Cell spreading and lamellipodial extension rate is regulated by membrane tension

Cell spreading and motility require the extension of the plasma membrane in association with the assembly of actin. In vitro, extension must overcome resistance from tension within the plasma membrane. We report here that the addition of either amphiphilic compounds or fluorescent lipids that expanded the plasma membrane increased the rate of cell spreading and lamellipodial extension, stimulated new lamellipodial extensions, and caused a decrease in the apparent membrane tension. Further, in PDGF-stimulated motility, the increase in the lamellipodial extension rate was associated with a decrease in the apparent membrane tension and decreased membrane-cytoskeleton adhesion through phosphatidylinositol diphosphate hydrolysis. Conversely, when membrane tension was increased by osmotically swelling cells, the extension rate decreased. Therefore, we suggest that the lamellipodial extension process can be activated by a physical signal (perhaps secondarily), and the rate of extension is directly dependent upon the tension in the plasma membrane. Quantitative analysis shows that the lamellipodial extension rate is inversely correlated with the apparent membrane tension. These studies describe a physical chemical mechanism involving changes in membrane-cytoskeleton adhesion through phosphatidylinositol 4,5-biphosphate-protein interactions for modulating and stimulating the biochemical processes that power lamellipodial extension.     

Spreading of cells on various substrates evaluated by fourier analysis of shape

Summary In order to evaluate in mathematical terms the morphological changes occurring in the course of cell spreading, Fourier analysis of shape was applied. Human urothelial Hu 961 b cells plated on type IV collagen, fibronectin, laminin, glass and bovine serum albumine (BSA) were studied. Fourier parameters describing cell shape as well as surface areas covered by the cells on the substrate were subjected to statistical analysis. Using analysis of variance and discriminant analysis it was found that parameters describing cell shape (both gross shape of cells and their fine scale contour foldings) possessed a higher power of discrinunation between the cells spread on various substrates than the differences in cell surface areas. In the course of observation (75 and 150 min) the highest number of attached cells and highest degree of spreading were found when cells were plated on type IV collagen. Moderate alterations in cell shape and moderate increase of surface area were seen in the group of cells seeded on fibronectin, whereas the cells plated on laminin, glass and BSA revealed a moderate increase of surface area, but no changes in their shape were observed. The differences in attachment of cells and in the degree of their spreading might be due to the variation in expression of plasma membrane receptors for various substrates. The Fourier analysis of cell shape coupled with measurement of surface area is a good tool for quantitative evaluation of cell spreading and can be used for discrimination between cells spread on different substrates.Supported in part by a grant (MZ-XIV) from the Polish Ministry of Health and Welfare     

Morphological and mechanical stability of bladder cancer cells in response to substrate rigidity

BackgroundMorphology of cells can be considered as an interplay between the accessibility of substrate anchoring sites, cytoskeleton properties and cellular deformability. To withstand tension induced by cell's environment, cells tend to spread out and, simultaneously, to remodel actin filament organization.MethodsIn this context, the use of polyacrylamide hydrogel substrates with a surface coated with laminin allows to trace remodeling of actin cytoskeleton during the interaction of cells with laminin-rich basement membrane. Reorganization of actin cortex can be quantified by a surface spreading area and deformability of single cells.ResultsIn our study, we demonstrated that morphological and mechanical alterations of bladder cancer cells in response to altered microenvironment stiffness are of biphasic nature. Threshold-dependent relations are induced by mechanical properties of cell microenvironment. Initially, fast alterations in cellular capability to spread and to deform are followed by slow-rate changes. A switch provided by cellular deformability threshold, in the case of non-malignant cells, triggers the formation of thick actin bundles accompanied by matured focal adhesions. For cancer cells, cell spreading and deformability thresholds switch between slow and fast rate of changes with weak reorganization of actin filaments and focal adhesions formation.ConclusionsThe presence of transition region enables the cells to achieve a morphological and mechanical stability, which together with altered expression of vinculin and integrins, can contribute to invasiveness of bladder cancers.General significanceOur findings show that morphological and mechanical stability is directly related to actin filament organization used by cancer cells to adapt to altered laminin-rich microenvironment.     

