Adhesion among neural cells of the chick embryo. III. Relationship of the surface molecule CAM to cell adhesion and the development of histotypic patterns

We have previously identified a molecule (named cell adhesion molecule [CAM]) that is involved in the in vitro aggregation of neural cells from chick embryos. In the present report, specific anti-CAM antibodies have been used to demonstrated that CAM is localized in neural tissues, and is associated with the plasma membrane of retinal cells and neurites. Furthermore, it has been shown by antibody absorption techniques that the decreased adhesiveness of cultured retinal cells obtained originally from older embryos is correlated with a decrease in the density or accessibility of cell adhesion molecules on the surface of these cells. The central role of CAM in neural cell aggregation has been established by the observation that anti-CAM Fab' fragments inhibit adhesion between neural cells in a variety of assays. To investigate the function of CAM and cell adhesion in developing tissues, aggregates of retinal cells that are capable of forming histotypic patterns in vitro were cultured in the presence and absence of anti-CAM Fab'. The Fab' was found to inhibit sorting out of cell bodies and neurites and to decrease the number of membrane-membrane contacts, suggesting that CAM is associated with cell-cell, cell- neurite, and neurite-neurite interactions.     

Atomic force microscope-based single cell force spectroscopy of breast cancer cell lines: An approach for evaluating cellular invasion

The adhesiveness of cancerous cells to their neighboring cells significantly contributes to tumor progression and metastasis. The single-cell force spectroscopy (SCFS) approach was implemented to survey the cell–cell adhesion force between cancerous cells in three cancerous breast cell lines (MCF-7, T47D, and MDA-MB-231). The gene expression levels of two dominant cell adhesion markers (E-cadherin and N-cadherin) were quantified by real-time PCR. Additionally, the local stiffness of the cell bodies was measured by atomic force microscopy (AFM), and the actin cytoskeletal organization was examined by confocal microscopy. Results indicated that the adhesion force between cells was conversely correlated with their invasion potential. The highest adhesion force was observed in the MCF-7 cells. A reduction in cell–cell adhesion, which is required for the detachment of cells from the main tumor during metastasis, is partly due to the loss of E-cadherin expression and the enhanced expression of N-cadherins. The reduced adhesion was accompanied by the softening of cells, as described by the rearrangement of actin filaments through confocal microscopy observations. The softening of the cell body and the reduced cellular adhesiveness are two adaptive mechanisms through which malignant cells achieve the increased deformability, motility, and strong metastasis potential necessary for passage through endothelial junctions and positioning in host tissue. This study presented application of SCFS to survey cell phenotype transformation during cancer progression. The results can be implemented as a platform for further investigations that target the manipulation of cellular adhesiveness and stiffness as a therapeutic choice.     

SMA-1 spectrin has essential roles in epithelial cell sheet morphogenesis in C. elegans

During Caenorhabditis elegans development, the embryo acquires its vermiform shape due to changes in the shape of epithelial cells, a process that requires an apically localized actin cytoskeleton. We show that SMA-1, an ortholog of beta(H)-spectrin required for normal morphogenesis, localizes to the apical membrane of epithelial cells when these cells are rapidly elongating. In spc-1 alpha-spectrin mutants, SMA-1 localizes to the apical membrane but its organization is altered, consistent with the hypothesis these proteins act together to form an apically localized spectrin-based membrane skeleton (SBMS). SMA-1 is required to maintain the association between actin and the apical membrane; sma-1 mutant embryos fail to elongate because actin, which provides the driving force for cell shape change, dissociates from the apical membrane skeleton during morphogenesis. Analysis of sma-1 expression constructs and mutant strains indicates SMA-1 maintains the association between actin and the apical membrane via interactions at its N-terminus and this activity is independent of alpha-spectrin. SMA-1 also preserves dynamic changes in the organization of the apical membrane skeleton. Taken together, our results show the SMA-1 SBMS plays a dynamic role in converting changes in actin organization into changes in epithelial cell shape during C. elegans embryogenesis.     

