Annexin-A5 and annexin-A6 silencing prevents metastasis of breast cancer cells in zebrafish.

BACKGROUND INFORMATION: During tumor invasion and metastasis processes, cancer cells are exposed to major compressive and shearing forces, due to their migration through extracellular matrix, dense cell areas, and complex fluids, which may lead to numerous plasma membrane damages. Cancer cells may survive to these mechanical stresses thanks to an efficient membrane repair machinery. Consequently, this machinery may constitute a relevant target to inhibit cancer cell dissemination. RESULTS: We show here that annexin-A5 (ANXA5) and ANXA6 participate in membrane repair of MDA-MB-231 cells, a highly invasive triple-negative breast cancer cell line. These crucial components of the membrane repair machinery are substantially expressed in breast cancer cells in correlation with their invasive properties. In addition, high expression of ANXA5 and ANXA6 predict poor prognosis in high-grade lung, gastric, and breast cancers. In zebrafish, the genetic inhibition of ANXA5 and ANXA6 leads to drastic reduction of tumor cell dissemination. CONCLUSION: We conclude that the inhibition of ANXA5 and ANXA6 prevents membrane repair in cancer cells, which are thus unable to survive to membrane damage during metastasis. SIGNIFICANCE: This result opens a new therapeutic strategy based on targeting membrane repair machinery to inhibit tumor invasion and metastasis.

Trafficking of Annexins during Membrane Repair in Human Skeletal Muscle Cells.

Defects in membrane repair contribute to the development of muscular dystrophies, such as Miyoshi muscular dystrophy 1, limb girdle muscular dystrophy (LGMD), type R2 or R12. Deciphering membrane repair dysfunctions in the development of muscular dystrophies requires precise and detailed knowledge of the membrane repair machinery in healthy human skeletal muscle cells. Using correlative light and electron microscopy (CLEM), we studied the trafficking of four members of the annexin (ANX) family, in myotubes damaged by laser ablation. Our data support a model in which ANXA4 and ANXA6 are recruited to the disruption site by propagating as a wave-like motion along the sarcolemma. They may act in membrane resealing by proceeding to sarcolemma remodeling. On the other hand, ANXA1 and A2 exhibit a progressive cytoplasmic recruitment, likely by interacting with intracellular vesicles, in order to form the lipid patch required for membrane resealing. Once the sarcolemma has been resealed, ANXA1 is released from the site of the membrane injury and returns to the cytosol, while ANXA2 remains accumulated close to the wounding site on the cytoplasmic side. On the other side of the repaired sarcolemma are ANXA4 and ANXA6 that face the extracellular milieu, where they are concentrated in a dense structure, the cap subdomain. The proposed model provides a basis for the identification of cellular dysregulations in the membrane repair of dystrophic human muscle cells.

Annexin-A6 in Membrane Repair of Human Skeletal Muscle Cell: A Role in the Cap Subdomain.

Defects in membrane repair contribute to the development of some muscular dystrophies, highlighting the importance to decipher the membrane repair mechanisms in human skeletal muscle. In murine myofibers, the formation of a cap subdomain composed notably by annexins (Anx) is critical for membrane repair. We applied membrane damage by laser ablation to human skeletal muscle cells and assessed the behavior of annexin-A6 (AnxA6) tagged with GFP by correlative light and electron microscopy (CLEM). We show that AnxA6 was recruited to the site of membrane injury within a few seconds after membrane injury. In addition, we show that the deficiency in AnxA6 compromises human sarcolemma repair, demonstrating the crucial role played by AnxA6 in this process. An AnxA6-containing cap-subdomain was formed in damaged human myotubes in about one minute. Through transmission electron microscopy (TEM), we observed that extension of the sarcolemma occurred during membrane resealing, which participated in forming a dense lipid structure in order to plug the hole. By properties of membrane folding and curvature, AnxA6 helped in the formation of this tight structure. The compaction of intracellular membranes-which are used for membrane resealing and engulfed in extensions of the sarcolemma-may also facilitate elimination of the excess of lipid and protein material once cell membrane has been repaired. These data reinforce the role played by AnxA6 and the cap subdomain in membrane repair of skeletal muscle cells.

