Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic cells.

Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression.

A human septin octamer complex sensitive to membrane curvature drives membrane deformation with a specific mesh-like organization.

Septins are cytoskeletal proteins interacting with the inner plasma membrane and other cytoskeletal partners. Being key in membrane remodeling processes, they often localize at specific micrometric curvatures. To analyze the behavior of human septins at the membrane and decouple their role from other partners, we used a combination of bottom-up in vitro methods. We assayed their ultrastructural organization, their curvature sensitivity, as well as their role in membrane reshaping. On membranes, human septins organize into a two-layered mesh of orthogonal filaments, instead of generating parallel sheets of filaments observed for budding yeast septins. This peculiar mesh organization is sensitive to micrometric curvature and drives membrane reshaping as well. The observed membrane deformations together with the filamentous organization are recapitulated in a coarse-grained computed simulation to understand their mechanisms. Our results highlight the specific organization and behavior of animal septins at the membrane as opposed to those of fungal proteins.

Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms.

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 subunit (also known as SEPTIN9). 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, for the first time, the 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 and microtubule cytoskeleton.

Characterization of the first tetrameric transcription factor of the GntR superfamily with allosteric regulation from the bacterial pathogen Agrobacterium fabrum.

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.

Synergistic role of nucleotides and lipids for the self-assembly of Shs1 septin oligomers.

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 micrometric long filaments formed by Shs1 protomers. In addition, molecular modeling experiments have revealed a potential molecular mechanism regarding the selectivity of septin subunits for phosphoinositide lipids.

Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents.

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.

Reduction-responsive cholesterol-based block copolymer vesicles for drug delivery.

We developed a new robust reduction-responsive polymersome based on the amphiphilic block copolymer PEG-SS-PAChol. The stability and robustness were achieved by the smectic physical cross-linking of cholesterol-containing liquid crystal polymer PAChol in the hydrophobic layer. The reduction-sensitivity was introduced by the disulfide bridge (-S-S-) that links the hydrophilic PEG block and the hydrophobic PAChol block. We used a versatile synthetic strategy based on atom transfer radical polymerization (ATRP) to synthesize the reduction-responsive amphiphilic block copolymers. The reductive cleavage of the disulfide bridge in the block copolymers was first evidenced in organic solution. The partial destruction of PEG-SS-PAChol polymersomes in the presence of a reducing agent was then demonstrated by cryo-electron microscopy. Finally, the calcein release from PEG-SS-PAChol polymersomes triggered by glutathione (GSH) was observed both in PBS suspension and in vitro inside the macrophage cells. High GSH concentrations (≥35 mM in PBS or artificially enhanced in macrophage cells by GSH-OEt pretreatment) and long incubation time (in the order of hours) were, however, necessary to get significant calcein release. These polymersomes could be used as drug carriers with very long circulation profiles and slow release kinetics.

Cool-contacts: Cryo-Electron Microscopy of Membrane Contact Sites and Their Components.

Electron microscopy has played a pivotal role in elucidating the ultrastructure of membrane contact sites between cellular organelles. The advent of cryo-electron microscopy has ushered in the ability to determine atomic models of constituent proteins or protein complexes within sites of membrane contact through single particle analysis. Furthermore, it enables the visualization of the three-dimensional architecture of membrane contact sites, encompassing numerous copies of proteins, whether in vitro reconstituted or directly observed in situ using cryo-electron tomography. Nevertheless, there exists a scarcity of cryo-electron microscopy studies focused on the site of membrane contact and their constitutive proteins. This review provides an overview of the contributions made by cryo-electron microscopy to our understanding of membrane contact sites, outlines the associated limitations, and explores prospects in this field.

Polymersomes with PEG corona: structural changes and controlled release induced by temperature variation.

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.

VAP-A intrinsically disordered regions enable versatile tethering at membrane contact sites.

Membrane contact sites (MCSs) 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 that involve 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 that VAP-A localization to different MCSs depends on its intrinsically disordered regions (IDRs) in human cells. 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 oxysterol-binding protein (OSBP) and CERT. However, removing IDRs from VAP-A restricts its distribution and function to ER-mitochondria MCS. Our data suggest that IDRs do not modulate VAP-A preference toward specific partners but do adjust their geometry to MCS organization and lifetime constraints. Thus, IDR-mediated VAP-A conformational flexibility ensures membrane tethering plasticity and efficiency.

