Thermal induced modification of the contact mechanics of adhering liposomes on cationic substrate

The correlation between the mechanical property and the thermotropic transition of the phospholipid bilayer has been recently demonstrated (Chem. Phys. Lipids 110 (2001) 27). However, the role of thermal induced mechanical responses of phospholipid bilayer on the contact mechanics of liposome adhering on a cationic substrate has not been determined. In this study, confocal-reflectance interference contrast microscopy, phase contrast microscopy and contact mechanics modeling are applied to probe the adhesion mechanisms of liposomes in the presence of electrostatic interactions during the thermotropic transition of the lipid bilayer. When temperature increases from 23 to 49 °C at pH 7.4, the degree of liposome deformation (a/R) and adhesion energy of dipalmitoyl-sn-glycero-3-phosphocholine liposome increases by 10% and remains constant, respectively, on 3-amino-propyl-triethoxy-silane (APTES) modified substrate. The extents of increase in these two parameters are highly dependent on the physicochemical properties of the rigid substrate. At pH 4, the adhesion energies above and below the phase transition temperature (T_m) are increased by one order of magnitude due to the formation of the free silanol groups on APTES substrate. In hypotonic condition, the degree of vesicle deformation remains constant and the adhesion energy reduces by 20% during sample heating. Under all conditions, the adhesion energy of the adhering liposome spans a few orders of magnitude against the increase of liposome size as the surface area to volume ratio is maximized in smallest vesicle.     

Biomechanics of smooth adhesive pads in insects: influence of tarsal secretion on attachment performance

Many insects possess smooth adhesive pads on their legs, which adhere by thin films of a two-phasic secretion. To understand the function of such fluid-based adhesive systems, we simultaneously measured adhesion, friction and contact area in single pads of stick insects (Carausius morosus). Shear stress was largely independent of normal force and increased with velocity, seemingly consistent with the viscosity-effect of a continuous fluid film. However, measurements of the remaining force 2 min after a sliding movement show that adhesive pads can sustain considerable static friction. Repeated sliding movements and multiple consecutive pull-offs to deplete adhesive secretion showed that on a smooth surface, friction and adhesion strongly increased with decreasing amount of fluid. In contrast, pull-off forces significantly decreased on a rough substrate. Thus, the secretion does not generally increase attachment but does so only on rough substrates, where it helps to maximize contact area. When slides were repeated at one position so that secretion could accumulate, sliding shear stress decreased but static friction remained clearly present. This suggests that static friction which is biologically important to prevent sliding is based on non-Newtonian properties of the adhesive emulsion rather than on a direct contact between the cuticle and the substrate.     

Thermodynamics of cell adhesion. II. Freely mobile repellers.

The equilibrium adhesion of a cell or vesicle to a substrate is analyzed in a theoretical model in which two types of mobile molecules in the cell membrane are of interest: receptors that can form bonds with fixed ligands in the substrate and repellers that repel the substrate. If the repulsion between the repeller molecule and substrate is greater than kT, there is substantial redistribution of the repellers from the contact area. Coexisting equilibrium states are observed having comparable free energies (a) with unstretched bonds and repeller redistribution and (b) with stretched bonds and partial redistribution.     

Force-controlled equilibria of specific vesicle-substrate adhesion

We have developed "vertical" magnetic tweezers capable of exerting controlled pico and subpico Newton forces. Using this apparatus, we apply a point-like force to a vesicle that is adhered by means of specific bonds between the vesicle-carrying oligosaccharide sialyl LewisX and the surface-grafted E-selectin. An exponential decrease of the bound vesicle area with the decay rate that is insensitive to the force and the composition of the system is observed. We measure an equilibrium under force that is associated with an increased binding in the center of the contact zone. We also show that the determination of the shape is potentially sufficient to determine the number of formed specific bonds.     