Mechanics of Biomimetic Liposomes Encapsulating an Actin Shell

Cell-shape changes are insured by a thin, dynamic, cortical layer of cytoskeleton underneath the plasma membrane. How this thin cortical structure impacts the mechanical properties of the whole cell is not fully understood. Here, we study the mechanics of liposomes or giant unilamellar vesicles, when a biomimetic actin cortex is grown at the inner layer of the lipid membrane via actin-nucleation-promoting factors. Using a hydrodynamic tube-pulling technique, we show that tube dynamics is clearly affected by the presence of an actin shell anchored to the lipid bilayer. The same force pulls much shorter tubes in the presence of the actin shell compared to bare membranes. However, in both cases, we observe that the dynamics of tube extrusion has two distinct features characteristic of viscoelastic materials: rapid elastic elongation, followed by a slower elongation phase at a constant rate. We interpret the initial elastic regime by an increase of membrane tension due to the loss of lipids into the tube. Tube length is considerably shorter for cortex liposomes at comparable pulling forces, resulting in a higher spring constant. The presence of the actin shell seems to restrict lipid mobility, as is observed in the corral effect in cells. The viscous regime for bare liposomes corresponds to a leakout of the internal liquid at constant membrane tension. The presence of the actin shell leads to a larger friction coefficient. As the tube is pulled from a patchy surface, membrane tension increases locally, leading to a Marangoni flow of lipids. As a conclusion, the presence of an actin shell is revealed by its action that alters membrane mechanics.     

ST6Gal-I–mediated sialylation of the epidermal growth factor receptor modulates cell mechanics and enhances invasion

Heterogeneity within the glycocalyx influences cell adhesion mechanics and signaling. However, the role of specific glycosylation subtypes in influencing cell mechanics via alterations of receptor function remains unexplored. It has been shown that the addition of sialic acid to terminal glycans impacts growth, development, and cancer progression. In addition, the sialyltransferase ST6Gal-I promotes epidermal growth factor receptor (EGFR) activity, and we have shown EGFR is an ‘allosteric mechano-organizer’ of integrin tension. Here, we investigated the impact of ST6Gal-I on cell mechanics. Using DNA-based tension gauge tether probes of variable thresholds, we found that high ST6Gal-I activity promotes increased integrin forces and spreading in Cos-7 and OVCAR3, OVCAR5, and OV4 cancer cells. Further, employing inhibitors and function-blocking antibodies against β1, β3, and β5 integrins and ST6Gal-I targets EGFR, tumor necrosis factor receptor, and Fas cell surface death receptor, we validated that the observed phenotypes are EGFR-specific. We found that while tension, contractility, and adhesion are extracellular-signal-regulated kinase pathway-dependent, spreading, proliferation, and invasion are phosphoinositide 3-kinase-Akt serine/threonine kinase dependent. Using total internal reflection fluorescence microscopy and flow cytometry, we also show that high ST6Gal-I activity leads to sustained EGFR membrane retention, making it a key regulator of cell mechanics. Our findings suggest a novel sialylation-dependent mechanism orchestrating cellular mechanics and enhancing cell motility via EGFR signaling.     

Cell Blebbing and Membrane Area Homeostasis in Spreading and Retracting Cells

Cells remodel their plasma membrane and cytoskeleton during numerous physiological processes, including spreading and motility. Morphological changes require the cell to adjust its membrane tension on different timescales. While it is known that endo- and exocytosis regulate the cell membrane area in a timescale of 1 h, faster processes, such as abrupt cell detachment, require faster regulation of the plasma membrane tension. In this article, we demonstrate that cell blebbing plays a critical role in the global mechanical homeostasis of the cell through regulation of membrane tension. Abrupt cell detachment leads to pronounced blebbing (which slow detachment does not), and blebbing decreases with time in a dynamin-dependent fashion. Cells only start spreading after a lag period whose duration depends on the cell's blebbing activity. Our model quantitatively reproduces the monotonic decay of the blebbing activity and accounts for the lag phase in the spreading of blebbing cells.     

