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The formation of surfaces decorated with biomacromolecules such as proteins, glycans, or nucleic acids with well-controlled orientations and densities is of critical importance for the design of in vitro models, e.g., synthetic cell membranes and interaction assays. To this effect, ligand molecules are often functionalized with an anchor that specifically binds to a surface with a high density of binding sites, providing control over the presentation of the molecules. Here, we present a method to robustly and quantitatively control the surface density of one or several types of anchor-bearing molecules by tuning the relative concentrations of target molecules and free anchors in the incubation solution. We provide a theoretical background that relates incubation concentrations to the final surface density of the molecules of interest and present effective guidelines toward optimizing incubation conditions for the quantitative control of surface densities. Focusing on the biotin anchor, a commonly used anchor for interaction studies, as a salient example, we experimentally demonstrate surface density control over a wide range of densities and target molecule sizes. Conversely, we show how the method can be adapted to quality control the purity of end-grafted biopolymers such as biotinylated glycosaminoglycans by quantifying the amount of residual free biotin reactant in the sample solution.
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Biotina , Biotina/química , Membrana Celular , BiopolímerosRESUMEN
Multivalent interactions are common in biological systems and are also widely deployed for targeting applications in biomedicine. A unique feature of multivalent binding is "superselectivity". Superselectivity refers to the sharp discrimination of surfaces (e.g., on cells or cell compartments) by their comparative surface densities of a given receptor. This feature is different from the conventional "type" selectivity, which discriminates surfaces by their distinct receptor types. In a broader definition, a probe is superselective if it converts a gradual change in any one interaction parameter into a sharp on/off dependency in probe binding.This Account describes our systematic experimental and theoretical efforts over the past decade to analyze the determinants of superselective binding. It aims to offer chemical biologists, biophysicists, biologists, and biomedical scientists a set of guidelines for the interpretation of multivalent binding data, and design rules for tuning superselective targeting. We first provide a basic introduction that identifies multiple low-affinity interactions and combinatorial entropy as the minimal set of conditions required for superselective recognition. We then introduce the main experimental and theoretical tools and analyze how salient features of the multivalent probes (i.e., their concentration, size, ligand valency, and scaffold type), of the surface receptors (i.e., their affinity for ligands, surface density, and mobility), and of competitors and cofactors (i.e., their concentration and affinity for the ligands and/or receptors) influence the sharpness and the position of the threshold for superselective recognition.Emerging from this work are a set of relatively simple yet quantitative data analysis guidelines and superselectivity design rules that apply to a broad range of probe types and interaction systems. The key finding is the scaling variable xS which faithfully predicts the influence of the surface receptor density, probe ligand valency, receptor-ligand affinity, and competitor/cofactor concentrations and affinities on superselective recognition. The scaling variable is a simple yet versatile tool to quantitatively tune the on/off threshold of superselective probes. We exemplify its application by reviewing and reinterpreting literature data for selected biological and biomedical interaction systems where superselectivity clearly is important.Our guidelines can be deployed to generate a new mechanistic understanding of multivalent recognition events inside and outside cells and the downstream physiological/pathological implications. Moreover, the design rules can be harnessed to develop novel superselective probes for analytical purposes in the life sciences and for diagnostic/therapeutic intervention in biomedicine.
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Biología , Ligandos , Unión ProteicaRESUMEN
Polymer brushes are attractive as surface coatings for a wide range of applications, from fundamental research to everyday life, and also play important roles in biological systems. How colloids (e.g., functional nanoparticles, proteins, viruses) bind and move across polymer brushes is an important yet under-studied problem. A mean-field theoretical approach is presented to analyze the binding and transport of colloids in planar polymer brushes. The theory explicitly considers the effect of solvent strength on brush conformation and of colloid-polymer affinity on colloid binding and transport. The position-dependent free energy of the colloid insertion into the polymer brush which controls the rate of colloid transport across the brush is derived. It is shown how the properties of the brush can be adjusted for brushes to be highly selective, effectively serving as tuneable gates with respect to colloid size and affinity to the brush-forming polymer. The most important parameter regime simultaneously allowing for high brush permeability and selectivity corresponds to a condition when the repulsive and attractive contributions to the colloid insertion free energy nearly cancel. This theory should be useful to design sensing and purification devices with enhanced selectivity and to better understand mechanisms underpinning the functions of biological polymer brushes.