Tandem NMR and Mass Spectrometry Analysis of Human Nuclear Membrane Lipids.

The human nuclear membrane is composed of a double bilayer, the inner membrane being linked to the protein lamina network and the outer nuclear membrane continuous with the endoplasmic reticulum. Nuclear membranes can form large invaginations inside the nucleus; their specific roles still remain unknown. Although much of the protein identification has been determined, their lipid composition remains largely undetermined. In order to understand the mechanical and dynamic properties of nuclear membranes we investigated their lipid composition by two quantitative methods, namely, 31P and 1H multidimensional NMR and mass spectrometry, using internal standards. We also developed a nondetergent nuclei extraction protocol allowing to produce milligram quantities of nuclear membrane lipids. We found that the nuclear membrane lipid extract is composed of a complex mixture of phospholipids with different phosphatidylcholine species present in large amounts. Negatively charged lipids, with elevated amounts of phosphatidylinositol (PI), were also present. Mass spectrometry confirmed the phospholipid composition and provided further information on acyl-chain length and unsaturation. Lipid chain lengths ranged between 30 and 38 carbon atoms (two chains summed up) with a high proportion of 34 carbon atom length for most species. PI lipids have high amounts of chain lengths with 36-38 carbons. Independent of the chain length unsaturations were highly elevated with one to two double bonds per lipid species.

Extracellular vesicles from activated platelets: a semiquantitative cryo-electron microscopy and immuno-gold labeling study.

Cells release membrane vesicles in their surrounding medium either constitutively or in response to activating signals. Two main types of extracellular vesicles (EVs) are commonly distinguished based on their mechanism of formation, membrane composition and size. According to the current model, EVs shed from the plasma membrane, often called microvesicles, expose phosphatidylserine (PS) and range in size from 100 nm to 1 µm, while EVs originating from endosomal multi-vesicular bodies, called exosomes, contain tetraspanin proteins, including CD63, and range in size from 50 to 100 nm. Heijnen et al. [1] have shown that activated platelets release EVs corresponding to these two types of vesicles, using negative staining electron microscopy (EM) and immuno-gold labeling. Here, we apply cryo-EM and immuno-gold labeling to provide a quantitative analysis of EVs released by platelets activated by thrombin, TRAP and CRP-XL, as well as EVs from serum. We show that EVs activated by these three agonists present a similar size distribution, the majority of them forming a broad peak extending from 50 nm to 1 µm, about 50% of them ranging from 50 to 400 nm. We show also that 60% of the EVs from TRAP or CRP-XL activation expose CD41, a majority of them exposing also PS. To explain the presence of large EVs CD41-negative or PS-negative, several alternative mechanisms of EV formation are proposed. We find also that the majority of EVs in activated platelet samples expose CD63, and distinguish two populations of CD63-positive EVs, namely large EVs with low labeling density and small EVs with high labeling density.

Imaging and Quantification of Extracellular Vesicles by Transmission Electron Microscopy.

Extracellular vesicles (EVs) are cell-derived vesicles that are present in blood and other body fluids. EVs raise major interest for their diverse physiopathological roles and their potential biomedical applications. However, the characterization and quantification of EVs constitute major challenges, mainly due to their small size and the lack of methods adapted for their study. Electron microscopy has made significant contributions to the EV field since their initial discovery. Here, we describe the use of two transmission electron microscopy (TEM) techniques for imaging and quantifying EVs. Cryo-TEM combined with receptor-specific gold labeling is applied to reveal the morphology, size, and phenotype of EVs, while their enumeration is achieved after high-speed sedimentation on EM grids.

Membrane repair of human skeletal muscle cells requires Annexin-A5.

