Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response

Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125- fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex.     

Immediate-early signaling induced by E-cadherin engagement and adhesion

Epithelial cell-cell interactions require localized adhesive interactions between E-cadherin on opposing membranes and the activation of downstream signaling pathways that affect membrane and actin dynamics. However, it is not known whether E-cadherin engagement and activation of these signaling pathways are locally coordinated or whether signaling is sustained or locally down-regulated like other receptor-mediated pathways. To obtain high spatiotemporal resolution of immediate-early signaling events upon E-cadherin engagement, we used laser tweezers to place beads coated with functional E-cadherin extracellular domain on cells. We show that cellular E-cadherin accumulated rapidly around beads, reaching a sustained plateau level in 1-3 min. Phosphoinositides and Rac1 co-accumulated with E-cadherin, reached peak levels with E-cadherin, but then rapidly dispersed. Both E-cadherin and Rac1 accumulated independently of Rac1 GTP binding/hydrolysis, but these activities were required for Rac1 dispersal. E-cadherin accumulation was dependent on membrane dynamics and actin polymerization, but actin did not stably co-accumulate with E-cadherin; mathematical modeling showed that diffusion-mediated trapping could account for the initial E-cadherin accumulation. We propose that initial E-cadherin accumulation requires active membrane dynamics and involves diffusion-mediated trapping at contact sites; to propagate further contacts, phosphatidylinositol 3-kinase and Rac1 are transiently activated by E-cadherin engagement and initiate a new round of membrane dynamics, but they are subsequently suppressed at that site to allow maintenance of weak E-cadherin mediated adhesion.     

Regulation of the assembly and adhesion activity of E-cadherin by nectin and afadin for the formation of adherens junctions in Madin-Darby canine kidney cells

The Ca2+-independent immunoglobulin-like molecule nectin first forms cell-cell adhesion and then assembles cadherin at nectin-based cell-cell adhesion sites, resulting in the formation of adherens junctions (AJs). Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Here, we studied the roles and modes of action of nectin and afadin in the formation of AJs in cultured MDCK cells. The trans-interaction of nectin assembled E-cadherin, which associated with p120(ctn), beta-catenin, and alpha-catenin, at the nectin-based cell-cell adhesion sites in an afadin-independent manner. However, the assembled E-cadherin showed weak cell-cell adhesion activity and might be the non-trans-interacting form. This assembly was mediated by the IQGAP1-dependent actin cytoskeleton, which was organized by Cdc42 and Rac small G proteins that were activated by the action of trans-interacting nectin through c-Src and Rap1 small G protein in an afadin-independent manner. However, Rap1 bound to afadin, and this Rap1-afadin complex then interacted with p120(ctn) associated with non-trans-interacting E-cadherin, thereby causing the trans-interaction of E-cadherin. Thus, nectin regulates the assembly and cell-cell adhesion activity of E-cadherin through afadin, nectin signaling, and p120(ctn) for the formation of AJs in Madin-Darby canine kidney cells.     

Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion

Epithelial cell-cell adhesion requires interactions between opposing extracellular domains of E-cadherin, and among the cytoplasmic domain of E-cadherin, catenins, and actin cytoskeleton. Little is known about how the cadherin-catenin-actin complex is assembled upon cell-cell contact, or how these complexes initiate and strengthen adhesion. We have used time-lapse differential interference contrast (DIC) imaging to observe the development of cell-cell contacts, and quantitative retrospective immunocytochemistry to measure recruitment of proteins to those contacts. We show that E-cadherin, alpha-catenin, and beta- catenin, but not plakoglobin, coassemble into Triton X-100 insoluble (TX-insoluble) structures at cell-cell contacts with kinetics similar to those for strengthening of E-cadherin-mediated cell adhesion (Angres, B., A. Barth, and W.J. Nelson. 1996. J. Cell Biol. 134:549- 557). TX-insoluble E-cadherin, alpha-catenin, and beta-catenin colocalize along cell-cell contacts in spatially discrete micro-domains which we designate "puncta," and the relative amounts of each protein in each punctum increase proportionally. As the length of the contact increases, the number of puncta increases proportionally along the contact and each punctum is associated with a bundle of actin filaments. These results indicate that localized clustering of E- cadherin/catenin complexes into puncta and their association with actin is involved in initiating cell contacts. Subsequently, the spatial ordering of additional puncta along the contact may be involved in zippering membranes together, resulting in rapid strengthening of adhesion.     