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Polímeros , Proteínas , Polímeros/química , Solventes/química , Conformación Molecular , Coloides/químicaRESUMEN
Cellular differentiation is directly determined by concentration gradients of morphogens. As a central model for gradient formation during development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in Drosophila wing and eye discs. What is not known is how extracellular Hh spread is achieved and how it translates into precise gradients. Here we show that two separate binding areas located on opposite sides of the Hh molecule can interact directly and simultaneously with two heparan sulfate (HS) chains to temporarily cross-link the chains. Mutated Hh lacking one fully functional binding site still binds HS but shows reduced HS cross-linking. This, in turn, impairs Hhs ability to switch between both chains in vitro and results in striking Hh gradient hypomorphs in vivo. The speed and propensity of direct Hh switching between HS therefore shapes the Hh gradient, revealing a scalable design principle in morphogen-patterned tissues.
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Proteínas de Drosophila , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Heparitina Sulfato/metabolismo , Drosophila/metabolismo , Sitios de Unión , Drosophila melanogaster/metabolismo , Alas de AnimalesRESUMEN
The present report assesses the capability of a soluble glycosyltransferase to modify glycolipids organized in two synthetic membrane systems that are attractive models to mimic cell membranes: giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs). The objective was to synthesize the Gb3 antigen (Galα1,4Galß1,4Glcß-Cer), a cancer biomarker, at the surface of these membrane models. A soluble form of LgtC that adds a galactose residue from UDP-Gal to lactose-containing acceptors was selected. Although less efficient than with lactose, the ability of LgtC to utilize lactosyl-ceramide as an acceptor was demonstrated on GUVs and SLBs. The reaction was monitored using the B-subunit of Shiga toxin as Gb3-binding lectin. Quartz crystal microbalance with dissipation analysis showed that transient binding of LgtC at the membrane surface was sufficient for a productive conversion of LacCer to Gb3. Molecular dynamics simulations provided structural elements to help rationalize experimental data.
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Glucolípidos , Lactosa , Lactosa/metabolismo , Glucolípidos/metabolismo , Membrana Dobles de Lípidos/química , Membrana Celular/metabolismo , Liposomas Unilamelares/químicaRESUMEN
Leukocyte recruitment from the vasculature into tissues is a crucial component of the immune system but is also key to inflammatory disease. Chemokines are central to this process but have yet to be therapeutically targeted during inflammation due to a lack of mechanistic understanding. Specifically, CXCL4 (Platelet Factor 4, PF4) has no established receptor that explains its function. Here, we use biophysical, in vitro, and in vivo techniques to determine the mechanism underlying CXCL4-mediated leukocyte recruitment. We demonstrate that CXCL4 binds to glycosaminoglycan (GAG) sugars on proteoglycans within the endothelial extracellular matrix, resulting in increased adhesion of leukocytes to the vasculature, increased vascular permeability, and non-specific recruitment of a range of leukocytes. Furthermore, GAG sulfation confers selectivity onto chemokine localization. These findings present mechanistic insights into chemokine biology and provide future therapeutic targets.
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Factor Plaquetario 4 , Proteoglicanos , Factor Plaquetario 4/metabolismo , Receptores de Quimiocina , Quimiocinas/metabolismo , Glicosaminoglicanos , Matriz Extracelular/metabolismoRESUMEN
Moieties that compete with multivalent interactions or act as cofactors are common in living systems, but their effect on multivalent binding remains poorly understood. We derive a theoretical model that shows how the superselectivity of multivalent interactions is modulated by the presence of cofactors or competitors. We find that the role of these participating moieties can be fully captured by a simple rescaling of the affinity constant of the individual ligand-receptor bonds. Theoretical predictions are supported by experimental data of the membrane repair protein annexin A5 binding to anionic lipid membranes in the presence of Ca2+ cofactors and of the extracellular matrix polysaccharide hyaluronan (HA) binding to CD44 cell surface receptors in the presence of HA oligosaccharide competitors. The obtained findings should facilitate understanding of multivalent recognition in biological systems and open new routes for fine-tuning the selectivity of multivalent nanoprobes in medicinal chemistry.
