Detachment of agglutinin-bonded red blood cells. II. Mechanical energies to separate large contact areas.

As detailed in a companion paper (Berk, D., and E. Evans. 1991. Biophys. J. 59:861-872), a method was developed to quantitate the strength of adhesion between agglutinin-bonded membranes without ambiguity due to mechanical compliance of the cell body. The experimental method and analysis were formulated around controlled assembly and detachment of a pair of macroscopically smooth red blood cell surfaces. The approach provides precise measurement of the membrane tension applied at the perimeter of an adhesive contact and the contact angle theta c between membrane surfaces which defines the mechanical leverage factor (1-cos theta c) important in the definition of the work to separate a unit area of contact. Here, the method was applied to adhesion and detachment of red cells bound together by different monoclonal antibodies to red cell membrane glycophorin and the snail-helix pomatia-lectin. For these tests, one of the two red cells was chemically prefixed in the form of a smooth sphere then equilibrated with the agglutinin before the adhesion-detachment procedure. The other cell was not exposed to the agglutinin until it was forced into contact with the rigid cell surface by mechanical impingement. Large regions of agglutinin bonding were produced by impingement but no spontaneous spreading was observed beyond the forced contact. Measurements of suction force to detach the deformable cell yielded consistent behavior for all of the agglutinins: i.e., the strength of adhesion increased progressively with reduction in contact diameter throughout detachment. This tension-contact diameter behavior was not altered over a ten-fold range of separation rates. In special cases, contacts separated smoothly after critical tensions were reached; these were the highest values attained for tension. Based on measurements reported in another paper (Evans et al. 1991. Biophys. J. 59:838-848) of the forces required to rupture molecular-point attachments, the density of cross-bridges was estimated with the assumption that the tension was proportional to the discrete rupture force x the number of attachments per unit length. These estimates showed that only a small fraction of agglutinin formed cross-bridges at initial assembly and increased progressively with separation. When critical tension levels were reached, it appeared that nearly all local agglutinin was involved as cross-bridges. Because one cell surface was chemically fixed, receptor accumulation was unlikely; thus, microscopic "roughness" and steric repulsion probably modulated formation of cross-bridges on initial contact.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lipid-glass adhesion in giga-sealed patch-clamped membranes.

Adhesion between patch-clamped lipid membranes and glass micropipettes is measured by high contrast video imaging of the mechanical response to the application of suction pressure across the patch. The free patch of membrane reversibly alters both its contact angle and radius of curvature on pressure changes. The assumption that an adhesive force between the membrane and the pipette can sustain normal tension up to a maximum Ta at the edge of the free patch accounts for the observed mechanical responses. When the normal component of the pressure-induced membrane tension exceeds Ta membrane at the contact point between the free patch and the lipid-glass interface is pulled away from the pipette wall, resulting in a decreased radius of curvature for the patch and an increased contact angle. Measurements of the membrane radius of curvature as a function of the suction pressure and pipette radius determine line adhesion tensions Ta which range from 0.5 to 4.0 dyn/cm. Similar behavior of patch-clamped cell membranes implies similar adhesion mechanics.     

Determination of bilayer membrane bending stiffness by tether formation from giant, thin-walled vesicles.

The curvature elastic modulus (bending stiffness) of stearoyloleoyl phosphatidylcholine (SOPC) bilayer membrane is determined from membrane tether formation experiments. R. E. Waugh and R. M. Hochmuth 1987. Biophys. J. 52:391-400) have shown that the radius of a bilayer cylinder (tether) is inversely related to the force supported along its axis. The coefficient that relates the axial force on the tether to the tether radius is the membrane bending stiffness. Thus, the bending stiffness can be calculated directly from measurements of the tether radius as a function of force. Giant (10-50-microns diam) thin-walled vesicles were aspirated into a micropipette and a tether was pulled out of the surface by gravitational forces on small glass beads that had adhered to the vesicle surface. Because the vesicle keeps constant surface area and volume, formation of the tether requires displacement of material from the projection of the vesicle in the pipette. Tethers can be made to grow longer or shorter or to maintain equilibrium by adjusting the aspiration pressure in the micropipette at constant tether force. The ratio of the change in the length of the tether to the change in the projection length is proportional to the ratio of the pipette radius to the tether radius. Thus, knowing the density and diameter of the glass beads and measuring the displacement of the projection as a function of tether length, independent determinations of the force on the tether and the tether radius were obtained. The bending stiffness for an SOPC bilayer obtained from these data is approximately 2.0 x 10(-12) dyn cm, for tether radii in the range of 20-100 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. I. Membrane separation from the cytoskeleton

