Deborah Leckband | University of Illinois at Urbana-Champaign (original) (raw)

Papers by Deborah Leckband

Lateral mobility and dimensionality have both been shown to influence cellular behavior, but have... more Lateral mobility and dimensionality have both been shown to influence cellular behavior, but have yet to be combined and applied in a single in vitro platform to address, e.g., cell adhesion in a setting mimicking the three-dimensional environment of neighboring cells in a reductionist way. To study the effect of the lateral mobility of cell adhesive ligands in three dimensions we present and characterize a platform, which enables patterning of single cells into microwells presenting a cell membrane mimetic interface pre-patterned to its walls. Soluble E-cadherin extracellular domains coupled through an optimized streptavidin-antibody linkage to lipids in a supported lipid bilayer (SPB) were presented on the microwell walls as either laterally mobile or immobile ligands. The fluidity was controlled through a small change in temperature by choosing phospholipids for the SPB with a lipid phase transition temperature around 30 C. The platform thus enabled the investigation of cell adhesion to either laterally immobile or mobile E-cadherin ligands presented on the same cell membrane mimetic surface. Chinese hamster ovary (CHO) cells engineered to express E-cadherin that were cultured on the platform demonstrated that enhanced cadherin lateral mobility significantly decreased the formation of actin bundles and resulted in more diffuse actin organization, while constraining the cell shape to that of the microwell. This example highlights the potential to use in vitro cell culture platforms to mimic direct cell-cell interaction in a controlled environment that nevertheless captures the dynamic nature of the native cell environment.

The water soluble anionic dye, cobalt phthalocyanine disulfonate (PcCoDS), was used to prepare π-... more The water soluble anionic dye, cobalt phthalocyanine disulfonate (PcCoDS), was used to prepare π-electron terminated model monolayers with a high surface free energy. We report the en face self-assembly of monomeric PcCoDS monolayers on dioctadecydiammonium bromide (DODAB). Direct surface force measurements showed that the phthalocyanine overlayers increased the adhesion between the surfactant membranes in water nearly 100-fold. This increased attraction correlated with the dye-induced aggregation of DODAB vesicles. Simultaneous force and electronic absorbance measurements indicate that the formation of strong adhesive contacts between the dye layers corresponds with phthalocyanine dimerization. Further, the adhesion increased in proportion to the dye coverage, and, at the maximum dye coverage, it is at least as strong as hydrophobic interactions that stabilize the membranes. The surface free energy of PcCoDS/ DODA membranes, determined from JKR analysis of the contact area vs applied load, is 5.2 ( 0.4 mN m -1 . Analysis of the intersurface attraction using Lifschitz theory for multilayered systems suggests that the dispersion force contributes substantially to the dye interactions. Such forces acting between assemblies of other aromatic compounds in water may similarly contribute to the stability of molecularly engineered materials.

The mechanism that drives the segregation of cells into tissuespecific subpopulations during deve... more The mechanism that drives the segregation of cells into tissuespecific 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.

This work describes quantitative force and bead aggregation measurements of the adhesion and bind... more 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.

This study demonstrates that the thermally induced collapse of end-grafted poly(N-isopropylacryla... more This study demonstrates that the thermally induced collapse of end-grafted poly(N-isopropylacrylamide) (PNIPAM) above the lower critical solution temperature (LCST) of 32°C depends on the chain grafting density and molecular weight. The polymer was grafted from the surface of a self-assembled monolayer containing the initiator (BrC-(CH 3 ) 2 COO(CH 2 ) 11 S) 2 , using surface-initiated atom transfer radical polymerization. Varying the reaction time and monomer concentration controlled the molecular weight, and diluting the initiator in the monolayer altered the grafting density. Surface force measurements of the polymer films showed that the chain collapse above the LCST decreases with decreasing grafting density and molecular weight. At T > LCST, the advancing water contact angle increases sharply on PNIPAM films of high molecular weight and grafting density, but the change is less pronounced with films of low-molecular-weight chains at lower densities. Below the LCST, the force-distance profiles exhibit nonideal polymer behavior and suggest that the brush architecture comprises dilute outer chains and much denser chains adjacent to the surface.