Antagonist-induced deadhesion of specifically adhered vesicles

By use of a model system consisting of giant vesicles adhering to flat substrates, we identified, both experimentally and theoretically, two new control mechanisms for antagonist-induced deadhesion. Adhesion is established by specific binding of surface-grafted E-selectin and vesicle-carrying oligosaccharide Lewis(X). Deadhesion is achieved by controlled titration of monoclonal antibodies against E-selectin. The first mechanism is characterized by a considerable retraction of the contact zone resulting in a loss of contact area between the vesicle and the substrate. Within the developed theoretical framework, the observed equilibrium state is understood as a balance between the spreading pressure of the vesicle and the antagonist-induced lateral pressure at the edge of the contact zone. In the second mechanism, the antibodies induce unbinding by penetrating the contact zone without significantly affecting its size. This process reveals the decomposition of the adhesion zone into microdomains of tight binding separated by strongly fluctuating sections of the membrane. Both experiment and theory show a sigmoidal decrease of the number of bound ligands as a function of the logarithm of antagonist concentration. The work presented herein also provides a new method for the determination of the receptor binding affinity of either the surface-embedded ligands or the competing antagonist molecules.     

A numerical analysis of forces exerted by laminar flow on spreading cells in a parallel plate flow chamber assay

Exposure of spreading anchorage-dependent cells to laminar flow is a common technique to measure the strength of cell adhesion. Since cells protrude into the flow stream, the force exerted by the fluid on the cells is a function of cell shape. To assess the relationship between cell shape and the hydrodynamic force on adherent cells, we obtained numerical solutions of the velocity and stress fields around bovine aortic endothelial cells during various stages of spreading and calculated the force required to detach the cells. Morphometric parameters were obtained from light and scanning electron microscopy measurements. Cells were assumed to have a constant volume, but the surface area increased during spreading until the membrane was stretched taut. Two-dimensional models of steady flow were generated using the software packages ANSYS (mesh generation) and FIDAP (problem solution). The validity of the numerical results was tested by comparison with published results for a semicircle in contact with the surface. The drag force and torque were greatest for round cells making initial contact with the surface. During spreading, the drag force and torque declined by factors of 2 and 20, respectively. The calculated forces and moments were used in adhesion models to predict the wall shear stress at which the cells detached. Based upon published values for the bond force and receptor number, round cells should detach at shear stresses between 2.5 and 6 dyn/cm(2), whereas substantially higher stresses are needed to detach spreading and fully spread cells. Results from the simulations indicate that (1) the drag force varies little with cell shape whereas the torque is very sensitive to cell shape, and (2) the increase in the strength of adhesion during spreading is due to increased contact area and receptor densities within the contact area. (c) 1993 John Wiley & Sons, Inc.     

Chemical force microscopy of cellulosic fibers

Atomic force microscopy with chemically modified cantilever tips (chemical force microscopy) was used to study the pull-off forces (adhesion forces) on cellulose model surfaces and bleached softwood kraft pulp fibers in aqueous media. It was found that for the –COOH terminated tips, the adhesion forces are dependent on pH, whereas for the –CH₃ and –OH terminated tips adhesion is not strongly affected by pH. Comparison between the cellulose model surfaces and cellulosic fibers under our experimental conditions reveal that surface roughness does not affect adhesion strongly. X-ray photoelectron spectroscopy (XPS) and Fourier Transformed Infrared (FTIR) spectroscopy reveal that both substrate surfaces have homogeneous chemical composition. The results show that chemical force microscopy can be used for the chemical characterization of cellulose surfaces at a nano-level.     

Neutrophil adhesive contact dependence on impingement force

Neutrophil capture and recruitment from the circulation requires the formation of specific receptor/ligand bonds under hydrodynamic forces. In the present study we examine bond formation between beta2-integrins on neutrophils and immobilized ICAM-1 while using micropipettes to control the force of contact between the cell and substrate. Magnesium was used to induce the high affinity conformation of the integrins, and bond formation was assessed by measuring the probability of adhesion during repeated contacts. Increasing the impingement force caused an increase in the contact area and led to a proportional increase in adhesion probability (from approximately 20 to 50%) over the range of forces tested (50-350 pN). In addition, different-sized beads were used to change the force per unit area in the contact zone (contact stress). We conclude that for a given contact stress, the rate of bond formation increases linearly with contact area, but that increasing contact stress results in higher intrinsic rates of bond formation.     