We have used a biomembrane force probe decorated with P-selectin to form point attachments with PSGL-1 receptors on a human neutrophil (PMN) in a calcium-containing medium and then to quantify the forces experienced by the attachment during retraction of the PMN at fixed speed. From first touch to final detachment, the typical force history exhibited the following sequence of events: i), an initial linear-elastic displacement of the PMN surface, ii), an abrupt crossover to viscoplastic flow that signaled membrane separation from the interior cytoskeleton and the beginning of a membrane tether, and iii), the final detachment from the probe tip by usually one precipitous step of P-selectin:PSGL-1 dissociation. In this first article I, we focus on the initial elastic response and its termination by membrane separation from the cytoskeleton, initiating tether formation. Quantifying membrane unbinding forces for rates of loading (force/time) in the elastic regime from 240 pN/s to 38,000 pN/s, we discovered that the force distributions agreed well with the theory for kinetically limited failure of a weak bond. The kinetic rate for membrane unbinding was found to increase as an exponential function of the pulling force, characterized by an e-fold scale in force of approximately 17 pN and a preexponential factor, or apparent unstressed off rate, of approximately 1/s. The rheological properties of tether growth subsequent to the membrane unbinding events are presented in a companion article II.     

Force measurements on single molecular contacts through evanescent wave microscopy

We introduce a new method to apply controlled forces on single molecules. The motion of a micron-sized bead attached to a solid surface through a single molecular contact is tracked by evanescent wave microscopy as a force is exerted through a flow. We report measurements of the streptavidin-biotin bond rupture force obtained with this technique. We also obtain detailed measurements of the balance of forces involved in detaching an adhering bead with a flow. A small lateral force translates into a much bigger normal force on the attachment point. This effect is relevant for the interpretation of common cell adhesion assays.     

Theoretical and experimental studies on viscoelastic properties of erythrocyte membrane.

The deformation of a portion of erythrocyte during aspirational entry into a micropipette has been analyzed on the basis of a constant area deformation of an infinite plane membrane into a cylindrical tube. Consideration of the equilibrium of the membrane at the tip of the pipette has generated the relation between the aspirated length and the dimensionless time during deformational entry as well as during relaxation after the removal of aspiration pressure. Experimental studies on deformation and relaxation of normal human erythrocytes were performed with the use of micropipettes and a video dimension analyzer which allowed the continuous recording of the time-courses. The deformation consisted of an initial rapid phase with a membrane viscosity (range 0.6 x 10(-4) to 4 x 10(-4) dyn.s/cm) varying inversely with the degree of deformation and a later slow phase with a high membrane viscosity (mean 2.06 x 10(-2) dyn.s/cm) which was not correlated with the degree of deformation. The membrane viscosity of the recovery phase after 20 s of deformation (mean 5.44 x 10(-4) dyn.s/cm) was also independent of the degree of deformation. When determined after a short period of deformation (e.g., 2 s), however, membrane viscosity of the recovery phase became lower and agreed with that of the deformation phase. These results suggest that the rheological properties of the membrane can undergo dynamic changes depending on the extent and duration of deformation, reflecting molecular rearrangement in response to membrane strain.     

Alterations of the apparent area expansivity modulus of red blood cell membrane by electric fields.

Red blood cell membrane exhibits a large resistance to changes in surface area. This resistance is characterized by the area expansivity modulus K, which relates the isotropic membrane force resultant, T, to the fractional change in membrane surface area delta A/Ao. The experimental technique commonly used to determine K is micropipette aspiration. Using this method, E. A. Evans and R. Waugh (1977, Biophys. J. 20:307-313) obtained a value of 450 dyn/cm for the modulus. In the present report, it is shown that the value of K, as determined using this method, is affected by electric potential differences applied across the tip of the pipette. Using Ag-AgCl electrodes and current clamping electronics, we obtained values for K ranging from 150 dyn/cm with -1.0 V applied, to 1,500 dyn/cm with 1.0 V applied. At 0.0 V the modulus obtained was approximately 500 dyn/cm. A reversible, voltage- and pressure-dependent change in the cell volume probably accounts for the effect of the voltage on the calculated value of the modulus. The use of lanthanum chloride or increasing the extra- and intracellular solute concentrations reduced the voltage dependence of the measurements. It was also found that when dissimilar metals were used to "ground" the pipette to the chamber to prevent lysis of cells by static charge, values for K ranged from 121 to 608 dyn/cm. Based on measurements made at zero applied volts, in the presence of 0.4 mM lanthanum and at high solute concentration, we conclude that the true value of the modulus is approximately 500 dyn/cm.     

