A novel Dictyostelium gene encoding multiple repeats of adhesion inhibitor-like domains has effects on cell-cell and cell-substrate adhesion

The Dictyostelium protein AmpA (adhesion modulation protein A) is encoded by the gene originally identified by the D11 cDNA clone. AmpA contains repeated domains homologous to a variety of proteins that influence cell adhesion. The protein accumulates during development, reaching a maximal level at the finger stage. Much of the AmpA protein is found extracellularly during development, and in culminants, AmpA is found in association with anterior-like cells. Characterization of an ampA- strain generated by gene replacement reveals a significant increase in cell-cell clumping when cells are starved in nonnutrient buffer suspensions. Developing ampA- cells are also more adhesive to the underlying substrate and are delayed in developmental progression, with the severity of the delay increasing as cells are grown in the presence of bacteria or on tissue culture dishes rather than in suspension culture. Reintroduction of the ampA gene rescues the developmental defects of ampA- cells; however, expression of additional copies of the gene in wild-type cells results in more severe developmental delays and decreased clumping in suspension culture. We propose that the AmpA protein functions as an anti-adhesive to limit cell-cell and cell-substrate adhesion during development and thus facilitates cell migration during morphogenesis.     

Wettability of substrata controls cell-substrate and cell-cell adhesions

The maintenance of endothelial cell (EC) monolayer architecture requires stable adhesions not only between neighboring cells but also between cells and the extracellular matrix. While the influence of biomaterials surface wettability on cell-substratum adhesion is rather well studied, its impact on cell-cell cohesion has not been extensively investigated. In the present study a model system consisting of hydrophilic and hydrophobic glass pre-coated with fibronectin and fibrinogen was used to study the influence of surface wettability on both types of cell adhesions. It was demonstrated that the substrate wettability controls the adhesion and cytoskeletal organization of endothelial cells, which has an impact on the subsequent ability of cells to establish stable cell-cell cohesions. These effects were related to the accessibility of specific domains of the adsorbed proteins. While the hydrophobic substratum promoted cell-cell cohesion, on hydrophilic substrata cell-substrate adhesion was dominant. In addition, evidence for an influence of surface wettability on the cross talk between integrins and cadherins was found.     

Assessment of cell-substrate adhesion by a centrifugal method

Summary A centrifugal method has been evaluated for measuring the strength of Vero Green Monkey kidney cell adhesion to growth surfaces. The centrifugal force necessary to remove cells gave a quantitative measure of cell adhesion and hence the quality of the growth surface. After being subjected to high gravity forces, both the remaining attached cells and the detached cells were viable, indicating the detachment process did not simply rupture the cell. Electron microscope examination of growth surfaces after cell detachment suggested that remnants related to filopodia remained.     

Differential regulation of endogenous cadherin expression in Madin-Darby canine kidney cells by cell-cell adhesion and activation of beta -catenin signaling

Cadherins mediate cell-cell adhesion, but little is known about how their expression is regulated. In Madin-Darby canine kidney (MDCK) cells, the cadherin-associated cytoplasmic proteins alpha- and beta-catenin form high molecular weight protein complexes with two glycoproteins (Stewart, D. B., and Nelson, W. J. (1997) J. Biol. Chem. 272, 29652-29662), one of which is E-cadherin and the other we show here is the type II cadherin, cadherin-6 (K-cadherin). In low density, motile MDCK cells, the steady-state level of cadherin-6 is low, but protein is synthesized. However, following cell-cell adhesion, cadherin-6 becomes stabilized and accumulates by >50-fold at cell-cell contacts while the E-cadherin level increases only 5-fold during the same period. To investigate a role of beta-catenin in regulation of cadherin expression in MDCK cells, we examined the effects of expressing signaling-active beta-catenin mutants (DeltaGSK, DeltaN90, and DeltaN131). In these cells, while levels of E-cadherin, alpha- and beta-catenin are similar to those in control cells, levels of cadherin-6 are significantly reduced due to rapid degradation of newly synthesized protein. Additionally, these cells appeared more motile and less cohesive, as expression of DeltaGSK-beta-catenin delayed the establishment of tight confluent cell monolayers compared with control cells. These results indicate that the level of cadherin-6, but not that of E-cadherin, is strictly regulated post-translationally in response to Wnt signaling, and that E-cadherin and cadherin-6 may contribute different properties to cell-cell adhesion and the epithelial phenotype.     

