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Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules
Journal of Cell Biology (JCB)
Abstract
A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E-cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types.
... RPE cells are distinctive in that they contain apical Na + , K + -ATPase (Miller and Steinberg, 1979;Gundersen et al., 1991). Nevertheless, depending on the RPE preparation studied, apical expression can be lost (Geisen et al., 2006) or accompanied by basolateral expression (Okami et al., 1990;Hu et al., 1994;Marrs et al., 1995). Despite many years of investigation, the sorting signals and mechanisms that mediate the apical polarization of Na + , K + -ATPase remain poorly understood (Cereijido et al., 2012). ...
... Our images taken from human eye sections (Figure 8) support this assumption. Accordingly, it is plausible that Na + , K + -ATPase is only detected at the apical domain of cultured RPE cells under very specific conditions (Hu et al., 1994;Marrs et al., 1995;Rizzolo and Zhou, 1995;Kannan et al., 2006;Sonoda et al., 2010) because of the lack of an interaction of RPE cells with photoreceptors in cultures. However, in various RPE models, Na + , K + -ATPase is observed in the apical domain, even in the absence of contact with the retina. ...
Na⁺, K⁺-ATPase, or the Na⁺ pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β1 subunit of Na⁺, K⁺-ATPase plays an important role in this mechanism because homotypic β1-β1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na⁺ pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na⁺, K⁺-ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na⁺ pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β2 subunit. qPCR results showed a time-dependent increase in the level of β2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α2 subunit in that domain. Our results demonstrate that the apical polarization of Na⁺, K⁺-ATPase in RPE cells depends on the expression of the β2 subunit.
... В клетках РПЭ цыпленка отсутствуют десмосомы и десмосомальные белки. Десмосомы отсутствуют у некоторых млекопитающих (мышей и крыс) (Marrs et al., 1995). Данные о наличии десмосом в РПЭ глаза человека противоречивы. ...
... Образование такого потенциала в норме характерно для нейронов. Na + / K + -ATФаза располагается на апикальной стороне клеточной мембраны РПЭ (Marrs et al., 1995), в отличие от других эпителиальных клеток, где данный фермент-насос располагается на базолатеральной стороне (Wilt, Rizzolo, 2001; Low et al., 2002). РПЭ также необходим для развития и поддержания сетчатки путем секретирования большого количества транспортных белков, например, транстиретина (TTR) для транспорта витамина А (Strauss, 2005). ...
The review summarizes the data on the structure and functions of retinal pigment epithelial cells in vertebrate animals and human. Main attention is focused on modern concepts of protein expression patterns and cell-cell and cell-matrix interactions in situ in human retinal pigment epithelium.
... Additionally, our results demonstrated that application of N and ND to the media counteracted these morphological changes. Earlier investigations demonstrated that vitamin D restored damaged tight junctions and increased ZO-1 protein levels in ARPE-19 cells under oxidative stress conditions [36,46], which suggests its role in blood-retinal barrier integrity through intercellular adherent junctions [47]. ...
Age-related macular degeneration (AMD) is a multifactorial disease of the retina featured by dysfunction of retinal pigmented epithelial (RPE) and loss of photoreceptor cells under oxidative stress and inflammatory conditions. Vitamin D and antioxidants have beneficial effects against retinal degenerative diseases, such as AMD. We investigated the impact of associating vitamin D (ND) with a nutritional antioxidant complex (Nutrof Total®; N) on oxidative stress and inflammation-like induced conditions by H2O2 and LPS, respectively, in human retinal epithelial (ARPE-19) and human retinal endothelial (HREC) cells. Application of either N or ND treatments to H2O2-induced media in ARPE-19 cells counteracted late apoptosis, attenuated oxidative DNA damage, and increased cell proliferation. Significant reduction in the expression levels of MCP1, IL-8, and IL6 cytokines was observed following application of either N or ND treatments under LPS-induced conditions in ARPE-19 cells and in MCP-1 and IL12p70 cytokine levels in HREC cells. ND and not N revealed significant downregulation of IFNγ in ARPE-19 cells, and of IL-6 and IL-18 in HREC cells. In conclusion, adding vitamin D to Nutrof Total® protects in a synergistic way against oxidative and inflammatory stress-induced conditions in retinal epithelial and endothelial cells.
... The extracellular domains of each isoform contain one to three sites for N-glycosylation. N-glycosylation is important for cellcell and cell-extracellular matrix interactions and is present in several cytoskeletal proteins, including laminins, integrins, and cadherins [ (416,720), reviewed in (118)]. The majority of disease-causing sarcoglycan mutations lie within the extracellular domains (70,71,378,433,435,556,592). ...
Cardiac and skeletal striated muscles are intricately designed machines responsible for muscle contraction. Coordination of the basic contractile unit, the sarcomere, and the complex cytoskeletal networks are critical for contractile activity. The sarcomere is comprised of precisely organized individual filament systems that include thin (actin), thick (myosin), titin, and nebulin. Connecting the sarcomere to other organelles (e.g., mitochondria and nucleus) and serving as the scaffold to maintain cellular integrity are the intermediate filaments. The costamere, on the other hand, tethers the sarcomere to the cell membrane. Unique structures like the intercalated disc in cardiac muscle and the myotendinous junction in skeletal muscle help synchronize and transmit force. Intense investigation has been done on many of the proteins that make up these cytoskeletal assemblies. Yet the details of their function and how they interconnect have just started to be elucidated. A vast number of human myopathies are contributed to mutations in muscle proteins; thus understanding their basic function provides a mechanistic understanding of muscle disorders. In this review, we highlight the components of striated muscle with respect to their interactions, signaling pathways, functions, and connections to disease.