Spreading of cells on various substrates evaluated by Fourier analysis of shape

In order to evaluate in mathematical terms the morphological changes occurring in the course of cell spreading, Fourier analysis of shape was applied. Human urothelial Hu 961 b cells plated on type IV collagen, fibronectin, laminin, glass and bovine serum albumin (BSA) were studied. Fourier parameters describing cell shape as well as surface areas covered by the cells on the substrate were subjected to statistical analysis. Using analysis of variance and discriminant analysis it was found that parameters describing cell shape (both gross shape of cells and their fine scale contour foldings) possessed a higher power of discrimination between the cells spread on various substrates than the differences in cell surface areas. In the course of observation (75 and 150 min) the highest number of attached cells and highest degree of spreading were found when cells were plated on type IV collagen. Moderate alterations in cell shape and moderate increase of surface area were seen in the group of cells seeded on fibronectin, whereas the cells plated on laminin, glass and BSA revealed a moderate increase of surface area, but no changes in their shape were observed. The differences in attachment of cells and in the degree of their spreading might be due to the variation in expression of plasma membrane receptors for various substrates. The Fourier analysis of cell shape coupled with measurement of surface area is a good tool for quantitative evaluation of cell spreading and can be used for discrimination between cells spread on different substrates.     

A Highlights from MBoC Selection: Spatial distribution of cell–cell and cell–ECM adhesions regulates force balance while main-taining E-cadherin molecular tension in cell pairs

Mechanical linkage between cell–cell and cell–extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell–cell and cell–ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell–cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell–cell and cell–ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell–cell pairs resulted in shorter junction lengths and constant cell–cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell–cell forces and was evenly distributed along cell–cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.     

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.     

The universal dynamics of cell spreading

Cell adhesion and motility depend strongly on the interactions between cells and extracellular matrix (ECM) substrates. When plated onto artificial adhesive surfaces, cells first flatten and deform extensively as they spread. At the molecular level, the interaction of membrane-based integrins with the ECM has been shown to initiate a complex cascade of signaling events [1], which subsequently triggers cellular morphological changes and results in the generation of contractile forces [2]. Here, we focus on the early stages of cell spreading and probe their dynamics by quantitative visualization and biochemical manipulation with a variety of cell types and adhesive surfaces, adhesion receptors, and cytoskeleton-altering drugs. We find that the dynamics of adhesion follows a universal power-law behavior. This is in sharp contrast with the common belief that spreading is regulated by either the diffusion of adhesion receptors toward the growing adhesive patch [3-5] or by actin polymerization [6-8]. To explain this, we propose a simple quantitative and predictive theory that models cells as viscous adhesive cortical shells enclosing a less viscous interior. Thus, although cell spreading is driven by well-identified biomolecular interactions, it is dynamically limited by its mesoscopic structure and material properties.     

Beta1 integrin-mediated T cell adhesion and cell spreading are regulated by calpain

To investigate the function of calpain in T cells, we sought to determine the role of this protease in cellular events mediated by beta1 integrins. T cell receptor cross-linked or phorbol ester-stimulated T cells binding to immobilized fibronectin induce the translocation of calpain to the cytoskeletal/membrane fraction of these cells. Such translocation of calpain is associated with proteolytic modification of protein tyrosine phosphatase 1B, increased cellular adhesion, and dramatic alterations in cellular morphology. However, affinity-related increases in T cell adhesion induced by the anti-beta1 integrin antibody 8A2 occur in a calpain-independent manner and in the absence of morphological shape changes. Furthermore, calpain undergoes activation in response to either alpha4beta1 or alpha5beta1 integrin binding to fibronectin in appropriately stimulated T cells, and calpain II as well as protein tyrosine phosphatase 1B accumulates at sites of focal contact formation. Inhibition of calpain activity not only inhibits the proteolytic modification of protein tyrosine phosphatase 1B, but also decreases the ability of T cells to adhere to and spread on immobilized fibronectin. Thus, we describe a potential regulatory role for calpain in beta1 integrin-mediated signaling events associated with T cell adhesion and cell spreading on fibronectin.     

A semianalytical model to study the effect of cortical tension on cell rolling

Cell rolling on the vascular endothelium plays an important role in trafficking of leukocytes, stem cells, and cancer cells. We describe a semianalytical model of cell rolling that focuses on the microvillus as the unit of cell-substrate interaction and integrates microvillus mechanics, receptor clustering, force-dependent receptor-ligand kinetics, and cortical tension that enables incorporation of cell body deformation. Using parameters obtained from independent experiments, the model showed excellent agreement with experimental studies of neutrophil rolling on P-selectin and predicted different regimes of cell rolling, including spreading of the cells on the substrate under high shear. The cortical tension affected the cell-surface contact area and influenced the rolling velocity, and modulated the dependence of rolling velocity on microvillus stiffness. Moreover, at the same shear stress, microvilli of cells with higher cortical tension carried a greater load compared to those with lower cortical tension. We also used the model to obtain a scaling dependence of the contact radius and cell rolling velocity under different conditions of shear stress, cortical tension, and ligand density. This model advances theoretical understanding of cell rolling by incorporating cortical tension and microvillus extension into a versatile, semianalytical framework.     

