Biomedical subjects

D E Leckband

Publications and source records attributed to D E Leckband.

16 recordsLinked to original sources

PubMed2006

Similarities between heterophilic and homophilic cadherin adhesion.

The mechanism that drives the segregation of cells into tissue-specific subpopulations during development is largely attributed to differences in intercellular adhesion. This process requires the cadherin family of calcium-dependent glycoproteins. A widely held view is that protein-level discrimination between different cadherins on cell surfaces drives this sorting process. Despite this postulated molecular selectivity, adhesion selectivity has not been quantitatively verified at the protein level. In this work, molecular force measurements and bead aggregation assays tested whether differences in cadherin bond strengths could account for cell sorting in vivo and in vitro. Studies were conducted with chicken N-cadherin, canine E-cadherin, and Xenopus C-cadherin. Both qualitative bead aggregation and quantitative force measurements show that the cadherins cross-react. Furthermore, heterophilic adhesion is not substantially weaker than homophilic adhesion, and the measured differences in adhesion do not correlate with cell sorting behavior. These results suggest that the basis for cell segregation during morphogenesis does not map exclusively to protein-level differences in cadherin adhesion.

Animals↗

PubMed2006

Calcium site mutations in cadherin: impact on adhesion and evidence of cooperativity.

This work describes quantitative force and bead aggregation measurements of the adhesion and binding mechanisms of canine E-cadherin mutants W2A, D134A, D103A, D216A, D325A, and D436A. The W2A mutation affects the formation of the N-terminal strand dimer, and the remaining mutations target calcium binding sites at the interdomain junctions. Surface force measurements show that the full ectodomain of canine E-cadherin forms two bound states that span two intermembrane gap distances. The outer bond coincides with adhesion between the N-terminal extracellular domains (EC1) and the inner bond corresponds to adhesion via extracellular domain 3 (EC3). The W2A, D103A, D134A, and D216A mutations all eliminated adhesion between the N-terminal domains, and they attenuated or nearly eliminated the inner bond. The W2A mutant, which does not destabilize the protein structure, attenuates binding via EC3, which is separated from the mutation by several hundred amino acids. This long-range effect suggests that the presence or absence of tryptophan-2 docking allosterically alters the adhesive function of distal sites on the protein. This finding appears to reconcile the multidomain binding mechanism with mutagenesis studies, which suggested that W2 is the sole binding interface. The effects of the calcium site mutations indicate that structural perturbations cooperatively impact large regions of the protein structure. However, the influence of the calcium sites on cadherin structure and function depends on their location in the protein.

Animals↗

PubMed2005

Multiple-bond kinetics from single-molecule pulling experiments: evidence for multiple NCAM bonds.

The kinetic parameters of single bonds between neural cell adhesion molecules were determined from atomic force microscope measurements of the forced dissociation of the homophilic protein-protein bonds. The analytical approach described provides a systematic procedure for obtaining rupture kinetics for single protein bonds from bond breakage frequency distributions obtained from single-molecule pulling experiments. For these studies, we used the neural cell adhesion molecule (NCAM), which was recently shown to form two independent protein bonds. The analysis of the bond rupture data at different loading rates, using the single-bond full microscopic model, indicates that the breakage frequency distribution is most sensitive to the distance to the transition state and least sensitive to the molecular spring constant. The analysis of bond failure data, however, motivates the use of a double-bond microscopic model that requires an additional kinetic parameter. This double-bond microscopic model assumes two independent NCAM-NCAM bonds, and more accurately describes the breakage frequency distribution, particularly at high loading rates. This finding agrees with recent surface-force measurements, which showed that NCAM forms two spatially distinct bonds between opposed proteins.

Animals↗

PubMed2001

A quantitative assessment of heterogeneity for surface-immobilized proteins.