Fibrosis represents a process characterized by excessive deposition of extracellular matrix (ECM) proteins. It often represents the evolution of pathological conditions, causes organ failure, and can, in extreme cases, compromise the functionality of organs to the point of causing death. In recent years, considerable efforts have been made to understand the molecular mechanisms underlying fibrotic evolution and to identify possible therapeutic strategies. Great interest has been aroused by the discovery of a molecular association between epithelial to mesenchymal plasticity (EMP), in particular epithelial to mesenchymal transition (EMT), and fibrogenesis, which has led to the identification of complex molecular mechanisms closely interconnected with each other, which could explain EMT-dependent fibrosis. However, the result remains unsatisfactory from a therapeutic point of view. In recent years, advances in epigenetics, based on chromatin remodeling through various histone modifications or through the intervention of non-coding RNAs (ncRNAs), have provided more information on the fibrotic process, and this could represent a promising path forward for the identification of innovative therapeutic strategies for organ fibrosis. In this review, we summarize current research on epigenetic mechanisms involved in organ fibrosis, with a focus on epigenetic regulation of EMP/EMT-dependent fibrosis.
Pulmonary fibrosis is a chronic and progressive fibrotic disease that results in impaired gas exchange, ventilation, and eventual death. The pro-fibrotic environment is instigated by various factors, leading to the transformation of epithelial cells into myofibroblasts and/or fibroblasts that trigger fibrosis. Epithelial mesenchymal transition (EMT) is a biological process that plays a critical role in the pathogenesis of pulmonary fibrosis. Epigenetic regulation of tissue-stromal crosstalk involving DNA methylation, histone modifications, non-coding RNA, and chromatin remodeling plays a key role in the control of EMT. The review investigates the epigenetic regulation of EMT and its significance in pulmonary fibrosis.
The proper localization of ion channels and transporters to the apical or basolateral membrane domains is essential for correct ion transport through epithelia. Polarized sorting is achieved through elaborate intracellular sorting pathways encompassing the trans-Golgi network, early endosomes, and recycling endosomes that are fine-tuned for epithelial function and thus different from the secretory and endocytic systems in non-polarized cells. Polarized cells express epithelial-cell specific molecules, including sorting adaptors like the cytosolic clathrin adaptor AP-1B, which are required for efficient segregation and targeting of proteins to specific plasma membrane domains. In this book chapter, we will first discuss the various sorting stations in the cells followed by a review of apical and basolateral sorting signals and their interpretation at different compartments. Specific emphasis is placed on the trafficking of ion transporters.
Human retinal pigment epithelial (RPE) cells at different population doublings (PDs) were cultured for 28 days to examine their phenotypic heterogeneity in a confluent state. In an early population (PD = 2.8), cells showed a cobblestone-like appearance (type I), which gradually became small and tight, and eventually exhibited dark pigmentation. Some cells showed a dome-like structure (type II), which detached from the culture surface during culture. With increasing PD, the cells showed active migration that caused a shift in phenotype from a single layer of large, flattened cells (type III) to a multiple cell layers (stratified) with flattened, irregularly shaped cells (type IV). Immunostaining of specific RPE markers, ZO-1 and Na(+)/K(+)-ATPase revealed that cells have markedly decreased expressions in a late population (PD = 10.1). RPE phenotypes were classified into four types by measuring the nuclear size and local density. The frequencies of type I cells decreased with increasing PD value, while the frequencies of type III and IV cells increased along with the decrease in type I. The frequencies of type IV cells at PD = 10.1 had increased by 10.3-fold compared with PD = 2.8. From these results, the nuclear size and local density were proposed as indicators for understanding phenotypic heterogeneity of RPE cells in the passaged cell population during cell expansion. It is concluded that the population doubling level is an important factor to affect the transition of RPE phenotype and thereby to modulate the quality of cultured cells.
Age-related macular degeneration (AMD) is a leading cause of blindness among the aging population. Currently, replacement of diseased retinal pigment epithelium (RPE) cells with transplanted healthy RPE cells could be a feasible approach for AMD therapy. However, maintaining cell-cell contact and good viability of RPE cells cultured in vitro is difficult and fundamentally determines the success of RPE cell transplantation. This study was conducted to examine the role of Matrigel and Activin A (MA) in regulating cell-cell contact and anti-apoptotic activity in human RPE (hRPE) cells, as assessed by atomic force microscopy (AFM), scanning electron microscope (SEM), immunofluorescence staining, quantitative polymerase chain reaction (qPCR) analysis, Annexin V/propidium iodide (PI) analysis, mitochondrial membrane potential (△Ψ m) assays, intracellular reactive oxygen species (ROS) assays and Western blotting. hRPE cells cultured in vitro could maintain their epithelioid morphology after MA treatment over at least 4 passages. The contact of N-cadherin to the lateral cell border was promoted in hRPE cells at P2 by MA. MA treatment also enhanced the expression of tight junction-associated genes and proteins, such as Claudin-1, Claudin-3, Occludin and ZO-1, as well as polarized ZO-1 protein distribution and barrier function, in cultured hRPE cells. Moreover, MA treatment decreased apoptotic cells, ROS and Bax and increased △Ψ m and Bcl2 in hRPE cells under serum withdrawal-induced apoptosis. In addition, MA treatment elevated the protein expression levels of β-catenin and its target proteins, including Cyclin D1, c-Myc and Survivin, as well as the gene expression levels of ZO-1, β-catenin, Survivin and TCF-4, all of which could be down-regulated by the Wnt/β-catenin pathway inhibitor XAV-939. Taken together, MA treatment could effectively promote cell-cell contact and anti-apoptotic activity in hRPE cells, partly involving the Wnt/β-catenin pathway. This study will benefit the understanding of hRPE cells and future cell therapy.