Clustering of the neural cell adhesion molecule (NCAM) at the neuronal cell surface induces caspase-8- and -3-dependent changes of the spectrin meshwork required for NCAM-mediated neurite outgrowth

Changes in neuronal morphology underlying neuronal differentiation depend on rapid and sustained cytoskeleton rearrangements in the growing neurites. Whereas cell adhesion molecules are well established as regulators of neuronal differentiation, less is known about the signaling mechanisms by which they influence the cytoskeleton. Here we show that the neural cell adhesion molecule (NCAM) associates with the active form of caspase-8 and that clustering of NCAM at the neuronal cell surface leads to activation of caspase-8 and -3 followed by the cleavage of the sub-membranous brain spectrin meshwork, but not of the actin or tubulin cytoskeleton. Inhibitors of caspase-8 and -3 specifically block the NCAM-dependent spectrin cleavage and abolish NCAM-dependent neurite outgrowth. NCAM-dependent rearrangements of the membrane associated spectrin meshwork via caspase-8 dependent caspase-3 activation are thus indispensable for NCAM-mediated neurite outgrowth.     

Antibodies to the L1 adhesion molecule inhibit Schwann cell ensheathment of neurons in vitro

To investigate whether neural adhesion molecules are involved in neuron- induced Schwann cell differentiation, cocultures of pure dorsal root ganglion neurons, and Schwann cells were maintained in the presence of antibodies to evaluate possible perturbing effects. Several parameters characteristic of differentiating Schwann cells were studied, such as transition of spindle-shaped to flattened, i.e., more epithelioid morphology, association with neuronal cell bodies, ensheathment of neurites, production of basal lamina and collagen fibrils, and expression of the myelin associated glycoprotein (MAG). A complete ablation of Schwann cell differentiation in all features studied was seen with antibodies to the neural adhesion molecule L1. Antibodies to N-CAM did not reduce the association of Schwann cells with neurites but abolished the interdigitation of Schwann cell processes into neurite bundles, while leaving the other parameters studied unaffected. Fab fragments of antibodies to J1, MAG, and mouse liver membranes did not interfere with the manifestation of any of these parameters. None of the antibodies changed incorporation of [3H]thymidine into Schwann cells.     

L1CAM ubiquitination facilitates its lysosomal degradation

The cell adhesion molecule L1 is implicated in several processes in the developing and adult nervous system. Intracellular trafficking of L1 is important for cell migration, neurite growth and adhesion. We demonstrate here that L1 is ubiquitinated at the plasma membrane and in early endosomes. Mono-ubiquitination regulates L1 intracellular trafficking by enhancing its lysosomal degradation. We propose that L1’s ubiquitination might be an additional mechanism to control its re-appearance at the cell surface thereby influencing processes like neurite growth and cell adhesion.     

Inhibition of cell adhesion by phosphorylated Ezrin/Radixin/Moesin

Altered phosphorylation status of the C-terminal Thr residues of Ezrin/Radixin/Moesin (ERM) is often linked to cell shape change. To determine the role of phophorylated ERM, we modified phosphorylation status of ERM and investigated changes in cell adhesion and morphology. Treatment with Calyculin-A (Cal-A), a protein phosphatase inhibitor, dramatically augmented phosphorylated ERM (phospho-ERM). Cal-A-treatment or expression of phospho-mimetic Moesin mutant (Moesin-TD) induced cell rounding in adherent cells. Moreover, reattachment of detached cells to substrate was inhibited by either treatment. Phospho-ERM, Moesin-TD and actin cytoskeleton were observed at the plasma membrane of such round cells. Augmented cell surface rigidity was also observed in both cases.

Meanwhile, non-adherent KG-1 cells were rather rich in phospho-ERM. Treatment with Staurosporine, a protein kinase inhibitor that dephosphorylates phospho-ERM, up-regulated the integrin-dependent adhesion of KG-1 cells to substrate.

These findings strongly suggest the followings: (1) Phospho-ERM inhibit cell adhesion, and therefore, dephosphorylation of ERM proteins is essential for cell adhesion. (2) Phospho-ERM induce formation and/or maintenance of spherical cell shape. (3) ERM are constitutively both phosphorylated and dephosphorylated in cultured adherent and non-adherent cells.     