The spatial distribution of molecular signals within cells is crucial for cellular functions. Her... more The spatial distribution of molecular signals within cells is crucial for cellular functions. Here, as a model to study the polarized spatial distribution of molecular activities, we used cells on micropatterned strips of fibronectin with one end free and the other end contacting a neighbouring cell. Phosphoinositide 3-kinase and the small GTPase Rac display greater activity at the free end, whereas myosin II light chain and actin filaments are enriched near the intercellular junction. Phosphoinositide 3-kinase and Rac polarization depend specifically on the N-cadherin-p120catenin complex, whereas myosin II light chain and actin filament polarization depend on the N-cadherin-b-catenin complex. Integrins promote high phosphoinositide 3-kinase/Rac activities at the free end, and the N-cadherin-p120catenin complex excludes integrin a5 at the junctions to suppress local phosphoinositide 3-kinase and Rac activity. We hence conclude that N-cadherin couples with distinct effectors to polarize phosphoinositide 3-kinase/Rac and myosin II light chain/actin filaments in migrating cells.

Adhesion is essential in biology. Intercellular interactions maintain the structural hierarchy of... more Adhesion is essential in biology. Intercellular interactions maintain the structural hierarchy of all multicellular organisms across all anatomical length scales. Cells transduce mechanical signals and respond by regulating adhesion, motility, and differentiation. Other adhesive interactions are central to immunity. Pathogenic microorganisms use adhesive interactions with cells in the first steps in infection. Determining the molecular mechanisms underlying these processes is central to understanding the fundamental basis of related diseases and to developing strategies to treating or preventing disease.

Poly(ethylene glycol) (PEG)-stabilized liposomes were recently shown to exhibit differences in ce... more 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.

This report describes an approach to extend indefinitely the duration of traction force measureme... more This report describes an approach to extend indefinitely the duration of traction force measurements with cells cultured on soft polyacrylamide gels. Typical observation times in traction force measurements on similar substrates have been limited to 24-48 h, but cell differentiation or responses to external stimuli often occur over much longer periods of several days or weeks. This study describes a method for covalently linking fluorescent marker beads to a polyacrylamide matrix that renders the hydrogels useful for traction force measurements over several days. This approach was validated by comparing the contractility of C2C12 murine skeletal muscle cells prior to myotube formation, after one day in culture, with that of myotubes after 7 days in culture. Measured tractions increased concurrent with the differentiation of C2C12 cells to the contractile, myotube phenotype. Covalent bead linkage thus extends the useful period during which traction force data can be obtained with cells cultured on optically transparent polyacrylamide hydrogels with controlled elastic moduli.

This study reports a theoretical analysis of the forced separation of two adhesive surfaces linke... more This study reports a theoretical analysis of the forced separation of two adhesive surfaces linked via a large number of parallel noncovalent bonds. To describe the bond kinetics, we implement a three-state reaction model with kinetic rates obtained from a simple integral expression of the mean first passage time for diffusive barrier crossing in a pulled-distance-dependent potential. We then compute the rupture force for the separation of adhesive surfaces at a constant rate. The results correspond well with a Brownian dynamics simulation of the same system. The separation rate relative to the intrinsic relaxation time of the bonds defines three loading regimes and the general dependence of the adhesion on kinetic or thermodynamic parameters of the bonds. In the equilibrium regime, the rupture force asymptotically approaches the equilibrium rupture force, which increases linearly with the equilibrium bond energy. In the near-equilibrium regime, the rupture force increases with the separation rate and increasingly correlates with the bond rupture barrier. In the far-from-equilibrium regime where rebinding is irrelevant, the rupture force varies linearly with the rupture barrier.

Apparatus. The surface force apparatus quantifies the force between 2 macroscopic surfaces as a f... more Apparatus. The surface force apparatus quantifies the force between 2 macroscopic surfaces as a function of their absolute separation, with a resolution of Ϯ0.1 nm (16-18). In these

Cell surface carbohydrates are important targets for many cell surface receptors, and they mediat... more Cell surface carbohydrates are important targets for many cell surface receptors, and they mediate crucial biological processes ranging from pathogen infectivity to neutrophil adhesion to drug targeting. A central challenge is to identify relationships between lectin architecture and function that influence the adhesion strength, avidity, and kinetics of receptor-glycan bonds. This information is central both to understanding recognition mechanisms and to developing effective therapeutic agents for drug targeting or for preventing infection. Increasingly, force probes are used to assess structure activity relationships of both the glycan ligands and the receptors that bind them, as well as molecular mechanisms underlying binding and adhesion. This review describes recent advances in the use of different force measurement techniques to quantify receptor-glycan bond parameters, and to identify novel features of molecular mechanisms underlying recognition and adhesion. The examples discussed focus in particular on single bond rupture, surface force measurements, and micropipette manipulation. This review emphasizes the often-unique information obtained from studies of lectin interactions with carbohydrate ligands that complement more common structure determinations and solution binding studies.