Mechanisms of Epithelial Cell–Cell Adhesion and Cell Compaction Revealed by High-resolution Tracking of E-Cadherin–?Green Fluorescent Protein

Cadherin-mediated adhesion initiates cell reorganization into tissues, but the mechanisms and dynamics of such adhesion are poorly understood. Using time-lapse imaging and photobleach recovery analyses of a fully functional E-cadherin/GFP fusion protein, we define three sequential stages in cell–cell adhesion and provide evidence for mechanisms involving E-cadherin and the actin cytoskeleton in transitions between these stages. In the first stage, membrane contacts between two cells initiate coalescence of a highly mobile, diffuse pool of cell surface E-cadherin into immobile punctate aggregates along contacting membranes. These E-cadherin aggregates are spatially coincident with membrane attachment sites for actin filaments branching off from circumferential actin cables that circumscribe each cell. In the second stage, circumferential actin cables near cell–cell contact sites separate, and the resulting two ends of the cable swing outwards to the perimeter of the contact. Concomitantly, subsets of E-cadherin puncta are also swept to the margins of the contact where they coalesce into large E-cadherin plaques. This reorganization results in the formation of a circumferential actin cable that circumscribes both cells, and is embedded into each E-cadherin plaque at the contact margin. At this stage, the two cells achieve maximum contact, a process referred to as compaction. These changes in E-cadherin and actin distributions are repeated when additional single cells adhere to large groups of cells. The third stage of adhesion occurs as additional cells are added to groups of >3 cells; circumferential actin cables linked to E-cadherin plaques on adjacent cells appear to constrict in a purse-string action, resulting in the further coalescence of individual plaques into the vertices of multicell contacts. The reorganization of E-cadherin and actin results in the condensation of cells into colonies. We propose a model to explain how, through strengthening and compaction, E-cadherin and actin cables coordinate to remodel initial cell–cell contacts to the final condensation of cells into colonies.     

Cell-specific association of heat shock-induced proton flux with actin ring formation in Chenopodium cells: comparison of auto- and heterotroph cultures

A comparison of the responses of extracellular pH, buffering capacity and actin cytoskeleton in autotroph and heterotroph Chenopodium rubrum cells to heat shock revealed cell-specific reactions: alkalinization caused by the heat shock at 25-35 degrees C was higher in heterotroph cells and characterized by heat shock-induced changes in the actin cytoskeleton and ring formation at 35-37 degrees C. Rings (diameter up to 3 mum) disappeared and extracellular pH recovered after the heat-shocked cells were transferred into control medium. At 41 degrees C, no rings but a network of coarse actin filaments were induced; at higher temperatures, fragmentation of the actin cytoskeleton and release of buffering compounds occurred, indicating sudden membrane leakage at 45-47 degrees C. The calcium chelator EGTA [ethylene-glycol-bis(beta-aminoethyl-ether)-N,N,N',N'-tetraacetic-acid] increased the frequency of heat shock-induced rings. Ionophore (10 microM nigericin) and the sodium/proton antiport blocker [100 microM 5-(N-ethyl-N-isopropyl)-amiloride] mimicked the effect of the 37 degrees C heat shock. The cytoskeleton inhibitors latrunculin B, cytochalasin D and 2,3-butanedione monoxime inhibited ring formation but not alkalinization. In autotroph cells, the treatment with nigericin (10 microM) produced rings, although the actin cytoskeleton was not affected by temperatures up to 45 degrees C. We conclude that Chenopodium cells express a specific temperature sensor that has ascendancy over the organization of the actin cytoskeleton; this is probably a temperature- and potential-sensitive proton-transporting mechanism that is dependent on the culture conditions of the heterotroph cells.     