The proper localization of ion channels and transporters to the apical or basolateral membrane domains is essential for correct ion transport through epithelia. Polarized sorting is achieved through elaborate intracellular sorting pathways encompassing the trans-Golgi network, early endosomes, and recycling endosomes that are fine-tuned for epithelial function and thus different from the secretory and endocytic systems in non-polarized cells. Polarized cells express epithelial cell-specific molecules, including sorting adaptors like the cytosolic clathrin adaptor AP-1B, that are required for efficient segregation and targeting of proteins to specific plasma membrane domains. In this book chapter, we will first discuss the various sorting stations in the cells followed by a review of apical and basolateral sorting signals and their interpretation at different compartments. Specific emphasis is placed on the trafficking of ion transporters.
The potential for Ksp-cadherin involvement in either the development or maintenance of the metanephric kidney was assessed by immunocytochemical localization of a monoclonal antibody directed against the rabbit isoform of Ksp-cadherin in both neonatal and adult, rabbit kidneys. In the adult kidney Ksp-caaherin expression was detected on the basolateral membrane of all cell, types in both the tubular nephron and the collecting system. Immunoelectron microscopy indicated that Ksp-cadherin was expressed at uniform levels along the entire length of both the lateral membranes and the basal infoldings of all tubular epithelial cell types. In the nephrogenic zone of the neonatal rabbit kidney Ksp-cadherin expression was detected exclusively on the basolateral membranes of epithelial cells in the more highly differentiated regions of the expanding ureteric duct. In the highly differentiated corticomedullary and medullary regions of the neonatal kidney, distinct basolateral staining was observed in all segments of the tubular nephron and the collecting system. The relatively late appearance of Ksp-cadherin expression in the developing metanephros indicates that Ksp-cadherin probably does not participate in the direction of renal morphogenesis. However, the high levels of Ksp-cadherin expression observed in all segments of the tubular nephron and the collecting system in the adult kidney suggests that it may play a role in the maintenance of the terminally differentiated tubular epithelial phenotype.
Retinal pigment epithelium (RPE) cells form a monolayer at the blood-retina barrier between the retina and choriocapillaries. The barrier function may be damaged by multiple stresses to the cell, including the repeated exposure to oxidants that are generated by photoreceptor cell turnover. The purpose of our study was to document the protective effect of pigment epithelium-derived factor (PEDF), a tropic factor produced by the RPE, on H2O2-induced RPE barrier dysfunction. When assayed by a FITC-labeled dextran transepithelial flux, the increased permeability of the RPE barrier (induced by H2O2) was prevented by PEDF pretreatment. To further explore the mechanism leading to this permeability change, we investigated the distribution of cytoskeleton and junctional proteins. The redistribution of the two junctional proteins occludin, and N-cadherin and actin reorganization in RPE, induced by H2O2, can be prevented by PEDF pretreatment. PEDF can also prevent H2O2-induced stress kinase p38/27-kDa heat shock protein signaling which is known to mediate actin rearrangement. These findings indicated that PEDF can stabilize actin, maintain normal membrane occludin and N-cadherin structure, and preserve the barrier function of RPE cells against oxidative stress. (c) 2006 Elsevier Inc. All rights reserved.
Cadherin function is required for normal keratinocyte intercellular adhesion and stratification, In the present study, we have investigated whether cadherin-cadherin interactions may also modulate keratinocyte differentiation, as evidenced by alterations in the levels of several differentiation markers, Confluent keratinocyte cultures, propagated in low Ca2+ medium in which cadherins are not active, were pre-incubated with antibodies that block the function of E-cadherin and/or P-cadherin; Ca2+ was then elevated to 1 mM to activate the cadherins and induce differentiation. In control cultures (incubated with no antibody or with antibodies to other cell surface molecules), Ca2+ elevation induced an increase in type 1 transglutaminase, profilaggrin, and loricrin, as measured by western blotting and in agreement with previous results. However, the concurrent addition of antibodies against both E- and P-cadherin prevented this increase in transglutaminase 1 protein, Incubation with either antibody alone had no consistent effect. Profilaggrin and loricrin, which are later markers of keratinocyte differentiation, responded differently from transglutaminase 1 to addition of antibodies. In the presence of anti-E-cadherin antibody, both loricrin and profilaggrin levels were dramatically enhanced compared to the high Ca2+ control cells, while addition of antibody to P-cadherin slightly attenuated the Ca2+-induced increase. In the presence of both antibodies, loricrin and profilaggrin protein levels were intermediate between those observed in the presence of either antibody alone, The expression of involucrin, however, was unaffected by addition of antibodies, In addition, effects of the anti-cadherin antibodies were not secondary to alterations in proliferation or programmed cell death, as determined by several independent assays of these processes. Thus, the consequences of cadherin inhibition depend upon both the particular cadherin and the differentiation marker under study. Taken together, these data suggest that E-cadherin and P-cadherin contribute to the orderly progression of terminal differentiation in the epidermis in multiple ways.
