A Highlights from MBoC Selection: Chloride Intracellular Channel 4 Is Critical for the Epithelial Morphogenesis of RPE Cells and Retinal Attachment

Retinal detachment is a sight-threatening condition. The molecular mechanism underlying the adhesion between the RPE and photoreceptors is poorly understood because the intimate interactions between these two cell types are impossible to model and study in vitro. In this article, we show that chloride intracellular channel 4 (CLIC4) is enriched at apical RPE microvilli, which are interdigitated with the photoreceptor outer segment. We used a novel plasmid-based transfection method to cell-autonomously suppress CLIC4 in RPE in situ. CLIC4 silenced RPE cells exhibited a significant loss of apical microvilli and basal infoldings, reduced retinal adhesion, and epithelial-mesenchymal transition. Ectopically expressing ezrin failed to rescue the morphological changes exerted by CLIC4 silencing. Neural retinas adjacent to the CLIC4-suppressed RPE cells display severe dysplasia. Finally, a high level of aquaporin 1 unexpectedly appeared at the apical surfaces of CLIC4-suppressed RPE cells, together with a concomitant loss of basal surface expression of monocarboxylate transporter MCT3. Our results suggested that CLIC4 plays an important role in RPE-photoreceptor adhesion, perhaps by modulating the activity of cell surface channels/transporters. We propose that these changes may be attributable to subretinal fluid accumulation in our novel retinal detachment animal model.     

Early adhesion of human mesenchymal stem cells on TiO(2) surfaces studied by single-cell force spectroscopy measurements

Understanding the interactions involved in the adhesion of living cells on surfaces is essential in the field of tissue engineering and biomaterials. In this study, we investigate the early adhesion of living human mesenchymal stem cells (hMSCs) on flat titanium dioxide (TiO(2) ) and on nanoporous crystallized TiO(2) surfaces with the use of atomic force microscopy-based single-cell force spectroscopy measurements. The choice of the substrate surfaces was motivated by the fact that implants widely used in orthopaedic and dental surgery are made in Ti and its alloys. Nanoporous TiO(2) surfaces were produced by anodization of Ti surfaces. In a typical force spectroscopy experiment, one living hMSC, immobilized onto a fibronectine-functionalized tipless lever is brought in contact with the surface of interest for 30 s before being detached while recording force-distance curves. Adhesion of hMSCs on nanoporous TiO(2) substrates having inner pore diameter of 45 nm was lower by approximately 25% than on TiO(2) flat surfaces. Force-distance curves exhibited also force steps that can be related to the pulling of membrane tethers from the cell membrane. The mean force step was equal to 35 pN for a given speed independently of the substrate surface probed. The number of tethers observed was substrate dependent. Our results suggest that the strength of the initial adhesion between hMSCs and flat or nanoporous TiO(2) surfaces is driven by the adsorption of proteins deposited from serum in the culture media.     

Application of fluid mechanic and kinetic models to characterize mammalian cell detachment in a radial-flow chamber

The strength of adhesion and dynamics of detachment of murine 3T3 fibroblasts from self-assembled monolayers were measured in a radial-flow chamber (RFC) by applying models for fluid mechanics, adhesion strength probability distributions, and detachment kinetics. Four models for predicting fluid mechanics in a RFC were compared to evaluate the accuracy of each model and the significance of inlet effects. Analysis of these models indicated an outer region at large radial positions consistent with creeping flow, an intermediate region influenced by inertial dampening, and an inner region dominated by entrance effects from the axially-oriented inlet. In accompanying experiments patterns of the fraction of cells resisting detachment were constructed for individual surfaces as a function of the applied shear stress and evaluated by comparison with integrals of both a normal and a log-normal distribution function. The two functions were equally appropriate, yielding similar estimates of the mean strength of adhesion. Further, varying the Reynolds number in the inlet, Re(d), between 630 and 1480 (corresponding to volumetric flow rates between 0.9 and 2.1 mL/s) did not affect the mean strength of adhesion. For these same experiments, analysis of the dynamics of detachment revealed three temporal phases: 1) rapid detachment of cells at the onset of flow, consistent with a first-order homogeneous kinetic model; 2) time-dependent rate of detachment during the first 30 sec. of exposure to hydrodynamic shear, consistent with the first-order heterogeneous kinetic model proposed by Dickinson and Cooper (1995); and 3) negligible detachment, indicative of pseudo-steady state after 60 sec. of flow. Our results provide rigorous guidelines for the measurement of adhesive interactions between mammalian cells and prospective biomaterial surfaces using a RFC. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 616-629, 1997.     