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Ácido Hialurónico , Receptores de Superficie Celular , Anexina A5 , Ácido Hialurónico/química , Ligandos , Lípidos , Oligosacáridos , Receptores de Superficie Celular/metabolismoRESUMEN
Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift (ΔD) and the negative frequency shift (- Δf) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from ~ 2 kDa tetrasaccharides to ~ 1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the ΔD/ - Δf ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.
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Glicosaminoglicanos , Ácido Hialurónico , Adhesión Celular , Sulfatos de Condroitina , Glicosaminoglicanos/química , Ácido Hialurónico/química , Tecnicas de Microbalanza del Cristal de CuarzoRESUMEN
To study conformational transition occuring upon inferior solvent strength in a brush formed by linear or dendritically branched macromolecules tethered to the inner surface of cylindrical or planar (slit-like) pore, a self-consistent field analytical approach is employed. Variations in the internal brush structure as a function of variable solvent strength and pore radius, and the onset of formation of a hollow channel in the pore center are analysed. The predictions of analytical theory are supported and complemented by numerical modelling by a self-consistent field Scheutjens-Fleer method. Scaling arguments are used to study microphase segregation under poor solvent conditions leading to formation of a laterally and longitudinally patterned structure in planar and cylindrical pores, respectively, and the effects of confinement on "octopus-like" clusters in the pores of different geometries.
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Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here, we show by cell-free reconstitution that binding to flat lipid membranes requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septin hexamers form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12- to 18-nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4-nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.
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Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Lípidos de la Membrana/metabolismo , Septinas/metabolismo , Animales , Animales Modificados Genéticamente , Membrana Celular/ultraestructura , Microscopía por Crioelectrón , Proteínas de Drosophila/genética , Proteínas de Drosophila/ultraestructura , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestructura , Humanos , Membrana Dobles de Lípidos , Lípidos de la Membrana/química , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/ultraestructura , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Tecnicas de Microbalanza del Cristal de Cuarzo , Septinas/genética , Septinas/ultraestructura , Relación Estructura-ActividadRESUMEN
The biological functions of natural polyelectrolytes are strongly influenced by the presence of ions, which bind to the polymer chains and thereby modify their properties. Although the biological impact of such modifications is well recognized, a detailed molecular picture of the binding process and of the mechanisms that drive the subsequent structural changes in the polymer is lacking. Here, we study the molecular mechanism of the condensation of calcium, a divalent cation, on hyaluronan, a ubiquitous polymer in human tissues. By combining two-dimensional infrared spectroscopy experiments with molecular dynamics simulations, we find that calcium specifically binds to hyaluronan at millimolar concentrations. Because of its large size and charge, the calcium cation can bind simultaneously to the negatively charged carboxylate group and the amide group of adjacent saccharide units. Molecular dynamics simulations and single-chain force spectroscopy measurements provide evidence that the binding of the calcium ions weakens the intramolecular hydrogen-bond network of hyaluronan, increasing the flexibility of the polymer chain. We also observe that the binding of calcium to hyaluronan saturates at a maximum binding fraction of â¼10-15 mol %. This saturation indicates that the binding of Ca2+ strongly reduces the probability of subsequent binding of Ca2+ at neighboring binding sites, possibly as a result of enhanced conformational fluctuations and/or electrostatic repulsion effects. Our findings provide a detailed molecular picture of ion condensation and reveal the severe effect of a few, selective and localized electrostatic interactions on the rigidity of a polyelectrolyte chain.
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We present a method to probe molecular and nanoparticle diffusion within thin, solvated polymer coatings. The device exploits the confinement with well-defined geometry that forms at the interface between a planar and a hemispherical surface (of which at least one is coated with polymers) in close contact and uses this confinement to analyze diffusion processes without interference of exchange with and diffusion in the bulk solution. With this method, which we call plane-sphere confinement microscopy (PSCM), information regarding the partitioning of molecules between the polymer coating and the bulk liquid is also obtained. Thanks to the shape of the confined geometry, diffusion and partitioning can be mapped as a function of compression and concentration of the coating in a single experiment. The method is versatile and can be integrated with conventional optical microscopes; thus it should find widespread use in the many application areas exploiting functional polymer coatings. We demonstrate the use of PSCM using brushes of natively unfolded nucleoporin domains rich in phenylalanine-glycine repeats (FG domains). A meshwork of FG domains is known to be responsible for the selective transport of nuclear transport receptors (NTRs) and their macromolecular cargos across the nuclear envelope that separates the cytosol and the nucleus of living cells. We find that the selectivity of NTR uptake by FG domain films depends sensitively on FG domain concentration and that the interaction of NTRs with FG domains obstructs NTR movement only moderately. These observations contribute important information to better understand the mechanisms of selective NTR transport.
