Intercellular adhesive selectivity. I. An improved assay for the measurement of embryonic chick intercellular adhesion (liver and other tissues)

An improved assay for measuring intercellular adhesive selectivity of embryonic chick liver cells is described. Three major improvements over earlier procedures are noted: (a) enhanced reproducibility of liver cell-liver cell aggregate adhesion (homotypic adhesion) was achieved; (b) 25-70% of the input cells adhered to the collecting aggregates during the course of routine experiments as compared to the 0.25% in earlier assays. This increase in cellular adhesion suggests that the observed cell pick-up is a characteristic of the majority of the dissociated liver cell population; (c) the rate of intercellular adhesion was increased 1,000-fold. The main feature of the assay is that it measures the tissue adhesive selectivities of the dissociated cell population. Studies were undertaken on three embryonic chick tissues (liver, neural retina, and mesencephalon) to determine the tissue selectivity of intercellular adhesion of these dissociated cell types. Some general properties of liver cell homotypic adhesion have been studied and are reported.     

Intercellular adhesive selectivity. III. Species selectivity of embryonic liver intercellular adhesion

A species difference in the intercellular adhesive selectivity of mixtures of embryonic liver cells is reported. This is first quantitative assessment of species differences in the intercellular adhesive properties of embryonic cells. A collecting aggregate assay, a new double-label assay procedure, and histological and autoradiographic procedures were used to elucidate the intercellular adhesive selectivity of developing mammalian and avian liver cells. Evidence is presented that the reported adhesive differences are not due to the different cell types composing the respective embryonic mammalian and avian livers. Finally, such heterolgous-homotypic selectivity of adhesion is not a property of all tissues, since it is shown that developing brain cells (mesencephalon) do not exhibit the avove intercellular adhesive selectivity (mammalian vs. avian). These findings provide further support for the hypothesis that generic identity as well as cell type may play an important part in determining the intercellular adhesive behavior of heterologous-homotypic mixtures of embryonic cells. A possible evolutionary divergence of morphogenetic mechanisms is discussed.     

Is cell sorting caused by differences in the work of intercellular adhesion? A critique of the steinberg hypothesis

The differential adhesion hypothesis, developed by Malcolm Steinberg, proposes that the histotypic sorting out behavior of aggregated cells is mechanistically equivalent to certain aspects of liquid surface tension, specifically the spontaneous separation of immiscible liquids of differing surface tension. According to Steinberg's hypothesis, the adhesive forces between aggregated cells play essentially the same role in cell sorting as are played by intermolecular attractive forces in liquid surface tension.In this paper I discuss a number of crucial distinctions between intermolecular attraction (in liquids) and intercellular adhesion (in aggregates). First, liquid drops are closed systems thermodynamically whereas aggregates of living cells can generate an indeterminate amount of metabolic energy capable of altering cell positions and adhesions. Secondly, intercellular adhesions are more than just close range attractions since cells can be held together by forces in addition to those which originally pulled them together. Third, the breakage of intercellular adhesions need not be simply the reverse, thermodynamically, of the formation of those adhesions. And fourthly, because intercellular adhesion is generally concentrated at relatively small foci such as desmosomes, a maximization of intercellular adhesion does not necessarily require a maximization of intercellular contact area, or vice versa.In addition, several alternative hypotheses are proposed, each of which is theoretically capable of explaining cell sorting and the other surface tension-like aspects of cell aggregate behavior which Steinberg has sought to explain as consequences of differential adhesion. In particular, a differential surface contraction hypothesis is proposed, according to which cell sorting and related phenomena are the results of cell surface contractions induced to occur over those portions of the cell surface which are exposed to the surrounding culture medium. Because of the evidence that various invagination type movements of embryonic epithelia are caused by cell surface contractions, it is suggested that differential surface contraction is the most likely explanation of histotypic cell sorting. A number of experiments are suggested by which these various hypotheses might be tested.     