Protrusive and Contractile Forces of Spreading Human Neutrophils

Human neutrophils are mediators of innate immunity and undergo dramatic shape changes at all stages of their functional life cycle. In this work, we quantified the forces associated with a neutrophil’s morphological transition from a nonadherent, quiescent sphere to its adherent and spread state. We did this by tracking, with high spatial and temporal resolution, the cell’s mechanical behavior during spreading on microfabricated post-array detectors printed with the extracellular matrix protein fibronectin. Two dominant mechanical regimes were observed: transient protrusion and steady-state contraction. During spreading, a wave of protrusive force (75 ± 8 pN/post) propagates radially outward from the cell center at a speed of 206 ± 28 nm/s. Once completed, the cells enter a sustained contractile state. Although post engagement during contraction was continuously varying, posts within the core of the contact zone were less contractile (−20 ± 10 pN/post) than those residing at the geometric perimeter (−106 ± 10 pN/post). The magnitude of the protrusive force was found to be unchanged in response to cytoskeletal inhibitors of lamellipodium formation and myosin II-mediated contractility. However, cytochalasin B, known to reduce cortical tension in neutrophils, slowed spreading velocity (61 ± 37 nm/s) without significantly reducing protrusive force. Relaxation of the actin cortical shell was a prerequisite for spreading on post arrays as demonstrated by stiffening in response to jasplakinolide and the abrogation of spreading. ROCK and myosin II inhibition reduced long-term contractility. Function blocking antibody studies revealed haptokinetic spreading was induced by β₂ integrin ligation. Neutrophils were found to moderately invaginate the post arrays to a depth of ∼1 μm as measured from spinning disk confocal microscopy. Our work suggests a competition of adhesion energy, cortical tension, and the relaxation of cortical tension is at play at the onset of neutrophil spreading.     

Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na(+) and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl(-) efflux through chloride channels and Na(+) influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.     

Rho-ROCK signaling differentially regulates chondrocyte spreading on fibronectin and bone sialoprotein

The mammalian growth plate is a dynamic structure rich in extracellular matrix (ECM). Interactions of growth plate chondrocytes with ECM proteins regulate cell behavior. In this study, we compared chondrocyte adhesion and spreading dynamics on fibronectin (FN) and bone sialoprotein (BSP). Chondrocyte adhesion and spreading were also compared with fibroblasts to analyze potential cell-type-specific effects. Chondrocyte adhesion to BSP is independent of posttranslational modifications but is dependent on the RGD sequence in BSP. Whereas chondrocytes and fibroblasts adhered at similar levels on FN and BSP, cells displayed more actin-dependent spread on FN despite a 16x molar excess of BSP adsorbed to plastic. To identify intracellular mediators responsible for this difference in spreading, we investigated focal adhesion kinase (FAK)-Src and Rho-Rho kinase (ROCK) signaling. Although activated FAK localized to the vertices of adhered chondrocytes, levels of FAK activation did not correlate with the extent of spreading. Furthermore, Src inhibition reduced chondrocyte spreading on both FN and BSP, suggesting that FAK-Src signaling is not responsible for less cell spreading on BSP. In contrast, inhibition of Rho and ROCK in chondrocytes increased cell spreading on BSP and membrane protrusiveness on FN but did not affect cell adhesion. In fibroblasts, Rho inhibition increased fibroblast spreading on BSP while ROCK inhibition changed membrane protrusiveness of FN and BSP. In summary, we identify a novel role for Rho-ROCK signaling in regulating chondrocyte spreading and demonstrate both cell- and matrix molecule-specific mechanisms controlling cell spreading.     