Depletion of protein kinase N3 (PKN3) impairs actin and adherens junctions dynamics and attenuates endothelial cell activation

Several pathways are involved in the control of endothelial cell morphology, endothelial permeability and function in order to maintain vascular homeostasis. Here we report that protein kinase N3 (PKN3) appears to play a pivotal role in maintaining endothelial cell morphology, cell-cell junctions and motility. An RNAi-based cell biological approach in cultured human endothelial cells (HUVEC) revealed that knockdown of PKN3 expression gave rise to cells with divergent cell morphology, impaired locomotion, disturbed adherens junctions (AJ) integrity and irregular actin organization. Notably, knockdown of PKN3 cells led to improper stress fiber formation and marked adhesiveness of intercellular adherens junctions when cells became stimulated with the pro-inflammatory cytokine TNF-α. Moreover, TNF-α-induced ICAM-1 expression on the cell surface was reduced in cells with suppressed PKN3 expression. Finally, loss-of-function for PKN3 appeared to affect Pyk2 phosphorylation in endothelial cells. These observations suggest that PKN3 can be considered a novel protein implicated in remodeling the actin-adherens junction, possibly by linking ICAM-1-signaling with actin/AJ dynamics. We propose that loss of PKN3 function and concomitant aberrations in actin rearrangement may attenuate pro-inflammatory activation of endothelial cells.     

Numb is a negative regulator of HGF dependent cell scattering and Rac1 activation

Numb is an endocytic adaptor protein that regulates internalization and post-endocytic trafficking of cell surface proteins. In polarized epithelial cells Numb is localized to the basolateral membrane, and recent work has implicated Numb in regulation of cell adhesion and migration, suggesting a role for Numb in epithelial–mesenchymal transition (EMT). We depleted MDCK cells of Numb and examined the effects downstream of EMT-promoting stimuli. While knockdown of Numb did not affect apicobasal polarity, we show that depletion of Numb destabilizes E-cadherin-based cell–cell adhesion and promotes loss of epithelial cell morphology. In addition, Numb knockdown in MDCK cells potentiates HGF-induced lamellipodia formation and cell dispersal. Examination of Rac1-GTP levels in Numb knockdown cells revealed hyperactivation of Rac1 following extracellular calcium depletion and HGF stimulation, which corresponds with enhanced loss of cell adhesions and lamellipodia formation. Furthermore, inhibition of Rac1 in Numb depleted cells stabilized cell–cell contacts following depletion of extracellular calcium. Together, these data indicate that Numb acts to suppress Rac1-GTP accumulation, and its loss leads to increased sensitivity toward extracellular signals that disrupt cell–cell adhesion to induce epithelial–mesenchymal transition (EMT) and cell dispersal.     

A zyxin-nectin interaction facilitates zyxin localization to cell-cell adhesions

Cell–cell junction remodeling is associated with dramatic actin reorganizations. Several actin regulatory systems have been implicated in actin remodeling events as cell–cell contacts are assembled and disassembled, including zyxin/LPP–VASP complexes. These complexes facilitate strong cell–cell adhesion by maintaining actin-membrane connections. It has been proposed that zyxin and LPP localize to cell–cell junctions via a well-defined interaction with alpha-actinin. This was recently confirmed for LPP, but zyxin localization at cell–cell contacts occurs independently of alpha-actinin binding. Here we seek to map the zyxin sequence responsible for localization to cell–cell contacts and identify the protein that docks zyxin at this cellular location. Previous results have shown that a zyxin fragment excluding the alpha-actin binding site and the LIM domains (amino acids 51–392) can independently localize to cell–cell contacts. Here, expression of smaller zyxin fragments show that zyxin localization requires amino acids 230–280. A yeast-two-hybrid screen, using the central region of zyxin as bait, resulted in the identification of the cell–cell adhesion receptor nectin-4 as a zyxin binding partner. Further demonstrating zyxin–nectin interactions, zyxin binds the intracellular domain of nectin-2 in vitro. Depletion of nectin-2 from L cells expressing E-cadherin results in a loss of zyxin localization to cell–cell contacts, demonstrating that the zyxin–nectin interaction plays a critical role in zyxin targeting to these sites.     

Identification of proteins bearing β1–6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis

The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary β1–6-N-acetylglucosamine (β1–6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing β1–6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35 — from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing β1–6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing β1–6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules.     

