Molecular basis for microbial adhesion to geochemical surfaces: computer simulation of Pseudomonas aeruginosa adhesion to goethite

The adhesion of Pseudomonas aeruginosa to the goethite mineral is investigated using classical molecular simulation. A fragment model for goethite has been integrated into a fully atomistic membrane model. Properties for the resulting system are evaluated for a 1.5-ns simulation in the isothermal-isobaric ensemble. The response of the membrane to the presence of the mineral is investigated. Radial distribution functions are used to present an average picture of the hydrogen bonding. Orientational vectors, assigned to the saccharide groups, reveal the extent of the mineral's perturbations on the membrane. Significant structural changes were observed for the outermost saccharide groups, several of which rotate to form hydrogen bonds with the mineral surface. The structure of the inner core, and the corresponding integrity of the membrane, is maintained. The mineral surface dehydrates slightly in the presence of the membrane as saccharide hydroxyl groups compete with water molecules for hydrogen-bonding sites on its surface.     

Gangliosides as components of lipid membrane domains

Cell membrane components are organized as specialized domains involved in membrane-associated events such as cell signaling, cell adhesion, and protein sorting. These membrane domains are enriched in sphingolipids and cholesterol but display a low protein content. Theoretical considerations and experimental data suggest that some properties of gangliosides play an important role in the formation and stabilization of specific cell lipid membrane domains. Gangliosides are glycolipids with strong amphiphilic character and are particularly abundant in the plasma membranes, where they are inserted into the external leaflet with the hydrophobic ceramide moiety and with the oligosaccharide chain protruding into the extracellular medium. The geometry of the monomer inserted into the membrane, largely determined by the very large surface area occupied by the oligosaccharide chain, the ability of the ceramide amide linkage to form a network of hydrogen bonds at the water-lipid interface of cell membranes, the Delta(4) double bond of sphingosine proximal to the water-lipid interface, the capability of the oligosaccharide chain to interact with water, and the absence of double bonds into the double-tailed hydrophobic moiety are the ganglioside features that will be discussed in this review, to show how gangliosides are responsible for the formation of cell lipid membrane domains characterized by a strong positive curvature.     

Mapping the oligomeric interface of diacylglycerol kinase by engineered thiol cross-linking: homologous sites in the transmembrane domain

This work represents the first stage of thiol-based cross-linking studies to map the oligomeric interface of the homotrimeric membrane protein diacylglycerol kinase (DAGK). A total of 53 single-cysteine mutants spanning DAGK's three transmembrane segments and the first part of a cytoplasmic domain were purified and subjected to catalytic oxidation in mixed micelles. Four mutants (A52C, I53C, A74C, and I75C) were observed to undergo intratrimer disulfide bond formation between homologous sites on adjacent subunits. To establish whether the homologous sites are proximal in the ground-state conformation of DAGK or whether the disulfide bonds formed as a result of motions that brought normally distal sites into transient proximity, additional cross-linking experiments were carried out in three different milieus of varying fluidity [mixed micelles, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles, and Escherichia coli membranes]. Cross-linking experiments included disulfide bond formation under three different catalytic conditions [Cu(II)-phenanthroline oxidation, I(2) oxidation, and thionitrobenzoate-based thiol exchange] and reactions with a set of bifunctional thiol-reactive chemical cross-linkers presenting two different reactive chemistries and several spacer lengths. On the basis of these studies, residues 53 and 75 are judged to be in stable proximity within the DAGK homotrimer, while position 52 appears to be more distal and forms disulfide bonds only as a result of protein motions. Results for position 74 were ambiguous. In lipid vesicles and mixed micelles DAGK appears to execute motions that are not present in native membranes, with mobility also being higher for DAGK in mixed micelles than in POPC vesicles.     

The distribution of Concanavalin A receptor sites on the membrane of chromaffin granules.

The distribution of concanavalin A (con A) receptor sites on the membranes of chromaffin granules has been investigated by binding studies using 125I-labelled con A and by electron-microscope studies using ferritin-labelled con A. In both experiments con A was observed to bind to chromaffin granule membranes but not to intact granules. The ferritin-con A particles bind to only one of the two possible surfaces of the chromaffin granule membranes. These results are in agreement with previous observations concerning the asymmetric distribution of saccharide residues on the surfaces of a number of different plasma membranes. They suggest that for the intracellular membrane of the chromaffin granule the saccharide sites, like those in plasma membranes, are not exposed to the cell cytoplasm. Further work is necessary to establish whether these sites are on the inner surface of the membrane or whether they are unmasked during the conversion of granules to membrane ghosts.     