Cellular responses to substrate topography: role of myosin II and focal adhesion kinase

Although two-dimensional cultures have been used extensively in cell biological research, most cells in vivo exist in a three-dimensional environment with complex topographical features, which may account for at least part of the striking differences between cells grown in vivo and in vitro. To investigate how substrate topography affects cell shape and movement, we plated fibroblasts on chemically identical polystyrene substrates with either flat surfaces or micron-sized pillars. Compared to cells on flat surfaces, 3T3 cells on pillar substrates showed a more branched shape, an increased linear speed, and a decreased directional stability. These responses may be attributed to stabilization of cell adhesion on pillars coupled to myosin II-dependent contractions toward pillars. Moreover, using FAK-/- fibroblasts we showed that focal adhesion kinase, or FAK, is essential for the responses to substrate topography. We propose that increased surface contact provided by topographic features guides cell migration by regulating the strength of local adhesions and contractions, through a FAK- and myosin II-dependent mechanism.     

The dynamics and mechanics of endothelial cell spreading

Cell adhesion to extracellular matrix is mediated by receptor-ligand interactions. When a cell first contacts a surface, it spreads, exerting traction forces against the surface and forming new bonds as its contact area expands. Here, we examined the changes in shape, actin polymerization, focal adhesion formation, and traction stress generation that accompany spreading of endothelial cells over a period of several hours. Bovine aortic endothelial cells were plated on polyacrylamide gels derivatized with a peptide containing the integrin binding sequence RGD, and changes in shape and traction force generation were measured. Notably, both the rate and extent of spreading increase with the density of substrate ligand. There are two prominent modes of spreading: at higher surface ligand densities cells tend to spread isotropically, whereas at lower densities of ligand the cells tend to spread anisotropically, by extending pseudopodia randomly distributed along the cell membrane. The extension of pseudopodia is followed by periods of growth in the cell body to interconnect these extensions. These cycles occur at very regular intervals and, furthermore, the extent of pseudopodial extension can be diminished by increasing the ligand density. Measurement of the traction forces exerted by the cell reveals that a cell is capable of exerting significant forces before either notable focal adhesion or stress fiber formation. Moreover, the total magnitude of force exerted by the cell is linearly related to the area of the cell during spreading. This study is the first to monitor the dynamic changes in the cell shape, spreading rate, and forces exerted during the early stages (first several hours) of endothelial cell adhesion.     

Substrate-induced deformation and adhesion of phospholipid vesicles at the main phase transition

The physiochemical properties of phospholipid vesicle, e.g. permeability, elasticity, etc., are directly modulated by the chain-melting transition of the lipid bilayer. Currently, there is a lack of understanding in the relationship between thermotropic transition, mechanical deformation and adhesion strength for an adherent vesicle at temperature close to main phase transition temperature T(m). In this study, the contact mechanics of dimyristoyl-phosphatidylcholine (DMPC) vesicle at the main phase transition are probed by confocal reflectance interference contrast microscopy in combination with phase contrast microscopy. It is shown that DMPC vesicles strongly adhere on pure fused silica substrate at T(m) and the degree of deformation as well as the adhesion energy is a decreasing function against the mid-plane diameter of the vesicles. Furthermore, an increase of osmotic pressure at the gel/liquid crystalline phase co-existence imposes insignificant changes in both the degree of deformation and adhesion energy of adherent vesicles when the lipid bilayer permeability is maximized. With the reverse of substrate charge, the mechanical deformation and adhesion strength for larger vesicles (mid-plane diameter >18 microm) are significantly reduced. By monitoring the parametric response of substrate-induced vesicle adhesion during main phase transition, it is shown that the degree of deformation and adhesion energy of adhering vesicle is increased and unchanged, respectively, against the increase of temperature.     

Nanoscale investigation on adhesion of E. coli to surface modified silicone using atomic force microscopy

Bacterial adhesion on biomaterial surfaces is the initial step in establishing infections and leads to the formation of biofilms. In this study, silicone was modified with different biopolymers and silanes, including: heparin, hyaluronan, and self-assembled octadecyltrichlorosilane (OTS), and fluoroalkylsilane (FAS). The aim was to provide a stable and bacteria-resistant surface by varying the degree of hydrophobicity and the surface structure. The adhesion of Escherichia coli (JM 109) on different modified silicone surfaces was investigated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Mica, an ideal hydrophilic and smooth surface, was employed as a control specimen to study the effect of hydrophobicity and surfaces roughness on bacterial adhesion. AFM probes were coated with E. coli and the force measurements between the bacteria-immobilized tip and various materials surfaces were obtained while approaching to and retracting from the surfaces. A short-range repulsive force was observed between the FAS coated silicone and bacteria. The pull-off force of bacteria to FAS was the smallest among coated surfaces. On the other hand, heparin exhibited a long-range attractive force during approach and required a higher pull-off force in retraction. Both AFM and SEM results indicated that FAS reduced bacterial adhesion whereas heparin enhanced the adhesion compared to pure silicone. The work demonstrates that hydrophobicity cannot be used as a criterion to predict bacterial adhesion. Rather, both the native properties of the individual strain of bacteria and the specific functional structure of the surfaces determine the strength of force interaction, and thus the extent of adhesion.     