Junctional complexes, including tight junctions, actin-associated adherens junctions, and intermediate-filament-associated desmosomes, are major structural components in epithelial cells that are essential for the establishment of epithelial cell polarity. Various signaling molecules, involving tyrosine kinases and phosphatases, small GTPases, and components of the Wnt signaling pathway regulate the assembly and disassembly of junctional complexes during embryonic development and cell differentiation. The levels of regulation involve the coordinated interactions of junctional proteins with the cytoskeleton, the interplay between different junctional complexes, and the active participation of junctional proteins in signaling cascades. The interaction of beta-catenin with both components of adherens junctions and proteins of the Wnt/wingless signaling pathway is the best characterized mechanism to date that links cell adhesion to gene expression and cell fate determination.
DefinitionApical-Basolateral Polarization in Epithelial CellsMechanisms to Localize PM Proteins Apically or BasolaterallyMechanisms Involved in the Sorting of Na, K-ATPaseProton-Coupled Monocarboxylate TransportersVariations in the Sorting Machinery Among EpitheliaFuture PerspectivesAbbreviationsReferences
The structure and functions of cadherins have been found to be much more complex and diverse than previously assumed. Cadherins are involved in the regulation of morphogenesis in many organisms, including invertebrate species, thus indicating the general importance of this family in organizing multicellular structures. In the classical view, cadherins function mainly in the adhesion of solid tissues, but they have been found also in mesenchymal tissues and on migrating cells as well, indicating that they may be involved in cell rearrangement of most if not all tissues. This seems to be achieved at least, in part, by the expression of a large number of cadherin subtypes with distinct adhesion specificities. Crystal structures from the parts of the extracellular domains of E- and N-cadherin reveal new insights in the interaction mechanisms of these proteins. However, it is still unclear whether each cadherin varies in its three-dimensional structure and what the consequences of its cytoplasmic anchorage are. Thus, a major area for future studies is to resolve the 3D structure of other cadherins and the complex cytoplasmic network underlying cadherin clusters. The knowledge about the proteins being differentially recruited into junctions mediated by different cadherins may answer the question about the signals cadherins are able to transmit.
During development cells of epithelial and mesenchymal origin convert between the two phenotypes in what has been described as Epithelial-Mesenchymal Transition (EMT) and Mesenchymal-Epithelial Transition (MET). The common characteristics exhibited during EMT are a loss of epithelial cell contacts, a reorganization of cytoskeletal components to promote a motile phenotype, and a remodeling of the surrounding extracellular matrix to allow for invasion. These events are tightly regulated and required for proper cellular organization and organogenesis during development. Studies in cancer models have identified an analogous plasticity of some epithelial cancer cells which acquire mesenchymal features as a means to escape the primary tumor mass. During the initial stages of tumor metastasis a complex series of events occur in which cancer cells leave the original tumor site and migrate to other parts of the body via the bloodstream and/or the lymphatic system. All metastatic cells must first acquire the abilities to disseminate, migrate and invade the surrounding tissue to allow for metastasis to occur. Thus, a reactivation of developmental pathways resulting in an EMT-like program is one possible mechanism by which cells acquire these capabilities and are able to form distal metastasis. Intriguingly, many similarities between developmental and oncogenic EMT have been identified and has led to our understanding of common signaling pathways (including TGF-beta, Ras and Wnt), transcriptional regulators (including the Snail, Zeb and Twist families) and microRNAs (including let-7 and miR-200 families) which regulate EMT. Aberrant regulation of these pathways and factors is associated with increased metastatic potential in vitro and in animal models and correlate with poor clinical outcomes. This chapter focuses on the EMT program in cancer, its regulation, its parallels to developmental EMT and its significance to the progression to metastatic disease.
β-Catenin is essential for the function of cadherins, a family of Ca²⁺-dependent cell–cell adhesion molecules, by linking them to α-catenin and the actin cytoskeleton. β-Catenin also binds to adenomatous polyposis coli (APC) protein, a cytosolic protein that is the product of a tumor suppressor gene mutated in colorectal adenomas. We have expressed mutant β-catenins in MDCK epithelial cells to gain insights into the regulation of β-catenin distribution between cadherin and APC protein complexes and the functions of these complexes. Full-length β-catenin, β-catenin mutant proteins with NH2-terminal deletions before (ΔN90) or after (ΔN131, ΔN151) the α-catenin binding site, or a mutant β-catenin with a COOH-terminal deletion (ΔC) were expressed in MDCK cells under the control of the tetracycline-repressible transactivator. All β-catenin mutant proteins form complexes and colocalize with E-cadherin at cell–cell contacts; ΔN90, but neither ΔN131 nor ΔN151, bind α-catenin. However, β-catenin mutant proteins containing NH2-terminal deletions also colocalize prominently with APC protein in clusters at the tips of plasma membrane protrusions; in contrast, full-length and COOH-terminal– deleted β-catenin poorly colocalize with APC protein. NH2-terminal deletions result in increased stability of β-catenin bound to APC protein and E-cadherin, compared with full-length β-catenin. At low density, MDCK cells expressing NH2-terminal–deleted β-catenin mutants are dispersed, more fibroblastic in morphology, and less efficient in forming colonies than parental MDCK cells. These results show that the NH2 terminus, but not the COOH terminus of β-catenin, regulates the dynamics of β-catenin binding to APC protein and E-cadherin. Changes in β-catenin binding to cadherin or APC protein, and the ensuing effects on cell morphology and adhesion, are independent of β-catenin binding to α-catenin. These results demonstrate that regulation of β-catenin binding to E-cadherin and APC protein is important in controlling epithelial cell adhesion.