Essential role of Src suppressed C kinase substrates in endothelial cell adhesion and spreading

Integrin-mediated substrate adhesion of endothelial cells leads to dynamic rearrangement of the actin cytoskeleton. Protein kinase C (PKC) stimulates reorganization of microfilaments and adhesion, but the mechanism by which this occurs is unknown. Src suppressed C kinase substrate (SSeCKS) is a PKC substrate that may play an important role in regulating actin cytoskeleton. We found that SSeCKS was localized to focal adhesion sites soon after cell adhesion and that SSeCKS translocated from the membrane to the cytosol during the process of cell spreading. Using small interfering RNAs specific to SSeCKS, we show that RPMVEC cells in which SSeCKS expression was inhibited reduce adhesion and spread on LN through blocking the formation of actin stress fibers and focal adhesions. These results demonstrated SSeCKS modulate endothelial cells adhesion and spreading by reorganization of the actin cytoskeleton.     

Essential role of SRC suppressed C kinase substrates in Schwann cells adhesion, spreading and migration

Integrin-mediated substrate adhesion of endothelial cells leads to dynamic rearrangement of the actin cytoskeleton. Protein kinase C (PKC) stimulates reorganization of microfilaments and adhesion, while the responses of Schwann cells during adhesion and migration are unknown, so we examined the expression changes of SSeCKS and F-actin in Schwann cells after exposure to fibronectin. Src (sarcoma) suppressed C kinase substrate (SSeCKS) is a PKC substrate that may play an important role in regulating actin cytoskeleton. We found that SSeCKS was localized to focal adhesion sites soon after Schwann cells adhesion and that SSeCKS increased during the process of cell spreading. Using small interfering RNAs specific to SSeCKS, we showed that Schwann cells in which SSeCKS expression was inhibited reduced cellular adhesion, spreading and promoted cellular migration on fibronectin through reorganization of actin stress fibers and blocking formation of focal adhesions. These results demonstrated SSeCKS modulate Schwann cells adhesion, spreading and migration by reorganization of the actin cytoskeleton.     

Casein kinase II phosphorylation of E-cadherin increases E-cadherin/beta-catenin interaction and strengthens cell-cell adhesion

Beta-catenin, a member of the Armadillo repeat protein family, binds directly to the cytoplasmic domain of E-cadherin, linking it via alpha-catenin to the actin cytoskeleton. A 30-amino acid region within the cytoplasmic domain of E-cadherin, conserved among all classical cadherins, has been shown to be essential for beta-catenin binding. This region harbors several putative casein kinase II (CKII) and glycogen synthase kinase-3beta (GSK-3beta) phosphorylation sites and is highly phosphorylated. Here we report that in vitro this region is indeed phosphorylated by CKII and GSK-3beta, which results in an increased binding of beta-catenin to E-cadherin. Additionally, in mouse NIH3T3 fibroblasts expression of E-cadherin with mutations in putative CKII sites resulted in reduced cell-cell contacts. Thus, phosphorylation of the E-cadherin cytoplasmic domain by CKII and GSK-3beta appears to modulate the affinity between beta-catenin and E-cadherin, ultimately modifying the strength of cell-cell adhesion.     

Influence of receptor lateral mobility on adhesion strengthening between membranes containing LFA-3 and CD2

We have used an in vitro model system of glass-supported planar membranes to study the effects of lateral mobility of membrane-bound receptors on cell adhesion. Egg phosphatidylcholine (PC) bilayers were reconstituted with two anchorage isoforms of the adhesion molecule lymphocyte function-associated antigen 3 (LFA-3). The diffusion coefficient of glycosyl phosphatidylinositol (GPI)-anchored LFA-3 approached that of phospholipids in the bilayers, whereas the transmembrane (TM)-anchored isoform of LFA-3 was immobile. Both static and laminar flow assays were used to quantify the strength of adherence to the lipid bilayers of the T lymphoma cell line Jurkat that expresses the counter-receptor CD2. Cell adhesion was dependent on LFA-3 density and was more efficient on membranes containing the GPI isoform than the TM isoform. Kinetic measurements demonstrated an influence of contact time on the strength of adhesion to the GPI isoform at lower site densities (25-50 sites/microns2), showing that the mobility of LFA-3 is important in adhesion strengthening. At higher site densities (1,500 sites/microns2) and longer contact times (20 min), Jurkat cell binding to the TM and GPI isoforms of LFA-3 showed equivalent adhesion strengths, although adhesion strength of the GPI isoform developed twofold more rapidly than the TM isoform. Reduction of CD2 mobility on Jurkat cells at 5 degrees C greatly decreased the rate of adhesion strengthening with the TM isoform of LFA-3, resulting in a 30-fold difference between the two LFA-3 isoforms. Our results demonstrate that the ability of a membrane receptor and its membrane-bound counter-receptor to diffuse laterally enhances cell adhesion both by allowing accumulation of ligands in the cell contact area and by increasing the rate of receptor-ligand bond formation.     

