Cation Type Specific Cell Remodeling Regulates Attachment Strength

Single-molecule experiments indicate that integrin affinity is cation-type-dependent, but in spread cells integrins are engaged in complex focal adhesions (FAs), which can also regulate affinity. To better understand cation-type-dependent adhesion in fully spread cells, we investigated attachment strength by application of external shear. While cell attachment strength is indeed modulated by cations, the regulation of integrin-mediated adhesion is also exceedingly complex, cell specific, and niche dependent. In the presence of magnesium only, fibroblasts and fibrosarcoma cells remodel their cytoskeleton to align in the direction of applied shear in an α₅-integrin/fibronectin-dependent manner, which allows them to withstand higher shear. In the presence of calcium or on collagen in modest shear, fibroblasts undergo piecewise detachment but fibrosarcoma cells exhibit increased attachment strength. These data augment the current understanding of force-mediated detachment by suggesting a dynamic interplay in situ between cell adhesion and integrins depending on local niche cation conditions.     

Investigating lasp-2 in cell adhesion: new binding partners and roles in motility

Focal adhesions are intricate protein complexes that facilitate cell attachment, migration, and cellular communication. Lasp-2 (LIM-nebulette), a member of the nebulin family of actin-binding proteins, is a newly identified component of these complexes. To gain further insights into the functional role of lasp-2, we identified two additional binding partners of lasp-2: the integral focal adhesion proteins vinculin and paxillin. Of interest, the interaction of lasp-2 with its binding partners vinculin and paxillin is significantly reduced in the presence of lasp-1, another nebulin family member. The presence of lasp-2 appears to enhance the interaction of vinculin and paxillin with each other; however, as with the interaction of lasp-2 with vinculin or paxillin, this effect is greatly diminished in the presence of excess lasp-1. This suggests that the interplay between lasp-2 and lasp-1 could be an adhesion regulatory mechanism. Lasp-2’s potential role in metastasis is revealed, as overexpression of lasp-2 in either SW620 or PC-3B1 cells—metastatic cancer cell lines—increases cell migration but impedes cell invasion, suggesting that the enhanced interaction of vinculin and paxillin may functionally destabilize focal adhesion composition. Taken together, these data suggest that lasp-2 has an important role in coordinating and regulating the composition and dynamics of focal adhesions.     

Divalent cations regulate the organization of integrins αvβ3 and αvβ5 on the cell surface

Extracellular divalent cations are important regulators of integrin ligand binding activity. In this study we evaluated how divalent cations affect the organization of integrins into focal adhesion sites. Integrins αvβ3 and αvβ5 were compared because they share a high degree of structural homology and because both integrins mediate cell adhesion to vitronectin. On MG-63 osteosarcoma cells, we found that both the extent and pattern of integrin organization was regulated by the type of extracellular divalent ion. Integrin αvβ3 organized in focal contacts when Mn²⁺ or Mg²⁺ was present, but not in Ca²⁺. In contrast, αvβ5 organized in focal contacts only when Ca²⁺ or Mg²⁺ was present. Integrin αvβ5 clustered in a centrally located punctate field on the ventral surface of the cell in the presence of Mn²⁺. These observations reveal a previously unappreciated role for divalent ions in regulating the organization of integrins into focal adhesion sites. © 1996 Wiley-Liss, Inc.     

Transglutaminase stabilizes melanoma adhesion under laminar flow

To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN). Such contacts may be stabilized by transglutaminase catalyzed-crosslinkage of cell focal adhesion proteins. We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN. At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling. Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT). Cells spread and remained adherent on immobilized FN at high WSDeTs (≥ 32.5 dynes/cm²). The high resistance of adherent cells to shear forces suggested that transglutaminase-mediated crosslinking might be involved. Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm²). In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN in a dose-dependent manner and prevented the formation of crosslinked¹²⁵I-FN complex that failed to enter a SDS-polyacrylamide gradient gel. The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN.     