Micromanipulation of adhesion of phorbol 12-myristate-13-acetate-stimulated T lymphocytes to planar membranes containing intercellular adhesion molecule-1.

This paper presents an analytical and experimental methodology to determine the physical strength of cell adhesion to a planar membrane containing one set of adhesion molecules. In particular, the T lymphocyte adhesion due to the interaction of the lymphocyte function associated molecule 1 on the surface of the cell, with its counter-receptor, intercellular adhesion molecule-1 (ICAM-1), on the planar membrane, was investigated. A micromanipulation method and mathematical analysis of cell deformation were used to determine (a) the area of conjugation between the cell and the substrate and (b) the energy that must be supplied to detach a unit area of the cell membrane from its substrate. T lymphocytes stimulated with phorbol 12-myristate-13-acetate (PMA) conjugated strongly with the planar membrane containing purified ICAM-1. The T lymphocytes attached to the planar membrane deviated occasionally from their round configuration by extending pseudopods but without changing the size of the contact area. These adherent cells were dramatically deformed and then detached when pulled away from the planar membrane by a micropipette. Detachment occurred by a gradual decrease in the radius of the contact area. The physical strength of adhesion between a PMA-stimulated T lymphocyte and a planar membrane containing 1,000 ICAM-1 molecules/micron 2 was comparable to the strength of adhesion between a cytotoxic T cell and its target cell. The comparison of the adhesive energy density, measured at constant cell shape, with the model predictions suggests that the physical strength of cell adhesion may increase significantly when the adhesion bonds in the contact area are immobilized by the actin cytoskeleton.     

Free energy potential for aggregation of giant, neutral lipid bilayer vesicles by Van der Waals attraction.

Here, we report the first direct observation of Van der Waals' attraction between biomembrane capsules using measurements of the free energy reduction per unit area of membrane-membrane contact formation. In these studies, the membrane capsules were reconstituted neutral (egg phosphatidylcholine) lipid bilayers of giant (greater than 10(-3) cm diam) vesicles. Micromanipulation methods were used to select and maneuver two vesicles into proximity for contact; after adhesion was allowed to occur, the extent of contact formation was regulated through the vesicle membrane tensions that were controlled by micropipette suction. The free energy reduction per unit area of contact formation was proportional to the membrane tension multiplied by a simple function of the pipette and vesicle dimensions. The free energy potential for Van der Waals attraction between the neutral bilayers in 120 mM NaCl solutions was 1.5 X 10(-2) ergs/cm2. Also, when human serum albumin was added to the medium in the range of 0-1 mg/ml, the free energy potential for bilayer-bilayer adhesion was not affected. Using published values for equilibrium spacing between lipid bilayers in multilamellar lipid-water dispersions and the theoretical equation for van der Waals attraction between continuous dielectric layers, we calculated the value for the Hamaker coefficient of the Van der Waals attraction to be 5.8 X 10(-14) ergs.     

Shear induced adhesion: contact mechanics of biological spatula-like attachment devices

Most biological hairy adhesive systems of insects, arachnids, and reptiles, involved in locomotion, rely not on flat punches on their tips, but rather on spatulate structures. Several hypotheses have been previously proposed to explain the functional importance of this particular contact geometry: (1) enhancement of adaptability to the rough substrate; (2) contact formation by shear force rather than by normal load; (3) increase in total peeling line due to the use of an array of multiple spatulae; (4) contact breakage by peeling off. In the present paper, we used numerical approach to study dynamics of spatulate tips during contact formation on rough substrates. The model clearly demonstrates that the contact area increases under applied shear force, especially when spatulae are misaligned prior to the contact formation. Applied shear force has an optimum describing the situation when maximal contact is formed but no slip occurs. At such equilibrium, maximal adhesion can be generated. This principle manifests the crucial role of spatulate terminal elements in biological fibrillar adhesion.     

