Polymer-cushioned bilayers. II. An investigation of interaction forces and fusion using the surface forces apparatus.

We have created phospholipid bilayers supported on soft polymer "cushions" which act as deformable substrates (see accompanying paper, Wong, J. Y., J. Majewski, M. Seitz, C. K. Park, J. N. Israelachvili, and G. S. Smith. 1999. Biophys. J. 77:1445-1457). In contrast to "solid-supported" membranes, such "soft-supported" membranes can exhibit more natural (higher) fluidity. Our bilayer system was constructed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles onto polyethylenimine (PEI)-supported Langmuir-Blodgett lipid monolayers on mica. We used the surface forces apparatus (SFA) to investigate the long-range forces, adhesion, and fusion of two DMPC bilayers both above and below their main transition temperature (T(m) approximately 24 degrees C). Above T(m), hemi-fusion activation pressures of apposing bilayers were considerably smaller than for solid-supported bilayers, e.g., directly supported on mica. After separation, the bilayers naturally re-formed after short healing times. Also, for the first time, complete fusion of two fluid (liquid crystalline) phospholipid bilayers was observed in the SFA. Below T(m) (gel state), very high pressures were needed for hemi-fusion and the healing process became very slow. The presence of the polymer cushion significantly alters the interaction potential, e.g., long-range forces as well as fusion pressures, when compared to solid-supported systems. These fluid model membranes should allow the future study of integral membrane proteins under more physiological conditions.     

Hip joint forces in sheep.

Testing orthopaedic implants at the proximal femur of sheep requires knowledge of the contact forces acting on this joint. Telemeterized implants were used for long-term measurements of these forces in four sheep, mostly during treadmill walking. Joint forces in the same sheep varied widely from day to day and interindividual differences were also pronounced. Forces during walking were mostly higher than in previous short-term measurements. At medium walking speed, loads in the range of 65-140% of the body weight were typical. Fast walking increased the forces by only 20%, compared to slow speed. Stomping on the ground at the beginning of the stance phase and starting to run freely led to very high forces. The highest values observed were nearly four times the body weight. As in humans, the directions of high forces varied only slightly in the frontal plane throughout the whole stance phase but much more in the transverse plane. With regard to the force magnitudes and their directions, sheep seem to be a good model for testing human implant at the proximal femur.     

The attractive forces between polar lipid bilayers.

Long-range attractive forces between lipid bilayers are not well described by the Lifshitz theory of Van der Waals forces between macroscopic media. It is shown that when correlations between polar headgroups are taken into account, the predicted forces take a qualitatively different form consistent with the measured data.     

Molecular forces in ribosomes and polynucleotide complexes.

The stability constant of the complex of tRNA with 50S subunits of ribosomes was compared in ordinary and heavy water. A considerable effect (about fourfold) was observed, showing the importance of hydrogen bonds in this interaction. In addition, the isotope effect of complementary polynucleotide interaction was measured for two examples. In the case of the binary complex of heptainosinic acid oligomers with poly(C) in the presence of 10^?3 M MgCl₂, the transfer from ordinary to heavy water gave an increase of the stability constant of about 5%. But in the case of a ternary complex of hexaadenylic acid with poly(U) under the same conditions, the stability constant in D₂O increased threefold. The isotope effect depends strongly on magnesium ion concentration and is possibly due to some specific mechanism of magnesium ion complexing involving water molecules.     

Repulsive forces in lecithin glycol lamellar phases.

The repulsive pressure vs. distance for phospholipid bilayers in glycol has been determined from vapor pressure measurements. The magnitude of this pressure is similar to the case when water is present between the lipid bilayers. Hence, an interaction directly corresponding to the previously reported hydration force is shown also for nonaqueous lecithin/solvent systems.     

Direct measurement of the intermolecular forces between counterion-condensed DNA double helices. Evidence for long range attractive hydration forces.

Rather than acting by modifying van der Waals or electrostatic double layer interactions or by directly bridging neighboring molecules, polyvalent ligands bound to DNA double helices appear to act by reconfiguring the water between macromolecular surfaces to create attractive long range hydration forces. We have reached this conclusion by directly measuring the repulsive forces between parallel B-form DNA double helices pushed together from the separations at which they have self organized into hexagonal arrays of parallel rods. For all of the wide variety of "condensing agents" from divalent Mn to polymeric protamines, the resulting intermolecular force varies exponentially with a decay rate of 1.4-1.5 A, exactly one-half that seen previously for hydration repulsion. Such behavior qualitatively contradicts the predictions of all electrostatic double layer and van der Waals force potentials previously suggested. It fits remarkably well with the idea, developed and tested here, that multivalent counterion adsorption reorganizes the water at discrete sites complementary to unadsorbed sites on the apposing surface. The measured strength and range of these attractive forces together with their apparent specificity suggest the presence of a previously unexpected force in molecular organization.     

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.     

Geometrical factors influencing muscle force development. II. Radial forces.