How myosin VI traps its off-state, is activated and dimerizes.

Myosin VI (Myo6) is the only minus-end directed nanomotor on actin, allowing it to uniquely contribute to numerous cellular functions. As for other nanomotors, the proper functioning of Myo6 relies on precise spatiotemporal control of motor activity via a poorly defined off-state and interactions with partners. Our structural, functional, and cellular studies reveal key features of myosin regulation and indicate that not all partners can activate Myo6. TOM1 and Dab2 cannot bind the off-state, while GIPC1 binds Myo6, releases its auto-inhibition and triggers proximal dimerization. Myo6 partners thus differentially recruit Myo6. We solved a crystal structure of the proximal dimerization domain, and show that its disruption compromises endocytosis in HeLa cells, emphasizing the importance of Myo6 dimerization. Finally, we show that the L926Q deafness mutation disrupts Myo6 auto-inhibition and indirectly impairs proximal dimerization. Our study thus demonstrates the importance of partners in the control of Myo6 auto-inhibition, localization, and activation.

Self-assembly of amphiphilic liquid crystal polymers obtained from a cyclopropane-1,1-dicarboxylate bearing a cholesteryl mesogen.

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-BuP(4) 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 polymers exhibit in bulk an interdigitated smectic A (SmA(d)) 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.

Solubilization and Purification of α5ß1 Integrin from Rat Liver for Reconstitution into Nanodiscs.

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 α5ß1 integrin from rat livers, its purification and reconstitution into nanodiscs. At the plasma membrane of many cells, including tumor metastases, this essential heterodimeric transmembrane protein mediates the communication between extracellular matrix and cytosolic cytoskeleton in processes of cell adhesion and migration. We expect that the protocols that are described here will provide new opportunities for the determination of the full structure of α5ß1 integrin, as well as for the understanding of how interacting partners can regulate function and activity of this transmembrane protein.

Galectin-3 Binding to α5ß1 Integrin in Pore Suspended Biomembranes.

Galectin-3 (Gal3) is a ß-galactoside binding lectin that mediates many physiological functions, including the binding of cells to the extracellular matrix for which the glycoprotein α5ß1 integrin is of critical importance. The mechanisms by which Gal3 interacts with membranes have not been widely explored to date due to the complexity of cell membranes and the difficulty of integrin reconstitution within model membranes. Herein, to study their interaction, Gal3 and α5ß1 were purified, and the latter reconstituted into pore-suspended lipid bilayers comprised eggPC:eggPA. Using electrochemical impedance and fluorescence lifetime correlation spectroscopy, we found that on incubation with low nanomolar concentrations of wild-type Gal3, the membrane's admittance and fluidity, as well as integrin's lateral diffusivity, were enhanced. These effects were diminished in the following conditions: (i) absence of integrin, (ii) presence of lactose as a competitive inhibitor of glycan-Gal3 interaction, and (iii) use of a Gal3 mutant that lacked the N-terminal oligomerization domain (Gal3ΔNter). These findings indicated that WTGal3 oligomerized on α5ß1 integrin in a glycan-dependent manner and that the N-terminal domain interacted directly with membranes in a way that is yet to be fully understood. At concentrations above 10 nM of WTGal3, membrane capacitance started to decrease and very slowly diffusing molecular species appeared, which indicated the formation of protein clusters made from WTGal3-α5ß1 integrin assemblies. Overall, our study demonstrates the capacity of WTGal3 to oligomerize in a cargo protein-dependent manner at low nanomolar concentrations. Of note, these WTGal3 oligomers appeared to have membrane active properties that could only be revealed using our sensitive methods. At slightly higher WTGal3 concentrations, the capacity to generate lateral assemblies between cargo proteins was observed. In cells, this could lead to the construction of tubular endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis or to the formation of galectin lattices, depending on cargo glycoprotein stability at the membrane, the local Gal3 concentration, or plasma membrane intrinsic parameters. The study also demonstrates the utility of microcavity array-suspended lipid bilayers to address the biophysics of transmembrane proteins.

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins.

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.

PI4P and BLOC-1 remodel endosomal membranes into tubules.

Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers contribute to the flux of membrane lipids and proteins to acceptor organelles, but how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. Using imaging approaches on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability, and functions of recycling endosomal tubules. In vitro, BLOC-1 binds and tubulates negatively charged membranes, including those containing PI4P. In cells, endosomal PI4P production by type II PI4-kinases is needed to form and stabilize BLOC-1-dependent recycling endosomal tubules. Decreased PI4KIIs expression impairs the recycling of endosomal cargoes and the life cycles of intracellular pathogens such as Chlamydia bacteria and influenza virus that exploit the membrane dynamics of recycling endosomes. This study demonstrates how a phospholipid and a protein complex coordinate the remodeling of cellular membranes into functional tubules.

The multidrug resistance half-transporter ABCG2 is purified as a tetramer upon selective extraction from membranes.

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 His(6)-ABCG2 expressed in Sf9 insect cells and in human Flp-In-293/ABCG2 cells after solubilization in mild detergents. His(6)-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.

[Cryo-electron microcopy for a new vision of the cell and its components]. / La cryo-microscopie électronique révèle une nouvelle vision de la cellule et de ses composants.

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.

TITLE: La cryo-microscopie électronique révèle une nouvelle vision de la cellule et de ses composants. ABSTRACT: 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.

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

Membrane contact sites (MCS) are subcellular regions where two organelles appose their membranes to exchange small molecules, including lipids. Structural information on how proteins form MCS is scarce. We designed an in vitro MCS with two membranes and a pair of tethering proteins suitable for cryo-tomography analysis. It includes VAP-A, an ER transmembrane protein interacting with a myriad of cytosolic proteins, and oxysterol-binding protein (OSBP), a lipid transfer protein that transports cholesterol from the ER to the trans Golgi network. We show that VAP-A is a highly flexible protein, allowing formation of MCS of variable intermembrane distance. The tethering part of OSBP contains a central, dimeric, and helical T-shape region. We propose that the molecular flexibility of VAP-A enables the recruitment of partners of different sizes within MCS of adjustable thickness, whereas the T geometry of the OSBP dimer facilitates the movement of the two lipid-transfer domains between membranes.

Self-assembly of linear-dendritic diblock copolymers: from nanofibers to polymersomes.

We report the formation of cylindrical micelles, sheet-like micelles, tubular micelles, as well as polymer vesicles by a new series of amphiphilic linear-dendritic block-copolymers (BCs). The BCs, noted as PEGm-AZOn, are composed of poly(ethylene glycol) (PEG) chains of different molecular weights as hydrophilic blocks and the first four generations of azobenzene-containing dendrons based on 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) as hydrophobic blocks (m represents the degree of polymerization of PEG, and n is the number of azobenzene units at the periphery of dendron). The polymeric aggregates were formed by adding water to solutions of the BCs in dioxane. The micellar dispersions in water were finally obtained by removing dioxane via dialysis against water. The morphology of the micellar self-assemblies was studied by transmission electron microscopy (TEM), cryo-electron microscopy (cryo-TEM), and atomic force microscopy (AFM). A generation-dependent aggregation behavior was found for the series of BCs PEG45-AZOn. Core-shell structured nanofibers with an inner diameter of 8 nm were observed for the copolymer PEG45-AZO2 (hydrophilic/hydrophobic weight ratio equal to 67/33). Lyotropic liquid crystalline behavior was detected for the aqueous solution of the nanofibers. The coexistence of sheet-like aggregates and tubular micelles was detected for the copolymer PEG45-AZO8 in which the number of cyanoazobenzene units is increased to 8 (hydrophilic/hydrophobic weight ratio equal to 33/67). The tubular micelles could be intermediates in the sheet-like aggregate-to-vesicle transition. Polymer vesicles (polymersomes) with a diameter in the range 300-800 nm were observed for the copolymer PEG45-AZO16 (hydrophilic/hydrophobic weight ratio equal to 20/80). The membrane of the sheet-like aggregates, tubular micelles, and polymersomes was shown to have a bilayer structure, as revealed by cryo-TEM. UV illumination of the aqueous polymersome dispersion induced the formation of wrinkles in the vesicle membrane, thus showing that this type of polymeric aggregate is photoresponsive.