Deformation and flow of membrane into tethers extracted from neuronal growth cones.

Membrane tethers are extracted at constant velocity from neuronal growth cones using a force generated by a laser tweezers trap. A thermodynamic analysis shows that as the tether is extended, energy is stored in the tether as bending and adhesion energies and in the cell body as "nonlocal" bending. It is postulated that energy is dissipated by three viscous mechanisms including membrane flow, slip between the two monolayers that form the bilayer, and slip between membrane and cytoskeleton. The analysis predicts and the experiments show a linear relation between tether force and tether velocity. Calculations based on the analytical results and the experimental measurements of a tether radius of approximately 0.2 micron and a tether force at zero velocity of approximately 8 pN give a bending modulus for the tether of 2.7 x 10(-19) N.m and an extraordinarily small "apparent surface tension" in the growth cone of 0.003 mN/m, where the apparent surface tension is the sum of the far-field, in-plane tension and the energy of adhesion. Treatments with cytochalasin B and D, ethanol, and nocodazole affect the apparent surface tension but not bending. ATP depletion affects neither, whereas large concentrations of DMSO affect both. Under conditions of flow, data are presented to show that the dominant viscous mechanism comes from the slip that occurs when the membrane flows over the cytoskeleton. ATP depletion and the treatment with DMSO cause a dramatic drop in the effective viscosity. If it is postulated that the slip between membrane and cytoskeleton occurs in a film of water, then this water film has a mean thickness of only approximately 10 A.     

The production and release of an extracellular polysaccharide during starvation of a marine Pseudomonas sp. and the effect thereof on adhesion

A marine Pseudomonas sp. S9 produced and released an extracellular polysaccharide during complete energy and nutrient starvation in static conditions. The presence of the polysaccharide on the cell surface, demonstrable by immune transmission electron microscopy, correlated with changes in the degree of adhesion to hydrophobic surfaces. Polysaccharide coated cells showed a lower degree of adhesion than did cells devoid of the polymer. After 10 h of starvation, no ruthenium red stained antibody stabilized polysaccharides could be observed on the cell surface. The polysaccharide was not produced during growth since lysates of mid-log phase cells did not precipitate the antiserum. The relative proportions of sugars in the polysaccharide were 28% glucose, 35% N-acetylglucosamine and 37% N-acetylgalactosamine. The released polysaccharide did not significantly alter the physical parameters of surface tension and viscosity of the starvation regime. Cells starved in agitated conditions did not produce any extracellular polysaccharides and exhibited a different adhesion pattern to hydrophobic surfaces.Non-standard abbreviations FSS Four salt solution - GLC gas liquid chromatography - MS Mass spectrometry - NSS nine salt solution     

Equilibrium mechanics of monolayered epithelium

In order to fully understand the epithelial mechanics it is essential to integrate different levels of epithelial organization. In this work, we propose a theoretical approach for connecting the macroscopic mechanical properties of a monolayered epithelium to the mechanical properties at the cellular level. The analysis is based on the established mechanical models—at the macroscopic scale the epithelium is described within the mechanics of thin layers, while the cellular level is modeled in terms of the cellular surface (cortical) tension and the intercellular adhesion. The macroscopic elastic energy of the epithelium is linked to the energy of an average epithelial cell. The epithelial equilibrium state is determined by energy minimization and the macroscopic elastic moduli are calculated from deformations around the equilibrium. The results indicate that the epithelial equilibrium state is defined by the ratio between the adhesion strength and the cellular surface tension. The lower and the upper bounds for this ratio are estimated. If the ratio is small, the epithelium is cuboidal, if it is large, the epithelium becomes columnar. Importantly, it is found that the cellular cortical tension and the intercellular adhesion alone cannot produce the flattened squamous epithelium. Any difference in the surface tension between the apical and basal cellular sides bends the epithelium towards the side with the larger surface tension. Interestingly, the analysis shows that the epithelial area expansivity modulus and the shear modulus depend only on the cellular surface tension and not on the intercellular adhesion. The results are presented in a general analytical form, and are thus applicable to a variety of monolayered epithelia, without relying on the specifics of numerical finite-element methods. In addition, by using the standard theoretical tools for multi-laminar systems, the results can be applied to epithelia consisting of layers with different mechanical properties.     

