymersomes turn into thick-walled capsules, whereas smectic LC polymersomes collapse into dense aggregates. As these drastic and irreversible changes decrease or remove the inner compartment volume of the vesicle, LC polymersomes can be used for thermal-responsive controlled release, as shown by a study of calcein release. Finally, toxicity studies proved that LC polymersomes were noncytotoxic and had no effect on cell morphology.

International audience; We study the self-assembly of a new family of amphiphilic liquid crystal (LC) copolymers synthesized by the anionic ring-opening polymerization of a new cholesterol-based LC monomer, 4-(cholesteryl)butyl ethyl cyclopropane-1,1-dicarboxylate. Using the t-BuP4 phosphazene base and thiophenol or a poly(ethylene glycol) (PEG) functionalized with thiol group to generate in situ the initiator during the polymerization, LC homopolymer and amphiphilic copolymers with narrow molecular weight distributions were obtained. The self-assemblies of the LC monomer, homopolymer, and block copolymers in bulk and in solution were studied by small- angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), polarizing optical microscopy (POM), and transmission electron microscopy (TEM). All polymersexhibit in bulk an interdigitated smectic A (SmAd) phase with a lamellar period of 4.6 nm. The amphiphilic copolymers self-organize in solution into vesicles with wavy membrane and nanoribbons with twisted and folded structures, depending on concentration and size of LC hydrophobic block. These new morphologies will help the comprehension of the fascinating organization of thermotropic mesophase in lyotropic structures.

Monolayer of functionalized lipid spread at the air/water interface is used for the structural analysis of soluble and membrane proteins by electron crystallography and single particle analysis. This powerful approach lacks of a method for the screening of the binding of proteins to the surface of the lipid layer. Here, we described an optical method based on the use of reflected light microscopy to image, without the use of any labeling, the lipid layer at the surface of buffers in the Teflon wells used for 2D crystallization. Images revealed that the lipid layer was made of a monolayer coexisting with liposomes or aggregates of lipids floating at the surface. Protein binding led to an increase of the contrast and the decrease of the fluidity of the lipid surface, as demonstrated with the binding of soluble Shiga toxin B subunit, of purple membrane and of solubilized His-BmrA, a bacterial ABC transporter. Moreover the reconstitution of membrane proteins bound to the lipidic surface upon detergent removal can be followed through the appearance of large recognizable domains at the surface. Proteins binding and reconstitution were further confirmed by electron microcopy. Overall, this method provides a quick evaluation of the monolayer trials, a significant reduction in screening by transmission electron microscopy and new insights in the proteins binding and 2D crystallogenesis at the lipid surface.

Self-assembled block copolymer nanofibers are attractive materials for multiple applications. We propose here a novel, very simple and straightforward method to prepare polymeric nanofibers at high solids contents directly in water. It is based on an aqueous emulsion polymerization process performed under living radical polymerization conditions, using the RAFT method.

The structural changes in liquid crystal (LC) polymer vesicles triggered by changes in temperature and the application of a magnetic field were investigated by SANS, cryo-TEM, SEM and high sensitivity DSC. PEG-b-PA444 and PEG-b-PMAazo444, two block copolymers with side-on nematic hydrophobic blocks, were used to form vesicles with a radius of 450–500 nm and bilayer membrane thickness of 10–15 nm at room temperature. Upon heating the membrane thickness, d, started to increase dramatically from a temperature (∼55 °C) above Tg but below TNI of the LC polymer block, and reached up to 120 nm at T > TNI. The vesicles transformed into thick-walled capsules. The thickness of the membrane was inconsistent with a bilayer structure and surprisingly the capsules were stable even for temperatures above TNI. As the PEG chains should partially dehydrate with temperature, we propose that the membrane reorganized into a structure consisting of microphase separated LC and PEG domains. Analysis of changes in structural parameters such as the internal aqueous volume and the polymer membrane volume suggest that capsule scission and fusion also occurred during this transition. The polymersomes were also sensitive to applied magnetic fields and for some temperatures the application of a strong magnetic field (1.4 T) substantially increased the membrane thickness due to the induced mesogen alignment.