Langmuir, 2005

This work describes the use of microfluidic tools to generate covalently immobilized counter grad... more This work describes the use of microfluidic tools to generate covalently immobilized counter gradients of extracellular matrix (ECM) proteins laminin and collagen I. Using these platforms, we demonstrate control of the expression levels of two proteins linked to cell cycle progression by virtue of the spatial location of cells on the gradients, and hence by the local ECM environments in these devices. In contrast to physisorbed gradients, covalently immobilized protein patterns preserved the gradient fidelity, making long term cell studies feasible. This method of precisely controlling local cell environments is simple and broadly portable to other cell types and to other ECM proteins or soluble factors. Our approach promises to enable new investigations in cell biology that will contribute to the establishment of biological design rules for controlling cell growth, differentiation, and function.

Langmuir, 2006

This paper explores the effects of the surface density and concentration profiles of extra cellul... more This paper explores the effects of the surface density and concentration profiles of extra cellular matrix proteins on the migration of rat intestinal IEC-6 cells. Microfluidic devices were used to create linear, immobilized gradients of laminin. This study investigated both the impact of the steepness and local concentrations on the directedness of cell migration. The bulk concentrations of proteins in the feed streams in the mixing device determined the gradient profile and the local concentration of laminin in the device. Two sets of gradients were used to explore cell migration directedness: (i) gradients with similar change in local concentration, i.e., the same gradient steepness, and (ii) different gradients with similar local concentrations. Cells migrated up the gradients, independent of the steepness of the gradients used in this study. At the same local laminin concentration, the migration rate was independent of the gradient steepness. However, cell directedness decreased significantly at high laminin densities.

Birth Defects Research Part C-embryo Today-reviews, 2005

DEVELOPMENT light on the function of Wnt signaling and epithelial-mesenchymal crosstalk during em... more DEVELOPMENT light on the function of Wnt signaling and epithelial-mesenchymal crosstalk during embryonic and postnatal development is examined, along with data on the interplay of heparan sulfate proteoglycans in the signaling process. Birth Defects Research (Part C) 75:58 -71, 2005.

Biochemistry, 2005

The structures of adhesion proteins play an important role in the formation of intercellular junc... more The structures of adhesion proteins play an important role in the formation of intercellular junctions and the control of intermembrane spacing. This paper describes the combination of neutron and X-ray specular reflectivity measurements to investigate the structure of the ectodomain of the neuralcell-adhesion molecule (NCAM). The measurements with unmodified NCAM suggest the presence of a bend in the extraceullar region. Measurements with the polysialic-acid-modified form of NCAM reveal that, at physiological ionic strength, the carbohydrate chains extend beyond the range of the unmodified protein. The excluded volume of the polymer is also ionic-strength-dependent, as expected for a polyelectrolyte. The structural characteristics obtained from these independent analyses of X-ray and neutron reflectivity data agree with each other, with prior reflectivity studies, and with molecular dimensions obtained from direct-force measurements. These results provide structural insights into the configuration of the NCAM ectodomain and the regulation of NCAM adhesion by post-translational modification. †

Analytical Chemistry, 1999

Optical, evanescent wave biosensors have become popular tools for quantitatively characterizing t... more Optical, evanescent wave biosensors have become popular tools for quantitatively characterizing the kinetic properties of biomolecular interactions. Analyzing data from biosensor experiments, however, is often complicated when mass-transfer influences the detection kinetics. We present a computational, transport-kinetic model that can be used to analyze transport-limited biosensor data. This model describes a typical biosensor experiment in which a soluble analyte diffuses through a flow chamber and binds to a receptor immobilized on the transducer surface. Analyte transport in the flow chamber is described by the diffusion equation while the kinetics of analyte-surface association and dissociation are captured by a reactive boundary condition at the sensor surface. Numerical integration of the model equations and nonlinear least-squares fitting are used to compare model kinetic data to experimental results and generate estimates for the rate constants that describe analyte detection. To demonstrate the feasibility of this model, we use it to analyze data collected for the binding of fluorescently labeled trinitrobenzene to immobilized monoclonal anti-TNT antibodies. A successful analysis of this antigenantibody interaction is presented for data collected with a fluorescence-based fiber-optic immunoassay. The results of this analysis are compared with the results obtained with existing methods for analyzing diffusionlimited kinetic data.