Ionic requirements for membrane-glass adhesion and giga seal formation in patch-clamp recording

Patch-clamp recording has revolutionized the study of ion channels, transporters, and the electrical activity of small cells. Vital to this method is formation of a tight seal between glass recording pipette and cell membrane. To better understand seal formation and improve practical application of this technique, we examine the effects of divalent ions, protons, ionic strength, and membrane proteins on adhesion of membrane to glass and on seal resistance using both patch-clamp recording and atomic force microscopy. We find that H(+), Ca(2+), and Mg(2+) increase adhesion force between glass and membrane (lipid and cellular), decrease the time required to form a tight seal, and increase seal resistance. In the absence of H(+) (10(-10) M) and divalent cations (<10(-8) M), adhesion forces are greatly reduced and tight seals are not formed. H(+) (10(-7) M) promotes seal formation in the absence of divalent cations. A positive correlation between adhesion force and seal formation indicates that high resistance seals are associated with increased adhesion between membrane and glass. A similar ionic dependence of the adhesion of lipid membranes and cell membranes to glass indicates that lipid membranes without proteins are sufficient for the action of ions on adhesion.     

Tight junction formation in early <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryos: identification and ultrastructural characterization of junctional crests and junctional vesicles

How tight junctions (TJ) form during early amphibian embryogenesis is still an open question. We used time-lapse video microscopy, scanning electron microscopy (SEM), TEM and freeze-fracture to gain new insight into TJ biogenesis in early clevages of Xenopus laevis. Video analysis suggests three phases in junction formation between blastomeres. A first "waiting" phase, where new unpigmented lateral membranes are generated. A second "mixing" phase, where the unpigmented lateral membrane is separated from the pigmented apical membrane by an area showing a limited degree of intermingling of cortical pigment. And a third "sealing" phase, characterized by the formation of cingulin-containing boundaries between membrane domains, and their rapid directional adhesion in a zipper-like fashion. By SEM, we characterized these boundaries ("junctional crests", JC) as arrays of villiform protrusions at the border between old and new membranes. In the 2-cell embryo, JC are deeply located, and thus not visible at the surface, but they become increasingly more superficial as cleavages progress. After adjacent blastomeres have adhered to each other, fractured JC display linear arrays of junctional vesicles (JV) of 1-3 mum diameter. TEM analysis shows that JV are symmetrically located near the apposed membranes of adjacent blastomeres, and that the membranes near the JV display focal sites of intimate contact, typical of TJ. Freeze-fracture analysis confirms that intramembrane fibrils, typical of TJ, are present at adhesion sites. We conclude that TJ are formed following the sealing of JC, through the recruitment, sorting and assembly of membrane and cytoplasmic proteins at or near JV.     

Influence of increased concentration of Ca2+, La3+, PVP and urea on primary adhesivity of embryonic brain cells. Ultrastructural pattern of adhering cells

With the help of a previously described experimental arrangement the influence of increased external concentration of Ca2+, La3+, PVP and urea was tested on the initial stages of brain cell adhesivity and its kinetics. Urea, an inhibitor of hydrogen bonds, significantly inhibited the adhesivity of the treated cells. PVP significantly increased cell adhesivity. The adhesivity was enhanced and speeded up by increased concentrations of Ca2+ and La3+. It is evident that the membrane surface potential, zeta potential and formation of H+ bonds and bridges are highly important for cellular adhesivity. EM control of freshly dissociated cells disclosed that a part of the cells had been damaged. According to the ultrastructural organisation, the surface membrane is damaged to a small or greater extent. Intercellular contacts were formed in vitro either between non damaged surfaces of membranes, or between fragments of membranes, or contacts were mediated by membrane debris. Because cellular debris disappeared during rotation from single adhesive complexes, it is probable, that disrupted membranes are used for restoration of membranes, or serve as a metabolic substrates, or are catabolized.     