If the subfragment-2 (S2) portion of the myosin cross-bridge to actin does not lie parallel to the myofilament axes then when a muscle fiber contracts, there will be a radial component to the cross-bridge force. When the subfragment-1 (S1) portion of the cross-bridge attaches to actin with its long axis projecting through the filament axis, the magnitude of the radial force depends upon the azimuthal location of the actin site, but when the attachment of the S1 to actin is slewed, as in the reconstruction of Moore et al. (J. Mol. Biol., 1970, 50:279-294), then for a single cross-bridge the radial component of the cross-bridge force is not quite so sensitive to actin site location and is approximately 0.1 the axial component. In both cases, the ratio of the radial to axial force decreases with decreasing filament separation. If the radial-axial force ratio for each cross-bridge is approximately 0.1, then at full overlap in a frog skeletal muscle fiber the radial component of the cross-bridge force accompanying full activation will exert a compressive pressure of approximately 5 X 10(-3) atm. This would have little effect upon an intact muscle fiber where the volume constraints are likely osmotic, but it might produce a 1-2% change in filament spacing in a "skinned" muscle fiber from which the sarcolemma had been removed. These computations assume that the S2 link between the S1 head and the myosin filament does not support a bending moment of shear. If it does, then the radial component of the cross-bridge will be either greater or less, depending on the specific cross-bridge geometry.     

Variability of ground reaction forces during treadmill walking.

The purpose of this study was to investigate whether or not the neuromuscular locomotor system is optimized at a unique speed by examining the variability of the ground reaction force (GRF) pattern during walking in relation to different constant speeds. Ten healthy male subjects were required to walk on a treadmill at 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 km/h. Three components [vertical (F(z)), anteroposterior (F(y)), and mediolateral (F(x)) force] of the GRF were independently measured for approximately 35 steps consecutively for each leg. To quantify the GRF pattern, five indexes (first and second peaks of F(z), first and second peaks of F(y), and F(x) peak) were defined. Coefficients of variation were calculated for these five indexes to evaluate the GRF variability for each walking speed. It became clear for first and second peaks of F(z) and F(x) peak that index variabilities increased in relation to increments in walking speed, whereas there was a speed (5.5-5.8 km/h) at which variability was minimum for first and second peaks of F(y), which were related to forward propulsion of the body. These results suggest that there is "an optimum speed" for the neuromuscular locomotor system but only for the propulsion control mechanism.     

Interaction forces between red cells agglutinated by antibody. III. Micromanipulation.

In the flow studies described in two previous papers (Tha, S. P., and H. L. Goldsmith, 1986, Biophys. J. 50:1109-1116; Tha, S. P., J. Shuster, and H. L. Goldsmith, 1986, Biophys. J. 50:1117-1126), hydrodynamic forces of the order of 10(-11) N (mu dyn) were applied to measure the force of separation of doublets of hardened, sphered human red blood cells cross-linked by anti-B antibody. The same cell preparation and hyperimmune antiserum has here been used to carry out experiments with micropipet aspiration techniques. One cell of a doublet was aspirated onto a holding pipet, and a second aspiration pipet was brought into proximity of the other cell so that the two pipets and the doublet were colinear. Suction was then raised until the two cells separated. Some doublets were assembled by aspiration of a singlet, bringing a second singlet into apposition with the first, and releasing it from the pipet which was then withdrawn. Cells could be repeatedly assembled and separated. At 3.56% vol/vol antiserum, the mean normal force of separation was 0.45 +/- 0.11 nN in phosphate-buffered saline suspensions containing 2.5 x 10(4) cells/microliter; at 1.22% vol/vol antiserum, the value was 0.22 +/- 0.11 nN. The above values of the force were approximately 2.5 x greater than those from the flow studies. The data could be fitted to a Poisson distribution with 0.05 nN as the force needed to break a single cross-bridge (c.f. 0.024 nN from the previous hydrodynamic data). The forces of separation of randomly assembled doublets were lower than those of preexisting doublets. Repeated assembly and separation of doublets showed that the cell surfaces are nonuniform in adhesion strength both over the local scale less than 0.25 micron2 and the cell population.     

Measurement and modification of forces between lecithin bilayers.

We probe in two different ways the competing attractive and repulsive forces that create lamellar arrays of the phospholipid lecithin when in equilibrium with pure water. The first probe involves the addition of low molecular weight solutes, glucose and sucrose, to a system where the phospholipid is immersed in a large excess of water. Small solutes can enter the aqueous region between bilayers. Their effect is first to increase and then to decrease the separation between bilayers as sugar concentration increases. We interpret this waxing and waning of the lattice spacing in terms of the successive weakening and strengthening of the attractive van der Waals forces originally responsible for creation of a stable lattice. The second probe is an "osmotic stress method," in which very high molecular weight neutral polymer is added to the pure water phase but is unable to enter the multilayers. The polymer competes for water with the lamellar lattice, and thereby compresses it. From the resulting spacing (determined by X-ray diffraction) and the directly measured osmotic pressure, we find a force vs. distance curve for compressing the lattice (or, equivalently, the free energy of transfer to bulk water of water between bilayers. This method reveals a very strong, exponentially varying "hydration force" with a decay distance of about 2 A.     

Mapping interaction forces with the atomic force microscope.

Force curves were recorded as the sample was raster-scanned under the tip. This opens new opportunities for imaging with the atomic force microscope: several characteristics of the samples can be measured simultaneously, for example, topography, adhesion forces, elasticity, van der Waals, and electrostatic interactions. The new opportunities are illustrated by images of several characteristics of thin metal films, aggregates of lysozyme, and single molecules of DNA.