Evidence that tubulobulbar complexes in the seminiferous epithelium are involved with internalization of adhesion junctions

Tubulobulbar complexes may be part of the mechanism by which intercellular adhesion junctions are internalized by Sertoli cells during sperm release. These complexes develop in regions where Sertoli cells are attached to adjacent cells by intercellular adhesion junctions termed ectoplasmic specializations. At sites where Sertoli cells are attached to spermatid heads, tubulobulbar complexes consist of fingerlike processes of the spermatid plasma membrane, corresponding invaginations of the Sertoli cell plasma membrane, and a surrounding cuff of modified Sertoli cell cytoplasm. At the terminal ends of the complexes occur clusters of vesicles. Here we show that tubulobulbar complexes develop in regions previously occupied by ectoplasmic specializations and that the structures share similar molecular components. In addition, the adhesion molecules nectin 2 and nectin 3, found in the Sertoli cell and spermatid plasma membranes, respectively, are concentrated at the distal ends of tubulobulbar complexes. We also demonstrate that double membrane bounded vesicles are associated with the ends of tubulobulbar complexes and nectin 3 is present on spermatids, but is absent from spermatozoa released from the epithelium. These results are consistent with the conclusion that Sertoli cell and spermatid membrane adhesion domains are internalized together by tubulobulbar complexes. PKCalpha, a kinase associated with endocytosis of adhesion domains in other systems, is concentrated at tubulobulbar complexes, and antibodies to endosomal and lysosomal (LAMP1, SGP1) markers label the cluster of vesicles associated with the ends of tubulobulbar complexes. Our results are consistent with the conclusion that tubulobulbar complexes are involved with the disassembly of ectoplasmic specializations and with the internalization of intercellular membrane adhesion domains during sperm release.     

Cell surface lipids and adhesion. III. The effects on cell adhesion of changes in plasmalemmal lipids.

The two preceding papers of this series suggest that the state of the plasmalemmal lipids affects cell adhesion. Plasmalemmal composition was altered by the experimental incorporation of fatty acids into R1 and R2 positions in the phosphatidyl components of the cell surface. In this paper we report that: (1) If the incorporation is of long chain length fatty acids (saturated) cell adhesion rises. (2) If the incorporation is of unsaturated fatty acids cell adhesion falls as the unsaturation increases. (3) Incorporation has to be extensive to produce a large change in adhesion. (4) Changes in adhesion parallel the plasmalemmal incorporation but do not follow the total cell incorporation. Item (4) argues that it is plasmalemmal and not other membrane lipids that are involved in cell adhesion. Item (3) suggests that bulk membrane properties and not some very specific grouping are involved in the effects of lipids on adhesion. The similar extents of incorporation of the various different fatty acids and the negligible amounts of lysophospholipids in the membranes of cells that have incorporated fatty acids argue that the effects are not due to differential accumulations of these lysolipids when incubations are done with different fatty acids. The changes in adhesion cannot be accounted for by changes in surface charge density since the electrophoretic mobility of the cells is unchanged by these incubations. It is suggested that these effects on adhesion due to changes in plasmalemmal lipids can be explained either in terms of the action of intermembrane van der Waals--London (electrodynamic) forces in cell adhesion or of changes in surface fluidity. These alternatives are discussed.     

In vitro inhibitory effect of IL-8 and other chemoattractants on neutrophil-endothelial adhesive interactions.

We have previously reported that cytokine- or LPS-activated human umbilical vein endothelial cell (HUVEC) monolayers secrete IL-8 that can act as a neutrophil-selective adhesion inhibitor. In our study we investigated the mechanisms involved in the leukocyte adhesion inhibitory action of IL-8. The leukocyte adhesion inhibitory effect appears to be mediated by the action of IL-8 on the neutrophil, does not involve down-regulation of relevant endothelial adhesion molecules such as endothelial-leukocyte adhesion molecule-1 or intercellular adhesion molecule-1, and is quantitatively similar in different endothelial activation states that are predominantly endothelial-leukocyte adhesion molecule-1 dependent or intercellular adhesion molecule-1 dependent. In addition to inhibiting the attachment of freshly isolated peripheral blood neutrophils to cytokine-activated HUVEC monolayers, IL-8 also promoted a rapid detachment of tightly adherent neutrophils from activated HUVEC, and abolished neutrophil transendothelial migration. Certain other chemoattractants, including FMLP and C5a, had similar inhibitory actions, indicating IL-8 was not unique in its ability to inhibit various neutrophil-endothelial interactions. In contrast, two other neutrophil agonists 1-0-alkyl-2-acetyl sn-glycero-3-phosphocholine and granulocyte-macrophage-CSF, which, like IL-8, are produced by activated HUVEC, as well as the leukocyte-derived chemoattractant leukotriene B4, exerted minimal inhibitory effects on adhesion. Regardless of their ability to modulate neutrophil-endothelial cell adhesion, all these agents induced altered leukocyte surface expression of functionally important adhesion molecules, including loss of L-selectin (leukocyte adhesion molecule-1, LECAM-1) and increase in CD11b/CD18. Thus, although the above agonists have been characterized primarily as chemoattractants, our findings demonstrate that these agents can exert a wide range of modulatory effects on neutrophil-endothelial adhesive interactions.     