Many biotechnological applications use protein receptors immobilized on solid supports. Although, in solution, these receptors display homogeneous binding affinities and association/dissociation kinetics for their complementary ligand, they often display heterogeneous binding characteristics after immobilization. In this study, a fluorescence-based fiber-optic biosensor was used to quantify the heterogeneity associated with the binding of a soluble analyte, fluorescently labeled trinitrobenzene, to surface-immobilized monoclonal anti-TNT antibodies. The antibodies were immobilized on silica fiber-optic probes via five different immobilization strategies. We used the Sips isotherm to assesses and compare the heterogeneity in the antibody binding affinity and kinetic rate parameters for these different immobilization schemes. In addition, we globally analyzed kinetic data with a two-compartment transport-kinetic model to analyze the heterogeneity in the analyte-antibody kinetics. These analyses provide a quantitative tool by which to evaluate the relative homogeneity of different antibody preparations. Our results demonstrate that the more homogeneous protein preparations exhibit more uniform affinities and kinetic constants.

Biosensing Techniques↗

PubMed2001

Long-term stability of grafted polyethylene glycol surfaces for use with microstamped substrates in neuronal cell culture.

Crucial to long-term stability of neuronal micropatterns is functional retention of the underlying substratum while exposed to cell culture conditions. We report on the ability of covalently bound PEG films in long-term cell culture to continually retard protein adhesion and cell growth. PDMS microstamps were used to create poly-d-lysine (PDL) substrates permissive to cell attachment and growth, and polyethylene glycol (PEG) substrates were used to minimize protein and cell adhesion. Film thickness was measured using null ellipsometry and atomic force microscopy (AFM). Organosilane film structure was examined using Fourier transform infrared (FT-IR) spectroscopy. Long-term film stability in cell culture conditions was tested by immersion in 0.1 M sodium phosphate buffer pH 7.4 for up to one month. Null ellipsometry and water contact measurements indicated that organosilane films were stable up to one month, whereas the PEG film thickness declined rapidly after day 25. Hippocampal cells plated at 200 cells/mm2 on uniform PEG substrates gave a steady increase in biofilm thickness on PEG films throughout the culture, possibly from proteins of neuronal origin. We found that all the layers in the cross-linking procedure were stable in cell culture conditions, with the exception of PEG, which degraded after day 25.

Animals↗

PubMed2000

Measurements of interbilayer forces and protein adsorption on uncharged lipid bilayers displaying poly(ethylene glycol) chains.

Poly(ethylene glycol) (PEG)-stabilized liposomes were recently shown to exhibit differences in cell uptake that were linked to the liposome charge. To determine the differences and similarities between charged and uncharged PEG-decorated liposomes, we directly measured the forces between two supported, neutral bilayers with terminally grafted PEG chains. The measurements were performed with the surface force apparatus. The force profiles were similar to those measured with negatively charged PEG conjugates of 1, 2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine (DSPE), except that they lacked the longer ranged electrostatic repulsion observed with the charged compound. Theories for simple polymers describe the forces between end-grafted polymer chains on neutral bilayers. The force measurements were complemented by surface plasmon resonance studies of protein adsorption onto these layers. The lack of electrostatic forces reduced the adsorption of positively charged proteins and enhanced the adsorption of negatively charged ones. The absence of charge also allowed us to determine how membrane charge and the polymer grafting density independently affect protein adsorption on the coated membranes. Such studies suggest the physical basis of the different interactions of charged and uncharged liposomes with proteins and cells.

Adsorption↗

PubMed2000

Force probe measurements of antibody-antigen interactions.

The surface force apparatus has been used to quantify directly the forces that govern the interactions between proteins and ligands. In this work, we describe the measured interactions between the antigen fluorescein and the Fab' fragment of the monoclonal 4-4-20 anti-fluorescyl IgG antibody. Here we first describe the use of the surface force apparatus to demonstrate directly the impact of the charge composition in the region of the antibody binding site on the antibody interactions. Several approaches are described for immobilizing antigens, antibodies, and proteins in general for direct force measurements. The measured force profiles presented are accompanied by an extensive discussion of protocols used to analyze the force-distance curves and to interpret them in terms of the antibody structure. In addition to long-range electrostatic forces, we also consider short-range forces that can affect the strength of adhesion between the Fab' and immobilized fluorescein. The latter investigations demonstrate the influence of interfacial properties on the recognition of surface-bound antigens.