Defect in membrane repair contributes to the development of limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. In healthy skeletal muscle, unraveling membrane repair mechanisms requires to establish an exhaustive list of the components of the resealing machinery. Here we show that human myotubes rendered deficient for Annexin-A5 (AnxA5) suffer from a severe defect in membrane resealing. This defect is rescued by the addition of recombinant AnxA5 while an AnxA5 mutant, which is unable to form 2D protein arrays, has no effect. Using correlative light and electron microscopy, we show that AnxA5 binds to the edges of the torn membrane, as early as a few seconds after sarcolemma injury, where it probably self-assembles into 2D arrays. In addition, we observed that membrane resealing is associated with the presence of a cluster of lipid vesicles at the wounded site. AnxA5 is present at the surface of these vesicles and may thus participate in plugging the cell membrane disruption. Finally, we show that AnxA5 behaves similarly in myotubes from a muscle cell line established from a patient suffering from LGMD2B, a myopathy due to dysferlin mutations, which indicates that trafficking of AnxA5 during sarcolemma damage is independent of the presence of dysferlin.

Tripartite assembly of RND multidrug efflux pumps.

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA-MexB-TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

Fluorescence triggering: A general strategy for enumerating and phenotyping extracellular vesicles by flow cytometry.

Plasma contains cell-derived extracellular vesicles (EVs) which participate in various physiopathological processes and have potential biomedical applications. Despite intense research activity, knowledge on EVs is limited mainly due to the difficulty of isolating and characterizing sub-micrometer particles like EVs. We have recently reported that a simple flow cytometry (FCM) approach based on triggering the detection on a fluorescence signal enabled the detection of 50× more Annexin-A5 binding EVs (Anx5+ EVs) in plasma than the conventional FCM approach based on light scattering triggering. Here, we present the application of the fluorescence triggering approach to the enumeration and phenotyping of EVs from platelet free plasma (PFP), focusing on CD41+ and CD235a+ EVs, as well as their sub-populations which bind or do not bind Anx5. Higher EV concentrations were detected by fluorescence triggering as compared to light scattering triggering, namely 40× for Anx5+ EVs, 75× for CD41+ EVs, and 15× for CD235a+ EVs. We found that about 30% of Anx5+ EVs were of platelet origin while only 3% of them were of erythrocyte origin. In addition, a majority of EVs from platelet and erythrocyte origin do not expose PS, in contrast to the classical theory of EV formation. Furthermore, the same PFP samples were analyzed fresh and after freeze-thawing, showing that freeze-thawing processes induce an increase, of about 35%, in the amount of Anx5+ EVs, while the other EV phenotypes remain unchanged. The method of EV detection and phenotyping by fluorescence triggering is simple, sensitive and reliable. We foresee that its application to EV studies will improve our understanding on the formation mechanisms and functions of EVs in health and disease and help the development of EV-based biomarkers.

High-speed centrifugation induces aggregation of extracellular vesicles.

Plasma and other body fluids contain cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential biomedical applications. In order to isolate, concentrate and purify EVs, high-speed centrifugation is often used. We show here, using electron microscopy, receptor-specific gold labelling and flow cytometry, that high-speed centrifugation induces the formation of EV aggregates composed of a mixture of EVs of various phenotypes and morphologies. The presence of aggregates made of EVs of different phenotypes may lead to erroneous interpretation concerning the existence of EVs harbouring surface antigens from different cell origins.

Annexin-A5 promotes membrane resealing in human trophoblasts.

Annexin-A5 (AnxA5) is the smallest member of the annexins, a group of soluble proteins that bind to membranes containing negatively-charged phospholipids, principally phosphatidylserine, in a Ca(2+)-dependent manner. AnxA5 presents unique properties of binding and self-assembling on membrane surfaces, forming highly ordered two-dimensional (2D) arrays. We showed previously that AnxA5 plays a central role in the machinery of cell membrane repair of murine perivascular cells, promoting the resealing of membrane damages via the formation of 2D protein arrays at membrane disrupted sites and preventing the extension of membrane ruptures. As the placenta is one of the richest source of AnxA5 in humans, we investigated whether AnxA5 was involved in membrane repair in this organ. We addressed this question at the level of human trophoblasts, either mononucleated cytotrophoblasts or multinucleated syncytiotrophoblasts, in choriocarcinoma cells and primary trophoblasts. Using established procedure of laser irradiation and fluorescence microscopy, we observed that both human cytotrophoblasts and syncytiotrophoblasts repair efficiently a µm²-size disruption. Compared to wild-type cells, AnxA5-deficient trophoblasts exhibit severe defect of membrane repair. Through specifically binding to the disrupted site as early as a few seconds after membrane wounding, AnxA5 promotes membrane resealing of injured human trophoblasts. In addition, we observed that a large membrane area containing the disrupted site was released in the extracellular milieu. We propose mechanisms ensuring membrane resealing and subsequent lesion removal in human trophoblasts. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Free-standing lipid films stabilized by Annexin-A5.