The effect of short-term temperature changes on the adhesivity of nerve cells. Participation of DNA molecules on non-specific cellular adhesion

Brain cells from 16 to 18-day-old mice embryos were dissociated by mild trypsinization and sieving. Immediately after dissociation the cells were preincubated in a PBS solution at -6 to +54 degrees C for 3 and 20 min. After this preincubation cells were rotated for 60 min at 37 degrees C in the PBS solution. Cellular adhesivity was estimated during this time period and EM pictures of organized in vitro aggregates after 24-28 h were taken. In a separate series of experiments, freshly dissociated were treated with DNAase before the rotation procedure. Preincubation in a cold or a warm medium did not alter the inhibition of cellular adhesivity significantly. Distinct inhibition of cellular adhesion was observed in cells preincubated above 53 degrees C. Adhesion was also inhibited below -5 degrees C, however, this effect was mainly dependent on the rate of freezing and thawing. Digestion of dissociated cells with DNAase (20 micrograms/ml) decreased cell adhesion. At 37 degrees C the adhesivity decreased by about 20%. Aggregates of cells preincubated at 0 degrees C for 20 min did not exhibit marked EM changes after 24-28 h in vitro. The present results have shown the rather high resistance of molecules responsible for cellular adhesion and its reversibility to temperature changes. Furthermore, non-specific cellular adhesion was shown on physically active DNA molecules.     

CD2AP contributes to cell migration and adhesion in cultured gastric epithelium

The potential association of CD2AP with the adherens junction protein E-cadherin, co-localization with the actin cytoskeleton, and involvement in cell migration was investigated in cultured rat gastric mucosal cells. In stationary cells, CD2AP was localized perinuclearly while E-cadherin was expressed along cell-cell contacts and F-actin formed a branched network and adhesion belts. In migrating cells, CD2AP appeared as thread-like accumulations in the leading edges, colocalizing with F-actin and occasionally with E-cadherin. Intracellular injection of anti-CD2AP significantly retarded the migration speed of the cells suggesting a crucial role for CD2AP in mucosal cell migration, possibly as a scaffolding protein between cell membrane proteins and actin cytoskeleton. Co-immunoprecipitation assays revealed that CD2AP and E-cadherin are in a complex in HGF stimulated cells. It is concluded that CD2AP interacts with E-cadherin and co-localizes with F-actin in the leading edge of migrating cells, and significantly contributes to cell migration in restituting gastric epithelium.     

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.     

Evidence for heterophilic adhesion of embryonic retinal cells and neuroblastoma cells to substratum-adsorbed NCAM

The adhesion of embryonic chicken retinal cells and mouse N2A neuroblastoma cells to purified embryonic chicken retinal NCAM adsorbed on a solid substratum was examined using a quantitative centrifugal adhesion assay. Both cell types adhered to NCAM and the adhesion was specifically inhibited by monovalent anti-NCAM antibody fragments. N2A cell adhesion depended on the amount of NCAM applied to the substratum, was cation independent, and was insensitive to treatment with the cytoskeletal perturbing drugs colchicine and cytochalasin D. These results indicated that the tubulin and actin cytoskeletons were not critically required for adhesion to NCAM and make it unlikely that the cell surface ligand for NCAM is an integrin. Adhesion was however temperature dependent, strengthening greatly after a brief incubation at 37 degrees C. CHO cells transfected with NCAM cDNAs did not adhere specifically to substratum-bound NCAM and pretreatment of N2A cells and retinal cells with anti-NCAM antibodies did not inhibit adhesion to substratum-bound NCAM. These results suggest that a heterophilic interaction between substratum-adsorbed NCAM and a non-NCAM ligand on the surface of the probe cells affects adhesion in this system and support the possibility that heterophilic adhesion may be a function of NCAM in vivo.     