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.     

Forces Required to Initiate Membrane Tether Extrusion from Cell Surface Depend on Cell Type But Not on the Surface Molecule

When a cell adhered to another cell or substratum via surface proteins is forced to detach, lipid membrane tethers are often extruded from the cell surface before the protein bond dissociates. For example, during the inflammatory reaction leukocytes roll on the surface of activated endothelial cells. The rolling adhesion is mediated by interactions of selectins with their ligands, e.g., P-selectin glycoprotein ligand (PSGL)-1, which extrudes membrane tethers from the surfaces of both leukocytes and endothelial cells. Membrane tether extrusion has been suggested to regulate leukocyte rolling. Here we examine several factors that may affect forces required to initiate membrane tethers, or initial tether force. It was found that initial tether forces were similar regardless of the presence or absence of the cytoplasmic tail of P-selectin and regardless of whether the tethers were extruded via binding to PSGL-1 or Fcγ receptors. Initial tether forces were found to depend on the cell types tested and were greatly reduced by treatment of latrunculin A, which inhibits actin polymerization. These data provide additional insights to the control of membrane tether extrusion, which should be taken into account when cellular functions such as rolling where tether extrusion plays a regulatory role are compared using different cell types expressing the same molecule.     

Tether-guided lamellipodia enable rapid wound healing

Adhesion between animal cells and the underlying extracellular matrix is challenged during wounding, cell division, and a variety of pathological processes. How cells recover adhesion in the immediate aftermath of detachment from the extracellular matrix remains incompletely understood, due in part to technical limitations. Here, we used acute chemical and mechanical perturbations to examine how epithelial cells respond to partial delamination events. In both cases, we found that cells extended lamellipodia to establish readhesion within seconds of detachment. These lamellipodia were guided by sparse membrane tethers whose tips remained attached to their original points of adhesion, yielding lamellipodia that appear to be qualitatively distinct from those observed during cell migration. In vivo measurements in the context of a zebrafish wound assay showed a similar behavior, in which membrane tethers guided rapidly extending lamellipodia. In the case of mechanical wounding events, cells selectively extended tether-guided lamellipodia in the direction opposite of the pulling force, resulting in the rapid reestablishment of contact with the substrate. We suggest that membrane tether-guided lamellipodial respreading may represent a general mechanism to reestablish tissue integrity in the face of acute disruption.     

Platelet-activating factor mediates procoagulant activity on the surface of endothelial cells by promoting leukocyte adhesion

Endothelial cells co-express platelet-activating factor and P-selectin on their surfaces after activation by certain receptor-mediated agonists. Together they mediate the adhesion of leukocytes to the endothelial cell surface. P-selectin tethers leukocytes to the endothelial cells surface allowing leukocyte activation by platelet-activating factor. Adhesion and activation are specific for leukocytes because they are the only cells known to express the ligand for P-selectin. Leukocytes adherent to the endothelial cell surface may promote thrombosis by three mechanisms: (1) they secrete factors that damage the underlying endothelium, (2) they secrete factors that directly initiate the coagulation cascade, and (3) they bind and activate platelets.     