Free-standing lipid bilayers in nano- and micro-pores are interesting membrane models and attractive for biotechnological applications. We describe here the controlled preparation of proteo-lipid mono- and bilayers using the Langmuir-Schaefer transfer or Langmuir-Blodgett technique, respectively on hydrophobic and hydrophilic surfaces. We demonstrate the formation of suspended proteo-lipid layers by Transmission Electron Microscopy (TEM) and in situ Atomic Force Microscopy (AFM) imaging. Using Annexin-A5 as a membrane-associated protein, continuous proteo-lipid mono- and bilayers were formed, which span pore arrays over areas of several square-micrometers. The 2D organization of proteins associated to lipid monolayer is well preserved during the transfer process and the protein association is Ca(2+)-dependent and therefore reversible. The simple formation and reliable transfer of stabilized free-standing lipid films is a first crucial step to create biomimetic membranes for biotechnological applications and membrane protein research.

Development of a platform of antibody-presenting liposomes.

Antibody-presenting liposomes present high interest as drug delivery systems. The association of antibodies to liposomes is usually realized by covalent coupling of IgGs or their antigen-binding fragments to lipid polar head groups by means of hetero-bifunctional crosslinkers. We present here an original platform of IgG-presenting liposomes which is based on a fusion protein between Annexin-A5 (Anx5) and the IgG-binding ZZ repeat derived from Staphylococcus aureus protein A. The Anx5ZZ fusion protein acts as a bi-functional adaptor that anchors IgGs to liposomes in a non covalent and highly versatile manner. The interactions between IgGs, Anx5ZZ and liposomes were characterized by PAGE, dynamic light scattering and fluorescence quenching assays, establishing that binding of Anx5ZZ to IgGs and of Anx5ZZ-IgG complexes to liposomes is complete with stoichiometric amounts of each species. We found that the sequence of assembly is important and that Anx5ZZ-IgG complexes need to be formed first in solution and then adsorbed to liposomes in order to avoid aggregation. The targeting capacity of Anx5ZZ-IgG-functionalized liposomes was demonstrated by electron microscopy on an ex vivo model system of atherosclerotic plaques. This study shows that the Anx5ZZ adaptor constitutes an efficient platform for functionalizing liposomes with IgGs. This platform may present potential applications in molecular imaging and drug delivery.

Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair.

Eukaryotic cells possess a universal repair machinery that ensures rapid resealing of plasma membrane disruptions. Before resealing, the torn membrane is submitted to considerable tension, which functions to expand the disruption. Here we show that annexin-A5 (AnxA5), a protein that self-assembles into two-dimensional (2D) arrays on membranes upon Ca(2+) activation, promotes membrane repair. Compared with wild-type mouse perivascular cells, AnxA5-null cells exhibit a severe membrane repair defect. Membrane repair in AnxA5-null cells is rescued by addition of AnxA5, which binds exclusively to disrupted membrane areas. In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair. We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca(2+), AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing.

Effect of Mg2+ versus Ca2+ on the behavior of annexin A5 in a membrane-bound state.