Real-time monitoring of epithelial cell-cell and cell-substrate interactions by infrared surface plasmon spectroscopy

The development of novel technologies capable of monitoring the dynamics of cell-cell and cell-substrate interactions in real time and a label-free manner is vital for gaining deeper insights into these most fundamental cellular processes. However, the label-free technologies available today provide only limited information on these processes. Here, we report a new (to our knowledge) infrared surface plasmon resonance (SPR)-based methodology that can resolve distinct phases of cell-cell and cell-substrate adhesion of polarized Madin Darby canine kidney epithelial cells. Due to the extended penetration depth of the infrared SP wave, the dynamics of cell adhesion can be detected with high accuracy and high temporal resolution. Analysis of the temporal variation of the SPR reflectivity spectrum revealed the existence of multiple phases in epithelial cell adhesion: initial contact of the cells with the substrate (cell deposition), cell spreading, formation of intercellular contacts, and subsequent generation of cell clusters. The final formation of a continuous cell monolayer could also be sensed. The SPR measurements were validated by optical microscopy imaging. However, in contrast to the SPR method, the optical analyses were laborious and less quantitative, and hence provided only limited information on the dynamics and phases of cell adhesion.     

NCAM polysialic acid can regulate both cell-cell and cell-substrate interactions

We have proposed previously that the polysialic acid (PSA) moiety of NCAM can influence membrane-membrane apposition, and thereby serve as a selective regulator of a variety of contact-dependent cell interactions. In this study, cell and tissue culture models are used to obtain direct evidence that the presence of PSA on the surface membrane can affect both cell-cell and cell-substrate interactions. Using a neuroblastoma/sensory neuron cell hybrid, it was found that removal of PSA with a specific neuraminidase (endo-N) augments cell-cell aggregation mediated by the L1 cell adhesion molecule as well as cell attachment to a variety of tissue culture substrates. In studies of embryonic spinal cord axon bundling, which involves both cell-cell and cell-substrate interactions, the pronounced defasciculation produced by removal of PSA is most easily explained by an increase in cell-substrate interaction. The fact that in both studies NCAM's intrinsic adhesion function was found not to be an important variable further illustrates that regulation of the cell surface by PSA can extend beyond binding mediated by the NCAM polypeptide.     

Differential thyroid hormone-regulated rat thyrotropin beta gene expression detected by blot hybridization

In the rat, there is a single TSH beta-subunit gene represented by three exons interrupted by two introns. This gene contains two promoters which determines the synthesis of two mRNAs with 5'-untranslated regions that differ by 43 base pairs. This study evaluates the steady state levels of these TSH beta mRNAs in various thyroidal states. Blot hybridization analyses of pituitary mRNA with synthetic probes designed to detect either one or both TSH beta mRNAs were performed. One probe corresponds to 24 bases in the 5'-untranslated region of mRNA1 and a second corresponds to 25 nucleotides in the coding region and detects both mRNA1 and mRNA2. These studies indicate the presence of TSH beta mRNA species of indistinguishable size consistent with the presence of two TSH beta mRNAs that contain slightly different 5'-untranslated regions. Comparison of pituitary RNA obtained from normal and hypothyroid rats reveals that the shorter mRNA (mRNA2) is increased approximately 6- to 8-fold with hypothyroidism while the abundance of the longer mRNA (mRNA1) is relatively unchanged. Treatment of either normal or hypothyroid animals with T3 decreases the abundance of mRNA2 while again mRNA1 is relatively unaffected. Thus, although both mRNAs are detected, only one mRNA is dramatically altered by thyroidal status. Therefore, the single rat TSH beta gene is transcribed into two mRNAs via the use of alternative promoters of which only one is markedly regulated by thyroid hormones.     