Lateral mobility and dimensionality have both been shown to influence cellular behavior, but have... more Lateral mobility and dimensionality have both been shown to influence cellular behavior, but have yet to be combined and applied in a single in vitro platform to address, e.g., cell adhesion in a setting mimicking the three-dimensional environment of neighboring cells in a reductionist way. To study the effect of the lateral mobility of cell adhesive ligands in three dimensions we present and characterize a platform, which enables patterning of single cells into microwells presenting a cell membrane mimetic interface pre-patterned to its walls. Soluble E-cadherin extracellular domains coupled through an optimized streptavidin-antibody linkage to lipids in a supported lipid bilayer (SPB) were presented on the microwell walls as either laterally mobile or immobile ligands. The fluidity was controlled through a small change in temperature by choosing phospholipids for the SPB with a lipid phase transition temperature around 30 C. The platform thus enabled the investigation of cell adhesion to either laterally immobile or mobile E-cadherin ligands presented on the same cell membrane mimetic surface. Chinese hamster ovary (CHO) cells engineered to express E-cadherin that were cultured on the platform demonstrated that enhanced cadherin lateral mobility significantly decreased the formation of actin bundles and resulted in more diffuse actin organization, while constraining the cell shape to that of the microwell. This example highlights the potential to use in vitro cell culture platforms to mimic direct cell-cell interaction in a controlled environment that nevertheless captures the dynamic nature of the native cell environment.

The water soluble anionic dye, cobalt phthalocyanine disulfonate (PcCoDS), was used to prepare π-... more The water soluble anionic dye, cobalt phthalocyanine disulfonate (PcCoDS), was used to prepare π-electron terminated model monolayers with a high surface free energy. We report the en face self-assembly of monomeric PcCoDS monolayers on dioctadecydiammonium bromide (DODAB). Direct surface force measurements showed that the phthalocyanine overlayers increased the adhesion between the surfactant membranes in water nearly 100-fold. This increased attraction correlated with the dye-induced aggregation of DODAB vesicles. Simultaneous force and electronic absorbance measurements indicate that the formation of strong adhesive contacts between the dye layers corresponds with phthalocyanine dimerization. Further, the adhesion increased in proportion to the dye coverage, and, at the maximum dye coverage, it is at least as strong as hydrophobic interactions that stabilize the membranes. The surface free energy of PcCoDS/ DODA membranes, determined from JKR analysis of the contact area vs applied load, is 5.2 ( 0.4 mN m -1 . Analysis of the intersurface attraction using Lifschitz theory for multilayered systems suggests that the dispersion force contributes substantially to the dye interactions. Such forces acting between assemblies of other aromatic compounds in water may similarly contribute to the stability of molecularly engineered materials.

The mechanism that drives the segregation of cells into tissuespecific subpopulations during deve... more The mechanism that drives the segregation of cells into tissuespecific 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.

This work describes quantitative force and bead aggregation measurements of the adhesion and bind... more 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.

This study demonstrates that the thermally induced collapse of end-grafted poly(N-isopropylacryla... more This study demonstrates that the thermally induced collapse of end-grafted poly(N-isopropylacrylamide) (PNIPAM) above the lower critical solution temperature (LCST) of 32°C depends on the chain grafting density and molecular weight. The polymer was grafted from the surface of a self-assembled monolayer containing the initiator (BrC-(CH 3 ) 2 COO(CH 2 ) 11 S) 2 , using surface-initiated atom transfer radical polymerization. Varying the reaction time and monomer concentration controlled the molecular weight, and diluting the initiator in the monolayer altered the grafting density. Surface force measurements of the polymer films showed that the chain collapse above the LCST decreases with decreasing grafting density and molecular weight. At T > LCST, the advancing water contact angle increases sharply on PNIPAM films of high molecular weight and grafting density, but the change is less pronounced with films of low-molecular-weight chains at lower densities. Below the LCST, the force-distance profiles exhibit nonideal polymer behavior and suggest that the brush architecture comprises dilute outer chains and much denser chains adjacent to the surface.