Cell shape and substrate rigidity both regulate cell stiffness

Cells from many different tissues sense the stiffness and spatial patterning of their microenvironment to modulate their shape and cortical stiffness. It is currently unknown how substrate stiffness, cell shape, and cell stiffness modulate or interact with one another. Here, we use microcontact printing and microfabricated arrays of elastomeric posts to independently and simultaneously control cell shape and substrate stiffness. Our experiments show that cell cortical stiffness increases as a function of both substrate stiffness and spread area. For soft substrates, the influence of substrate stiffness on cell cortical stiffness is more prominent than that of cell shape, since increasing adherent area does not lead to cell stiffening. On the other hand, for cells constrained to a small area, cell shape effects are more dominant than substrate stiffness, since increasing substrate stiffness no longer affects cell stiffness. These results suggest that cell size and substrate stiffness can interact in a complex fashion to either enhance or antagonize each other's effect on cell morphology and mechanics.     

Biomechanics of cell rolling: shear flow, cell-surface adhesion, and cell deformability

The mechanics of leukocyte (white blood cell; WBC) deformation and adhesion to endothelial cells (EC) has been investigated using a novel in vitro side-view flow assay. HL-60 cell rolling adhesion to surface-immobilized P-selectin was used to model the WBC-EC adhesion process. Changes in flow shear stress, cell deformability, or substrate ligand strength resulted in significant changes in the characteristic adhesion binding time, cell-surface contact and cell rolling velocity. A 2-D model indicated that cell-substrate contact area under a high wall shear stress (20 dyn/cm2) could be nearly twice of that under a low stress (0.5 dyn/cm2) due to shear flow-induced cell deformation. An increase in contact area resulted in more energy dissipation to both adhesion bonds and viscous cytoplasm, whereas the fluid energy that inputs to a cell decreased due to a flattened cell shape. The model also predicted a plateau of WBC rolling velocity as flow shear stresses further increased. Both experimental and computational studies have described how WBC deformation influences the WBC-EC adhesion process in shear flow.     

Role of lamellar membrane structure in tether formation from bilayer vesicles.

A theoretical analysis is presented of the formation of membrane tethers from micropipette-aspirated phospholipid vesicles. In particular, it is taken into account that the phospholipid membrane is composed of two layers which are in contact but unconnected. The elastic energy of the bilayer is taken to be the sum of contributions from area expansivity, relative expansivity of the two monolayers, and bending. The vesicle is aspirated into a pipette and a constant point force is applied at the opposite side in the direction away from the pipette. The shape of the vesicle in approximated as a cylindrical projection into the pipette with a hemispherical cap, a spherical section, and a cylindrical tether with a hemispherical cap. The dimensions of the different regions of the vesicle are obtained by minimizing its elastic energy subject to the condition that the volume of the vesicle is fixed. The range of values for the parameters of the system is determined at which the existence of a tether is possible. Stability analysis is performed showing which of these configurations are stable. The importance of the relative expansion and compression of the constituent monolayers is established by recognizing that local bending energy by itself does not stabilize the vesicle geometry, and that in the limit as the relative expansivity modulus becomes infinitely large, a tether cannot be formed. Predictions are made for the functional relationships among experimentally observable quantities. In a companion report, the results of this analysis are applied to experimental measurements of tether formation, and used to calculate values for the membrane material coefficients.     

Fabrication of a dual substrate display to test roles of cell adhesion proteins in vesicle targeting to plasma membrane domains

While much is known of the molecular machinery involved in protein sorting during exocytosis, less is known about the spatial regulation of exocytosis at the plasma membrane (PM). This study outlines a novel method, dual substrate display, used to formally test the hypothesis that E-cadherin-mediated adhesion directs basolateral vesicle exocytosis to specific sites at the PM. We show that vesicles containing the basolateral marker protein VSV-G preferentially target to sites of adhesion to E-cadherin rather than collagen VI or a control peptide. These results support the hypothesis that E-cadherin adhesion initiates signaling at the PM resulting in targeted sites for exocytosis.