Regulation of cell-substrate adhesion by proteoglycans immobilized on extracellular substrates

We have demonstrated previously that chick embryo fibroblasts synthesize and secrete a large chondroitin sulfate proteoglycan (designated PG-M) that binds to fibronectin. We now report the possibility that PG-M interactions with cell surfaces can modulate cell-substrate adhesion. When PG-M was added to the medium, various types of trypsinized cells failed to adhere not only to fibronectin-coated substrates but also to collagen- or vitronectin-coated substrates. Adhesion of the cells to laminin or glycyl-arginyl-glycyl-aspartyl-serine derivatized serum albumin (arginyl-glycyl-aspartic acid-containing molecules with no capacity to bind PG-M) was also inhibited by PG-M. Treatment of the proteoglycan with either proteolytic enzymes or chondroitinase abolished its inhibitory effects on the cell adhesion. These results suggest that direct binding between PG-M and fibronectin, if any, is not a cause of the inhibition by PG-M and that only the proteoglycan form is responsible for the activity. When the immobilization of added PG-M to available plastic surfaces of coated dishes was blocked by pretreating the dishes with serum albumin, the inhibitory effect of PG-M was abolished, suggesting that the immobilized fraction of PG-M can act as a cell adhesion inhibitor. In immobilized form, both cartilage chondroitin sulfate proteoglycan (designated PG-H) and chondroitin sulfate-derivatized serum albumin also inhibited cell adhesion. In contrast, heparan sulfate proteoglycan form LD and heparan sulfate-derivatized serum albumin had far lower inhibitory activities, indicating that the active site for the interaction between cells and PG-M is on the chondroitin sulfate chains.     

Differential regulation of cell-cell contact, invasion and anoikis by hScrib and hDlg in keratinocytes

The components of the Scrib/Dlg tumour suppressor complex have complementary roles in Drosophila and loss of both proteins is a common event in many different human tumours. However no studies have directly addressed the respective contributions of loss of hScrib and hDlg in the same human cell background to cellular phenotypes associated with cell transformation. In human HaCaT keratinocytes we show that removal of hScrib greatly reduces cell-cell contact and cell-matrix interactions, and promotes an invasive phenotype. Conversely, in cells lacking hDlg1 cell-cell contacts are maintained and there are decreases in both cell growth and invasion. However, hDlg-depleted cells show increased resistance to a specialized form of apoptosis known as anoikis, to which cells lacking hScrib are highly susceptible. Thus whilst it has been widely assumed that hScrib and hDlg have complementary roles, these studies in fact demonstrate that hScrib and hDlg1 have distinct and opposing functions in human keratinocytes.     

Structural basis of cell-cell adhesion by NCAM

The neural cell adhesion molecule NCAM, a member of the immunoglobulin superfamily, mediates cell-cell recognition and adhesion via a homophilic interaction. NCAM plays a key role during development and regeneration of the nervous system and is involved in synaptic plasticity associated with memory and learning. The 1.85 A crystal structure of the two N-terminal extracellular domains of NCAM reported here provides a structural basis for the homophilic interaction. The molecular packing of the two-domain structure reveals a cross shaped antiparallel dimer, and provides fundamental insight into trans-cellular recognition mediated by NCAM.     