Inhibition of cell adhesion by phosphorylated Ezrin/Radixin/Moesin

Altered phosphorylation status of the C-terminal Thr residues of Ezrin/Radixin/Moesin (ERM) is often linked to cell shape change. To determine the role of phophorylated ERM, we modified phosphorylation status of ERM and investigated changes in cell adhesion and morphology. Treatment with Calyculin-A (Cal-A), a protein phosphatase inhibitor, dramatically augmented phosphorylated ERM (phospho-ERM). Cal-A-treatment or expression of phospho-mimetic Moesin mutant (Moesin-TD) induced cell rounding in adherent cells. Moreover, reattachment of detached cells to substrate was inhibited by either treatment. Phospho-ERM, Moesin-TD and actin cytoskeleton were observed at the plasma membrane of such round cells. Augmented cell surface rigidity was also observed in both cases.Meanwhile, non-adherent KG-1 cells were rather rich in phospho-ERM. Treatment with Staurosporine, a protein kinase inhibitor that dephosphorylates phospho-ERM, up-regulated the integrin-dependent adhesion of KG-1 cells to substrate.These findings strongly suggest the followings: (1) Phospho-ERM inhibit cell adhesion, and therefore, dephosphorylation of ERM proteins is essential for cell adhesion. (2) Phospho-ERM induce formation and/or maintenance of spherical cell shape. (3) ERM are constitutively both phosphorylated and dephosphorylated in cultured adherent and non-adherent cells.     

Interaction of the NG2 proteoglycan with the actin cytoskeleton

The NG2 chondroitin sulfate proteoglycan is a membrane-spanning molecule expressed by immature precursor cells in a variety of developing tissues. In tightly adherent cell lines with a flattened morphology, NG2 is organized on the cell surface in linear arrays that are highly co-localized with actin and myosin-containing stress fibers in the cytoskeleton. In contrast, microtubules and intermediate filaments in the cytoskeleton exhibit completely different patterns of organization, suggesting that NG2 may use microfilamentous stress fibers as a means of cytoskeletal anchorage. Consistent with this is the observation that cytochalasin D disrupts the organization of both stress fibers in the cytoskeleton and NG2 on the cell surface. Very similar linear cell surface arrays are also seen with three other cell surface molecules thought to interact with the actin cytoskeleton: the α₅β₁ integrin, the CD44 proteoglycan, and the L1 neuronal cell adhesion molecule. Since the cytoplasmic domains of these four molecules are dissimilar, it seems possible that cytoskeletal anchorage in each case may occur via different mechanisms. One indication of such differences can be seen in colchicine-treated cells which have lost their flattened morphology but still retain long actin-positive tendrils as remnants of the actin cytoskeleton. NG2 and α₅β₁ are associated with these tendrils while CD44 and L1 are not, suggesting that at least two subclasses of cell surface molecules exist which can interact with different subdomains of the actin cytoskeleton. © 1996 Wiley-Liss, Inc.     

IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor, or IFN-gamma.

Alteration in the surface membrane of endothelial cells (EC) is a feature of endothelial activation both at sites of inflammation in vivo and after stimulation with cytokines in vitro. The effects of stimulating EC with IL-1 or TNF include enhanced adhesiveness for polymorphonuclear leukocytes (PMN) and T cells, the induction of EC leukocyte adhesion molecule-1 (ELAM-1) expression, and the increased expression of intercellular adhesion molecule-1 (ICAM-1) and the 1.4C3 Ag. In contrast, IFN-gamma stimulation increases EC binding of T cells but not PMN and enhances ICAM-1 expression but not ELAM-1 or 1.4C3 Ag expression. Recently we have reported that the T cell-derived cytokine IL-4 also increases EC adhesiveness for T cells but not PMN. In this study we have examined the effect of IL-4 on the expression of several cytokine-inducible EC activation Ag, by using a previously described ELISA technique. IL-4 modulation of activation Ag expression was concentration dependent, optimal at around 100 U/ml, and exhibited a unique pattern compared to that seen with the other cytokines. Although, IL-4 stimulation increased 1.4C3 Ag expression (p less than 0.001), it significantly inhibited constitutive ICAM-1 expression (p less than 0.01) and did not induce ELAM-1. Furthermore, IL-4 exhibited significant synergy with IL-1 or TNF in inducing 1.4C3 Ag expression (p less than 0.001) but inhibited the increased expression of ICAM-1 produced by IL-1, TNF, or IFN-gamma (p less than 0.01) and inhibited the induction of ELAM-1 by IL-1 and TNF (p less than 0.001). In contrast, IL-4 had no effect on the expression of EC HLA-class I, -DR, -DP, or -DQ and neither enhanced nor inhibited the effect of IFN-gamma on the expression of these molecules. Finally, although IL-4 alone caused little if any shape change in EC monolayers, it strongly synergized with TNF or IFN-gamma in causing a change in shape to a more fibroblastic morphology. These observations indicate that IL-4 increases EC adhesiveness for T cells by the induction of a different adhesion molecule to ICAM-1. Furthermore, the ability of IL-4 to both enhance and inhibit the expression of activation Ag on EC already activated by IL-1, TNF, or IFN-gamma suggests that it may be important in altering the quality of inflammatory responses such as may occur during the development and maintenance of chronic or immune-mediated inflammation.     