Variability of the Influence of Physicochemical Factors Affecting Bacterial Adhesion to Polystyrene Substrata

The role of electrostatic and hydrophobic interactions and solid and liquid surface tensions in the adhesion of four bacterial species (Pseudomonas fluorescens, Enterobacter cloacae, Chromobacterium sp., and Flexibacter sp.) to hydrophobic polystyrene petri dishes and to more hydrophilic polystyrene tissue culture dishes was investigated. The effect of electrostatic interactions was investigated by determining the effects of different electrolyte solutions on attachment to and of different electrolyte and pH solutions on detachment from the polystyrene substrate. The significance of solid and liquid surface tensions and hydrophobic interactions was investigated by measuring the effects of different surfactants (including a concentration series of dimethyl sulfoxide) on adhesion and detachment. Adhesion varied with bacterial species, substratum, and electrolyte type and concentration, with no apparent correlation between adhesion and electrolyte valence or concentration. The influence of different pH and detergent solutions on bacterial detachment also varied with species, substratum, pH, and detergent type; however, the greatest degree of detachment of all strains from the surfaces was produced by detergent treatment. The results suggest that adhesion cannot be attributed to any one type of adhesive interaction. There was some evidence for both electrostatic and hydrophobic interactions, but neither interaction could wholly account for the data.     

Gangliosides support neural retina cell adhesion

Cell surface carbohydrates and complementary carbohydrate receptors may mediate cell-cell recognition during neuronal development. To model such interactions, we developed a technique to test the ability of cell surface lipids (particularly glycosphingolipids) to mediate specific cell recognition and adhesion (Blackburn, C.C., and Schnaar, R.L. (1983) J. Biol. Chem. 258, 1180-1188). When cells were incubated on plastic microwells adsorbed with various glycolipids, carbohydrate-specific cell adhesion was readily detected. We report here the use of this method to study adhesion of embryonic chick neural retina cells to purified cell surface lipids. Rapid and specific cell adhesion was observed when the neural retina cells were incubated on surfaces adsorbed with gangliosides (an important class of neuronal cell surface glycoconjugates) but not on surfaces adsorbed with various neutral glycosphingolipids, phospholipids, or sulfatide. This suggests that the observed cell adhesion was specific for the carbohydrate moiety of the adsorbed ganglioside and was not due to nonspecific ionic or hydrophobic interactions. Although the surface density of adsorbed lipid required to support cell adhesion was the same for all gangliosides examined, the extent of adhesion varied when different purified gangliosides were used. Ganglioside-specific adhesion was not dependent on the presence of calcium (at 37 degrees C) and was attenuated by pretreatment of the cells with trypsin. The extent of ganglioside-directed neural retinal cell adhesion varied with embryonic age. These results imply that gangliosides may play a role in cell-cell recognition in the developing nervous system.     

Comparison of the fouling release properties of hydrophobic fluorinated and hydrophilic PEGylated block copolymer surfaces: attachment strength of the diatom Navicula and the green alga Ulva

To understand the role of surface wettability in adhesion of cells, the attachment of two different marine algae was studied on hydrophobic and hydrophilic polymer surfaces. Adhesion of cells of the diatom Navicula and sporelings (young plants) of the green macroalga Ulva to an underwater surface is mainly by interactions between the surface and the adhesive exopolymers, which the cells secrete upon settlement and during subsequent colonization and growth. Two types of block copolymers, one with poly(ethylene glycol) side-chains and the other with liquid crystalline, fluorinated side-chains, were used to prepare the hydrophilic and hydrophobic surfaces, respectively. The formation of a liquid crystalline smectic phase in the latter inhibited molecular reorganization at the surface, which is generally an issue when a highly hydrophobic surface is in contact with water. The adhesion strength was assessed by the fraction of settled cells (Navicula) or biomass (Ulva) that detached from the surface in a water flow channel with a wall shear stress of 53 Pa. The two species exhibited opposite adhesion behavior on the same sets of surfaces. While Navicula cells released more easily from hydrophilic surfaces, Ulva sporelings showed higher removal from hydrophobic surfaces. This highlights the importance of differences in cell-surface interactions in determining the strength of adhesion of cells to substrates.     

