Epithelial Cell Adhesion Molecule (Ep-CAM) Modulates Cell–Cell Interactions Mediated by Classic Cadherins

The contribution of noncadherin-type, Ca²⁺-independent cell–cell adhesion molecules to the organization of epithelial tissues is, as yet, unclear. A homophilic, epithelial Ca²⁺-independent adhesion molecule (Ep-CAM) is expressed in most epithelia, benign or malignant proliferative lesions, or during embryogenesis. Here we demonstrate that ectopic Ep-CAM, when expressed in cells interconnected by classic cadherins (E- or N-cadherin), induces segregation of the transfectants from the parental cell type in coaggregation assays and in cultured mixed aggregates, respectively. In the latter assay, Ep-CAM–positive transfectants behave like cells with a decreased strength of cell–cell adhesion as compared to the parental cells. Using transfectants with an inducible Ep-CAM–cDNA construct, we demonstrate that increasing expression of Ep-CAM in cadherin-positive cells leads to the gradual abrogation of adherens junctions. Overexpression of Ep-CAM has no influence on the total amount of cellular cadherin, but affects the interaction of cadherins with the cytoskeleton since a substantial decrease in the detergent-insoluble fraction of cadherin molecules was observed. Similarly, the detergent-insoluble fractions of α- and β-catenins decreased in cells overexpressing Ep-CAM. While the total β-catenin content remains unchanged, a reduction in total cellular α-catenin is observed as Ep-CAM expression increases. As the cadherin-mediated cell–cell adhesions diminish, Ep-CAM–mediated intercellular connections become predominant. An adhesion-defective mutant of Ep-CAM lacking the cytoplasmic domain has no effect on the cadherin-mediated cell–cell adhesions. The ability of Ep-CAM to modulate the cadherin-mediated cell–cell interactions, as demonstrated in the present study, suggests a role for this molecule in development of the proliferative, and probably malignant, phenotype of epithelial cells, since an increase of Ep-CAM expression was observed in vivo in association with hyperplastic and malignant proliferation of epithelial cells.Tissue and organ morphogenesis can be viewed as the result of interactions of various cell populations. One important type of intercellular interaction involved in the processes of tissue morphogenesis, morphogenetic movements of cells, and segregation of cell types, are adhesions mediated by cell adhesion molecules (Steinberg and Pool, 1982; Edelman, 1986; Cunningham, 1995; Takeichi, 1995; Gumbiner, 1996). Except for their direct mechanical role as interconnectors of cells and connectors of cells to substrates, cell adhesion molecules are also believed to be responsible for a variety of dynamic processes including cell locomotion, proliferation, and differentiation. There is also evidence that the adhesion systems within a cell may act as regulators of other cell adhesions, thereby offering a means of signaling that is relevant for rearrangements in cell or tissue organization (Edelman, 1993; Rosales et al., 1995; Gumbiner, 1996).In many tissues, a critical role in the maintenance of multicellular structures is assigned to cadherins, a family of Ca²⁺-dependent, homophilic cell–cell adhesion molecules (Takeichi, 1991, 1995; Gumbiner, 1996). In epithelia this critical role belongs to E-cadherin, which is crucial for the establishment and maintenance of epithelial cell polarity (McNeil et al., 1990; Näthke et al., 1993), morphogenesis of epithelial tissues (Wheelock and Jensen, 1992; Larue et al., 1996), and regulation of cell proliferation and programmed cell death (Hermiston and Gordon, 1995; Hermiston et al., 1996; Takahashi and Suzuki, 1996; Wilding et al., 1996; Zhu and Watt, 1996). Expression of different types of classic cadherin molecules (Nose et al., 1988; Friedlander et al., 1989; Daniel et al., 1995), and even quantitative differences in the levels of the same type of cadherin (Steinberg and Takeichi, 1994), may be responsible for segregation of cell types in epithelial tissues. The phenotype of epithelial cells may be modulated by expression of combinations of different types of cadherins (Marrs et al., 1995; Islam et al., 1996). However, cadherins represent only one of the intercellular adhesion systems that are present in epithelia, along with adhesion molecules of the immunoglobulin superfamily, such as carcinoembryonic antigen (Benchimol et al., 1989), and others. The actual contribution of Ca²⁺-independent