The spatial distribution of molecular signals within cells is crucial for cellular functions. Her... more The spatial distribution of molecular signals within cells is crucial for cellular functions. Here, as a model to study the polarized spatial distribution of molecular activities, we used cells on micropatterned strips of fibronectin with one end free and the other end contacting a neighbouring cell. Phosphoinositide 3-kinase and the small GTPase Rac display greater activity at the free end, whereas myosin II light chain and actin filaments are enriched near the intercellular junction. Phosphoinositide 3-kinase and Rac polarization depend specifically on the N-cadherin-p120catenin complex, whereas myosin II light chain and actin filament polarization depend on the N-cadherin-b-catenin complex. Integrins promote high phosphoinositide 3-kinase/Rac activities at the free end, and the N-cadherin-p120catenin complex excludes integrin a5 at the junctions to suppress local phosphoinositide 3-kinase and Rac activity. We hence conclude that N-cadherin couples with distinct effectors to polarize phosphoinositide 3-kinase/Rac and myosin II light chain/actin filaments in migrating cells.

Adhesion is essential in biology. Intercellular interactions maintain the structural hierarchy of... more Adhesion is essential in biology. Intercellular interactions maintain the structural hierarchy of all multicellular organisms across all anatomical length scales. Cells transduce mechanical signals and respond by regulating adhesion, motility, and differentiation. Other adhesive interactions are central to immunity. Pathogenic microorganisms use adhesive interactions with cells in the first steps in infection. Determining the molecular mechanisms underlying these processes is central to understanding the fundamental basis of related diseases and to developing strategies to treating or preventing disease.

Poly(ethylene glycol) (PEG)-stabilized liposomes were recently shown to exhibit differences in ce... more 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.

This report describes an approach to extend indefinitely the duration of traction force measureme... more This report describes an approach to extend indefinitely the duration of traction force measurements with cells cultured on soft polyacrylamide gels. Typical observation times in traction force measurements on similar substrates have been limited to 24-48 h, but cell differentiation or responses to external stimuli often occur over much longer periods of several days or weeks. This study describes a method for covalently linking fluorescent marker beads to a polyacrylamide matrix that renders the hydrogels useful for traction force measurements over several days. This approach was validated by comparing the contractility of C2C12 murine skeletal muscle cells prior to myotube formation, after one day in culture, with that of myotubes after 7 days in culture. Measured tractions increased concurrent with the differentiation of C2C12 cells to the contractile, myotube phenotype. Covalent bead linkage thus extends the useful period during which traction force data can be obtained with cells cultured on optically transparent polyacrylamide hydrogels with controlled elastic moduli.

This study reports a theoretical analysis of the forced separation of two adhesive surfaces linke... more This study reports a theoretical analysis of the forced separation of two adhesive surfaces linked via a large number of parallel noncovalent bonds. To describe the bond kinetics, we implement a three-state reaction model with kinetic rates obtained from a simple integral expression of the mean first passage time for diffusive barrier crossing in a pulled-distance-dependent potential. We then compute the rupture force for the separation of adhesive surfaces at a constant rate. The results correspond well with a Brownian dynamics simulation of the same system. The separation rate relative to the intrinsic relaxation time of the bonds defines three loading regimes and the general dependence of the adhesion on kinetic or thermodynamic parameters of the bonds. In the equilibrium regime, the rupture force asymptotically approaches the equilibrium rupture force, which increases linearly with the equilibrium bond energy. In the near-equilibrium regime, the rupture force increases with the separation rate and increasingly correlates with the bond rupture barrier. In the far-from-equilibrium regime where rebinding is irrelevant, the rupture force varies linearly with the rupture barrier.

Apparatus. The surface force apparatus quantifies the force between 2 macroscopic surfaces as a f... more Apparatus. The surface force apparatus quantifies the force between 2 macroscopic surfaces as a function of their absolute separation, with a resolution of Ϯ0.1 nm (16-18). In these

Cell surface carbohydrates are important targets for many cell surface receptors, and they mediat... more Cell surface carbohydrates are important targets for many cell surface receptors, and they mediate crucial biological processes ranging from pathogen infectivity to neutrophil adhesion to drug targeting. A central challenge is to identify relationships between lectin architecture and function that influence the adhesion strength, avidity, and kinetics of receptor-glycan bonds. This information is central both to understanding recognition mechanisms and to developing effective therapeutic agents for drug targeting or for preventing infection. Increasingly, force probes are used to assess structure activity relationships of both the glycan ligands and the receptors that bind them, as well as molecular mechanisms underlying binding and adhesion. This review describes recent advances in the use of different force measurement techniques to quantify receptor-glycan bond parameters, and to identify novel features of molecular mechanisms underlying recognition and adhesion. The examples discussed focus in particular on single bond rupture, surface force measurements, and micropipette manipulation. This review emphasizes the often-unique information obtained from studies of lectin interactions with carbohydrate ligands that complement more common structure determinations and solution binding studies.