Lymphocyte adhesion mediated by lymphocyte function-associated antigen-1. I. Long term augmentation by transient increases in intracellular cAMP.

Lymphocyte adhesion to target cells is mediated, in part, by the interaction of lymphocyte function-associated Ag-1 (LFA-1) with intercellular adhesion molecule-1 (ICAM-1). Cells of the B cell line, JY, express both coreceptors and have been used as a model for intercellular adhesion mediated by these molecules. Elevation of the intracellular cAMP concentration ([cAMP]i), by any of several reagents, for periods as brief as 30 min, led to enhanced intercellular adhesion in a concentration dependent manner 5 to 8 h later. Two protein kinase A inhibitors, KT5720 and H-89, but not the protein kinase C inhibitor calphostin C, blocked the effects of elevated [cAMP]i. These data suggest a role for protein kinase A in this response. The adhesion augmented by increased [cAMP]i was due to LFA-1/ICAM-1 interactions between cells because it was blocked by either anti-LFA-1 or anti-ICAM-1 mAb. Elevated [cAMP]i induced cell surface patching of LFA-1, but not ICAM-1, and this redistribution preceded intercellular adhesion. Finally, redistribution of LFA-1 was not mediated by the cytoskeleton. These results suggest a model in which transient activation of protein kinase A results in increased local concentration of LFA-1 at the cell surface and in augmented long term adhesion mediated by this integrin.     

Intercellular communication and carcinogenesis

Two types of intercellular communication (humoral and cell contact-mediated) are involved in control of cellular function in multicellular organisms, both of them mediated by membrane-embedded proteins. Involvement of aberrant humoral communication in carcinogenesis has been well documented and genes coding for some growth factors and their receptors have been classified as oncogenes. More recently, cell contact-mediated communication has been found to have an important role in carcinogenesis, and some genes coding for proteins involved in this type of communication appear to form a family of tumor-suppressor genes. Both homologous (among normal or (pre-)cancerous cells) as well as heterologous (between normal and (pre)cancerous cells) communications appear to play important roles in cell growth control. Gap junctional intercellular communication (GJIC) is the only means by which multicellular organisms can exchange low molecular weight signals directly from within one cell to the interior of neighboring cells. GJIC is altered by many tumor-promoting agents and in many human and rodent tumors. We have recently shown that liver tumor-promoting agents inhibit GJIC in the rat liver in vivo. Molecular mechanisms which could lead to aberrant GJIC include: (1) mutation of connexin genes; (2) reduced and/or aberrant expression of connexin mRNA; (3) aberrant localization of connexin proteins, i.e., intracytoplasmic rather than in the cytoplasmic membrane; and (4) modulation of connexin functions by other proteins, such as those involved in extracellular matrix and cell adhesion. Whilst mutations of the cx 32 gene appear to be rare in tumors, cx 37 gene mutations have been reported in a mouse lung tumor cell line. Our results suggest that aberrant connexin localization is rather common in cancer cells and that possible molecular mechanisms include aberrant phosphorylation of connexin proteins and lack of cell adhesion molecules. Studies on transfection of connexin genes into tumor cells suggest that certain connexin genes (e.g., cx 26, cx 43 and cx 32) act as tumor-suppressor genes.     

Cell adhesion in rat fibroblasts: effect of tumor promoters

In a study performed on transformed (SGS/3A) and normal syngeneic rat cells (FG/2) to identify the molecular mechanisms which regulate cell adhesion and contact inhibition in cell transformation, we investigated the effects of tumor promoters on cell to cell adhesion of rat fibroblasts. As tumor promoters we used 12-tetradecanoyl-phorbole-13-acetate (TPA) and the melittin, a polypeptide from bee venom, both substances capable of stimulating the neoplastic transformation. The intercellular adhesion assay consists in determining the percent of single cells labeled with 3H-L-leucine adhering to a confluent monolayer of unlabeled cells at different incubation times. The increase of cell-cell adhesion caused by TPA and melittin confirms what we have constantly observed in other experiments, i.e. that neoplastic cells SGS/3A always have a higher intercellular adhesion capacity than corresponding normal syngenic cells FG/2. Since one of the effects of the tumor promoters is also induction of a reversible alteration of the cytoskeleton, it is likely that their action on intercellular adhesion is regulated by a mechanism analogous to that proposed for explaining the increased intercellular adhesion observed after treatment with antimicrotubular compounds.     