nonjunctional adhesion molecules to the formation and maintenance of the epithelial tissue architecture and epithelial cell morphology is not clear.We have recently demonstrated that a 40-kD epithelial glycoprotein, which we have designated epithelial cell adhesion molecule (Ep-CAM)¹ (Litvinov et al., 1994a ), may perform as a homophilic, Ca²⁺-independent intercellular adhesion molecule, capable of mediating cell aggregation, preventing cell scattering, and directing cell segregation. This type I transmembrane glycoprotein consists of two EGF-like domains followed by a cysteine-poor region, a transmembrane domain, and a short (26-amino acid) cytoplasmic tail, and is not structurally related to the four major types of CAMs, such as cadherins, integrins, selectins, and the immunoglobulin superfamily (for review see Litvinov, 1995). Ep-CAM demonstrates adhesion properties when introduced into cell systems that are deficient in intercellular adhesive interactions (Litvinov et al., 1994a ). However, the participation of the Ep-CAM molecule in supporting cell–cell interactions of epithelial cells was not evident (Litvinov et al., 1994b ).Most epithelial cell types coexpress E-cadherin (and sometimes other classic cadherins) and Ep-CAM (for review see Litvinov, 1995) during some stage of embryogenesis. In adult squamous epithelia, which are Ep-CAM negative, de novo expression of this molecule is associated with metaplastic or neoplastic changes. Thus, in ectocervical epithelia, expression of Ep-CAM occurs in early preneoplastic lesions (Litvinov et al., 1996); most squamous carcinomas of the head and neck region are Ep-CAM positive (Quak et al., 1990), and basal cell carcinomas are Ep-CAM positive in contrast to the normal epidermis (Tsubura et al., 1992).In many tumors that express Ep-CAM heterogeneously, an Ep-CAM–positive cell population may be found within an Ep-CAM–negative cell population, with both cell types expressing approximately equal levels of cadherins, as illustrated in Fig. ​Fig.11 A by a case of basal cell carcinoma. In glandular tissues such as gastric epithelium, which are low/ negative for Ep-CAM, expression of Ep-CAM is related to the development of early stages of intestinal metaplasia (our unpublished observation). Even in tissues with relatively high Ep-CAM expression, such as colon, the development of polyps is accompanied by an increase in Ep-CAM expression (Salem et al., 1993). In intestinal metaplasia one may observe Ep-CAM–positive cells bordering morphologically identical normal cells that are Ep-CAM–negative (as illustrated in Fig. ​Fig.11 B) Ep-CAM–positive cells bordering Ep-CAM–negative epithelial cells may also be found in some normal tissues such as hair follicles (Tsubura et al., 1992). Open in a separate windowFigure 1Examples of Ep-CAM expression by some cells within the E-cadherin–positive cell population. (A) Heterogeneous expression of Ep-CAM in a basal cell carcinoma, as detected by immunofluorescent staining with mAb 323/A3 to Ep-CAM (green fluorescence); the red fluorescence indicates the expression of E-cadherin (mAb HECD-1). (B) The de novo expression of Ep-CAM in gastric mucosa in relation to the development of intestinal metaplasia; immunohistochemical staining with mAb 323/A3. Note the bordering Ep-CAM–positive and –negative cells. Bars, 30 μM.From the examples presented, an increased or de novo expression of Ep-CAM is often observed in epithelial tissues in vivo. Expression of an additional molecule that may participate in cell adhesion in the context of other adhesion systems may have certain effects on the cell–cell interactions. Therefore, we have investigated whether the increased/de novo expression of Ep-CAM in epithelial cells (a) has any impact on interactions of positive cells with the parental Ep-CAM–negative cells, and (b) modulates in any way intercellular adhesive interactions of cells interconnected by E-cadherin, which is the major morphoregulatory molecule in epithelia.Here we demonstrate that expression of Ep-CAM by some cells in a mixed cell population expressing classical cadherins induces segregation of the Ep-CAM–positive cells from the parental cell population due to a negative effect on cadherin junctions caused by expression of Ep-CAM. The cadherin-modulating properties observed for Ep-CAM suggest a role for this molecule in the development of a proliferative and metaplastic cell phenotype, and probably in the development and progression of malignancies.     