On the relative importance of specific and non-specific approaches to oral microbial adhesion

Abstract In this paper, it is suggested that specificity and non-specificity in (oral) microbial adhesion are different expressions for the same phenomena. It is argued that the same basic, physico-chemical forces are responsible for so-called 'non-specific' and 'specific' binding and that from a physico-chemical point of view the distinction between the two is an artificial one. Non-specific interactions arise from Van der Waals and electrostatic forces and hydrogen bonding, and originate from the entire cell. A specific bond consists of a combination of the same type of Van der Waals and electrostatic forces and hydrogen bonding, now originating from highly localized chemical groups, which together form a stereo-chemical combination. The absence or presence of specific receptor sites on microbial cell surfaces must therefore be reflected in the overall, non-specific surface properties of cells as well. This point is illustrated by showing that glucanbinding lectins on mutans streptococcal strains may determine the pH dependence of the zeta potentials of these cells. When studying microbial adhesion, a non-specific approach may be better suited to explain adhesion to inert substrata, whereas a specific approach may be preferred in case of adhesion to adsorbed protein films. Adhesion is, however, not as important in plaque formation in the human oral cavity as is retention, because low shear force periods. during which adhesion presumably occurs, are followed by high shear force periods, during which adhering cells must withstand these detachment forces. Evidence is provided that such detachment will be through cohesive failure in the pellicle mass, the properties of which are conditioned by the overall, non-specific substratum properties. Therefore, in vivo plaque formation may be more readily explained by a non-specific approach.     

Measurements of growth cone adhesion to culture surfaces by micromanipulation

Neurons were grown on plastic surfaces that were untreated, or treated with polylysine, laminin, or L1 and their growth cones were detached from their culture surface by applying known forces with calibrated glass needles. This detachment force was taken as a measure of the force of adhesion of the growth cone. We find that on all surfaces, lamellipodial growth cones require significantly greater detachment force than filopodial growth cones, but this differences is, in general, due to the greater area of lamellipodial growth cones compared to filopodial growth cones. That is, the stress (force/unit area) required for detachment was similar for growth cones of lamellipodial and filopodial morphology on all surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces, which had a significantly lower stress of detachment than on other surfaces. Surprisingly, the forces required for detachment (760-3,340 mudynes) were three to 15 times greater than the typical resting axonal tension, the force exerted by advancing growth cones, or the forces of retraction previously measured by essentially the same method. Nor did we observe significant differences in detachment force among growth cones of similar morphology on different culture surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces. These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture. Our micromanipulations revealed that the most mechanically resistant regions of growth cone attachment were confined to quite small regions typically located at the ends of filopodia and lamellipodia. Detached growth cones remained connected to the substratum at these regions by highly elastic retraction fibers. The closeness of contact of growth cones to the substratum as revealed by interference reflection microscopy (IRM) did not correlate with our mechanical measurements of adhesion, suggesting that IRM cannot be used as a reliable estimator of growth cone adhesion.     

Shear stress increases the residence time of adhesion of Pseudomonas aeruginosa

Although ubiquitous, the processes by which bacteria colonize surfaces remain poorly understood. Here we report results for the influence of the wall shear stress on the early-stage adhesion of Pseudomonas aeruginosa PA14 on glass and polydimethylsiloxane surfaces. We use image analysis to measure the residence time of each adhering bacterium under flow. Our main finding is that, on either surface, the characteristic residence time of bacteria increases approximately linearly as the shear stress increases (∼0–3.5 Pa). To investigate this phenomenon, we used mutant strains defective in surface organelles (type I pili, type IV pili, or the flagellum) or extracellular matrix production. Our results show that, although these bacterial surface features influence the frequency of adhesion events and the early-stage detachment probability, none of them is responsible for the trend in the shear-enhanced adhesion time. These observations bring what we believe are new insights into the mechanism of bacterial attachment in shear flows, and suggest a role for other intrinsic features of the cell surface, or a dynamic cell response to shear stress.     

On the relative importance of specific and non-specific approaches to oral microbial adhesion.