The retinal pigment epithelium (RPE) plays a key role in the development of many eye diseases leading to visual impairment and even blindness. Cell culture models of pathological changes in the RPE make it possible to study factors responsible for these changes and signaling pathways coordinating cellular and molecular mechanisms of cell interactions under pathological conditions. Moreover, they give an opportunity to reveal target cells and develop effective specific treatment for degenerative and dystrophic diseases of the retina. In this review, data are presented on RPE cell sources for culture models, approaches to RPE cell culturing, phenotypic changes of RPE cells in vitro, the role of signal pathways, and possibilities for their regulation in pathological processes.
Epithelial cell carcinogenesis involves the loss of cell polarity, alteration of polarized protein presentation, dynamic cell morphology changes, increased proliferation, and increased cell motility and invasion. Membrane vesicle trafficking underlies all of these processes. Specific membrane trafficking regulators, including RAB small GTPases, through the coordinated dynamics of intracellular trafficking along cytoskeletal pathways, determine the cell surface presentation of proteins and the overall function of both differentiated and neoplastic cells. Although mutations in vesicle trafficking proteins may not be direct drivers of transformation, components of the machinery of vesicle movement have crucial roles in the phenotypes of neoplastic cells. Therefore, the regulators of membrane vesicle trafficking decisions are essential mediators of the full range of cell physiologies that drive cancer cell biology, including initial loss of cell polarity, invasion and metastasis. Targeting of these fundamental intracellular processes may permit the manipulation of cancer cell behaviour.
Light toxicity is suspected to enhance certain retinal degenerative processes such as age-related macular degeneration. Death of photoreceptors can be induced by their exposure to the visible light, and although cellular processes within photoreceptors have been characterized extensively, the role of the retinal pigment epithelium (RPE) in this model is less well understood. We demonstrate that exposition to intense light causes the immediate breakdown of the outer blood-retinal barrier (BRB). In a molecular level, we observed the slackening of adherens junctions tying up the RPE and massive leakage of albumin into the neural retina. Retinal pigment epithelial cells normally secrete vascular endothelial growth factor (VEGF) at their basolateral side; light damage in contrast leads to VEGF increase on the apical side - that is, in the neuroretina. Blocking VEGF, by means of lentiviral gene transfer to express an anti-VEGF antibody in RPE cells, inhibits outer BRB breakdown and retinal degeneration, as illustrated by functional, behavioral and morphometric analysis. Our data show that exposure to high levels of visible light induces hyperpermeability of the RPE, likely involving VEGF signaling. The resulting retinal edema contributes to irreversible damage to photoreceptors. These data suggest that anti-VEGF compounds are of therapeutic interest when the outer BRB is altered by retinal stresses.
This chapter summarizes the important role that sorting of membrane proteins plays in generating cell polarity that has been characterized in recent years. The chapter presents the current understanding of how structural elements (cytoskeleton and junctional complexes) in epithelial cells function and give rise to the polarized cell phenotype. The generation and maintenance of polarized distributions of ion transporting proteins is required for the many specialized cellular functions of epithelial cells. Differentiation of epithelial cells, and thus the generation of polarized distribution of membrane proteins, is accompanied by the reorganization and remodeling of the cytoskeleton. Evidence suggests that cadherin-mediated cell-cell adhesion and the membrane-cytoskeleton serve to generate and maintain polarity of Na+-K+-ATPase in epithelial cells. A general mechanism for establishing the polarity of other membrane proteins may involve similar roles for the membrane-cytoskeleton in the remodeling of epithelial cells during cellular differentiation.
Functional studies divided cell adhesion mechanisms into two systems: (1) the Ca2+-dependent and (2) the Ca2+-independent systems. Ca2+-dependent systems are disrupted by trypsin but can be protected from proteolysis by Ca2+, whereas Ca2+-independent ones are inactivated only with high concentrations of trypsin and are not protected by Ca2+. This chapter focuses on the Ca2+-dependent cadherin adhesion molecules. This includes the classical cadherins that localize in cell-cell adherens junctions associated with actin microfilaments and the desmosomal cadherins that localize in desmosomal junctions and are involved in linking the intermediate filament cytoskeleton to cell membranes. A distinction is made between cell-cell adherens junctions where cadherins are localized and cell-matrix adherens junctions where adhesion molecules of the integrin family are generally found. In the mouse preimplantation embryo, a striking demonstration of Ca2+-dependent adhesion can be seen at the eight-cell stage when E-cadherin becomes functional and compaction occurs causing the cell membranes to flatten against each other and become indistinct.
This chapter describes the role of the membrane cytoskeleton in regulating protein sorting and retention in membranes. Protein sorting is a complex process occurring at all stages of the secretory cycle. Newly synthesized membrane proteins are transported through a series of common compartments,—the endoplasmic reticulum and Golgi cisternae—regardless of their ultimate destination. Upon arrival at the plasma membrane, proteins have different fates: Some proteins are internalized and then, in the endosome, are either sorted for degradation in lysosomes or recycled back to the plasma membrane; other resident proteins are sequestered from internalization pathways and retained in the plasma membrane. However, several known protein complexes that form membrane coat structures can be viewed as candidate membrane protein-sorting machines. These include the clathrin–AP complex, the coatamer (COP) complex, caveolae, p200, and spectrin. The chapter examines the structural and functional properties of the spectrin-based membrane skeleton to demonstrate its potential as a protein-sorting machine.
Cadherins comprise a large family of membrane glycoproteins that mediate Ca+-dependent cell adhesion during development and in the adult. Although the extracellular domain controls homotypic recognition and binding between cadherins on adjacent cells, proteins that bind to the cytoplasmic domain (catenins) also regulate cell adhesivity. Assembly of the cadherin/catenin complex is temporally and spatially regulated during transport to the cell surface, and in polarized epithelial cells, different cadherin/catenin and catenin complexes have specialized sub-cellular distributions. Changes in the levels of expression, dynamics of assembly and phosphorylation of catenins directly affect cadherin function. Taken together, catenins are emerging as important linkers in cellular processes involved in adhesion, proliferation and morphogenesis.