Blue light‐induced apoplastic acidification of Arabidopsis thaliana guard cells: Inhibition by ABA is mediated through protein phosphatases

The phytohormone abscisic acid (ABA) inhibits blue light‐induced apoplastic acidification of guard cells. The signal transduction pathway of ABA, mediating this response, was studied using ABA‐insensitive ( abi ) mutants of Arabidopsis thaliana . Apoplastic acidification was monitored with a flat tipped pH‐electrode placed on epidermal strips, in which only guard cells were viable. Blue light‐induced apoplastic acidification was reduced by vanadate and diethylstilbestrol (DES), indicating involvement of plasma membrane‐bound H⁺‐ATPases. In wild type epidermal strips, ABA reduced blue light‐induced acidification to 63%. The inhibition did not result from an increased cytoplasmic free Ca²⁺ concentration in guard cells, since factors that increase the Ca²⁺ concentration stimulated apoplastic acidification. Apoplastic acidification was not inhibited by ABA in abi1 and abi2 mutants. In abi1 epidermal strips ABA had no effect on the acidification rate, while it stimulated apoplastic acidification in abi2 . The ABA response in both mutants could be partially restored with protein kinase and phosphatase inhibitors. The abi1 guard cells became ABA responsive in the presence of okadaic acid, a protein phosphatase inhibitor. In abi2 guard cells the wild type ABA response was partially restored by K‐252a, a protein kinase inhibitor. Apoplastic inhibition is thus mediated through the protein phosphatases encoded by ABI1 and ABI2 . The results with protein kinase and protein phosphatase inhibitors indicate that ABI1 and ABI2 are involved in separate signal transduction pathways.     

Red and blue light-stimulated proton efflux by epidermal leaf cells of the Argenteum mutant of Pisum sativum

Light stimulates leaf expansion in dicotyledons by increasingapoplastic acidification, cell wall loosening and solute accumulationfor turgor maintenance. Red and blue light enhance growth viadifferent photo-systems, but the cellular location and modesof action of these systems is not known. Here, the effect of red and blue light was studied on transportprocesses in epidermal cells of expanding leaves of the Argenteummutant of Pisum satlvum. Both red and blue light caused extraceiiuiaracidification by isolated epidermal tissue, which was stimulatedby extracellular K⁺ and inhibited by DCCD at 0.1 mol m^–3.Acidification induced by red compared with blue light showeddifferent saturating kinetics in fluence rate-response curves.Under near saturating light conditions the effects of red andblue light were additive. The red light-induced acidificationwas inhibited by far-red light while the blue light-inducedacidification was not. Light caused a hyperpoianzation of themembrane potential in epidermal strips, and stimulated ⁸⁶Rb⁺uptake by epidermal protoplasts. These results show that phytochromeand an additional blue light-photoreceptor function in isolatedepidermal cells to promote proton efflux, hyperpolarization,and cation uptake. Key words: Pisum sativum, light-induced acidification, ion transport, epidermis, photoreceptor     

Selective single cell isolation for genomics using microraft arrays

Genomic methods are used increasingly to interrogate the individual cells that compose specific tissues. However, current methods for single cell isolation struggle to phenotypically differentiate specific cells in a heterogeneous population and rely primarily on the use of fluorescent markers. Many cellular phenotypes of interest are too complex to be measured by this approach, making it difficult to connect genotype and phenotype at the level of individual cells. Here we demonstrate that microraft arrays, which are arrays containing thousands of individual cell culture sites, can be used to select single cells based on a variety of phenotypes, such as cell surface markers, cell proliferation and drug response. We then show that a common genomic procedure, RNA-seq, can be readily adapted to the single cells isolated from these rafts. We show that data generated using microrafts and our modified RNA-seq protocol compared favorably with the Fluidigm C1. We then used microraft arrays to select pancreatic cancer cells that proliferate in spite of cytotoxic drug treatment. Our single cell RNA-seq data identified several expected and novel gene expression changes associated with early drug resistance.     