Rapid suppression of activated Rac1 by cadherins and nectins during de novo cell-cell adhesion

Cell-cell adhesion in simple epithelia involves the engagement of E-cadherin and nectins, and the reorganization of the actin cytoskeleton and membrane dynamics by Rho GTPases, particularly Rac1. However, it remains unclear whether E-cadherin and nectins up-regulate, maintain or suppress Rac1 activity during cell-cell adhesion. Roles for Rho GTPases are complicated by cell spreading and integrin-based adhesions to the extracellular matrix that occur concurrently with cell-cell adhesion, and which also require Rho GTPases. Here, we designed a simple approach to examine Rac1 activity upon cell-cell adhesion by MDCK epithelial cells, without cell spreading or integrin-based adhesion. Upon initiation of cell-cell contact in 3-D cell aggregates, we observed an initial peak of Rac1 activity that rapidly decreased by ～66% within 5 minutes, and further decreased to a low baseline level after 30 minutes. Inhibition of E-cadherin engagement with DECMA-1 Fab fragments or competitive binding of soluble E-cadherin, or nectin2alpha extracellular domain completely inhibited Rac1 activity. These results indicate that cadherins and nectins cooperate to induce and then rapidly suppress Rac1 activity during initial cell-cell adhesion, which may be important in inhibiting the migratory cell phenotype and allowing the establishment of initially weak cell-cell adhesions.     

Thrombin-induced expression of endothelial P-selectin and intercellular adhesion molecule-1: a mechanism for stabilizing neutrophil adhesion

Thrombin-induced expression of endothelial adhesivity toward neutrophils (PMN) was studied using human umbilical vein endothelial cells (HUVEC). HUVEC were challenged with human alpha-thrombin for varying durations up to 120 min, after which the cells were fixed with 1% paraformaldehyde and 51Cr-labeled human PMN were added to determine PMN adhesion. Endothelial adhesivity increased within 15 min after alpha-thrombin exposure, and the response persisted up to 120 min. Expression of endothelial adhesion proteins, P-selectin (GMP-140, PADGEM, CD62), and intercellular adhesion molecule-1 (ICAM-1; CD54) on the endothelial surface was quantitated by increase in the specific binding of anti-P-selectin mAb G1 and anti-ICAM-1 mAb RR1/1 labeled with 125I. P-selectin expression was maximal at 5-15 min alpha-thrombin exposure and decayed to basal levels within 90 min. In contrast, ICAM-1 activity increased at 30 min and remained elevated for 120 min after alpha-thrombin challenge. The initial endothelial adhesivity was dependent on P-selectin expression since PMN adhesion occurring within the first 30 min after alpha-thrombin challenge was inhibited by mAb G1. The later prolonged PMN adhesion was ICAM-1 dependent since this response was inhibited by mAb RR1/1 and to the same degree by the anti-CD18 mAb IB4. Anti-ELAM-1 mAb BB11 had no effect on adhesion of PMN to the alpha-thrombin-challenged cells. The initial P-selectin expression and PMN adhesion responses were reproduced by the 14-amino peptide (SFLLRNPNDKYEPF) (thrombin-receptor activity peptide; TRP-14) which comprised the NH2 terminus created by thrombin's proteolytic action on its receptors. However, TRP-14-induced PMN adhesion was transient, and TRP-14 did not cause ICAM-1 expression. The ICAM-1-dependent PMN adhesion mediated by alpha-thrombin was protein synthesis independent since ICAM-1 expression and PMN adhesion were not inhibited by cycloheximide pretreatment of HUVEC. Moreover, Northern blot analysis indicated absence of ICAM-1 mRNA signal up to 180 min after alpha-thrombin challenge. In conclusion, thrombin-induced endothelial adhesivity involves early- and late-phase responses. The initial reversible PMN adhesion is mediated by rapid P-selectin expression via TRP-14 generation. Thrombin-induced PMN adhesion is stabilized by a protein synthesis-independent upregulation of the constitutive ICAM-1 activity which enables the interaction of ICAM-1 with the CD18 beta 2 integrin on PMN.     