Micropipette suction for measuring piconewton forces of adhesion and tether formation from neutrophil membranes.

A new method for measuring piconewton-scale forces that employs micropipette suction is presented here. Spherical cells or beads are used directly as force transducers, and forces as small as 10-20 pN can be imposed. When the transducer is stationary in the pipette, the force is simply the product of the suction pressure and the cross-sectional area of the pipette minus a small correction for the narrow gap that exists between the transducer and the pipette wall. When the transducer is moving along the pipette, the force on it is corrected by a factor that is proportional to the ratio of its velocity relative to its drag-free velocity. With this technique, the minimum force required to form a membrane tether from neutrophils is determined (45 pN), and the length of the microvilli on the surface of neutrophils is inferred. The strength of this technique is in its simplicity and its ability to measure forces between cells without requiring a separate theory or a calibration against an external standard and without requiring the use of a solid surface.     

Molecular basis of fibrin clot elasticity

Blood clots must be stiff to stop hemorrhage yet elastic to buffer blood's shear forces. Upsetting this balance results in clot rupture and life-threatening thromboembolism. Fibrin, the main component of a blood clot, is formed from molecules of fibrinogen activated by thrombin. Although it is well known that fibrin possesses considerable elasticity, the molecular basis of this elasticity is unknown. Here, we use atomic force microscopy (AFM) and steered molecular dynamics (SMD) to probe the mechanical properties of single fibrinogen molecules and fibrin protofibrils, showing that the mechanical unfolding of their coiled-coil alpha helices is characterized by a distinctive intermediate force plateau in the systems' force-extension curve. We relate this plateau force to a stepwise unfolding of fibrinogen's coiled alpha helices and of its central domain. AFM data show that varying pH and calcium ion concentrations alters the mechanical resilience of fibrinogen. This study provides direct evidence for the coiled alpha helices of fibrinogen to bring about fibrin elasticity.     

Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. II. Tether flow terminated by P-selectin dissociation from PSGL-1

We have used a biomembrane force probe decorated with P-selectin to form point attachments with PSGL-1 receptors on a human neutrophil (PMN) in a calcium-containing medium and then to quantify the forces experienced by the attachment during retraction of the PMN at fixed speed. From first touch to final detachment, the typical force history exhibited the following sequence of events: i), an initial linear-elastic displacement of the PMN surface, ii), an abrupt crossover to viscoplastic flow that signaled membrane separation from the interior cytoskeleton and the beginning of a membrane tether, and iii), the final detachment from the probe tip most often by one precipitous step of P-selectin:PSGL-1 dissociation. Analyzing the initial elastic response and membrane unbinding from the cytoskeleton in our companion article I, we focus in this article on the regime of tether extrusion that nearly always occurred before release of the extracellular adhesion bond at pulling speeds > or =1 microm/s. The force during tether growth appeared to approach a plateau at long times. Examined over a large range of pulling speeds up to 150 microm/s, the plateau force exhibited a significant shear thinning as indicated by a weak power-law dependence on pulling speed, f(infinity) = 60 pN(nu(pull)/microm/s)(0.25). Using this shear-thinning response to describe the viscous element in a nonlinear Maxwell-like fluid model, we show that a weak serial-elastic component with a stiffness of approximately 0.07 pN/nm provides good agreement with the time course of the tether force approach to the plateau under constant pulling speed.     

Extraction of membrane proteins from a living cell surface using the atomic force microscope and covalent crosslinkers

The force curve mode of the atomic force microscope (AFM) was applied to extract intrinsic membrane proteins from the surface of live cells using AFM tips modified by amino reactive bifunctional covalent crosslinkers. The modified AFM tips were individually brought into brief contact with the living cell surface to form covalent bonds with cell surface molecules. The force curves recorded during the detachment process from the cell surface were often characterized by an extension of a few hundred nanometers followed mostly by a single step jump to the zero force level. Collection and analysis of the final rupture force revealed that the most frequent force values (of the force) were in the range of 0.4–0.6 nN. The observed rupture force most likely represented extraction events of intrinsic membrane proteins from the cell membrane because the rupture force of a covalent crosslinking system was expected to be significantly larger than 1.0 nN, and the separation force of noncovalent ligand-receptor pairs to be less than 0.2 nN, under similar experimental conditions. The transfer of cell surface proteins to the AFM tip was verified by recording characteristic force curves of protein stretching between the AFM tips used on the cell surface and a silicon surface modified with amino reactive bifunctional crosslinkers. This method will be a useful addition to bionanotechnological research for the application of AFM.     

Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces.

Adhesion and cytoskeletal structure are intimately related in biological cell function. Even with the vast amount of biological and biochemical data that exist, little is known at the molecular level about physical mechanisms involved in attachments between cells or about consequences of adhesion on the material structure. To expose physical actions at soft biological interfaces, we have combined an ultrasensitive transducer and reflection interference microscopy to image submicroscopic displacements of probe contact with a test surface under minuscule forces. The transducer is a cell-size membrane capsule pressurized by micropipette suction where displacement normal to the membrane under tension is proportional to the applied force. Pressure control of the tension tunes the sensitivity in operation over four orders of magnitude through a range of force from 0.01 pN up to the strength of covalent bonds (approximately 1000 pN)! As the surface probe, a microscopic bead is biochemically glued to the transducer with a densely-bound ligand that is indifferent to the test surface. Movements of the probe under applied force are resolved down to an accuracy of approximately 5 nm from the interference fringe pattern created by light reflected from the bead. With this arrangement, we show that local mechanical compliance of a cell surface can be measured at a displacement resolution set by structural fluctuations. When desired, a second ligand is bound sparsely to the probe for focal adhesion to specific receptors in the test surface. We demonstrate that monitoring fluctuations in probe position at low transducer stiffness enhances detection of molecular adhesion and activation of cytoskeletal structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Revealing early steps of alpha2beta1 integrin-mediated adhesion to collagen type I by using single-cell force spectroscopy

We have characterized early steps of alpha(2)beta(1) integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of alpha(2)beta(1) as a collagen type I receptor, alpha(2)beta(1)-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas alpha(2)beta(1)-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin-collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin-collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of alpha(2)beta(1)-mediated adhesion as weak initial, single-integrin-mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility.     

Locomotion forces generated by a polymorphonuclear leukocyte.

There have been very few studies which have measured the physical forces generated by cells during active movements. A special micropipette system has been designed to make it possible to observe cell motion within the pipette and to apply a pressure to counter the chemotactic migration of the cell. This provides a direct measure of the locomotion force generated by the cell. The average velocity of forward motion is 0.33 microns/s in the absence of counter-pressure. The application of a positive counter-pressure (C-P) causes a decrease in the velocity of the forward motion of the cell. At 17 cm H2O of C-P, the cell velocity drops to zero and even moves backward with a higher C-P. The results show that the decrement of velocity is linearly related to the magnitude of the C-P with a complete stoppage at a pressure of 17 cm H2O which corresponds to a force of 0.003 dyn. The maximum work rate of the cell is approximately 2.5 x 10(-8) erg/s.     

Multi-bead-and-spring model to interpret protein detachment studied by AFM force spectroscopy

This article deals with the detachment of molecules (fibrinogen) from a surface studied experimentally with an atomic force microscope. The detachment (or rupture) forces are measured as a function of the retraction velocity and exhibit a clear dependence on this parameter, even though the interaction between the molecules and the surface are nonspecific. To interpret these data, a mechanical multi-bead-and-spring model is developed. It consists of one to several parallel, "molecular" springs connected to an extra spring representing the cantilever that is moved at constant velocity. The free end of each molecular spring terminates with a particle that interacts with the surface through a Lennard-Jones potential. This Brownian dynamics model is used to analyze the experimental findings. In the framework of this model, it appears that the fibrinogen molecule must be ascribed a stiffness much smaller than that of the cantilever. In addition, several bonds between the molecule and the surface must be taken into account for the range of the molecule-surface interaction not to be unrealistically small. In future work, this model will be extended to more complex mechanisms such as the detachment of cells from a surface.     

Extensional flow of erythrocyte membrane from cell body to elastic tether. II. Experiment.