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Proteínas de Complejo Poro Nuclear , Polímeros , Transporte Activo de Núcleo Celular , Difusión , Proteínas de Complejo Poro Nuclear/metabolismo , Fenilalanina/metabolismoRESUMEN
End-grafting of polyelectrolyte chains to conducting substrates offers an opportunity to fabricate electro-responsive surfaces capable of changing their physical/chemical properties (adhesion, wettability) in response to applied electrical voltage. We use a self-consistent field numerical approach to compare the equilibrium properties of tethered strong and weak (pH-sensitive) polyelectrolytes to applied electrical field in both salt-free and salt-containing solutions. We demonstrate that both strong and weak polyelectrolyte brushes exhibit segregation of polyions in two populations if the surface is oppositely charged with respect to the brush. This segregation gives rise to complex patterns in the dependence of the brush thickness on salt concentration. We demonstrate that adjustable ionization of weak polyelectrolytes weakens their conformational response in terms of the dependence of brush thickness on the amplitude of the applied voltage.
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Understanding antigen-antibody interactions is important to many emerging medical and bioanalytical applications. In particular, the levels of antigen expression at the cell surface may determine antibody-mediated cell death. This parameter has a clear effect on outcome in patients undergoing immunotherapy. In this context, CD20 which is expressed in the membrane of B cells has received significant attention as target for immunotherapy of leukemia and lymphoma using the monoclonal antibody rituximab. To systematically study the impact of CD20 density on antibody recognition, we designed self-assembled monolayers that display tunable CD20 epitope densities. For this purpose, we developed in situ click chemistry to functionalize SPR sensor chips. We find that the rituximab binding affinity depends sensitively and nonmonotonously on CD20 surface density. Strongest binding, with an equilibrium dissociation constant (KD = 32 nM) close to values previously reported from in vitro analysis with B cells (apparent KD between 5 and 19 nM), was obtained for an average inter-antigen spacing of 2 nm. This distance is required for improving rituximab recognition, and in agreement with the known requirement of CD20 to form clusters to elicit a biological response. More generally, this study offers an interesting outlook in the understanding of the necessity of epitope clusters for effective mAb recognition.
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Anticuerpos Monoclonales/inmunología , Antígenos/inmunología , Química Clic , Cinética , Rituximab/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
In most bacteria, the early step of septum formation implies the association of soluble FtsZ polymers with the cytoplasmic membrane. ZipA, together with FtsA, provides membrane tethering to FtsZ in Escherichia coli, forming a dynamic proto-ring that serves as an assembly scaffold for the remaining elements of the divisome. Despite their importance for bacterial cell division, multivalent interactions between proto-ring elements at membrane surfaces remain poorly characterized in quantitative terms. We measured the binding of FtsZ to ZipA incorporated in supported lipid bilayers at controlled densities by using a combination of biophysical surface-sensitive techniques (quartz crystal microbalance and spectroscopic ellipsometry) and analyzed how ZipA density and FtsZ concentration control the state of assembly of FtsZ. We found that ZipA attachment enables FtsZ-GMPCPP (where GMPCPP is a GTP analogue with a reduced level of hydrolysis) to assemble in several distinct ways: (i) two-dimensional polymerization at the membrane and (ii) three-dimensional polymerization from the membrane into the solution phase where this may be associated with the formation of higher-order complexes. In these processes, ZipA is required to enrich FtsZ at the surface but the FtsZ bulk concentration defines which morphology is being formed. Moreover, we report a strong effect of the nucleotide (GDP vs GMPCPP/GTP) on the kinetics of ZipA association/dissociation of FtsZ. These results provide insights into the mode of interaction of proto-ring elements in minimal membrane systems and contribute to the completion of our understanding of the initial events of bacterial division.
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Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , División Celular , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Hidrólisis , Cinética , Membrana Dobles de Lípidos/metabolismo , Nucleótidos/metabolismo , Multimerización de ProteínaRESUMEN
The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b-7 interacts with the lipid bilayer prior to recruiting C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion.