Changes in the integrin-mediated adhesion of human neutrophils in the cold and after rewarming

Changes in the mechanical and adhesive properties of neutrophils may modify perfusion of the microcirculation in cooled tissue. We tested how integrin-mediated adhesion of isolated human neutrophils was altered by cooling, or cooling and rewarming. First, adhesion was tested in a static assay. In the presence or absence of integrin-activating agents (formyl peptide, fMLP or Mn(++)), there were significant reductions in adhesion to immobilised albumin at 10 degrees C or 0 degrees C compared to 37 degrees C, although a slight increase in adhesion was induced by fMLP or Mn(++) at 10 degrees C or 0 degrees C. If cells were cooled for 5 or 20 min at 10 degrees C and rewarmed (in the absence of activators) there was >100% increase in adhesion compared to cells held at 37 degrees C. In a flow assay, neutrophils perfused over P-selectin at 37 degrees C formed rolling attachments, but if neutrophils were cooled to 10 degrees C and rewarmed for 1 or 5 min, there was transformation to stationary adhesion, which was reversed by antibody against CD18. After 20 minutes of rewarming, rolling was restored. Cooling and rewarming did not cause de novo expression of CD11b/CD18, and so appears to transiently activate constitutively-expressed integrin. Thus, integrin-mediated adhesion may be impaired in cold tissue but on return to normal temperature, neutrophils may transiently adhere locally or in remote vessels.     

Regulation of intercellular adhesion strength in fibroblasts

The regulation of adherens junction formation in cells of mesenchymal lineage is of critical importance in tumorigenesis but is poorly characterized. As actin filaments are crucial components of adherens junction assembly, we studied the role of gelsolin, a calcium-dependent, actin severing protein, in the formation of N-cadherin-mediated intercellular adhesions. With a homotypic, donor-acceptor cell model and plates or beads coated with recombinant N-cadherin-Fc chimeric protein, we found that gelsolin spatially co-localizes to, and is transiently associated with, cadherin adhesion complexes. Fibroblasts from gelsolin-null mice exhibited marked reductions in kinetics and strengthening of N-cadherin-dependent junctions when compared with wild-type cells. Experiments with lanthanum chloride (250 microm) showed that adhesion strength was dependent on entry of calcium ions subsequent to N-cadherin ligation. Cadherin-associated gelsolin severing activity was required for localized actin assembly as determined by rhodamine actin monomer incorporation onto actin barbed ends at intercellular adhesion sites. Scanning electron microscopy showed that gelsolin was an important determinant of actin filament architecture of adherens junctions at nascent N-cadherin-mediated contacts. These data indicate that increased actin barbed end generation by the severing activity of gelsolin associated with N-cadherin regulates intercellular adhesion strength.     

Two cell adhesion molecules, nectin and cadherin, interact through their cytoplasmic domain-associated proteins

We have found a new cell-cell adhesion system at cadherin-based cell-cell adherens junctions (AJs) consisting of at least nectin and l-afadin. Nectin is a Ca(2+)-independent homophilic immunoglobulin-like adhesion molecule, and l-afadin is an actin filament-binding protein that connects the cytoplasmic region of nectin to the actin cytoskeleton. Both the trans-interaction of nectin and the interaction of nectin with l-afadin are necessary for their colocalization with E-cadherin and catenins at AJs. Here, we examined the mechanism of interaction between these two cell-cell adhesion systems at AJs by the use of alpha-catenin-deficient F9 cell lines and cadherin-deficient L cell lines stably expressing their various components. We showed here that nectin and E-cadherin were colocalized through l-afadin and the COOH-terminal half of alpha-catenin at AJs. Nectin trans-interacted independently of E-cadherin, and the complex of E-cadherin and alpha- and beta-catenins was recruited to nectin-based cell-cell adhesion sites through l-afadin without the trans-interaction of E-cadherin. Our results indicate that nectin and cadherin interact through their cytoplasmic domain-associated proteins and suggest that these two cell-cell adhesion systems cooperatively organize cell-cell AJs.