Minimum energy analysis of membrane deformation applied to pipet aspiration and surface adhesion of red blood cells.

An experimental procedure is demonstrated which can be used to determine the interfacial free energy density for red cell membrane adhesion and membrane elastic properties. The experiment involves micropipet aspiration of a flaccid red blood cell and manipulation of the cell proximal to a surface where adhesion occurs. A minimum free energy method is developed to model the equilibrium contour of unsupported membrane regions and to evaluate the partial derivatives of the total free energy, which correspond to the micropipet suction force and the interfacial free energy density of adhesion. It is shown that the bending elasticity of the red cell membrane does not contribute significantly to the pressure required to aspirate a flaccid red cell. Based on experimental evidence, the upper bound for the bending or curvature elastic modulus of the red cell membranes is 10-12 ergs (dyn-cm). Analysis of the adhesion experiment shows that interfacial free energy densities for red cell adhesion can be measured from a lower limit of 10-4 ergs/cm2 to an upper limit established by the membrane tension for lysis of 5-10 ergs/cm2.     

Thermodynamic approach to explain cell adhesion to air-medium interfaces

Cell damage has been observed in suspension cell cultures with air sparging, especially in the absence of any protective additives. This damage is associated with cells adhering to bubbles, and it has been shown that if this adhesion is prevented, cell damage is prevented. This article presents a thermodynamic approach for predicting cell adhesion at the air-medium interface. With this relationship it can be shown that cell-gas adhesion can be prevented by lowering the surface tension of the liquid growth medium through the addition of surface-active protective additives. The thermodynamic relationship describes the change in free energy as a function of the interfacial tensions between the (i) gas and liquid phases, (ii) gas and cell phases, and (iii) liquid and cell phases. Experimental data, along with theoretical and empirical equations, are used to quantify the changes in free energy that predict the process of cell-gas adhesion. The thermodynamic model is nonspecific in nature and, consequently, results are equally valid for all types of cells. (c) 1995 John Wiley & Sons, Inc.     

Stretch activation and nonlinear elasticity of muscle cross-bridges.

When active insect fibrillar flight muscle is stretched, its ATPase rate increases and it develops "negative viscosity," which allows it to perform oscillatory work. We use a six-state model for the cross-bridge cycle to show that such "stretch activation" may arise naturally as a nonlinear property of a cross-bridge interacting with a single attachment site on a thin filament. Attachment is treated as a thermally activated process in which elastic energy must be supplied to stretch or compress the cross-bridge spring. We find that stretch activation occurs at filament displacements where, before the power stroke, the spring is initially in compression rather than in tension. In that case, pulling the filaments relieves the initial compression and reduces the elastic energy required for attachment. The result is that the attachment rate is enhanced by stretching. The model also displays the "delayed tension" effect observed in length-step experiments. When the muscle is stretched suddenly, the power stroke responds very quickly, but there is a time lag before dissociation at the end of the cycle catches up with the increased attachment rate. This lag is responsible for the delayed tension and hence also for the negative viscosity.     

Line tension and stability of domains in cell-adhesion zones mediated by long and short receptor-ligand complexes

Submicron scale domains of membrane-anchored receptors play an important role in cell signaling. Central questions concern the stability of these microdomains, and the mechanisms leading to the domain formation. In immune-cell adhesion zones, microdomains of short receptor-ligand complexes form next to domains of significantly longer receptor-ligand complexes. The length mismatch between the receptor-ligand complexes leads to membrane deformations and has been suggested as a possible cause of the domain formation. The domain formation is a nucleation and growth process that depends on the line tension and free energy of the domains. Using a combination of analytical calculations and Monte Carlo simulations, we derive here general expressions for the line tension between domains of long and short receptor-ligand complexes and for the adhesion free energy of the domains. We argue that the length mismatch of receptor-ligand complexes alone is sufficient to drive the domain formation, and obtain submicron-scale minimum sizes for stable domains that are consistent with the domain sizes observed during immune-cell adhesion.     