Probing the function of ionotropic and G protein-coupled receptors in surface-confined membranes

This article reports on recent electrical and optical techniques for investigating cellular signaling reactions in artificial and native membranes immobilized on solid supports. The first part describes the formation of planar artificial lipid bilayers on gold electrodes, which reveal giga-ohm electrical resistance and the insertion and characterization of ionotropic receptors therein. These membranes are suited to record a few or even single ion channels by impedance spectroscopy. Such tethered membranes on planar arrays of microelectrodes offer mechanically robust, long-lasting measuring devices to probe the influence of different chemistries on biologically important ionotropic receptors and therefore will have a future impact to probe the function of channel proteins in basic science and in biosensor applications. In a second part, we present complementary approaches to form inside-out native membrane sheets that are immobilized on micrometer-sized beads or across submicrometer-sized holes machined in a planar support. Because the native membrane sheets are plasma membranes detached from live cells, these approaches offer a unique possibility to investigate cellular signaling processes, such as those mediated by ionotropic or G protein-coupled receptors, with original composition of lipids and proteins.     

In vitro demonstration by the rate assay of the presence of small pore in the outer membrane of Pseudomonas aeruginosa

Determination of the rates of saccharide diffusions by the proteoliposomes showed that the outer membrane of Pseudomonas aeruginosa only possesses small diffusion pores and that protein F might have not been involved in the pore formation. Proteoliposomes containing stachyose or Dextan T-10 showed the same relative diffusion rates as measured by the liposome swelling method. Slopes of the lines, diffusion rate vs saccharide Mr, in the liposomes made of the P. aeruginosa and E. coli B outer membranes appeared to be -7.4 and -3.5, respectively. Intercepts of the lines with x-axis in the liposomes containing the P. aeruginosa and E. coli B outer membrane appeared to be about Mr, 220 and 320, respectively. Relative diffusion rates of saccharides through the liposome membranes reconstituted from the protein F-deficient outer membrane were superimposable with that of the protein F-sufficient outer membrane.     

Protein 4.1B in mouse islets of Langerhans and β-cell tumorigenesis

Protein 4.1 family proteins are thought to interact with membrane proteins and membrane skeletons. Immunohistochemical studies by light and electron microscopy were performed on mouse pancreas with a specific antibody against protein 4.1B. Specific protein 4.1B immunolabeling was observed on endocrine cells in the islets of Langerhans. Protein 4.1B localized along the plasma membranes facing adjacent cells. By immunoelectron microscopy, the immunolabeling of the cells was restricted to the cytoplasmic side just beneath their plasma membrane, including the membranes adjacent to neighboring cells, while the plasma membranes facing endothelial cells were not immunolabeled for protein 4.1B. The immunolocalization of E-cadherin was similar, if not identical, to that of protein 4.1B supporting the idea that protein 4.1B may be functionally interconnected with adhesion molecules. In a transgenic mouse model of pancreatic -cell carcinogenesis (Rip1Tag2), the loss of protein 4.1B expression coincided with the phenotypic transition from adenoma to carcinoma. Therefore, we propose a role of protein 4.1B as a connecting and/or signaling molecule between membrane architecture, cell adhesion, and tumor cell invasion in mouse pancreatic endocrine cells.     

Regulation of chloroplast membrane function: protein phosphorylation changes the spatial organization of membrane components

A chlorophyll-protein complex of chloroplast membranes, which simultaneously serves as light-harvesting antenna and membrane adhesion factor, undergoes reversible, lateral diffusion between appressed and nonappressed membrane regions under the control of a protein kinase. The phosphorylation-dependent migration process regulates the amount of light energy that is delivered to the reaction centers of photosystems I and II (PS I and PS II), and thereby regulates their rate of turnover. This regulatory mechanism provides a rationale for the finding that the two photosystems are physically separated in chloroplast membranes (PS II in appressed, grana membranes, and PS I in nonappressed, stroma membranes). The feedback system involves the following steps: a membrane-bound kinase senses the rate of PS II vs. PS I turnover via the oxidation-reduction state of the plastoquinone pool, which shuttles electrons from PS II via cytochrome f to PS I. If activated, the kinase adds negative charge (phosphate) to a grana- localized pigment-protein complex. The change in its surface charge at a site critical for promoting membrane adhesion results in increased electrostatic repulsion between the membranes, unstacking, the lateral movement of the complex to adjacent stroma membranes, which differ in their functional composition. The general significance of this type of membrane regulatory mechanism is discussed.     