Defining Extracellular Integrin α-Chain Sites That Affect Cell Adhesion and Adhesion Strengthening without Altering Soluble Ligand Binding

It was previously shown that mutations of integrin α4 chain sites, within putative EF-hand-type divalent cation-binding domains, each caused a marked reduction in α4β1-dependent cell adhesion. Some reports have suggested that α-chain “EF-hand” sites may interact directly with ligands. However, we show here that mutations of three different α4 “EF-hand” sites each had no effect on binding of soluble monovalent or bivalent vascular cell adhesion molecule 1 whether measured indirectly or directly. Furthermore, these mutations had minimal effect on α4β1-dependent cell tethering to vascular cell adhesion molecule 1 under shear. However, EF-hand mutants did show severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin α4 “EF-hand-like” sites may impair 1) static cell adhesion and 2) adhesion strengthening under shear flow by a mechanism that does not involve alterations of initial ligand binding.     

Shark cartilage extract interferes with cell adhesion and induces reorganization of focal adhesions in cultured endothelial cells

In this study, we examined the effects of shark cartilage extract on the attachment and spreading properties and the focal adhesion structure of cultured bovine pulmonary artery endothelial cells. Treatment with cartilage extract resulted in cell detachment from the substratum. Immunofluorescence staining of those treated cells that remained attached showed that, instead of being present in both central and peripheral focal adhesions as in control cells, both integrin alpha(v)beta(3) and vinculin were found only in peripheral focal adhesion and thinner actin filament bundles were seen. In addition to causing cell detachment, cartilage extract partially inhibited the initial adherence of the cells to the substratum in a dose-dependent manner. Integrin alpha(v)beta(3) and vinculin staining of these cells also showed a peripheral focal adhesion distribution pattern. Vitronectin induced cell spreading in the absence of serum, but was blocked by simultaneous incubation with cartilage extract, which was shown to inhibit both integrin alpha(v)beta(3) and vinculin recruitment to focal adhesion and the formation of stress fibers. Dot binding assays showed that these inhibitory effects on cell attachment and spreading were not due to direct binding of cartilage extract components to integrin alpha(v)beta(3) or vitronectin. Shark cartilage chondroitin sulfate had no inhibitory effect on either cell attachment or spreading of endothelial cells. These results show that the inhibitory effects of cartilage extract on cell attachment and spreading are mediated by modification of the organization of focal adhesion proteins.     

Measurement of Cationic and Intracellular Modulation of Integrin Binding Affinity by AFM-Based Nanorobot

Integrins are dynamic transmembrane cation-dependent heterodimers that both anchor cells in position and transduce signals into and out of cells. We used an atomic force microscope (AFM)-based nanorobotic system to measure integrin-binding forces in intact human intestinal epithelial Caco-2 cells. The AFM-based nanorobot enables human-directed, high-accuracy probe positioning and site-specific investigations. Functionalizing the AFM probe with an arginine-glycine-aspartate (RGD)-containing sequence (consensus binding sequence for integrins) allowed us to detect a series of peptide-cell membrane interactions with a median binding force of 115.1 ± 4.9 pN that were not detected in control interactions. Chelating divalent cations from the culture medium abolished these interactions, as did inhibiting intracellular focal adhesion kinase (FAK) using Y15. Adding 1 mM Mg²⁺ to the medium caused a rightward shift in the force-binding curve. Adding 1 mM Ca²⁺ virtually abolished the RGD-membrane specific interactions and blocked the Mg²⁺ effects. Cell adhesion assays demonstrated parallel effects of divalent cations and the FAK inhibitor on cell adhesion. These results demonstrate direct modulation of integrin-binding affinity by both divalent cations and intracellular signal inhibition. Additionally, three binding states (nonspecific, specific inactivated, and specific activated) were delineated from affinity measurements. Although other research has assumed that this process of integrin conformational change causes altered ligand binding, in this work we directly measured these three states in individual integrins in a physiologically based study.     

Transglutaminase stabilizes melanoma adhesion under laminar flow.

To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN). Such contacts may be stabilized by transglutaminase catalyzed-cross-linkage of cell focal adhesion proteins. We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN. At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling. Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT). Cells spread and remained adherent on immobilized FN at high WSDeTs (greater than or equal to 32.5 dynes/cm2). The high resistance of adherent cells to shear forces suggested that transglutaminase-mediated crosslinking might be involved. Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm2). In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN in a dose-dependent manner and prevented the formation of crosslinked 125I-FN complex that failed to enter a SDS-polyacrylamide gradient gel. The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN.     