Is cell sorting caused by differences in the work of intercellular adhesion? A critique of the steinberg hypothesis

The differential adhesion hypothesis, developed by Malcolm Steinberg, proposes that the histotypic sorting out behavior of aggregated cells is mechanistically equivalent to certain aspects of liquid surface tension, specifically the spontaneous separation of immiscible liquids of differing surface tension. According to Steinberg's hypothesis, the adhesive forces between aggregated cells play essentially the same role in cell sorting as are played by intermolecular attractive forces in liquid surface tension.In this paper I discuss a number of crucial distinctions between intermolecular attraction (in liquids) and intercellular adhesion (in aggregates). First, liquid drops are closed systems thermodynamically whereas aggregates of living cells can generate an indeterminate amount of metabolic energy capable of altering cell positions and adhesions. Secondly, intercellular adhesions are more than just close range attractions since cells can be held together by forces in addition to those which originally pulled them together. Third, the breakage of intercellular adhesions need not be simply the reverse, thermodynamically, of the formation of those adhesions. And fourthly, because intercellular adhesion is generally concentrated at relatively small foci such as desmosomes, a maximization of intercellular adhesion does not necessarily require a maximization of intercellular contact area, or vice versa.In addition, several alternative hypotheses are proposed, each of which is theoretically capable of explaining cell sorting and the other surface tension-like aspects of cell aggregate behavior which Steinberg has sought to explain as consequences of differential adhesion. In particular, a differential surface contraction hypothesis is proposed, according to which cell sorting and related phenomena are the results of cell surface contractions induced to occur over those portions of the cell surface which are exposed to the surrounding culture medium. Because of the evidence that various invagination type movements of embryonic epithelia are caused by cell surface contractions, it is suggested that differential surface contraction is the most likely explanation of histotypic cell sorting. A number of experiments are suggested by which these various hypotheses might be tested.     

Perfluorocarbon induces alveolar epithelial cell response through structural and mechanical remodeling

During total liquid ventilation, lung cells are exposed to perfluorocarbon (PFC) whose chemophysical properties highly differ from standard aqueous cell feeding medium (DMEM). We herein perform a systematic study of structural and mechanical properties of A549 alveolar epithelial cells in order to characterize their response to PFC exposure, using DMEM as control condition. Changes in F-actin structure, focal adhesion density and glycocalyx distribution are evaluated by confocal fluorescent microscopy. Changes in cell mechanics and adhesion are measured by multiscale magnetic twisting cytometry (MTC). Two different microrheological models (single Voigt and power law) are used to analyze the cell mechanics characterized by cytoskeleton (CSK) stiffness and characteristic relaxation times. Cell–matrix adhesion is analyzed using a stochastic multibond deadhesion model taking into account the non-reversible character of the cell response, allowing us to quantify the adhesion weakness and the number of associated bonds. The roles of F-actin structure and glycocalyx layer are evaluated by depolymerizing F-actin and degrading glycocalyx, respectively. Results show that PFC exposure consistently induces F-actin remodeling, CSK softening and adhesion weakening. These results demonstrate that PFC triggers an alveolar epithelial cell response herein evidenced by a decay in intracellular CSK tension, an adhesion weakening and a glycocalyx layer redistribution. These PFC-induced cell adjustments are consistent with the hypothesis that cells respond to a decrease in adhesion energy at cell surface. This adhesion energy can be even further reduced in the presence of surfactant adsorbed at the cell surface.     

Mapping cell-matrix stresses during stretch reveals inelastic reorganization of the cytoskeleton

The mechanical properties of the living cell are intimately related to cell signaling biology through cytoskeletal tension. The tension borne by the cytoskeleton (CSK) is in part generated internally by the actomyosin machinery and externally by stretch. Here we studied how cytoskeletal tension is modified during stretch and the tensional changes undergone by the sites of cell-matrix interaction. To this end we developed a novel technique to map cell-matrix stresses during application of stretch. We found that cell-matrix stresses increased with imposition of stretch but dropped below baseline levels on stretch release. Inhibition of the actomyosin machinery resulted in a larger relative increase in CSK tension with stretch and in a smaller drop in tension after stretch release. Cell-matrix stress maps showed that the loci of cell adhesion initially bearing greater stress also exhibited larger drops in traction forces after stretch removal. Our results suggest that stretch partially disrupts the actin-myosin apparatus and the cytoskeletal structures that support the largest CSK tension. These findings indicate that cells use the mechanical energy injected by stretch to rapidly reorganize their structure and redistribute tension.     