Antibodies, Monoclonal↗

PubMed2000

Long-term maintenance of patterns of hippocampal pyramidal cells on substrates of polyethylene glycol and microstamped polylysine.

For neurons to attach and remain in precise micropatterns for weeks in culture, background molecules that remain nonpermissive for extended culture durations need to be identified. Nonpermissive background molecules of either polyethylene glycol (PEG) or the amino acid serine (C3H7NO3) were evaluated. The foreground regions were microstamped with 3-, 5-, or 10-micron lines of poly-D-lysine (PDL), which promotes neural attachment and growth. After 29 days in culture the foreground compliance, or the fraction of all live somata which rested on the desired PDL surface, averaged 86% for serine and 90% for PEG, with only a small decline. The background compliance, or the fraction of square areas in the pattern background which were free of neurite extension, declined from highs of 40% and 55% (midculture) to 5.5% and 12% (29 days) for serine and PEG, respectively. Images of the cultures suggest that PEG is significantly more effective as a nonpermissive substrate. We conclude that these materials, especially PEG, are adequate for the maintenance of long-term patterned cultures of neurons. We believe that this is the first report of high-quality long-term patterning of cultured neurons.

Adsorption↗

PubMed1998

A neutron reflectivity study of polymer-modified phospholipid monolayers at the solid-solution interface: polyethylene glycol-lipids on silane-modified substrates.

The structure of polymer-decorated phospholipid monolayers at the solid-solution interface was investigated using neutron reflectometry. The monolayers were composed of distearoylphosphatidylethanolamine (DSPE) matrixed with varying amounts of DSPE-PEG (DSPE with polyethylene glycol covalently grafted to its headgroup). Mixed lipid monolayers were Langmuir-Blodgett deposited onto hydrophobic quartz or silicon substrates, previously hydrophobized by chemically grafting a robust monolayer of octadecyltrichlorosilane (OTS). We show that this method results in homogeneous and continuous phospholipid monolayers on the silanated substrates and determine that the grafted PEG chains extend away from the monolayers into the solvent phase as a function of their density, as expected from scaling theories. In addition, ligands were coupled to the end of the PEG chains and selective binding was demonstrated using fluorescence microscopy. Our results demonstrate that these constructs are ideal for further characterization and studies with well-defined monomolecular films.

Adsorption↗

PubMed1997

The influence of protein and interfacial structure on the self-assembly of oriented protein arrays.

These results demonstrate the complexity of factors that impact the self-assembly of protein arrays via the specific binding to receptor-functionalized interfaces. Both the composition and colloidal properties of the protein and target membrane surfaces will affect the protein-surface interactions. However, different structural features control the interactions over different distance regimes and with different consequences. The long-range interactions that control the adsorption kinetics are sensitive not only to the charge on the target surface but also by the topological charge distribution on the protein exterior. Short-range repulsive interactions rooted in both the protein topology and in the membrane structure, by contrast, can significantly alter both the rates and strengths binding. Consequently, the effective design of self-assembling protein arrays must consider not only the recognition interactions that drive such self-organization, but also the details of the micro-environment and their impact on molecular recognition events.

Avidin↗

PubMed1995

4-4-20 anti-fluorescyl IgG Fab' recognition of membrane bound hapten: direct evidence for the role of protein and interfacial structure.