Cysteine-disulfide cross-linking to monitor SNARE complex assembly during endoplasmic reticulum-Golgi transport

Assembly of cognate SNARE proteins into SNARE complexes is required for many intracellular membrane fusion reactions. However, the mechanisms that govern SNARE complex assembly and disassembly during fusion are not well understood. We have devised a new in vitro cross-linking assay to monitor SNARE complex assembly during fusion of endoplasmic reticulum (ER)-derived vesicles with Golgi-acceptor membranes. In Saccharomyces cerevisiae, anterograde ER-Golgi transport requires four SNARE proteins: Sec22p, Bos1p, Bet1p, and Sed5p. After tethering of ER-derived vesicles to Golgi-acceptor membranes, SNARE proteins are thought to assemble into a four-helix coiled-coil bundle analogous to the structurally characterized neuronal and endosomal SNARE complexes. Molecular modeling was used to generate a structure of the four-helix ER-Golgi SNARE complex. Based on this structure, cysteine residues were introduced into adjacent SNARE proteins such that disulfide bonds would form if assembled into a SNARE complex. Our initial studies focused on disulfide bond formation between the SNARE motifs of Bet1p and Sec22p. Expression of SNARE cysteine derivatives in the same strain produced a cross-linked heterodimer of Bet1p and Sec22p under oxidizing conditions. Moreover, this Bet1p-Sec22p heterodimer formed during in vitro transport reactions when ER-derived vesicles containing the Bet1p derivative fused with Golgi membranes containing the Sec22p derivative. Using this disulfide cross-linking assay, we show that inhibition of transport with anti-Sly1p antibodies blocked formation of the Bet1p-Sec22p heterodimer. In contrast, chelation of divalent cations did not inhibit formation of the Bet1p-Sec22p heterodimer during in vitro transport but potently inhibited Golgi-specific carbohydrate modification of glyco-pro-alpha factor. This data suggests that Ca(2+) is not directly required for membrane fusion between ER-derived vesicles and Golgi-acceptor membranes.     

Changes in membrane lipid composition cause alterations in epithelial cell-cell adhesion structures in renal papillary collecting duct cells

In epithelial tissues, adherens junctions (AJ) mediate cell-cell adhesion by using proteins called E-cadherins, which span the plasma membrane, contact E-cadherin on other cells and connect with the actin cytoskeleton inside the cell. Although AJ protein complexes are inserted in detergent-resistant membrane microdomains, the influence of membrane lipid composition in the preservation of AJ structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell-cell adhesion structures. We biochemically characterized the lipid composition of membranes containing AJ complexes. By using lipid membrane-affecting agents, we found that such agents induced the formation of new AJ protein-containing domains of different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that the membrane phospholipid composition plays an essential role in the in vivo maintenance of AJ structures involved in cell-cell adhesion structures in renal papillary collecting duct cells.     

Detailed mechanics of membrane-membrane adhesion and separation. I. Continuum of molecular cross-bridges

The mechanics of membrane-membrane adhesion are developed for the approximation that the molecular cross-bridging forces are continuously distributed as a normal stress (force per unit area). The significance of the analysis is that the finite range of the cross-bridging forces and the microscopic contact angle are not assumed negligible. Since the cross-bridging and adhesion forces are finite range interactions, there are two membrane regions: a free zone where the membranes are not subject to attractive forces; and an adherent zone where the membranes are held together by attractive stresses. The membrane is treated as an elastic continuum. The approach is to analyze the mechanics for each zone separately and then to require continuity of the solutions at the interface between the zones. Final solution yields the membrane contour and stresses proximal to and within the contact zone as well as the microscopic contact angle at the edge of the contact zone. It is demonstrated that the classical Young equation is consistent with this model. The results show that the microscopic contact angle becomes appreciable when the strength of adhesion is large or the length of the cross-bridge is large; however, the microscopic contact angle approaches zero as the membrane elastic stiffness increases. The solution predicts the width of the contact zone over which molecular bonds are stretched. It is this boundary region where increased biochemical activity is expected. In the classical model presented here, the level of tension necessary to oppose spreading of the contact is equal to the minimal level of tension required to separate the adherent membranes. This behavior is in contrast with that derived for the case of discrete molecular cross-bridges where the possibility of different levels of tension associated with adhesion and separation is introduced. The discrete cross-bridge case is the subject of a companion paper.     