Quantifying Cell Adhesion through Impingement of a Controlled Microjet

The impingement of a submerged, liquid jet onto a cell-covered surface allows assessing cell attachment on surfaces in a straightforward and quantitative manner and in real time, yielding valuable information on cell adhesion. However, this approach is insufficiently characterized for reliable and routine use. In this work, we both model and measure the shear stress exerted by the jet on the impingement surface in the micrometer-domain, and subsequently correlate this to jet-induced cell detachment. The measured and numerically calculated shear stress data are in good agreement with each other, and with previously published values. Real-time monitoring of the cell detachment reveals the creation of a circular cell-free area upon jet impingement, with two successive detachment regimes: 1), a dynamic regime, during which the cell-free area grows as a function of both the maximum shear stress exerted by the jet and the jet diameter; followed by 2), a stationary regime, with no further evolution of the cell-free area. For the latter regime, which is relevant for cell adhesion strength assessment, a relationship between the jet Reynolds number, the cell-free area, and the cell adhesion strength is proposed. To illustrate the capability of the technique, the adhesion strength of HeLa cervical cancer cells is determined ((34 ± 14) N/m²). Real-time visualization of cell detachment in the dynamic regime shows that cells detach either cell-by-cell or by collectively (for which intact parts of the monolayer detach as cell sheets). This process is dictated by the cell monolayer density, with a typical threshold of (1.8 ± 0.2) × 10⁹ cells/m², above which the collective behavior is mostly observed. The jet impingement method presents great promises for the field of tissue engineering, as the influence of both the shear stress and the surface characteristics on cell adhesion can be systematically studied.     

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.     

Effect of streptavidin RGD mutant on the adhesion of endothelial cells

Adhesion of endothelial cells (EC) to surfaces can be enhanced by supplementing the integrin-mediated adhesion with high-affinity streptavidin (SA) that links a biotinylated EC to a biotinylated surface. Biotin pullout from the EC membrane limits the effectiveness of this treatment, leading to a predominance of EC detachment by cohesive failure. In this study we investigated whether a RGD-SA mutant that links SA to EC integrin receptors, and eliminates EC biotinylation, improves EC adhesion. Suspended EC were incubated with the RGD-SA mutant prior to cell seeding, primarily via attachment to the RGD binding site on alpha(v)beta(3) integrin. RGD-SA-incubated EC were subsequently seeded onto a surface preadsorbed with a mixture of fibronectin (Fn) and biotinylated bovine serum albumin (b-BSA). Results showed EC adhesion supplemented with the RGD-SA-biotin system significantly increased cell retention under flow, critical shear stresses for detachment, focal contact area, and force per bond relative to SA used with biotinylated EC. These increases were accompanied by significant reductions in membrane fragments left behind following EC detachment, which suggested cohesive failure via cell membrane rupture was significantly reduced, and enhanced phosphorylation of focal adhesion kinase, which suggested activation and clustering of integrin receptors. Together, these results show that the integrin-independent augmentation of EC adhesion using SA-biotin can be further improved through use of an RGD-SA mutant.     

Effects of Physicochemical Factors on the Adhesion to Cellulose Avicel of the Ruminal Bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes subsp. succinogenes

Ruminococcus flavefaciens adhered instantly to cellulose, while Fibrobacter succinogenes had the highest percentage of adherent cells after about 25 min of contact between bacteria and cellulose. Adhesion of R. flavefaciens was unaffected by high concentrations of sugars (5%), temperature, pH, oxygen, metabolic inhibitors, and lack of Na⁺. In contrast, the attachment was affected by the removal of divalent cations (Mg²⁺ and Ca²⁺), the presence of cellulose derivatives (methylcellulose and hydroxyethylcellulose), and cystine. Adhesion of F. succinogenes was sensitive to low and high temperatures, high concentrations of glucose and cellobiose (5%), hydroxyethylcellulose (0.1%), redox potential, pH, lack of monovalent cations, and the presence of an inhibitor of membrane ATPases or lasalocid and monensin. Cells of F. succinogenes heated at 100°C no longer were adherent. On the other hand, adhesion was insensitive to the lack of divalent cations (Mg²⁺ and Ca²⁺), the presence of 2,4-dinitrophenol, tetrachlorosalicylanilide, or inhibitors of the electron transfer chains. Adhesion of F. succinogenes seems to be related to the metabolic functions of the cell. External proteins and/or cellulases themselves might play a part in the attachment process. Several mechanisms are probably involved in the adhesion of R. flavefaciens, the main one being the interaction between the large glycocalyx and the divalent cations Ca²⁺ and Mg²⁺. Hydrophobic bonds and enzymes may also be involved.     