Use of preformed cell aggregates and layers to measure tissue-specific differences in intercellular adhesion

The binding of aggregates formed from various 7-day chick embryo tissues to cultured cell layers was analyzed 24 hr following trypsin dissociation of the tissues. The proprotion of aggregates binding is independent of the number of aggregates added, and changes with time over 60 min in a manner consistent with a first-order process. The adhesive parameter measured, the percentage of aggregates binding to cell layers per unit time, varies slightly with aggregate size but is not dependent upon the probability of collision of the aggregate with the layer. The rate of binding and the effect of modifiers of binding (temperature, inhibitors of oxidative metabolism and glutaraldehyde) are substantially different for neural retina interactions than for liver or heart interactions, suggesting that retina cells may form intercellular bonds via a mechanism distinct from that of liver or heart cells. The rate of binding between like tissue types is, with one exception, greater than between unlike types. Glutaraldehyde treatment of only one of the reactants abolishes this adhesive specificity. Aggregate binding provides a means of quantitatively assessing intercellular adhesion which has the advantage of reducing the effects of trypsinization on measurements of adhesion, and therefore lends itself to the investigation of cellular consequences of adhesion.     

A quantitative method for determining polarization of neutrophil adhesion molecules associated with ischemia reperfusion

Ischemia-reperfusion-induced neutrophil adhesion to endothelium is CD18-dependent, but information regarding polarity of CD18 adhesion molecules remains speculative. This study evaluated neutrophil adhesion using an in vitro cell adhesion assay and introduces a quantitative method of measuring CD18 membrane distribution using confocal microscopy. Neutrophils from normal animals were isolated from whole blood and incubated with plasma from rat gracilis muscle flaps with no ischemia and reperfusion (nonischemic control, n = 10) or 4 hours of ischemia and 90 minutes of reperfusion (ischemia/reperfusion, n = 10), on coverslips pretreated with and without (phosphate-buffered saline) soluble intercellular adhesion molecules. Coverslips without intercellular adhesion molecules represented a negative control (intercellular adhesion molecules were required for adhesion). Percent adherence to intercellular adhesion molecules was expressed as a ratio of adherent cells/total cells. CD18 polarization was assessed by staining neutrophils with fluorescein isothiocyanate-labeled anti-CD11b, followed by confocal microscopy and Z-stack analysis. Membrane-associated CD18 was expressed as fluorescence intensity units in three equal areas of the cell membrane. Capping was defined as twice as much fluorescence in 33 percent of the cell membrane as in the remaining 67 percent. Neutrophils exposed to ischemia and reperfusion plasma showed a significant increase in adhesion (0.8 +/- 0.1 percent versus 16.7 +/- 2.2 percent, p < 0.001) and CD18 polarization (6.2 +/- 1.7 percent versus 43.9 +/- 12.2 percent, p = 0.0206) compared with controls. This article describes an in vitro assay that reliably reproduces the neutrophil adhesion phenomenon associated with ischemia-reperfusion injury. Results from confocal microscopy allowed for quantitative estimation of membrane-associated receptor polarization.     

Glycolipid-anchored form of CD4 increases intercellular adhesion but is unable to enhance T cell activation.

CD4 functions to enhance T cell activation by increasing intercellular adhesion and/or by transduction of an intracellular signal. To study the role of human CD4 in T cell activation we have used a murine T cell hybridoma, By 155.16, which produces IL-2 when stimulated by HLA-DR-bearing cells. Previously, we have shown that expression of human CD4 by this hybridoma enhances its ability to produce IL-2 in response to HLA-DR-bearing cells. Furthermore, deletion of the majority of the cytoplasmic domain renders CD4 less efficient at enhancing IL-2 production. We describe studies of a glycolipid-anchored mutant of the CD4 molecule, CD4PI. This mutant is composed of the entire extracellular domain of CD4 anchored to the outlet leaflet of the membrane via a covalent bond to glycosylphosphatidylinositol and, therefore, has no transmembrane or cytoplasmic domains. When expressed in By155.16, CD4PI shows no defect in its ability to increase intercellular adhesion but is unable to augment IL-2 production. These results clearly demonstrate that CD4 enhances T cell activation by mechanisms other than increasing intercellular adhesion.     