Kinetics and mechanics of cell adhesion

Cell adhesion is mediated by specific interaction between receptors and ligands. Such interaction provides not only physical linkage but also communication between the cell and its environment. The kinetics and mechanics of cell adhesion are coupled, because force can influence the formation and dissociation of receptor-ligand bonds. The kinetic rates and their force dependence determine how likely, how rapidly and how strongly cells bind as well as how long they remain bound. Since adhesion molecules are linked to apposing cellular membranes, their interaction is governed by two-dimensional (2D) kinetics. This is in contrast to the three-dimensional (3D) binding of soluble ligands to cell surface receptors. Unlike the 3D case in which many methods are available for measuring kinetic rates, not until recently have the 2D kinetic rates become experimentally measurable. In this review, I will discuss the recent progress in the experimental methods that enable quantification of the relevant kinetic and mechanical parameters, the fundamental concepts that underlie the physics of the biological phenomena, and the mathematical models that relate functions to the intrinsic properties of the adhesion molecules.     

Morphology of cell-substratum adhesion

Many cell types modulate growth, differentiation, and motility through changes in cell substrate adhesion, including regulation of focal contact formation. Clustering of cell surface adhesion receptors is an essential early step in the development of focal contacts, and thus may influence cell physiology. In this paper, we present a theoretical framework to examine how cell surface chemistry affects receptor clustering. Our one-dimensional tape-peeling model couples the equations of mechanical equilibrium for a cell membrane with kinetic receptor-ligand binding relations. We considered two distinct model scenarios: Adhesion mediated by multiple receptor-ligand interactions of different length and specific binding of a single receptor type occurs in the presence of van der Waals attraction and nonspecific repulsion. In each case, nonuniform (wave-like) membrane morphologies are observed in certain parameter ranges that support the clustering of adhesion receptors. The formation of these morphologies is described in terms of a balance of membrane stresses; when cell-surface potential as a function of separation distance is symmetric between two potential energy minima, nonuniform morphologies are obtained. Increases in the chemical binding energy between receptor and ligand (e.g., increases in ligand density) or decreases in the membrane rigidity result in smaller wavelengths for nonuniform interfaces. Additionally, we show wave-like geometries appear only when the mechanical compliance of receptor-ligand bonds is within an intermediate range, and examine how the mobility of “repellers”—glycocalyx molecules that exert a nonspecific repulsive force—influences membrane morphology. We find fully mobile repellers always redistribute to prevent nonuniform morphologies.     

Morphology of cell-substratum adhesion. Influence of receptor heterogeneity and nonspecific forces.

Many cell types modulate growth, differentiation, and motility through changes in cell substrate adhesion, including regulation of focal contact formation. Clustering of cell surface adhesion receptors is an essential early step in the development of focal contacts, and thus may influence cell physiology. In this paper, we present a theoretical framework to examine how cell surface chemistry affects receptor clustering. Our one-dimensional tape-peeling model couples the equations of mechanical equilibrium for a cell membrane with kinetic receptor-ligand binding relations. We considered two distinct model scenarios: Adhesion mediated by multiple receptor-ligand interactions of different length and specific binding of a single receptor type occurs in the presence of van der Waals attraction and nonspecific repulsion. In each case, nonuniform (wave-like) membrane morphologies are observed in certain parameter ranges that support the clustering of adhesion receptors. The formation of these morphologies is described in terms of a balance of membrane stresses; when cell-surface potential as a function of separation distance is symmetric between two potential energy minima, nonuniform morphologies are obtained. Increases in the chemical binding energy between receptor and ligand (e.g., increases in ligand density) or decreases in the membrane rigidity result in smaller wavelengths for nonuniform interfaces. Additionally, we show wave-like geometries appear only when the mechanical compliance of receptor-ligand bonds is within an intermediate range, and examine how the mobility of "repellers"--glycocalyx molecules that exert a nonspecific repulsive force--influences membrane morphology. We find fully mobile repellers always redistribute to prevent nonuniform morphologies.     