Monitoring of integrin-mediated adhesion of human ovarian cancer cells to model protein surfaces by quartz crystal resonators: evaluation in the impedance analysis mode

The quartz crystal microbalance (QCM) was used to monitor specific, integrin-mediated adhesion of human ovarian cancer cells to distinct extracellular matrix (ECM) proteins immobilized on gold-coated quartz crystal resonators. The QCM was operated in the impedance analysis mode, where frequency shift as well as bandwidth are accessible on a broad range of overtones. The increase in bandwidth caused by covering the quartz resonator with cells was reproducible and largely independent of overtone order, whereas the frequency shift displayed some variability. Thus the bandwidth proved to be the more robust parameter for sensing cell adhesive events. The bandwidth increased in proportion to the number of seeded cells to the quartz crystal as long as the number was below 150,000 cells/ml. Comparing the resonance parameters on different harmonics, one finds that viscoelastic modeling with homogeneous layer systems cannot reproduce the results: lateral heterogeneity has to be taken into account. The differences in adhesive strength of human ovarian cancer cells towards selected ECM proteins monitored by QCM was in good agreement with data obtained by conventional cell adhesion assays. Strong cell adhesion was observed to the ECM proteins vitronectin (VN) and fibronectin (FN), while only weak attachment occurred on laminin. In order to prove specific, integrin alphavbeta3-mediated cell adhesion to its ligands FN and VN, the cyclic integrin alphavbeta3-directed peptide c(RGDfV) was used as competitor and significantly reversed cell adhesion. Since integrin interaction with ECM proteins is dependent on the presence of bivalent cations, cell detachment was also seen after treatment of cell monolayers with the chelator ethylene-dinitro-tetra-acetic acid (EDTA). The QCM technique is a reliable method to monitor cell adsorption to ECM-pretreated surfaces in real time. It may be an alternative tool for screening specific and selective antagonists of integrin/ECM interaction.     

Analysis of the Behaviours Mediating Barnacle Cyprid Reversible Adhesion

When exploring immersed surfaces the cypris larvae of barnacles employ a tenacious and rapidly reversible adhesion mechanism to facilitate their characteristic ‘walking’ behaviour. Although of direct relevance to the fields of marine biofouling and bio-inspired adhesive development, the mechanism of temporary adhesion in cyprids remains poorly understood. Cyprids secrete deposits of a proteinaceous substance during surface attachment and these are often visible as ‘footprints’ on previously explored surfaces. The attachment structures, the antennular discs, of cyprids also present a complex morphology reminiscent of both the hairy appendages used by some terrestrial invertebrates for temporary adhesion and a classic ‘suction cup’. Despite the numerous analytical approaches so-far employed, it has not been possible to resolve conclusively the respective contributions of viscoelastic adhesion via the proteinaceous ‘temporary adhesive’, ‘dry’ adhesion via the cuticular villi present on the disc and the behavioural contribution by the organism. In this study, high-speed photography was used for the first time to capture the behaviour of cyprids at the instant of temporary attachment and detachment. Attachment is facilitated by a constantly sticky disc surface – presumably due to the presence of the proteinaceous temporary adhesive. The tenacity of the resulting bond, however, is mediated behaviourally. For weak attachment the disc is constantly moved on the surface, whereas for a strong attachment the disc is spread out on the surface. Voluntary detachment is by force, requiring twisting or peeling of the bond – seemingly without any more subtle detachment behaviours. Micro-bubbles were observed at the adhesive interface as the cyprid detached, possibly an adaptation for energy dissipation. These observations will allow future work to focus more specifically on the cyprid temporary adhesive proteins, which appear to be fundamental to adhesion, inherently sticky and exquisitely adapted for reversible adhesion underwater.     