Distinct timing of neutrophil spreading and stiffening during phagocytosis

Phagocytic cells form the first line of defense in an organism, engulfing microbial pathogens. Phagocytosis involves cell mechanical changes that are not yet well understood. Understanding these mechanical modifications promises to shed light on the immune processes that trigger pathological complications. Previous studies showed that phagocytes undergo a sequence of spreading events around their target followed by an increase in cell tension. Seemingly in contradiction, other studies observed an increase in cell tension concomitant with membrane expansion. Even though phagocytes are viscoelastic, few studies have quantified viscous changes during phagocytosis. It is also unclear whether cell lines behave mechanically similarly to primary neutrophils. We addressed the question of simultaneous versus sequential spreading and mechanical changes during phagocytosis by using immunoglobulin-G-coated 8- and 20-μm-diameter beads as targets. We used a micropipette-based single-cell rheometer to monitor viscoelastic properties during phagocytosis by both neutrophil-like PLB cells and primary human neutrophils. We show that the faster expansion of PLB cells on larger beads is a geometrical effect reflecting a constant advancing speed of the phagocytic cup. Cells become stiffer on 20- than on 8-μm beads, and the relative timing of spreading and stiffening of PLB cells depends on target size: on larger beads, stiffening starts before maximal spreading area is reached but ends after reaching maximal area. On smaller beads, the stiffness begins to increase after cells have engulfed the bead. Similar to PLB cells, primary cells become stiffer on larger beads but start spreading and stiffen faster, and the stiffening begins before the end of spreading on both bead sizes. Our results show that mechanical changes in phagocytes are not a direct consequence of cell spreading and that models of phagocytosis should be amended to account for causes of cell stiffening other than membrane expansion.     

Guard cells undergo constitutive and pressure-driven membrane turnover

Summary. During stomatal movement, guard cells undergo large and reversible changes in cell volume and consequently surface area. These alterations in surface area require addition and removal of plasma membrane material. How this is achieved is largely unknown. Here we summarize recent studies of membrane turnover in guard cells using electrophysiology and fluorescent imaging techniques. The results implicate that membrane turnover in guard cells and most likely in plant cells in general is sensitive to changes in membrane tension. We suggest that this provides a mechanism for the adaptation of surface area of guard cells to osmotically driven changes in cell volume. In addition, guard cells also exhibit constitutive membrane turnover. Constitutive and pressure-driven membrane turnover were found to be associated with addition and removal of K⁺ channels. This implies that some of the exo- and endocytic vesicles carry K⁺ channels. Together the results demonstrate that exo- and endocytosis is an essential process in guard cell functioning. Correspondence and reprints: Institute of Botany, Darmstadt University of Technology, Schnittspahnstrasse 3, 64287 Darmstadt, Federal Republic of Germany.     

Microfilament-associated proteins in tissue culture cells viewed by stereo immunofluorescence microscopy.

Stereo immunofluorescence microscopy avoids the problem of juxtaposition of structures often encountered in normal fluorescence microscopy. The procedure has been used in conjunction with antibodies against microfilament associated proteins to reveal the arrangement of microfilaments in a rat mammary cell line both in the fully spread state and in cells during the process of spreading on the substratum. use of antibodies to myosin, tropomyosin, alpha-actinin and filamin emphasizes that at early times during the spreading process these proteins are abundantly present underneath the upper plasma membrane, suggesting that the cortical layer present underneath this membrane may be contractile. In addition the results emphasize that even in well spread cells microfilament bundles are expressed both above and below the nucleus, in agreement with the assumption that microfilaments may form a supporting layer underneath the plasma membrane.     

Molecular mechanisms of cell segregation and boundary formation in development and tumorigenesis

The establishment and maintenance of precisely organized tissues requires the formation of sharp borders between distinct cell populations. The maintenance of segregated cell populations is also required for tissue homeostasis in the adult, and deficiencies in segregation underlie the metastatic spreading of tumor cells. Three classes of mechanisms that underlie cell segregation and border formation have been uncovered. The first involves differences in cadherin-mediated cell-cell adhesion that establishes interfacial tension at the border between distinct cell populations. A second mechanism involves the induction of actomyosin-mediated contraction by intercellular signaling, such that cortical tension is generated at the border. Third, activation of Eph receptors and ephrins can lead to both decreased adhesion by triggering cleavage of E-cadherin, and to repulsion of cells by regulation of the actin cytoskeleton, thus preventing intermingling between cell populations. These mechanisms play crucial roles at distinct boundaries during development, and alterations in cadherin or Eph/ephrin expression have been implicated in tumor metastasis.     

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.