The cadherin/catenin complexes expressed by a murine epidermal keratinocyte cell line PDV, expressing E- and P-cadherin, have been analysed using a combination of biochemical and confocal microscopy analysis. Two types of E-cadherin complexes, containing β-catenin or plakoglobin and α-catenin, were detected in PDV cells as in other cell types, while β-cadherin was mainly detected in complexes containing β-catenin and α-catenin in PDV and other murine epidermal keratinocytes. Bio tin-labelling studies have shown that both types of E-cadherin complexes are present at the surface of confluent cells. Furthermore, confocal microscopy analysis indicated that E-cadherin/ plakoglobin complexes are located in stable cell-cell contacts at the middle lateral membranes and associated with α-catenin and the actin cytoskeleton, with a similar distribution to that of the E-cadherin/β-catenin complexes. In addition, E-cadherin/ plakoglobin complexes not associated with α-catenin or the actin cytoskeleton were detected in lower planes of the lateral contacting membranes as well as E-cadherin non-associated with catenins in the more basal planes. These studies support that in murine epidermal keratinocytes both β-catenin- and plakoglobin-containing E-cadherin complexes contribute to the maintenance of stable cell-cell contacts and suggest a differential role of the plakoglobin containing complexes in different epithelial cell types.
Cell-to-cell junction structures play a key role in cell growth rate control and cell polarization. In endothelial cells
(EC), these structures are also involved in regulation of vascular permeability and leukocyte extravasation. To identify novel
components in EC intercellular junctions, mAbs against these cells were produced and selected using a morphological screening
by immunofluorescence microscopy. Two novel mAbs, LIA1/1 and VJ1/16, specifically recognized a 25-kD protein that was selectively
localized at cell–cell junctions of EC, both in the primary formation of cell monolayers and when EC reorganized in the
process of wound healing. This antigen corresponded to the recently cloned platelet-endothelial tetraspan antigen CD151/PETA-3
(platelet-endothelial tetraspan antigen-3), and was consistently detected at EC cell–cell contact sites. In addition to CD151/PETA-3,
two other members of the tetraspan superfamily, CD9 and CD81/ TAPA-1 (target of antiproliferative antibody-1), localized
at endothelial cell-to-cell junctions. Biochemical analysis demonstrated molecular associations among tetraspan molecules
themselves and those of CD151/ PETA-3 and CD9 with α3β1 integrin. Interestingly, mAbs directed to both CD151/PETA-3 and CD81/
TAPA-1 as well as mAb specific for α3 integrin, were able to inhibit the migration of ECs in the process of wound healing.
The engagement of CD151/PETA-3 and CD81/TAPA-1 inhibited the movement of individual ECs, as determined by quantitative time-lapse
video microscopy studies. Furthermore, mAbs against the CD151/PETA-3 molecule diminished the rate of EC invasion into collagen
gels. In addition, these mAbs were able to increase the adhesion of EC to extracellular matrix proteins. Together these results
indicate that CD81/TAPA-1 and CD151/PETA-3 tetraspan molecules are components of the endothelial lateral junctions implicated
in the regulation of cell motility, either directly or by modulation of the function of the associated integrin heterodimers.
Background
Cerebrospinal fluid (CSF) sodium concentration increases during migraine attacks, and both CSF and vitreous humor sodium increase in the rat migraine model. The Na,K-ATPase is a probable source of these sodium fluxes. Since Na,K-ATPase isoforms have different locations and physiological roles, our objective was to establish which alpha isoforms are present at sites where sodium homeostasis is disrupted.
Methods
Specific Na,K-ATPase alpha isoforms were identified in rat tissues by immunohistochemistry at the blood-CSF barrier at the choroid plexus, at the blood-CSF-trigeminal barrier at the meninges, at the blood-retina barrier, and at the blood-aqueous barrier at the ciliary body. Calcitonin gene-related peptide (CGRP), occludin, or von Willibrand factor (vWF) were co-localized with Na,K-ATPase to identify trigeminal nociceptor fibers, tight junctions, and capillary endothelial cells respectively.
Results
The Na,K-ATPase alpha-2 isoform is located on capillaries and intensely at nociceptive trigeminal nerve fibers at the meningeal blood-CSF-trigeminal barrier. Alpha-1 and −3 are lightly expressed on the trigeminal nerve fibers but not at capillaries. Alpha-2 is expressed at the blood-retina barriers and, with alpha-1, at the ciliary body blood aqueous barrier. Intense apical membrane alpha-1 was associated with moderate cytoplasmic alpha-2 expression at the choroid plexus blood-CSF barrier.
Conclusion
Na,K-ATPase alpha isoforms are present at the meningeal, choroid plexus, and retinal barriers. Alpha-2 predominates at the capillary endothelial cells in the meninges and retinal ganglion cell layer.
Rab25 is a tumor suppressor for colon cancer in humans and mice. To identify elements of intestinal polarity regulated by Rab25, we have developed Caco2-BBE cell lines stably expressing shRNA for Rab25 and lines rescuing Rab25 knockdown with reexpression of rabbit Rab25. Rab25 knockdown decreased α2, α5 and β1-integrin expression. We observed colocalization and direct association of Rab25 with α5β1-integrins. Rab25 knockdown also up-regulated Claudin-1 expression, increased transepithelial resistance and increased invasive behavior. Rab25 knockdown cells showed disorganized brush border microvilli with decreases in villin expression. All of these changes were reversed by reintroduction of rabbit Rab25. Rab25 knockdown altered the expression of 29 gene transcripts including the loss of α5-integrin transcripts. Rab25 loss decreased expression of one transcription factor, ETV4, and overexpression of ETV4 in Rab25 knockdown cells reversed losses of α5β1-integrin. The results suggest that Rab25 controls intestinal cell polarity through the regulation of gene expression.
Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness in working-age Americans. DR is traditionally regarded as a disorder of blood-retina barriers, and the leakage of blood content is a major pathological characteristic of the disease. While the breakdown of the endothelial barrier in DR has been investigated extensively, the vascular leakage through the retinal pigment epithelium (RPE) barrier in the disease has not been widely acknowledged. As the blood content leaked through the RPE barrier causes excessive water influx to the retina, the breakdown of the RPE barrier is likely to play a causative role in the development of some forms of diabetic macular edema, a major cause of vision loss in DR. In this article, we will discuss the clinical evidences of the diabetes-induced RPE barrier breakdown, the alteration of the RPE in diabetes, the molecular and cellular mechanism of RPE barrier breakdown, and the research tools for the analysis of RPE barrier leakage. Finally, we will discuss the methodology and potential applications of our recently developed fluorescent microscopic imaging for the diabetes- or ischemia-induced RPE barrier breakdown in rodents.
The sections in this article are:
Questions
Tools and Techniques with Which to Study Epithelial Polarity
Epithelial Monolayers Grown on Permeable Substrata
Morphological Techniques
Biochemical Techniques
Protein Trafficking Pathways in Epithelial Cells
Biogenetic Pathways
Transcytosis
Recycling Pathways
Tissue‐Specific Variation of Protein Polarity and Targeting Pathways in Epithelial Cells
Sorting Signals and Mechanisms
Establishment of Epithelial Polarity
The Role of E‐Cadherin
Future Prospects: In Vitro Systems, Genetic Models, and the Search for Sorting Machinery
The sections in this article are:
The Red Cell Membrane Skeleton
How Does the Spectrin Membrane Skeleton Stabilize the Red Cell?
The Trilayer Couple—Spectrin as A Membrane Organizer
Components of the Erythrocyte Membrane Skeleton
Spectrin
Actin
Ankyrin
Protein 4.1
Adducin
Dematin (Protein 4.9)
Pallidin (Protein 4.2)
P55 (an Erythrocyte Membrane‐Associated Guanylate Kinase)
Stomatin
Tropomyosin and Tropomodulin
Dynamin
Interactions with Phospholipids
The Spectrin Skeleton of Non‐Erythroid Cells
Spatial and Temporal Polarization
Proteins Interacting with Spectrin in Non‐Erythroid Cells
Cytoskeletal Elements
Adhesion Proteins
Evolving Concepts
Conclusions: The Linked Mosaic Model
The diversity of epithelia in the body permits a multitude of organ-specific functions. One of the foremost examples of this is the retinal pigment epithelium. Located between the photoreceptors of the retina and their principal blood supply, the choriocapillaris, the retinal pigment epithelium is critical for the survival and function of retinal photoreceptors. To serve this purpose, the retinal pigment epithelium cell has adapted the classic Golgi-to-cell-surface targeting pathways first described in such prototypic epithelial cell models as the Madin-Darby canine kidney cell, to arrive at a unique distribution of membrane and secreted proteins. More recent data suggest that the retinal pigment epithelium also takes advantage of its inherent asymmetry to augment the classical pathways of Golgi-to-cell-surface traffic. As retinal pigment epithelium transplants and gene therapy represent potential cures for retinal degenerative diseases, understanding the basis of the unique polarity properties of retinal pigment epithelium cells will be a critical issue for the development of future therapies.
With heterologous antibodies raised against animal N-cadherin, -catenin, and -catenin, we have visualized their reactive proteins within cells of maize root apices. Embedding using Steedman's wax allowed us to accomplish tissue-specific analysis which revealed that cells of epidermis, endodermis/pericycle, and outer stele tissues, all of which are tightly associated to each other, are especially enriched with presumed plant homologues of N-cadherin and both catenins. In the root epidermis, trichoblasts initiating root hairs showed prominent accumulations of cadherin-like antigens at outgrowing domains where they co-localize with actin. Close associations of cadherin-like proteins with F-actin were detected in parenchymatic cells of the stele, also at the immunogold electron microscopy level. A possible role of these interesting proteins in membrane-membrane interactions is indicated by their prominent accumulations at endoplasmic-reticulum-enriched pit-field-based plant cell adhesion domains in plasmolyzing cells of maize root apices exposed to mannitol. Intriguingly, these unique adhesion domains of plasmolyzing cells are enriched with endoplasmic-reticulum-resident calreticulin. Cadherin-like, but not catenin-like, proteins were abundant also within the nucleoplasm.
Calreticulin is a major Ca2+ binding protein in the endoplasmic reticulum of non-muscle cells. In this report we show that calreticulin protein is strongly induced by heat shock. Activation and attenuation of the heat shock transcriptional response is caused by heat shock factor that binds to 5'-flanking sequences of heat shock responsive genes, the heat shock element. The smallest stretch of DNA that shows detectable binding of heat shock factor in vitro contains a two-sequence unit nGAAnnTTCn which exists in the 5'-flanking region of calreticulin DNA (5'-gGAAccCAGcgTTC-3'). The present data provide direct evidence that calreticulin expression can be modulated by heat shock. Thus, our results strengthen the hypothesis that calreticulin, in addition to its function as a cellular Ca2+ store, is a multifunctional protein which performs at least some of its functions from the lumen of the ER.