The surface forces apparatus was used to identify the molecular forces that control the interactions of monoclonal 4-4-20 antifluorescyl IgG Fab' fragments with fluorescein-presenting supported planar bilayers. At long range, the electrostatic force between oriented Fab' and fluorescein monolayers was controlled by the composition of the protein exterior surrounding the antigen-combining site rather than by the overall protein charge. The measured positive electrostatic potential of the Fab' monolayer at pH > pI(Fab') was consistent with the structure of the exposed Fab' surface in which a ring of positive charge at the mouth of the antigen-combining site dominates the local electrostatic surface properties. Substantial differences in the electrostatic forces measured with denatured Fab' further demonstrated that the measured electrostatic surface properties and the consequent long-range interaction forces are controlled by the protein surface composition. At short range, the strength of the Fab'-mediated adhesion was modulated not only by the length of the fluorescein tether but also by membrane hydration. Steric hydration barriers at the membrane surface reduced the adhesion strength in proportion to their range of influence. These results provide direct evidence that long-range protein interactions with immobilized ligands are controlled by both the protein and the membrane surface compositions, while short-range, specific binding is modulated by both the protein structure and the membrane interfacial properties.

Antibodies, Monoclonal↗

PubMed1995

Transport effects on the kinetics of protein-surface binding.

A detailed model is presented for protein binding to active surfaces, with application to the binding of avidin molecules to a biotin-functionalized fiber optic sensor in experiments reported by S. Zhao and W. M. Reichert (American Chemical Society Symposium Series 493, 1992). Kinetic data for binding in solution are used to assign an intrinsic catalytic rate coefficient k to the biotin-avidin pair, deconvoluted from transport and electrostatic factors via application of coagulation theory. This intrinsic chemical constant is built into a reaction-diffusion analysis of surface binding where activity is restricted to localized sites (representing immobilized biotin molecules). The analysis leads to an effective catalytic rate coefficient keff characterizing the active surface. Thereafter, solution of the transport problem describing absorption of avidin molecules by the macroscopic sensor surface leads to predictions of the avidin flux, which are found to be in good agreement with the experimental data. The analysis suggests the following conclusions. 1) Translational diffusion limitations are negligible for avidin-biotin binding in solution owing to the small (kinetically limiting) value k = 0.00045 m/s. 2) The sparse distribution of biotin molecules and the presence of a repulsive hydration force produce an effective surface-average catalytic rate coefficient keff of order 10(-7) m/s, much smaller than k. 3) Avidin binding to the fiber optic sensor occurs in an intermediate regime where the rate is influenced by both kinetics and diffusion.

Avidin↗

PubMed1994

Direct force measurements of specific and nonspecific protein interactions.

Streptavidin-biotin (receptor-ligand) interaction forces were measured directly as a function of their intermolecular separation in various salt solutions and at various temperatures with a surface forces apparatus. Electrostatic and van der Waals forces were found to dominate the long-range streptavidin-biotin interaction at > 20 A. At intermediate separations, down to approximately 10 A, the interaction is governed by repulsive steric and attractive van der Waals and hydrophobic forces. A much stronger short-range attraction giving rise to the strong, specific adhesive binding was measured at molecular separations of less than 5 A. A decrease in the pH from 7.2 to 6.0 resulted in complete charge reversal on the binding surface of streptavidin (pK approximately 6) from net negative to net positive, while leaving the negatively charged biotin surface (pK approximately 3.0) unchanged, and the long-range interaction switched from repulsive to attractive. This observed behavior can be attributed to the titration of two histidines on the biotin binding surface of streptavidin. These results reveal a strong sensitivity of the long-range interaction forces to the detailed amino acid composition of the biotin binding surface. They also demonstrate the powerful regulatory potential conferred by small changes in local surface ionic conditions on protein interaction forces over different distance regimes. The effects of temperature on receptor-ligand dynamics and on the strength of intermembrane adhesion forces were studied by measuring the long-range force profiles and short-range adhesion forces above and below the chain melting temperature (Tc approximately 30 degrees C) of the lipids in the supporting bilayers. Increased bilayer fluidity due to a temperature increase to 33 degrees C (T > Tc) increased short-range adhesion by 7-fold relative to bilayers in the gel state at 25 degrees C (T < Tc). This effect was attributed to the enhanced rates of lateral diffusion and molecular rearrangements on the more fluid bilayer surfaces, which resulted in greater and more rapid intermembrane bond formation. A change in the rates of molecular rearrangements was also found to affect the repulsive part of the interaction potential at intermediate separations (10-20 A) via modulation of the steric repulsion between streptavidin and the highly flexible, polymer-like biotin molecules. This is expected to have a large effect on the association rates of receptor-ligand binding, even if it does not change the equilibrium binding energy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗

PubMed1994

Modulation of interaction forces between bilayers exposing short-chained ethylene oxide headgroups.

The use of liposomes as drug delivery systems has been limited by their rapid clearance from circulation by the mononuclear phagocyte system. Recent studies have found that circulation times can be greatly enhanced by incorporating a small amount of modified lipids whose headgroups are derivatized with a bulky water soluble polymeric chain of poly ethylene oxide. We report here a systematic study using the Surface Forces Apparatus to measure directly the interactions between two phosphatidyl ethanolamine lipid bilayers, exposing this polymeric headgroup at different concentrations in the bilayer. We found that the force becomes repulsive at all separations and that the thickness of the steric barrier could be controlled easily by adjusting the concentration of the modified lipids. Equilibrium force profiles were measured that were reversible and largely insensitive to changes in electrolyte concentration and temperature. The results have enabled the Dolan and Edwards theory for the steric forces of low coverage polymer surfaces and the Alexander de Gennes theory for high coverage surfaces to be tested, and both were found to apply. We conclude that these simple theories can be used to model the interactions of surprisingly short segments and, hence, apply to such systems as lipids with bulky headgroups and liposomes containing a sterically stabilizing polymer.

Biophysical Phenomena↗

PubMed1993

Role of calcium in the adhesion and fusion of bilayers.

The interaction forces and fusion mechanisms of mixed zwitterionic-anionic phospholipid bilayers were measured with the surface forces apparatus. The bilayers were 3:1 mixtures of either dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol (DMPC/DMPG) or dilauroylphosphatidylcholine and dilauroylphosphatidylglycerol (DLPC/DLPG), and experiments were carried out in NaCl solutions with and without CaCl2. In NaCl solutions, the forces between either mixed bilayer system were consistent with the DLVO (Derjaguin-Landau-Verwey-Overbeek) theory of repulsive electrostatic and attractive van der Waals forces, and fusion did not occur. At high pH (> 6) and in high (20 mM) NaCl concentrations, a short-range hydration force extending about 13 A was evident, indicative of Na+ binding to the surfaces. In the presence of this large hydration repulsion, the interbilayer adhesion was abolished. When CaCl2 was added to the bathing solutions in the presence or absence of NaCl, the bilayers phase separate into small domains, coinciding with the occurrence of a large, long-range attractive force. Fusion occurred readily between the more fluid domains. The phase separations and fusion events could be directly visualized by observing the shapes of the optical fringes used to measure the surface separation and the change in surface profiles with time. The ease of fusion between mixed bilayers in the presence of calcium correlated closely with the strength of the long-range attractive force. This force is attributed to the additional hydrophobic force between domains or domain boundaries due to the exposure of excess hydrophobic groups resulting from the Ca(2+)-induced condensation of the PG- headgroups.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗

PubMed1992

Long-range attraction and molecular rearrangements in receptor-ligand interactions.

A surface force apparatus was used to measure a long-range attractive protein-ligand force at separations D less than 85 angstroms. This force may effectively "steer" ligand trajectories, resulting in a greater than 27-fold enhancement of the association rate. A much stronger specific attraction is measured at contact (D less than 4 angstroms). A sevenfold increase in intermembrane adhesion resulted from increased lateral mobility of the receptors and molecular rearrangements in membranes above the solid-fluid transition temperature.

Bacterial Proteins↗