Alpha 1 beta 1 integrin on neural crest cells recognizes some laminin substrata in a Ca(2+)-independent manner

Neural crest cells migrate along pathways containing laminin and other extracellular matrix molecules. In the present study, we functionally and biochemically identify an alpha 1 beta 1 integrin heterodimer which bears the HNK-1 epitope on neural crest cells. Using a quantitative cell adhesion assay, we find that this heterodimer mediates attachment to laminin substrata prepared in the presence of Ca2+. Interestingly, neural crest cells bind to laminin-Ca2+ substrata in the presence or absence of divalent cations in the cell attachment medium. In contrast, the attachment of neural crest cells to laminin substrata prepared in the presence of EDTA, heparin, Mg2+, or Mn2+ requires divalent cations. Interactions with these laminin substrata are mediated by a different integrin heterodimer, since antibodies against beta 1 but not alpha 1 integrins inhibit neural crest cell attachment. Thus, the type of laminin substratum appears to dictate the choice of laminin receptor used by neural crest cells. The laminin conformation is determined by the ratio of laminin to Ca2+, though incorporation of heparin during substratum polymerization alters the conformation even in the presence of Ca2+. Once polymerized, the substratum appears stable, not being altered by soaking in either EDTA or divalent cations. Our findings demonstrate: (a) that the alpha 1 beta 1 integrin can bind to some forms of laminin in the absence of soluble divalent cations; (b) that substratum preparation conditions alter the conformation of laminin such that plating laminin in the presence of Ca2+ and/or heparin modulates its configuration; and (c) that neural crest cells utilize different integrins to recognize different laminin conformations.     

A theoretical analysis for the effect of focal contact formation on cell-substrate attachment strength.

For many cell types, growth, differentiation, and motility are dependent on receptor-mediated adhesion to ligand-coated surfaces. Focal contacts are strong, specialized, adhesive connections between cell and substrate in which receptors aggregate and connect extracellular ligand to intracellular cytoskeletal molecules. In this paper, we present a mathematical model to examine how focal contact formation affects cellular adhesive strength. To calculate adhesive strength with and without focal contacts, we use a one-dimensional tape peeling analysis to determine the critical tension necessary to peel the membrane. Receptor-ligand bonds are modeled as adhesive springs. In the absence of focal contacts, we derive analytic expressions for the critical tension at low and high ligand densities and show how membrane morphology affects adhesion. Then, focal contacts are modeled as cytoplasmic nucleation centers which bind adhesion receptors. The extent of adhesive strengthening upon focal contact formation depends on the elastic rigidity of the cytoskeletal connections, which determines the structural integrity of the focal contact itself. We consider two limits to this elasticity, very weak and rigid. Rigid cytoskeletal connections give much greater attachment strengths. The dependence of attachment strength on measurable model parameters is quite different in these two limits, which suggests focal contact structure might be deduced from properly performed adhesion experiments. Finally, we compare our model to the adhesive strengthening response reported for glioma cell adhesion to fibronectin (Lotz et al., 1989. J. Cell Biol. 109:1795-1805). Our model successfully predicts the observed detachment forces at 4 degrees C and yields values for the number of fibronectin receptors per glioma cell and the density of cytoskeletal connection molecules (talin) involved in receptor clusters which are consistent with measurements for other cell types. Comparison of the model with data at 37 degrees C suggests that while cytoskeletal cross-linking and clustering of fibronectin receptors significantly increases adhesion strength, specific glioma cell-substratum attachment sites possess little mechanical rigidity and detach through a peeling mechanism, consistent with the view that these sites of < or = 15 nm cell-substrate separation are precursors to fully formed, elastically rigid focal contacts.     