The effect of the lipid-binding site of the ankyrin-binding domain of erythroid β-spectrin on the properties of natural membranes and skeletal structures

It was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of β-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482–94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na⁺K⁺ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the β-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.     

Effect of mechanical deformation of neutrophils on their CD18/ICAM-1-dependent adhesion.

Mechanical deformation of polymorphonuclear leukocytes (PMN) changes their expression of the surface adhesion molecule CD11b/CD18. We tested the hypothesis that mechanical deformation of PMN enhances their adhesiveness. Purified human PMN were deformed through either 5- or 3-microm polycarbonate membrane filters and allowed to adhere to 96-well plates coated with human recombinant intercellular adhesion molecule-1 (ICAM-1). Flow cytometric studies showed that deformation of PMN increased CD11b/CD18 expression (P < 0.01). PMN adhesion to ICAM-1-coated plates was dependent on the magnitude of cell deformation (5 microm, 63.8 +/- 8.1%, P < 0.04; 3 microm, 232.4 +/- 20.9%, P < 0.01). Priming of PMN (0.5 nM N-formyl-methionyl-leucyl-phenylalanine) before deformation (5 microm) increased PMN adhesion (63.8 +/- 8.1 vs. 105.3 +/- 16.4%; P < 0.04). Stimulation (5% zymosan-activated plasma) of PMN after deformation resulted in increased adhesion, and the degree of increase was dependent on the magnitude of PMN deformation (stimulation, 50.6 +/- 4%; 5-microm filtration and stimulation, 62.9 +/- 6.6%; 3-microm filtration and stimulation, 249.9 +/- 24.2%; P < 0.01). This study shows that mechanical deformation of PMN causes an increase in PMN adhesiveness to ICAM-1 that was enhanced by both priming of PMN before deformation and stimulation after cell deformation.     

Stimulation of endothelial cell binding of lymphocytes by tumor necrosis factor

Lymphokines and monokines have been reported to affect endothelial cell (EC) morphology and function. In experiments here described, we have demonstrated that recombinant tumor necrosis factor (TNF) stimulates the adhesion of T lymphocytes to confluent monolayers of human umbilical vein EC. The increase in adhesion induced by TNF was EC-specific inasmuch as preincubation of the lymphocytes with TNF did not alter binding, and preincubation of human dermal fibroblasts with TNF did not increase their inherently low adhesiveness for lymphocytes. Stimulation of T-EC binding occurred after treatment of the EC with as little as 0.01 U/ml (1 pg/ml) of TNF. In kinetic experiments, preincubation of EC with TNF for 4 hr resulted in optimal adhesion. TNF-treated EC retained their increased adhesiveness after fixation with paraformaldehyde, suggesting that TNF stimulated binding by increasing the expression or accessibility of EC surface receptors for lymphocytes. Although antibodies to the lymphocyte function-associated antigen 1 alpha- or beta-chains on the T cell markedly inhibited unstimulated T-EC binding, such antibodies had no effect on the increase in EC adhesiveness induced by TNF, indicating that the increased binding resulted from the generation of an alternate binding receptor on the EC membrane. These findings provide additional evidence that cytokines participate in the mobilization of mononuclear cells in the chronic inflammatory reaction by stimulation of the adhesiveness of endothelium for circulating lymphocytes.     