Comparison of two cell-adhesion molecules, uvomorulin and cell-CAM 105

Two cell adhesion molecules, cell-CAM 105 and uvomorulin (UM), were compared by analysing their antigenic structures, their activity in cell aggregation assays and their expression in various tissues. Cell-CAM 105 is a membrane glycoprotein which mediates the intercellular adhesion of reaggregating rat hepatocytes, and UM was first described to be involved in the compaction of preimplantation mouse embryos and embryonal carcinoma cells. UM is not only expressed during embryonic development but also in various adult tissues including liver, epithelia of lung, gut, kidney and uterus. A similar distribution for UM was found in rat tissues on cell types where cell-CAM 105 is known to be present. Our studies show that (i) cell-CAM 105 and UM are distinct and different proteins; (ii) uvomorulin is involved in the compaction of rat preimplantation embryos but Fab anti-UM has no effect on reaggregating rat hepatocytes, where Fab anti-cell CAM is effective; (iii) distribution studies show that UM is expressed on a broader range of epithelial cells while cell-CAM 105 is more restricted to hepatocytes and simple epithelia. In cases where both cell adhesion molecules are expressed on the same cell types they can be localized to different parts of the cell surface.     

Plasma-membrane-Bound mcromoleculas are dynamically aggregated to form non-random codistribution patterns of selected functional elements. Do pattern recognition processes govern antigen presentation and intercellular interactions?

Molecular recognition processes between cell surface elements are discussed with special reference to cell surface pattern formation of membrane-bound integral proteins. The existence, as detected by flow cytometric resonance energy transfer (Appendix), and significance of cell surface patterns involving the interleukin-2 receptor, the T-cell receptor–CD3 system, the intercellular adhesion molecule ICAM-1, and the major histocompatilibilty complex class I and II molecules in the plasma membrane of lymphocytes are described. The modulation of antigen presentation by transmembrane potential changes is discussed, and a general role of transmembrane potential changes, and therefore of icon channel activities, adduced as one of the major regulatory mechanisms of cell–cell communications. A general role in the mediation and regulation of intercellular interactions is suggested for cell-surface macromolecular patterns. The dynamic pattern of protein and lipid molecules in the plasma membrane is generated by the genetic code, but has a remarkable flexibility and may be one of the major instruments of accomodation and recognition processes at the cellular level.     

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.     

An Epstein-Barr virus transformation-associated membrane protein interacts with src family tyrosine kinases.

In latently infected growth-transformed human lymphocytes, Epstein-Barr virus (EBV) encodes two integral plasma membrane proteins: LMP1, which constitutively induces B-lymphocyte activation and intercellular adhesion, and LMP2A, which associates with LMP1 and is a tyrosine kinase substrate. We now demonstrate that LMP2A associates with src family protein tyrosine kinases, particularly lyn kinase, in nonionic detergent extracts of transfected B lymphoma cells or in extracts of EBV-transformed B lymphocytes. The LMP2A and tyrosine kinase association is stable in nonionic detergents and includes a 70-kDa cell protein which is also an in vitro or in vivo kinase substrate. This LMP2A association with B-lymphocyte src family tyrosine kinases is likely to be an important pathway in EBV's effects on cell growth.     

Tumor necrosis factor induction of endothelial cell surface antigens is independent of protein kinase C activation or inactivation. Studies with phorbol myristate acetate and staurosporine.

We have investigated whether TNF-induced changes in human endothelial cell (EC) surface Ag expression are mediated by protein kinase C (PKC). This suggestion arose from the observations that PMA, a potent PKC activator, can mimic TNF by inducing expression of endothelial leukocyte adhesion molecule 1, intercellular adhesion molecule 1 (ICAM-1), and class I MHC molecules on human EC. However, in contrast to the actions of PMA, TNF neither causes membrane translocation of PKC nor induces the phosphorylation of the myristoylated alanine-rich C kinase substrate, two measures of PKC activation. Moreover, the PKC inhibitor staurosporine can block PMA-induced endothelial leukocyte adhesion molecule 1 expression at 4 h, but does not inhibit the actions of TNF. At 24 h, staurosporine itself induces intercellular adhesion molecule 1 and class I MHC, and acts additively with TNF. Twenty four hour treatment with PMA causes loss of PKC. We propose that at 24 h, staurosporine and PMA share a mechanism of action, namely diminution of PKC activity. However, 24 h treatment with TNF does not reduce the amount of PKC nor does it prevent activation of PKC by PMA. We conclude that TNF effects in EC are not mediated by PKC activation or inactivation.     