Adhesion-induced domain formation by interplay of long-range repulsion and short-range attraction force: a model membrane study.

We study the role of the interplay of specific and universal forces for the adhesion of giant vesicles on solid supported membranes. To model the situation of cell adhesion, we incorporated lipopolymers (phospholipids with polyethyleneoxide headgroups) as artificial glycocalix, whereas attractive lock-and-key forces are mimicked by incorporating biotinylated lipids into both membranes and by mediating the strong coupling through streptavidin. Adhesion is studied by quantitative reflection interference contrast microscopy (RICM), which enables visualization of the contact zone and reconstruction of the height profile of the membrane beyond the contact line (outside the contact zone) up to a height of 1 micron. We demonstrate that adhesion is accompanied by lateral phase separation, leading to the formation of domains of tight adhesion (adhesion plaques) separated by areas of weak adhesion exhibiting pronounced flickering. By analyzing the height profile S(x) near the contact line in terms of the tension equilibrium (Young equation) and the moment equilibrium, respectively, the adhesion energy and membrane tension can be approximately measured locally. We show that the adhesion energy is about three orders of magnitude larger for the adhesion plaques than for the weekly adhering regions. The adhesion is studied as a function of the excess area of the vesicle generated by temperature variation. A very remarkable finding is that increased excess area is not always stored in the contact area, but leads to the formation of microbuds (diameter approximately 2 microns).     

Membrane tether formation from blebbing cells

Membrane tension has been proposed to be important in regulating cell functions such as endocytosis and cell motility. The apparent membrane tension has been calculated from tether forces measured with laser tweezers. Both membrane-cytoskeleton adhesion and membrane tension contribute to the tether force. Separation of the plasma membrane from the cytoskeleton occurs in membrane blebs, which could remove the membrane-cytoskeleton adhesion term. In renal epithelial cells, tether forces are significantly lower on blebs than on membranes that are supported by cytoskeleton. Furthermore, the tether forces are equal on apical and basolateral blebs. In contrast, tether forces from membranes supported by the cytoskeleton are greater in apical than in basolateral regions, which is consistent with the greater apparent cytoskeletal density in the apical region. We suggest that the tether force on blebs primarily contains only the membrane tension term and that the membrane tension may be uniform over the cell surface. Additional support for this hypothesis comes from observations of melanoma cells that spontaneously bleb. In melanoma cells, tether forces on blebs are proportional to the radius of the bleb, and as large blebs form, there are spikes in the tether force in other cell regions. We suggest that an internal osmotic pressure inflates the blebs, and the pressure calculated from the Law of Laplace is similar to independent measurements of intracellular pressures. When the membrane tension term is subtracted from the apparent membrane tension over the cytoskeleton, the membrane-cytoskeleton adhesion term can be estimated. In both cell systems, membrane-cytoskeleton adhesion was the major factor in generating the tether force.     

Membrane viscoelasticity.

In this paper, we develop a theory for viscoelastic behavior of large membrane deformations and apply the analysis to the relaxation of projections produced by small micropipette aspiration of red cell discocytes. We show that this relaxation is dominated by the membrane viscosity and that the cytoplasmic and extracellular fluid flow have negligible influence on the relaxation time and can be neglected. From preliminary data, we estimate the total membrane "viscosity" when the membrane material behaves in an elastic solid manner. The total membrane viscosity is calculated to be 10(-3) dyn-s/cm, which is a surface viscosity that is about three orders of magnitude greater than the surface viscosity of lipid membrane components (as determined by "fluidity" measurements). It is apparent that the lipid bilayer contributes little to the fluid dynamic behavior of the whole plasma membrane and that a structural matrix dominates the viscous dissipation. However, we show that viscous flow in the membrane is not responsible for the temporal dependence of the isotropic membrane tension required to produce lysis and that the previous estimates of Rand, Katchalsky, et al., for "viscosity" are six to eight orders of magnitude too large.     