Annexin A5 (AnxA5) binds to negatively charged phospholipid membranes in a Ca(2+) dependent manner. Several studies already demonstrate that Mg(2+) ions cannot induce the binding. In this paper, quartz crystal microbalance with dissipation monitoring (QCM-D), Brewster angle microscopy (BAM), polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) and molecular dynamics (MD) were performed to elucidate the high specificity of Ca(2+) versus Mg(2+) on AnxA5 binding to membrane models. In the presence of Ca(2+), AnxA5 showed a strong interaction with lipids, the protein is adsorbed mainly in α-helix under the DMPS monolayer, with an orientation of the α-helices axes slightly tilted with respect to the normal of the phospholipid monolayer as revealed by PMIRRAS. The Ca(2+) ions interact strongly with the phosphate group of the phospholipid monolayer. In the presence of Mg(2+), instead of Ca(2+), no interaction of AnxA5 with lipids was detected. Molecular dynamics simulations allow us to explain the high specificity of calcium. Ca(2+) ions are well exposed and surrounded by labile water molecules at the surface of the protein, which then favour their binding to the phosphate group of the membrane, explaining their specificity. To the contrary, Mg(2+) ions are embedded in the protein structure, with a smaller number of water molecules strongly bound. We conclude that the embedded Mg(2+) ions inside the AnxA5 structure are not able to link the protein to the phosphate group of the phospholipids for this reason.

Analysis of CD44-hyaluronan interactions in an artificial membrane system: insights into the distinct binding properties of high and low molecular weight hyaluronan.

CD44 is a major cell surface receptor for the large polydisperse glycosaminoglycan hyaluronan (HA). Binding of the long and flexible HA chains is thought to be stabilized by the multivalent nature of the sugar molecule. In addition, high and low molecular weight forms of HA provoke distinct proinflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or antiproliferative signals in appropriate cell types. Despite the importance of such interactions, however, neither the stoichiometry of multivalent HA binding at the cell surface nor the molecular basis for functional distinction between different HA size categories is understood. Here we report on the design of a supported lipid bilayer system that permits quantitative analysis of multivalent binding through presentation of CD44 in a stable, natively oriented manner and at controlled density. Using this system in combination with biophysical techniques, we show that the amount of HA binding to bilayers that are densely coated with CD44 increases as a function of HA size, with half-maximal saturation at ∼30 kDa. Moreover, reversible binding was confined to the smaller HA species (molecular weight of ≤10 kDa), whereas the interaction was essentially irreversible with larger polymers. The amount of bound HA decreased with decreasing receptor surface density, but the stability of binding was not affected. From a physico-chemical perspective, the binding properties of HA share many similarities with the typical behavior of a flexible polymer as it adsorbs onto a homogeneously attractive surface. These findings provide new insight into the multivalent nature of CD44-HA interactions and suggest a molecular basis for the distinct biological properties of different size fractions of hyaluronan.

Annexin A5-functionalized liposomes for targeting phosphatidylserine-exposing membranes.

Long-circulating liposomes functionalized with cell-targeting elements and loaded with bioactive compounds present high interest as drug delivery nanosystems. We present here the synthesis and physicochemical characterization of liposomes containing PEGylated lipids covalently linked to oriented Annexin-A5 (Anx5) proteins, and we show that Anx5-functionalized liposomes are able to target phosphatidylserine (PS)-exposing membranes. The covalent coupling of Anx5 to liposomes is almost quantitative, which is mainly due to the high accessibility of the reacting groups. The influence of Anx5 functionalization on liposome aggregation was investigated by dynamic light scattering, showing that Anx5-functionalized liposomes are stable below a threshold density of 250 Anx5 molecules per liposome. Anx5-functionalized liposomes bind PS-containing membranes with very high efficacy, which is mainly due to the controlled orientation of the Anx5 at the liposome surface. A striking result, obtained by quartz crystal microbalance with dissipation monitoring, is that one single Anx5 molecule is able to anchor a liposome to a PS-containing supported membrane. Finally, we show by fluorescence microscopy that Anx5-functionalized liposomes bind PS-exposing apoptotic K562 cells with high specificity. This study demonstrates that Anx5-functionalized liposomes bind specifically to PS membranes and are thus potential candidates to deliver drug or imaging agents to sites of apoptosis or thrombosis.

Annexin-A6 presents two modes of association with phospholipid membranes. A combined QCM-D, AFM and cryo-TEM study.