Stability in a multi-species assemblage of large herbivores in East Africa

Summary Animal census data from Lake Manyara National Park in northern Tanzania are presented. The data refer to large mammalian herbivores, that is individually heavier than twenty kg, of which the numbers were counted in nine different years between 1959 and 1984. The total biomass of these herbivores was comprised mainly of African buffalo and African elephant. Five functional groups of herbivores were distinguished (buffalo, elephant-as-grazer, elephant-as-browser, other grazers, and other browsers). The pressures of all these groups were constant over time with the exception of that by buffalo. Buffalo numbers increased since the last outbreak of rinderpest in 1959. There was no correlation between herbivore biomass and rainfall fluctuations. Individual species showed large fluctuations in their numbers but within the total herbivore assemblage the different species compensated the fluctuations of the other species. This resulted in an overall constancy of herbivore biomass, and, thus, the carrying capacity of the system has to be viewed on the level of all species combined and not on that of the individual species. This view is supported by the result that size of the stability index showed that the system of herbivore species was stable.     

Large herbivores that strive mightily but eat and drink as friends

Summary Grazing in patches of Cynodon dactylon and of Sporobolus spicatus by four large herbivores, and the interaction between these sedentary herbivores was studied in Lake Manyara National Park, northern Tanzania. The herbivores were the African buffalo, Syncerus caffer; the African elephan, Loxodonta africana; the Burchell's zebra, Equus burchelli; and the wildebeest, Connochaetus taurinus. Four different hypotheses of the interactions between the herbivores were tested, viz., increased predator detection/protection through association of species, facilitation of the food intake through the influence of other species, use by other species of the food manipulation strategy of buffalo, and interspecific competition for food. On the level of a single day, zebra and wildebeest were symbiotic, which could have been caused by an increased chance of predator detection. A similar association between buffalo and wildebeest or zebra was also detected on C. dactylon grasslands. There was no indication of facilitation between any of the herbivores. Buffalo had a despotic relationship with elephant, that is the elephant's consumption was lowered when buffalo had visited a patch prior to their arrival. When elephant and buffalo arrived at the same time there appeared to be scramble competition between them.Habitat overlap was calculated for four pairs of species. In conjunction with the analyses of the patch visits, it was concluded that a small overlap was associated with interspecific competition and a large habitat overlap was associated with symbiosis.     

New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast microscopy

Summary One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.     

General Mechanisms for Solute Transport Across the Tonoplast of Plant Vacuoles: a Patch-Clamp Survey of Ion Channels and Proton Pumps

The electrical properties of the tonoplast from a large variety of plant materials such as mesophyll cells, storage cells, tumor cells, suspension cultured cells, guard cells, coleoptile cells, and liverwort cells have been investigated using the patch-clamp technique. Whole-vacuole recordings were employed to study the dynamics of an ATP-dependent proton pump by directly measuring the electrogenic currents. The addition of Mg-ATP induced an inwardly directed current which depolarized the tonoplast (the vacuole becoming positive inside). Furthermore, voltage-dependent passive ion fluxes were analyzed using whole vacuoles and isolated membrane patches. Whole-vacuolar currents and single-channel currents were induced at hyperpolarizing potentials, whereas currents decreased at positive trans-tonoplast potentials. The electrical properties of the tonoplast of vacuoles from various plant tissues were similar and it was concluded that ion fluxes across the tonoplast follow the same general mechanisms.