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Antígenos CD59/metabolismo , Membrana Celular/ultraestructura , Complemento C9/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Multimerización de Proteína , Membrana Celular/metabolismo , Complemento C5/metabolismo , Complemento C8/metabolismo , Humanos , Cinética , Microscopía de Fuerza Atómica/métodos , Imagen Individual de Molécula/métodosRESUMEN
Hyaluronan (HA) is a linear, regular polysaccharide that plays as a chief structural and functional component in peri- and extracellular matrices, thus contributing significantly to many basic cellular processes. To understand more comprehensively the response of the supramolecular organization of HA polymers to changes in their aqueous environment, we study the effects of Ca2+ concentration and pH on the morphology and rigidity of films of end-grafted HA polymers on planar supports (HA brushes), as a well-defined in vitro model system of HA-rich matrices, by reflection interference contrast microscopy and quartz crystal microbalance. The thickness and softness of HA brushes decrease significantly with Ca2+ concentration but do not change with pH, within the physiological ranges of these parameters. The effect of Ca2+ on HA brush thickness is virtually identical to the effect of Na+ at 10-fold higher concentrations. Moreover, the thickness and softness of HA brushes decrease appreciably upon HA protonation at pH less than 6. Effects of pH and calcium ions are fully reversible over large parameter ranges. These findings are relevant for understanding the supramolecular organization and dynamics of HA-rich matrices in biological systems and will also benefit the rational design of synthetic HA-rich materials with tailored properties.
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Cell-cell and cell-glycocalyx interactions under flow are important for the behaviour of circulating cells in blood and lymphatic vessels. However, such interactions are not well understood due in part to a lack of tools to study them in defined environments. Here, we develop a versatile in vitro platform for the study of cell-glycocalyx interactions in well-defined physical and chemical settings under flow. Our approach is demonstrated with the interaction between hyaluronan (HA, a key component of the endothelial glycocalyx) and its cell receptor CD44. We generate HA brushes in situ within a microfluidic device, and demonstrate the tuning of their physical (thickness and softness) and chemical (density of CD44 binding sites) properties using characterisation with reflection interference contrast microscopy (RICM) and application of polymer theory. We highlight the interactions of HA brushes with CD44-displaying beads and cells under flow. Observations of CD44+ beads on a HA brush with RICM enabled the 3-dimensional trajectories to be generated, and revealed interactions in the form of stop and go phases with reduced rolling velocity and reduced distance between the bead and the HA brush, compared to uncoated beads. Combined RICM and bright-field microscopy of CD44+ AKR1 T-lymphocytes revealed complementary information about the dynamics of cell rolling and cell morphology, and highlighted the formation of tethers and slings, as they interacted with a HA brush under flow. This platform can readily incorporate more complex models of the glycocalyx, and should permit the study of how mechanical and biochemical factors are orchestrated to enable highly selective blood cell-vessel wall interactions under flow.
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Glicocálix/metabolismo , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Linfocitos T/citología , Fenómenos Biomecánicos , Comunicación Celular , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Receptores de Hialuranos/genética , Técnicas Analíticas Microfluídicas/métodos , Microscopía de Interferencia , Linfocitos T/metabolismo , TransfecciónRESUMEN
The interaction between a biological membrane and its environment is a complex process, as it involves multivalent binding between ligand/receptor pairs, which can self-organize in patches. Any description of the specific binding of biomolecules to membranes must account for the key characteristics of multivalent binding, namely, its unique ability to discriminate sharply between high and low receptor densities (superselectivity), but also for the effect of the lateral mobility of membrane-bound receptors to cluster upon binding. Here we present an experimental model system that allows us to compare systematically the effects of multivalent interactions on fluid and immobile surfaces. A crucial feature of our model system is that it allows us to control the membrane surface chemistry, the properties of the multivalent binder, and the binding affinity. We find that multivalent probes retain their superselective binding behavior at fluid interfaces. Supported by numerical simulations, we demonstrate that, as a consequence of receptor clustering, superselective binding is enhanced and shifted to lower receptor densities at fluid interfaces. To translate our findings into a simple, predictive tool, we propose an analytical model that enables rapid predictions of how the superselective binding behavior is affected by the lateral receptor mobility as a function of the physicochemical characteristics of the multivalent probe. We believe that our model, which captures the key physical mechanisms underpinning multivalent binding to biological membranes, will greatly facilitate the rational design of nanoprobes for the superselective targeting of cells.