The role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells

We have examined cultures of neonatal human foreskin keratinocytes (HFKs) to determine the ligands and functions of integrins alpha 2 beta 1, and alpha 3 beta 1 in normal epidermal stratification and adhesion to the basement membrane zone (BMZ) in skin. We used three assay systems, HFK adhesion to purified extracellular matrix (ECM) ligands and endogenous secreted ECM, localization of integrins in focal adhesions (FAs), and inhibition of HFK adhesion with mAbs to conclude: (a) A new anti-alpha 3 beta 1 mAb, P1F2, localized alpha 3 beta 1 in FAs on purified laminin greater than fibronectin/collagen, indicating that laminin was the best exogeneous ligand for alpha 3 beta 1. However, in long term culture, alpha 3 beta 1 preferentially codistributed in and around FAs with secreted laminin-containing ECM, in preference to exogenous laminin. Anti-alpha 3 beta 1, mAb P1B5, detached prolonged cultures of HFKs from culture plates or from partially purified HFK ECM indicating that interaction of alpha 3 beta 1 with the secreted laminin-containing ECM was primarily responsible for HFK adhesion in long term culture. (b) In FA assays, alpha 2 beta 1 localized in FAs conincident with initial HFK adhesion to exogenous collagen, but not laminin or fibronectin. However, in inhibition assays, anti-alpha 2 beta 1 inhibited initial HFK adhesion to both laminin and collagen. Thus, alpha 2 beta 1 contributes to initial HFK adhesion to laminin but alpha 3 beta 1 is primarily responsible for long-term HFK adhesion to secreted laminin-containing ECM. (c) Serum or Ca2(+)-induced aggregation of HFKs resulted in relocation of alpha 2 beta 1 and alpha 3 beta 1 from FAs to cell-cell contacts. Further, cell-cell adhesion was inhibited by anti-alpha 3 beta 1 (P1B5) and a new anti-beta 1 mAb (P4C10). Thus, interaction of alpha 3 beta 1 with either ECM or membrane coreceptors at cell-cell contacts may facilitate Ca2(+)-induced HFK aggregation. (d) It is suggested that interaction of alpha 3 beta 1 with a secreted, laminin-containing ECM in cultured HFKs, duplicates the role of alpha 3 beta 1 in basal cell adhesion to the BMZ in skin. Further, relocation of alpha 2 beta 1 and alpha 3 beta 1 to cell-cell contacts may result in detachment of cells from the BMZ and increased cell-cell adhesion in the suprabasal cells contributing to stratification of the skin.     

Spatio-temporal regulation of Rac1 localization and lamellipodia dynamics during epithelial cell-cell adhesion

Cadherin-dependent epithelial cell-cell adhesion is thought to be regulated by Rho family small GTPases and PI 3-kinase, but the mechanisms involved are poorly understood. Using time-lapse microscopy and quantitative image analysis, we show that cell-cell contact in MDCK epithelial cells coincides with a spatio-temporal reorganization of plasma membrane Rac1 and lamellipodia from noncontacting to contacting surfaces. Within contacts, Rac1 and lamellipodia transiently concentrate at newest sites, but decrease at older, stabilized sites. Significantly, Rac1 mutants alter kinetics of cell-cell adhesion and strengthening, but not the eventual generation of cell-cell contacts. Products of PI 3-kinase activity also accumulate dynamically at contacts, but are not essential for either initiation or development of cell-cell adhesion. These results define a role for Rac1 in regulating the rates of initiation and strengthening of cell-cell adhesion.     

Differential modulation of cadherin-mediated cell-cell adhesion by platelet endothelial cell adhesion molecule-1 isoforms through activation of extracellular regulated kinases

The role of platelet endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cell-cell interactions and its contribution to cadherin-mediated cell adhesion are poorly understood. Such studies have been difficult because all known endothelial cells express PECAM-1. We have used Madin-Darby canine kidney (MDCK) cells as a model system in which to evaluate the role of PECAM-1 isoforms that differ in their cytoplasmic domains in cell-cell interactions. MDCK cells lack endogenous PECAM-1 but form cell-cell junctions similar to those of endothelial cells, in which PECAM-1 is concentrated. MDCK cells were transfected with two isoforms of murine PECAM-1, Delta15 and Delta14&15, the predominant isoforms expressed in vivo. Expression of the Delta15 isoform resulted in apparent dedifferentiation of MDCK cells concomitant with the loss of adherens junctions, down-regulation of E-cadherin, alpha- and beta-catenin expression, and sustained activation of extracellular regulated kinases. The Delta15 isoform was not concentrated at cell-cell contacts. In contrast, the Delta14&15 isoform localized to sites of cell-cell contact and had no effect on MDCK cell morphology, cadherin/catenin expression, or extracellular regulated kinase activity. Thus, the presence of exon 14 in the cytoplasmic domain of PECAM-1 has dramatic effects on the ability of cells to maintain adherens junctions and an epithelial phenotype. Therefore, changes in the expression of exon 14 containing PECAM-1 isoforms, which we have observed during development, may have profound functional consequences.