In this paper, it is suggested that specificity and non-specificity in (oral) microbial adhesion are different expressions for the same phenomena. It is argued that the same basic, physicochemical forces are responsible for so-called 'non-specific' and 'specific' binding and that from a physico-chemical point of view the distinction between the two is an artificial one. Non-specific interactions arise from Van der Waals and electrostatic forces and hydrogen bonding, and originate from the entire cell. A specific bond consists of a combination of the same type of Van der Waals and electrostatic forces and hydrogen bonding, now originating from highly localized chemical groups, which together form a stereochemical combination. The absence or presence of specific receptor sites on microbial cell surfaces must therefore be reflected in the overall, non-specific surface properties of cells as well. This point is illustrated by showing that glucan-binding lectins on mutans streptococcal strains may determine the pH dependence of the zeta potentials of these cells. When studying microbial adhesion, a non-specific approach may be better suited to explain adhesion to inert substrata, whereas a specific approach may be preferred in case of adhesion to adsorbed protein films. Adhesion is, however, not as important in plaque formation in the human oral cavity as is retention, because low shear force periods, during which adhesion presumably occurs, are followed by high shear force periods, during which adhering cells must withstand these detachment forces. Evidence is provided that such detachment will be through cohesive failure in the pellicle mass, the properties of which are conditioned by the overall, non-specific substratum properties. Therefore, in vivo plaque formation may be more readily explained by a non-specific approach.     

Adhesion and detachment characteristics of Chinese hamster cell membrane mutants

We have investigated the adhesion and detachment properties of wild-type Chinese hamster cells and of variant lines, which possess altered cell surface glycoproteins as detected by galactose oxidase-[3H]borohydride labeling. The wild-type and variant lines tested all adhered to protein-coated glass surfaces at the same rate; however, the variant cells differed from wild type and from each other in terms of the ease with which they were detached by trypsinization. Morphological differences between the various lines were also apparent. Our results suggest that the carbohydrate moieties of the terminal region of surface glycoproteins are not directly involved in the initial phase of cell-to-substratum attachment, but that they may modulate the proteolytic susceptibility of surface components which are involved in cell detachment.     

Depletion interactions in polymer solutions promote red blood cell adhesion to albumin-coated surfaces

The effects of concentration and molecular weight of neutral dextrans on the adhesion of human red blood cells (RBC) to albumin-coated glass have been investigated using a parallel-plate flow chamber. Results indicate that the adhesion is markedly increased in the presence of 70 kDa and 500 kDa dextran, with this increase reflected by both the number of cells adhering and the strength of the adhesion. This increased adhesiveness is attributed to reduced surface concentrations of the large polymers and hence attractive forces due to depletion interaction. Depletion interaction brings the adjacent surfaces closer, leading to an increased number of binding sites available to the cell and thus more efficient and stronger adhesion of single cells. Our results suggest that depletion might play a role in other specific cell-cell or cell-surface interactions via initiating close contacts to allow specific binding.     

Impact of surface energy and roughness on cell distribution and viability

Abstract

The aim of this study was to assess the respective impacts of the surface energy and surface roughness of bare and coated steels on biofouling and sanitisation. Bioadhesion of Staphylococcus aureus CIP 53.154 was studied on two stainless steel surfaces with smooth or specific micro-topography. Two coatings were also studied: silicon oxide (hydrophilic) and polysiloxane (hydrophobic). On smooth surfaces, adhesion was reduced on an apolar coating and cell viability increased with the surface polarity. A specific micro-topography decreased the level of bacterial adhesion on bare surfaces by a factor ten. On this surface, only single adherent cells were observed, contrasting with cells in clusters on smoother surfaces. As a consequence, cell repartition influenced bacterial viability. Most isolated adherent cells were dead whereas cells in clusters were still alive. In addition, the quaternary ammonium chloride used in sanitisation, acted at once both as a tensio-active molecule and a biocide. It only displaced adherent cells but did not remove them.     