Membrane tethers formed from blood cells with available area and determination of their adhesion energy

Fundamental to all mammalian cells is the adherence of the lipid bilayer membrane to the underlying membrane associated cytoskeleton. To investigate this adhesion, we physically detach the lipid membrane from the cell by mechanically forming membrane tethers. For the most part these have been tethers formed from either neutrophils or red cells. Here we do a simple thermodynamic analysis of the tether formation process using the entire cell, including tether, as the control volume. For a neutrophil, we show that the total adhesion energy per unit area between lipid membrane and cytoskeleton depends on the square of the tether force. For a flaccid red cell, we show that the total adhesion energy minus the tension in the spectrin cytoskeleton depends also on the square of the tether force. Finally, we discuss briefly the viscous flow of membrane. Using published data we calculate and compare values for the various adhesion energies and viscosities.     

Adhesive Contact in Animal: Morphology, Mechanism and Bio-Inspired Application

Many animals possess adhesive pads on their feet,which are able to attach to various substrates while controlling adhesive forces during locomotion.This review article studies the morphology of adhesive devices in animals,and the physical mechanisms of wet adhesion and dry adhesion.The adhesive pads are either ‘smooth' or densely covered with special adhesive setae.Smooth pads adhere by wet adhesion,which is facilitated by fluid secreted from the pads,whereas hairy pads can adhere by dry adhesion or wet adhesion.Contact area,distance between pad and substrate,viscosity and surface tension of the liquid filling the gap between pad and substrate are the most important factors which determine the wet adhesion.Dry adhesion was found only in hairy pads,which occurs in geckos and spiders.It was demonstrated that van der Waals interaction is the dominant adhesive force in geckos' adhesion.The bio-inspired applications derived from adhesive pads are also reviewed.     

The differential adhesion hypothesis: a direct evaluation

The differential adhesion hypothesis (DAH), advanced in the 1960s, proposed that the liquid-like tissue-spreading and cell segregation phenomena of development arise from tissue surface tensions that in turn arise from differences in intercellular adhesiveness. Our earlier measurements of liquid-like cell aggregate surface tensions have shown that, without exception, a cell aggregate of lower surface tension tends to envelop one of higher surface tension to which it adheres. We here measure the surface tensions of L cell aggregates transfected to express N-, P- or E-cadherin in varied, measured amounts. We report that in these aggregates, in which cadherins are essentially the only cell-cell adhesion molecules, the aggregate surface tensions are a direct, linear function of cadherin expression level. Taken together with our earlier results, the conclusion follows that the liquid-like morphogenetic cell and tissue rearrangements of cell sorting, tissue spreading and segregation represent self-assembly processes guided by the diminution of adhesive-free energy as cells tend to maximize their mutual binding. This conclusion relates to the physics governing these morphogenetic phenomena and applies independently of issues such as the specificities of intercellular adhesives.     

Spatiotemporal constraints on the force-dependent growth of focal adhesions

Focal adhesions (FAs) are the predominant mechanism by which cells mechanically couple to and exert traction forces on their extracellular matrix (ECM). It is widely presumed that FA size is modulated by force to mediate changes in adhesion strength at different levels of cellular tension. However, previous studies seeking correlations between force and FA morphology have yielded variable and often conflicting results. Here we show that a strong correlation between adhesion size and traction force exists only during the initial stages of myosin-mediated adhesion maturation and growth. For mature adhesions, no correlation between traction stress and size is observed. Rather, the tension that is sustained at mature adhesions is more strongly influenced by proximity to the cell edge, with peripheral adhesions transmitting higher tension than adhesions near the cell center. Finally, we show that mature adhesions can withstand sixfold increases in tension without changes in size. Thus, although a strong correlation between adhesion size and mechanical tension is observed during the initial stages of myosin-mediated adhesion maturation, no correlation is observed in mature, elongated adhesions. This work places spatiotemporal constraints on the force-dependent growth of adhesions and provides insight into the mechanical regulation of cell-ECM adhesion.     

Passive mechanical behavior of human neutrophils: effect of cytochalasin B.