This is the second of two papers on an analytical and experimental study of the flow of erythrocyte membrane. In the experiments discussed here, preswollen human erythrocytes are sphered by aspirating a portion of the cell membrane into a small micropipette; and long, thin, membrane filaments or tethers are steadily withdrawn from the cell at a point diametrically opposite to the point of aspiration. The aspirated portion of the membrane furnishes a reservoir of material that replaces the membrane as it flows as a liquid from the nearly spherical cell body to the cylindrical tether. The application of the principle of conservation of mass permits the tether radius Rt to be measured with the light microscope as the tether is formed and extended at a constant rate. The tether behaves as an elastic solid such that the tether radius decreases as the force or axial tension acting on the tether is increased. For the range of values for Rt is these experiments (100 A less than or equal to Rt less than or equal to 200 A), the slope of the tether-force, tether-radius line is -1.32 dyn/cm. The surface viscosity of the membrane as it flows from cell body to tether is 3 x 10(-3) dyn.s/cm. This viscosity is essentially constant for characteristic rates of deformation between 10 and 200 s-1.     

Red blood cells experience electrostatic repulsion but make molecular adhesions with glass.

We have studied the detachment of unfixed red cells from glass coverslips under unit gravity and by centrifugation in buffered isotonic solutions over a range of ionic strengths. Cell-glass contact areas and separation distances were measured by quantitative interference reflection microscopy. Detachment under unit gravity is highly dependent on ionic strength: dilution increases electrostatic repulsion and greatly reduces the proportion of adherent cells. However, even at 1.5 mM some cells stick. Over the range 3-110 mM such adherent cells are progressively removed by increasing centrifugal forces, but in a manner virtually independent of ionic strength. This fact, together with the irreversibility of pre-adherent cells as ionic strength is progressively reduced, as well as the resistance of cells to lateral shearing forces, provide evidence sufficient to reject the notion of secondary minimum adhesion for unfixed cells at any ionic strength down to 1.5 mM. We conclude that all unfixed cells that stick at ionic strengths from 157 to 1.5 mM make molecular contacts with glass. Comparison with long range force calculations suggests that to penetrate the electrostatic repulsion barrier the contact regions are unlikely to have average surface properties. A new method that compares frequency distributions of contact areas with responses to detachment forces shows that detachment forces are not linearly related to contact areas. This lack of relationship is less clearly evident for rigid glutaraldehyde-fixed cells and may therefore depend on the degree of cellular deformability.     

Flow-induced detachment of red blood cells adhering to surfaces by specific antigen-antibody bonds.

Fixed spherical swollen human red blood cells of blood type B adhering on a glass surface through antigen-antibody bonds to monoclonal mouse antihuman IgM, adsorbed or covalently linked on the surface, were detached by known hydrodynamic forces created in an impinging jet. The dynamic process of detachment of the specifically bound cells was recorded and analyzed. The fraction of adherent cells remaining on the surface decreased with increasing hydrodynamic force. For an IgM coverage of 0.26%, a tangential force on the order of 100 pN was able to detach almost all of the cells from the surface within 20 min. After a given time of exposure to hydrodynamic force, the fraction of adherent cells remaining increased with time, reflecting an increase in adhesion strength. The characteristic time for effective aging was approximately 4 h. Results from experiments in which the adsorbed antibody molecules were immobilized through covalent coupling and from evanescent wave light scattering of adherent cells, imply that deformation of red cells at the contact area was the principal cause for aging, rather than local clustering of the antibody through surface diffusion. Experiments with latex beads specifically bound to red blood cells suggest that, instead of breaking the antigen-antibody bonds, antigen molecules were extracted from the cell membrane during detachment.     

What is the biological relevance of the specific bond properties revealed by single-molecule studies?

During the last decade, many authors took advantage of new methodologies based on atomic force microscopy (AFM), biomembrane force probes (BFPs), laminar flow chambers or optical traps to study at the single-molecule level the formation and dissociation of bonds between receptors and ligands attached to surfaces. Experiments provided a wealth of data revealing the complexity of bond response to mechanical forces and the dependence of bond rupture on bond history. These results supported the existence of multiple binding states and/or reaction pathways. Also, single bond studies allowed us to monitor attachments mediated by a few bonds. The aim of this review is to discuss the impact of this new information on our understanding of biological molecules and phenomena. The following points are discussed: (i) which parameters do we need to know in order to predict the behaviour of an encounter between receptors and ligands, (ii) which information is actually yielded by single-molecule studies and (iii) is it possible to relate this information to molecular structure?