Langmuir, 2005

This work describes the use of microfluidic tools to generate covalently immobilized counter grad... more This work describes the use of microfluidic tools to generate covalently immobilized counter gradients of extracellular matrix (ECM) proteins laminin and collagen I. Using these platforms, we demonstrate control of the expression levels of two proteins linked to cell cycle progression by virtue of the spatial location of cells on the gradients, and hence by the local ECM environments in these devices. In contrast to physisorbed gradients, covalently immobilized protein patterns preserved the gradient fidelity, making long term cell studies feasible. This method of precisely controlling local cell environments is simple and broadly portable to other cell types and to other ECM proteins or soluble factors. Our approach promises to enable new investigations in cell biology that will contribute to the establishment of biological design rules for controlling cell growth, differentiation, and function.

Langmuir, 2006

This paper explores the effects of the surface density and concentration profiles of extra cellul... more This paper explores the effects of the surface density and concentration profiles of extra cellular matrix proteins on the migration of rat intestinal IEC-6 cells. Microfluidic devices were used to create linear, immobilized gradients of laminin. This study investigated both the impact of the steepness and local concentrations on the directedness of cell migration. The bulk concentrations of proteins in the feed streams in the mixing device determined the gradient profile and the local concentration of laminin in the device. Two sets of gradients were used to explore cell migration directedness: (i) gradients with similar change in local concentration, i.e., the same gradient steepness, and (ii) different gradients with similar local concentrations. Cells migrated up the gradients, independent of the steepness of the gradients used in this study. At the same local laminin concentration, the migration rate was independent of the gradient steepness. However, cell directedness decreased significantly at high laminin densities.

Birth Defects Research Part C-embryo Today-reviews, 2005

DEVELOPMENT light on the function of Wnt signaling and epithelial-mesenchymal crosstalk during em... more DEVELOPMENT light on the function of Wnt signaling and epithelial-mesenchymal crosstalk during embryonic and postnatal development is examined, along with data on the interplay of heparan sulfate proteoglycans in the signaling process. Birth Defects Research (Part C) 75:58 -71, 2005.

Biochemistry, 2005

The structures of adhesion proteins play an important role in the formation of intercellular junc... more The structures of adhesion proteins play an important role in the formation of intercellular junctions and the control of intermembrane spacing. This paper describes the combination of neutron and X-ray specular reflectivity measurements to investigate the structure of the ectodomain of the neuralcell-adhesion molecule (NCAM). The measurements with unmodified NCAM suggest the presence of a bend in the extraceullar region. Measurements with the polysialic-acid-modified form of NCAM reveal that, at physiological ionic strength, the carbohydrate chains extend beyond the range of the unmodified protein. The excluded volume of the polymer is also ionic-strength-dependent, as expected for a polyelectrolyte. The structural characteristics obtained from these independent analyses of X-ray and neutron reflectivity data agree with each other, with prior reflectivity studies, and with molecular dimensions obtained from direct-force measurements. These results provide structural insights into the configuration of the NCAM ectodomain and the regulation of NCAM adhesion by post-translational modification. †

Analytical Chemistry, 1999

Optical, evanescent wave biosensors have become popular tools for quantitatively characterizing t... more Optical, evanescent wave biosensors have become popular tools for quantitatively characterizing the kinetic properties of biomolecular interactions. Analyzing data from biosensor experiments, however, is often complicated when mass-transfer influences the detection kinetics. We present a computational, transport-kinetic model that can be used to analyze transport-limited biosensor data. This model describes a typical biosensor experiment in which a soluble analyte diffuses through a flow chamber and binds to a receptor immobilized on the transducer surface. Analyte transport in the flow chamber is described by the diffusion equation while the kinetics of analyte-surface association and dissociation are captured by a reactive boundary condition at the sensor surface. Numerical integration of the model equations and nonlinear least-squares fitting are used to compare model kinetic data to experimental results and generate estimates for the rate constants that describe analyte detection. To demonstrate the feasibility of this model, we use it to analyze data collected for the binding of fluorescently labeled trinitrobenzene to immobilized monoclonal anti-TNT antibodies. A successful analysis of this antigenantibody interaction is presented for data collected with a fluorescence-based fiber-optic immunoassay. The results of this analysis are compared with the results obtained with existing methods for analyzing diffusionlimited kinetic data.