A novel four transmembrane spanning protein, CLP24. A hypoxically regulated cell junction protein.

A novel hypoxically regulated intercellular junction protein (claudin-like protein of 24 kDa, CLP24) has been identified that shows homology to the myelin protein 22/epithelial membrane protein 1/claudin family of cell junction proteins, which are involved in the modulation of paracellular permeability. The CLP24 protein contains four predicted transmembrane domains and a C-terminal protein-protein interaction domain. These domains are characteristic of the four transmembrane spanning (tetraspan) family of proteins, which includes myelin protein 22, and are involved in cell adhesion at tight, gap and adherens junctions. Expression profiling analyses show that CLP24 is highly expressed in lung, heart, kidney and placental tissues. Cellular studies confirm that the CLP24 protein localizes to cell-cell junctions and co-localizes with the beta-catenin adherens junction-associated protein but not with tight junctions. Over-expression of CLP24 results in decreased adhesion between cells, and functional paracellular flux studies confirm that over-expression of the CLP24 protein modulates the junctional barrier function. These data therefore suggest that CLP24 is a novel, hypoxically regulated tetraspan adherens junction protein that modulates cell adhesion, paracellular permeability and angiogenesis.     

Cell surface localization and tissue distribution of a hepatocyte cell-cell adhesion glycoprotein (cell-CAM 105)

We recently identified a 105,000-dalton plasma membrane glycoprotein, denoted cell-CAM 105 (CAM, cell adhesion molecule), that is involved in intercellular adhesion of reaggregating rat hepatocytes (Ocklind, C., and B. Obrink, 1982, J. Biol. Chem., 257:6788-6795). In this communication we used a monospecific rabbit antiserum against cell-CAM 105 to localize the antigen by indirect immunofluorescence on isolated rat cells and on frozen rat tissue sections. This antiserum stained the surface of freshly isolated hepatocytes. In liver sections, however, the fluorescence seemed to be located exclusively along the bile canaliculi. In addition, cell-CAM 105 showed a very specific tissue distribution. Thus a specific fluorescence was seen only in the epithelia of the stomach, the small intestine, the large intestine, the glandular epithelium of the parotid gland, and the tubules of the kidney. No specific fluorescence was found in variety of other tissues, including cartilage, interstitial connective tissue, smooth muscle, skeletal muscle, heart muscle, eye, brain, skin, the epithelia of oesophagus, bladder, uterin mucosa, thyroid follicles, prostate gland, or collecting ducts of the kidney. In the simple epithelia of the intestine and the kidney tubules the fluorescence was confined to the apical, luminal portion. Thus, both in these epithelia and in liver, cell-CAM 105 was confined to the apical, luminal portion. Thus, both in these epithelia and in liver, cell-CAM 105 was located where the typical junctional complexes between cells are found. These findings taken together with the fact that cell-CAM 105 is involved in intercellular adhesion between hepatocytes suggest with the fac that cell-CAM 105 is involved in intercellular adhesion between hepatocytes suggest that cell-CAM 105 is a member of the junctional complexes of hepatocytes and some simple epithelia.     

Rapid Functional Analysis in Xenopus Oocytes of Po Protein Adhesive Interactions

We have developed a coupled Xenopus oocyte expression system for evaluating the functional effects of mutations in known or suspected adhesion molecules, which allows for a very rapid assessment of intercellular adhesion. As a model protein, we first used Protein zero (Po), an adhesion molecule that mediates self-adhesion of the Schwann cell plasma membrane to form compact myelin in the mammalian PNS. A wide variety of mutations in Po cause certain human peripheral neuropathies, such as the Charcot-Marie-Tooth disease (CMT) type 1B and Dejerine-Sottas syndrome (DSS). After wild-type Po mRNA is injected, the protein is synthesized and correctly targeted to the oocyte cell surface. When two oocytes are paired, wild-type Po redistributes and concentrates at the cell-cell apposition region, and by electron microscopy, the oocyte pairs show close cell-cell appositions and are devoid of the microvilli that are observed in uninjected oocyte pairs. These are hallmark features of highly adhesive cell:cell interfaces. Several point mutations in Po were engineered, corresponding to the molecular defects in the CMT type 1B or DSS. The proteins encoded by these mutations reached the cell surface but failed to concentrate at the oocyte interface. Po carrying a point mutation that is found in DSS is not targeted on the plasma membrane and fail to accumulate at the cell-cell contact site.