Agonist Leukadherin-1 Increases CD11b/CD18-Dependent Adhesion Via Membrane Tethers

Integrin CD11b/CD18 is a key adhesion receptor that mediates leukocyte migration and immune functions. Leukadherin-1 (LA1) is a small molecule agonist that enhances CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. Here, we used single-molecule force spectroscopy to investigate the biophysical mechanism by which LA1-activated CD11b/CD18 mediates leukocyte adhesion. Between the two distinct populations of CD11b/CD18:ICAM-1 complex that participate in cell adhesion, the cytoskeleton(CSK)-anchored elastic elements and the membrane tethers, we found that LA1 enhanced binding of CD11b/CD18 on K562 cells to ICAM-1 via the formation of long membrane tethers, whereas Mn²⁺ additionally increased ICAM-1 binding via CSK-anchored bonds. LA1 activated wild-type and LFA1^−/− neutrophils also showed longer detachment distances and time from ICAM-1-coated atomic force microscopy tips, but significantly lower detachment force, as compared to the Mn²⁺-activated cells, confirming that LA1 primarily increased membrane-tether bonds to enhance CD11b/CD18:ICAM-1 binding, whereas Mn²⁺ induced additional CSK-anchored bond formation. The results suggest that the two types of agonists differentially activate integrins and couple them to the cellular machinery, providing what we feel are new insights into signal mechanotransduction by such agents.     

Studies on cell adhesion and recognition. I. Extent and specificity of cell adhesion triggered by carbohydrate-reactive proteins (glycosidases and lectins) and by fibronectin

The extent and the specificity of the initial cell attachment induced by various proteins coated on plastic surfaces have been studied with the following results: (a) Cell adhesion on the surfaces coated with sialidase and beta-galactosidase was as strong as on concanavalin A and limulus lectin-coated surfaces and the reactions were strongly inhibited by glycosidase inhibitors or by competitive substrates. The adhesion on sialidase was inhibited by 2-deoxy-2,3-dehydro-N- acetylneuraminic acid and by polysialoganglioside (GT1b) at low concentration (0.05-0.1 mM). The cell adhesion on beta-galactosidase coat was inhibited by 1,4-D-galactonolactone and beta-methylgalactoside but not by alpha-methylgalactoside. Thus, the initiation of cell adhesion on glycosidase surfaces could be mediated through the interactions of the specific binding sites of the enzyme surface with the cell surface substrates under physiological conditions. (b) Cell adhesion on various lectins could be blocked by various competing monosaccharides at the concentrations similar to the inhibitory concentrations for binding of lectins from solution to the cells. (c) Cell adhesion on fibronectin surfaces as well as on gelatin-coated surfaces was equally inhibited by GT1b at relatively high concentrations (0.25-0.5 mM). Lower concentrations of GT1b (0.05-0.1 mM) inhibited the cell adhesion on surfaces of Limulus lectin and sialidase. It is suggested that the cell adhesion mediated by fibronectin is based on yet unknown interactions in contrast to a specific cell adhesion through glycosidases and lectins.     

Agonist Leukadherin-1 Increases CD11b/CD18-Dependent Adhesion Via Membrane Tethers

Integrin CD11b/CD18 is a key adhesion receptor that mediates leukocyte migration and immune functions. Leukadherin-1 (LA1) is a small molecule agonist that enhances CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. Here, we used single-molecule force spectroscopy to investigate the biophysical mechanism by which LA1-activated CD11b/CD18 mediates leukocyte adhesion. Between the two distinct populations of CD11b/CD18:ICAM-1 complex that participate in cell adhesion, the cytoskeleton(CSK)-anchored elastic elements and the membrane tethers, we found that LA1 enhanced binding of CD11b/CD18 on K562 cells to ICAM-1 via the formation of long membrane tethers, whereas Mn²⁺ additionally increased ICAM-1 binding via CSK-anchored bonds. LA1 activated wild-type and LFA1^−/− neutrophils also showed longer detachment distances and time from ICAM-1-coated atomic force microscopy tips, but significantly lower detachment force, as compared to the Mn²⁺-activated cells, confirming that LA1 primarily increased membrane-tether bonds to enhance CD11b/CD18:ICAM-1 binding, whereas Mn²⁺ induced additional CSK-anchored bond formation. The results suggest that the two types of agonists differentially activate integrins and couple them to the cellular machinery, providing what we feel are new insights into signal mechanotransduction by such agents.