Actin is a ubiquitous protein in eukaryotic cells. It plays a major role in cell motility and in the maintenance and control of cell shape. In this article, we intend to address the contribution of actin to the passive mechanical properties of human neutrophils. As a framework for assessing this contribution, the neutrophil is modeled as a simple viscous fluid drop with a constant cortical ("surface") tension. The reagent cytochalasin B (CTB) was used to disrupt the F-actin structure, and the neutrophil cortical tension and cytoplasmic viscosity were evaluated by single-cell micropipette aspiration. The cortical tension was calculated by simple force balance, and the viscosity was calculated according to a numerical analysis of the cell entry into the micropipette. CTB reduced the cell cortical tension in a dose-dependent fashion: by 19% at a concentration of 3 microM and by 49% at 30 microM. CTB also reduced the cytoplasmic viscosity by approximately -25% at a concentration of 3 microM and by approximately 65% at a concentration of 30 microM when compared at the same aspiration pressures. All three groups of neutrophils, normal cells, and cells treated with either 3 or 30 microM CTB, exhibited non-Newtonian behavior, in that the apparent viscosity decreased with increasing shear rate. The dependence of the cytoplasmic viscosity on deformation rate can be described empirically by mu = mu c(gamma m/gamma c)-b, where mu is cytoplasmic viscosity, gamma m is mean shear rate, mu c is the characteristic viscosity at the characteristic shear rate gamma c, and b is a material coefficient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct observation of α-actinin tension and recruitment at focal adhesions during contact growth

Adherent cells interact with extracellular matrix via cell–substrate contacts at focal adhesions. The dynamic assembly and disassembly of focal adhesions enables cell attachment, migration and growth. While the influence of mechanical forces on the formation and growth of focal adhesions has been widely observed, the force loading on specific proteins at focal adhesion complex is not clear. By co-expressing force sensitive α-actinin FRET probes and fluorescence labeled paxillin in MDCK cells, we have simultaneously observed the time-dependent changes in tension in α-actinin and the dynamics of focal adhesion during cell migration. We show that increase in tension in α-actinin at the focal adhesion coincides with elongation of the adhesion in its growth phase. The enlargement of focal adhesion is through a force sensitive recruitment of α-actinin and paxillin to the adhesion sites. Changes in α-actinin tension and correlated relocation of α-actinin in an active adhesion also guide the growth direction of the adhesion. The results support the model that cytoskeletal tension is coupled to focal adhesion via the linking protein, α-actinin at the adhesion complex. Lysophosphatidic acid caused an immediate increase in α-actinin tension followed by drastic focal adhesion formation and elongation. Application of Rho-ROCK inhibitor, Y27632, resulted in reversible reduction in tension in α-actinin and disassociation of focal adhesion, suggesting the involvement of myosin-II mediated contractile force in the focal adhesion dynamics. These findings suggest that α-actinin not only serves as a physical linker between cytoskeleton and integrin, but also participates in force transmission at adhesion sites to facilitate adhesion?s growth.     

Mechanics of spreading cells probed by atomic force microscopy

Cellular adhesion and motility are fundamental processes in biological systems such as morphogenesis and tissue homeostasis. During these processes, cells heavily rely on the ability to deform and supply plasma membrane from pre-existing membrane reservoirs, allowing the cell to cope with substantial morphological changes. While morphological changes during single cell adhesion and spreading are well characterized, the accompanying alterations in cellular mechanics are scarcely addressed. Using the atomic force microscope, we measured changes in cortical and plasma membrane mechanics during the transition from early adhesion to a fully spread cell. During the initial adhesion step, we found that tremendous changes occur in cortical and membrane tension as well as in membrane area. Monitoring the spreading progress by means of force measurements over 2.5 h reveals that cortical and membrane tension become constant at the expense of excess membrane area. This was confirmed by fluorescence microscopy, which shows a rougher plasma membrane of cells in suspension compared with spread ones, allowing the cell to draw excess membrane from reservoirs such as invaginations or protrusions while attaching to the substrate and forming a first contact zone. Concretely, we found that cell spreading is initiated by a transient drop in tension, which is compensated by a decrease in excess area. Finally, all mechanical parameters become almost constant although morphological changes continue. Our study shows how a single cell responds to alterations in membrane tension by adjusting its overall membrane area. Interference with cytoskeletal integrity, membrane tension and excess surface area by administration of corresponding small molecular inhibitors leads to perturbations of the spreading process.