The role of the cytoskeleton in cellular adhesion molecule expression in tumor necrosis factor-stimulated endothelial cells

Leukocyte infiltration is a hallmark of the atherosclerotic lesion. These cells are captured by cellular adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-endothelial cell adhesion molecule (PECAM), and E-selectin, on endothelial cells (EC). We examined the role of the actin cytoskeleton in tumor necrosis factor-alpha (TNF-alpha)-induced translocation of CAMs to the cell surface. Human aortic EC were grown on 96-well plates and an ELISA was used to assess surface expression of the CAMs. TNF-alpha increased VCAM-1, ICAM-1, and E-selectin by 4 h but had no affect on the expression of PECAM. A functioning actin cytoskeleton was important for VCAM-1 and ICAM-1 expression as both cytochalasin D, an actin filament disruptor, and jasplakinolide, an actin filament stabilizer, attenuated the expression of these CAMs. These compounds were ineffective in altering E-selectin surface expression. Myosin light chains are phosphorylated in response to TNF-alpha and this appears to be regulated by Rho kinase instead of myosin light chain kinase. However, the Rho kinase inhibitor, Y27632, had no affect on TNF-alpha-induced CAM expression. ML-7, a myosin light chain kinase inhibitor, had a modest inhibitory effect on the translocation of VCAM-1 but not on ICAM-1 or E-selectin. These data suggest that the surface expression of VCAM-1 and ICAM-1 is dependent on cycling of the actin cytoskeleton. Nevertheless, modulation of actin filaments via myosin light chain phosphorylation is not necessary. The regulation of E-selectin surface expression differs from that of the other CAMs.     

Micromanipulation of adhesion of phorbol 12-myristate-13-acetate-stimulated T lymphocytes to planar membranes containing intercellular adhesion molecule-1.

This paper presents an analytical and experimental methodology to determine the physical strength of cell adhesion to a planar membrane containing one set of adhesion molecules. In particular, the T lymphocyte adhesion due to the interaction of the lymphocyte function associated molecule 1 on the surface of the cell, with its counter-receptor, intercellular adhesion molecule-1 (ICAM-1), on the planar membrane, was investigated. A micromanipulation method and mathematical analysis of cell deformation were used to determine (a) the area of conjugation between the cell and the substrate and (b) the energy that must be supplied to detach a unit area of the cell membrane from its substrate. T lymphocytes stimulated with phorbol 12-myristate-13-acetate (PMA) conjugated strongly with the planar membrane containing purified ICAM-1. The T lymphocytes attached to the planar membrane deviated occasionally from their round configuration by extending pseudopods but without changing the size of the contact area. These adherent cells were dramatically deformed and then detached when pulled away from the planar membrane by a micropipette. Detachment occurred by a gradual decrease in the radius of the contact area. The physical strength of adhesion between a PMA-stimulated T lymphocyte and a planar membrane containing 1,000 ICAM-1 molecules/micron 2 was comparable to the strength of adhesion between a cytotoxic T cell and its target cell. The comparison of the adhesive energy density, measured at constant cell shape, with the model predictions suggests that the physical strength of cell adhesion may increase significantly when the adhesion bonds in the contact area are immobilized by the actin cytoskeleton.     

Cell surface energy and membrane associated actin in lymphocytes

We have shown previously that membrane associated actin correlates with the migratory abilities of lymphocytes during recirculation, and that cell surface energy correlates with the adhesiveness of lymphocytes to other cells. In this study, measurements of actin content and cell surface energy have been made for various lymphocyte subpopulations to examine the possibility that recirculation ability may be related to nonspecific adhesiveness. We have found that: both cell surface energy and actin content combine to provide a consistent explanation for the relative rates of recirculation of various lymphocyte subpopulations, and cell surface energies and actin contents vary independently in these lymphocyte subpopulations. Comparison of the actin contents and cell surface energies of metastatic and nonmetastatic lymphoma cell lines indicated that the differences in metastatic potential were more likely attributable to specific receptor-ligand interactions than to nonspecific adhesiveness. Cell surface energy and actin content are consistent with the greater adhesiveness of B cells than T cells to nylon wool, providing a physical basis for this common cell separation technique.