Annexins are soluble proteins that bind to biological membranes in a Ca(2+)-dependent manner. Annexin-A6 (AnxA6) is unique in the annexin family as it consists of the repeat of two annexin core modules, while all other annexins consist of a single module. AnxA6 has been proposed to participate in various membrane-related processes, including endocytosis and exocytosis, yet the molecular mechanism of association of AnxA6 with biological membranes, especially its ability to aggregate membranes, is still unclear. To address this question, we studied the association of AnxA6 with model phospholipid membranes by combining the techniques of quartz crystal microbalance with dissipation monitoring (QCM-D), (cryo-) transmission electron microscopy (TEM) and atomic force microscopy (AFM). The properties of membrane binding and membrane aggregation of AnxA6 were compared to two reference systems, annexin A5 (AnxA5), which is the annexin prototype, and a chimerical AnxA5-dimer molecule, which is able to aggregate two membranes in a symmetrical manner. We show that AnxA6 presents two modes of association with lipid membranes depending on Ca(2+)-concentration. At low Ca(2+)-concentration ( approximately 60-150microM), AnxA6 binds to membranes via its two coplanar annexin modules and is not able to associate two separate membranes. At high Ca(2+)-concentration ( approximately 2mM), AnxA6 molecules are able to bind two adjacent phospholipid membranes and present a conformation similar to the AnxA6 3D crystallographic structure. Possible biological implications of these novel membrane-binding properties of AnxA6 are discussed.

Formation of annexin-a5 protein/block copolymer micelle complexes: QCM-D and PAGE experiments.

The Annexin-A5 (Anx5) protein is a specific marker of the exposure of phosphatidylserine molecules at the surface of cells, which occurs in processes such as apoptosis and platelet activation. Decoration of self-assembled block copolymer nanostructures by Anx5 is of particular interest in micelle-mediated target drug delivery or in vivo magnetic resonance imaging, the Anx5 imparting (bio)functionality to the system. In this work, the reversible binding of the Anx5 onto polystyrene-b-poly(2-phosphatethyl methacrylate-co-2-hydroxyethyl methacrylate) (PS-b-P(PEMA-co-HEMA)) block copolymer micelles in the presence of Ca2+ ions is described using Quartz crystal microbalance with dissipation monitoring (QCM-D) and polyacrylamide gel electrophoresis (PAGE) analysis. QCM-D experiments confirmed the binding process as well as its reversibility and dependence on the characteristics of macromolecular assemblies, such as the number of phosphonic diacid groups (Pmic) and hydrodynamic diameter (2RH). A linear relationship between the amount of micelles and the amount of protein bound onto the micelle surface until a saturation point was established by QCM-D. The amount of Anx5 bound to PS-b-P(PEMA-co-HEMA) micelles was successfully quantified by PAGE experiments in nondenaturing conditions, which also corroborated that the binding process is mediated by Ca2+ ions. The ability of such surface (bio)-functionalized nanoparticle systems to stabilize and transport hydrophobic loads was highlighted by transmission electron microscopy (TEM) of assemblies with entrapped iron oxide particles.

Peptide-presenting two-dimensional protein matrix on supported lipid bilayers: an efficient platform for cell adhesion.

Understanding and controlling cell adhesion to biomaterials and synthetic materials are important issues in basic research and applied sciences. Supported lipid bilayers (SLBs) functionalized with cell adhesion peptides linked to lipid molecules are popular platforms of cell adhesion. In this paper, an alternative approach of peptide presentation is presented in which peptides are stereo-selectively linked to proteins self-assembling in a rigid two-dimensional (2D) matrix on SLBs. Annexin-A5 (Anx5) was used as prototype protein for its known properties of forming stable and rigid 2D matrices on lipid surfaces. Two types of Anx5-peptide complexes, containing either a RGD or an IKVAV sequence, were synthesized. The authors show that both Anx5-peptide complexes present the same properties of binding and 2D organization on lipid surfaces as Anx5, when investigated by quartz crystal microbalance with dissipation monitoring, atomic force microscopy, and transmission electron microscopy techniques. Anx5-RGD and Anx5-IKVAV 2D matrices were found to promote specific adhesion of human saphenous vein endothelial cells and mouse embryonic stem cells, respectively. The influence of the surface density of exposed peptides on cell adhesion was investigated, showing that cells attach to Anx5-peptide matrices when the average distance between peptides is smaller than about 60 nm. This cell adhesion platform provides control of the orientation and density of cell ligands, opening interesting possibilities for future applications.