Hsp72 interacts with paxillin and facilitates the reassembly of focal adhesions during recovery from ATP depletion

The cytoprotective effect of heat stress proteins on epithelial cell detachment, an important cause of acute, ischemic renal failure, was examined after ATP depletion by evaluating focal adhesion complex (FAC) integrity. The intracellular distribution of FAC proteins (paxillin, talin, and vinculin) was assessed by immunohistochemistry before, during, and after exposure of renal epithelial cells to metabolic inhibitors. The resulting ATP depletion caused reversible re-distribution of all three proteins from focal adhesions to the cytosol. Paxillin, a key adaptor protein, was selected as a surrogate marker for FAC integrity in subsequent studies. Prior heat stress increased hsp72, a molecular chaperone, in both the Triton X-100-soluble and -insoluble protein fractions. Compared with ATP depleted control, heat stress significantly decreased paxillin and hsp72 shift from the Triton X-100 soluble to the insoluble protein fraction (an established marker of denaturation and aggregation); increased paxillin-hsp72 interaction detected by co-immunoprecipitation; enhanced paxillin extractability from Triton X-100-insoluble precipitates, increased the reformation of focal adhesions, and improved cell attachment (p < 0.05). To determine whether hsp72 mediates protection afforded by heat stress, cells were infected with adenovirus containing human hsp72 or empty vector. Hsp72 overexpression increased its interaction with paxillin and improved focal adhesion reformation during recovery, mimicking the protective effects of heat stress. These data suggest that hsp72 facilitates the reassembly of focal adhesions and improves cell attachment by reducing paxillin denaturation and increasing its re-solubilization after ATP depletion.     

Initial adhesion of murine fibroblasts to collagen and fibronectin occurs by two mechanisms

The adhesion of Balb/c 3T12 cells to fibronectin (FN) and to denatured (DC) or native (NC) collagen is differentially sensitive to divalent cations and to sodium azide. Short-time adhesion (10 min) to FN requires either Mg2+ or Mn2+, whereas only Mn2+ stimulates attachment to DC and NC. Azide treatment only slightly affects adhesion of cells to FN, but strongly inhibits cell attachment to DC and NC. Attachment to any of these substrata is unaffected by monensin and by treatment of the cells with an intracellular fraction, making unlikely the possibility that molecules released by secretion or cell lysis participate in the adhesive process. Soluble collagen inhibits the adhesion of cells to DC and NC, but does not affect adhesion to FN. Finally, rabbit antiserum against collagen binding proteins inhibits cell attachment to NC and DC; the cells, however, attach normally to FN in presence of this antiserum. Taken together, our results support the view that 3T12 cells attach directly to native or denatured collagens and that FN is not required for this process.     

Cell Adhesion Strength Is Controlled by Intermolecular Spacing of Adhesion Receptors

Spatial patterning of biochemical cues on the micro- and nanometer scale controls numerous cellular processes such as spreading, adhesion, migration, and proliferation. Using force microscopy we show that the lateral spacing of individual integrin receptor-ligand bonds determines the strength of cell adhesion. For spacings ≥90 nm, focal contact formation was inhibited and the detachment forces as well as the stiffness of the cell body were significantly decreased compared to spacings ≤50 nm. Analyzing cell detachment at the subcellular level revealed that rupture forces of focal contacts increase with loading rate as predicted by a theoretical model for adhesion clusters. Furthermore, we show that the weak link between the intra- and extracellular space is at the intracellular side of a focal contact. Our results show that cells can amplify small differences in adhesive cues to large differences in cell adhesion strength.     

Removal kinetics of Bacillus cereus spores from a stainless steel surface exposed to constant shear stress 1 ‐ experimental system

The efficiency of cleaning in place procedures in dairy industries can be greatly affected by the presence of spore‐forming bacteria, which are able to adhere strongly to surfaces and to survive disinfection procedures. Microbial adhesion has been extensively studied, but very few studies have yet reported on the hydrodynamic removal of microorganisms, due to the lack of simple, routinely performable techniques. In this paper, a methodology using a coaxial cylinder double gap viscometer is described, to study the removal kinetics of Bacillus cereus spores from a stainless steel support under hydrodynamic conditions. This method was shown to be reproducible, sensitive and easy to perform, and allowed spore hydrodynamic removal kinetics to be studied as a function of both adhesion and detachment conditions. A high ionic strength attachment medium was shown to enhance adhesion forces, provided it did not contain macromolecules. An increase in shear stress was found to be favorable to spore detachment (4 to 5 times more spores were detached at 28 Pa than at 2 Pa), but removal kinetics were not found to be significantly different for 2 and 15 Pa. Thus, the effect of shear stress on spore removal kinetics may not be linear.