Role of gangliosides in adhesion and conductance changes in large spherical model membranes

The formation of two spherical model membranes at the tips of two syringes has allowed us to study the role of gangliosides in membrane adhesion and look for changes in conductance between two such membranes during the process of adhesion. Membranes were formed in aqueous 100 mM NaCl, 10 mM KCl, 1 mM CaCl2 from 1% (w/v) egg phosphatidylcholine in n-decane, with or without mixed bovine brain gangliosides. After thinning to the 'black' bilayer state, two membranes were moved into contact. With gangliosides, the contact area and conductance increased colinearly with time over a 5 to 20 min period of adhesion. The role of electrostatic bridging by calcium was investigated. In the absence of calcium or in the presence of 2 mM EDTA, adhesion proceeded after a longer lag time at about one-half the normal rate. As the ganglioside concentration was increased from 0 to 15 mol%, the electrical conductance of individual membranes decreased 3-fold from 48 +/- 30 nS/cm2 to 17 +/- 13 nS/cm2. The conductance was pH dependent with a minimum at neutral values. At neutral pH, when two membranes containing 4.1 mol% gangliosides adhered, the region of adhesion had a specific conductance three times that of the nonadhering regions of membranes. Without gangliosides, the specific conductance of the contact region was the same as that of non-adhering regions of the membrane. These data suggest that mixed gangliosides can mediate an adhesion-dependent increase in conductance.     

Mitochondrial creatine kinase mediates contact formation between mitochondrial membranes

Purified mitochondrial creatine kinase (Mi-CK) (EC 2.7.3.2) from chicken heart was shown to interact simultaneously with purified inner and outer mitochondrial membranes, thereby creating an intermembrane chondrial membranes, thereby creating an intermembrane were purified from rat liver and thus were fully devoid of Mi-CK. Intermembrane contact formation was demonstrated by measuring the binding of inner membrane vesicles to outer membranes spread at the air-water interface. Mi-CK also mediated intermembrane adhesion when membranes formed with total lipid extracts of both membranes were used, pointing to the role of lipids as potential membrane anchors of Mi-CK in the mitochondrial intermembrane space. Other enzymes of the intermembrane space that (like Mi-CK) are also cationic, as well as cytosolic isoenzymes of creatine kinase, failed to induce contact formation. Thus, of the proteins tested, membrane contact formation was specific for Mi-CK. The two oligomeric forms of Mi-CK (octamer and dimer) differed in their ability to mediate intermembrane adhesion, the octamer being more potent. Highly basic peptides, i.e. poly-L-lysines, were shown to strongly interact with membranes formed with lipid extracts of mitochondrial membranes: they both induced intermembrane binding and fusion. Interestingly, the extent of contact formation mediated by poly-L-lysines was lower than that of octameric Mi-CK. The implications of these findings on the function and localization of Mi-CK and on the structure of the mitochondrial intermembrane compartment are discussed.     

Vinculin and Cell-Cell Adhesion

Vinculin, a 117-kDa protein, is a constituent of adhesion plaques and adherence junctions in non-muscle cells. We investigated the role of vinculin on the physical strength of cell-cell adhesion by conducting disaggregation assays on aggregates of parental wild-type F9 mouse embryonal carcinoma cells (clone BIM), two vinculin-depleted F9 cell lines, γ227 and γ229, and a reconstituted γ229 cell line (R3) that re-express vinculin. Immunoblotting demonstrated that the four cell lines used in the study had similar expressions of the cell-cell adhesion molecule E-cadherin and associated membrane proteins α- and β-catenin. Double immunofluorescence analysis showed that, in contrast to the vinculin-null cell lines, BIM and R3 cells expressed abundant vinculin at the cell margins in adhesion plaques and in cell-cell margins that also contained actin. Laminar flow assays showed that both the vinculin-positive and vinculinnegative cell aggregates that were formed in culture in the course of 24 to 48 hours largely remained intact despite the imposition of shear flow at high shear rates. Since laminar flow imposed on cell aggregates act to separate cells from each other, our data indicate that F9 cells that were adherent to a substrate formed strong cell-cell adhesion bonds independent of vinculin expression. On the other hand, aggregates of vinculin-depleted γ229 and γ227 cells that were formed in suspension during a two-hour static incubation at 37°C were desegregated more easily with the imposition of shear flow than the BIM and R3 cell aggregates formed under identical conditions. Loss of vinculin was associated with a reduction in cell-cell adhesion strength only among those cells lacking contact to a substrate. Overall, the results indicate that vinculin is not needed for forming strong cell-cell adhesion bonds between neighboring carcinoma cells which are adherent to the basal lamina.