The protein kinase liver kinase B1 (LKB1) regulates cell polarity and intercellular junction stability. Also, LKB1 controls the activity of salt-inducible kinase 1 (SIK1). The role and relevance of SIK1 and its downstream effectors in linking the LKB1 signals within these processes are partially understood. We hypothesize that SIK1 may link LKB1 signals to the maintenance of epithelial junction stability by regulating E-cadherin expression. Results from our studies using a mouse lung alveolar epithelial (MLE-12) cell line or human renal proximal tubule (HK2) cell line transiently or stably lacking the expression of SIK1 (using SIK1 siRNAs or shRNAs), or with its expression abrogated (sik1(+/+) vs. sik1(-/-) mice), indicate that suppression of SIK1 (∼40%) increases the expression of the transcriptional repressors Snail2 (∼12-fold), Zeb1 (∼100%), Zeb2 (∼50%), and TWIST (∼20-fold) by activating cAMP-response element binding protein. The lack of SIK1 and activation of transcriptional repressors decreases the availability of E-cadherin (mRNA and protein expression by ∼100 and 80%, respectively) and the stability of intercellular junctions in epithelia (decreases in transepithelial resistance). Furthermore, LKB1-mediated increases in E-cadherin expression are impaired in cells where SIK1 has been disabled. We conclude that SIK1 is a key regulator of E-cadherin expression, and thereby contributes to the stability of intercellular junctions.
Cell-cell adhesion is thought to play important roles in development, in tissue morphogenesis, and in the regulation of cell migration and proliferation. Desmosomes are adhesive intercellular junctions that anchor the intermediate filament network to the plasma membrane. By functioning both as an adhesive complex and as a cell-surface attachment site for intermediate filaments, desmosomes integrate the intermediate filament cytoskeleton between cells and play an important role in maintaining tissue integrity. Recent observations indicate that tissue integrity is severely compromised in autoimmune and genetic diseases in which the function of desmosomal molecules is impaired. In addition, the structure and function of many of the desmosomal molecules have been determined, and a number of the molecular interactions between desmosomal proteins have now been elucidated. Finally, the molecular constituents of desmosomes and other adhesive complexes are now known to function not only in cell adhesion, but also in the transduction of intracellular signals that regulate cell behavior.
Although the pulmonary epithelia seem to be naturally permeable to a variety of peptides and proteins, the mechanisms of absorption are poorly understood. Possible mechanisms of absorption include: transcytosis; paracellular absorption between two cells; paracellular absorption at the tricellular junction at the intersection of three cells; and absorption through epithelial defects caused by senescent (apoptopic) cells. Evidence to support the role of any of these mechanisms in the absorption of a specific therapeutic by the healthy lung is weak. An emerging hypothesis is that for small peptides (i.e., 〈40 kDa) paracellular transport may dominate, while for larger proteins (i.e., 〉40 kDa) transcytosis may be more important. There is some evidence for receptor-mediated transcytosis of albumin. Complicating factors include osmolality, lung volume, smoking and disease states, macromolecule properties and regional differences.
Cadherins play important roles in cell—cell adhesion during tissue differentiation. Cadherins are linked to the actin cytoskeleton by catenins (β-catenin/armadillo, plakoglobin, and α-catenin). Recent results show that β-catenin also binds to another cytoskeletal complex containing the adenomatous polyposis coli protein and microtubules, and interacts with several signaling pathways that include tyrosine kinases and phosphatases and Wnt/Wingless. Interplay between these cytoskeletal complexes and signaling pathways may regulate morphogenesis.
NF2 is the most commonly mutated gene in benign tumours of the human nervous system. The NF2 protein, called schwannomin or merlin, is absent in virtually all schwannomas, and many meningiomas and ependymomas. Using the yeast two-hybrid system, we identified betaII-spectrin (also known as fodrin) as a schwannomin-binding protein. Interaction occurred between the carboxy-terminal domain of schwannomin isoform 2 and the ankyrin-binding region of betaII-spectrin. Isoform 1 of schwannomin, in contrast, interacted weakly with betaII-spectrin, presumably because of its strong self-interaction. Thus, alternative splicing of NF2 may regulate betaII-spectrin binding. Schwannomin co-immunoprecipitated with betaII-spectrin at physiological concentrations. The two proteins interacted in vitro and co-localized in several target tissues and in STS26T cells. Three naturally occurring NF2 missense mutations showed reduced, but not absent, betaII-spectrin binding, suggesting an explanation for the milder phenotypes seen in patients with missense mutations. STS26T cells treated with NF2 antisense oligonucleotides showed alterations of the actin cytoskeleton. Schwannomin itself lacks the actin binding sites found in ezrin, radixin and moesin, suggesting that signalling to the actin cytoskeleton occurs via actin-binding sites on betaII-spectrin. Thus, schwannomin is a tumour suppressor directly involved in actin-cytoskeleton organization, which suggests that alterations in the cytoskeleton are an early event in the pathogenesis of some tumour types.
The cadherins are a gene superfamily of calcium-dependent cell adhesion molecules. To date, the role(s) of the cadherins in human implantation remains poorly defined.
The spatiotemporal expression of the type 2 cadherins, known as cadherin-11 and cadherin-6, in the endometrium and placenta was examined using the reverse transcriptase-polymerase chain reaction.
Cadherin-6 and cadherin-11 are differentially expressed in the endometrial stroma during the menstrual cycle. The switch between cadherin-6 and cadherin-11 expression in the endometrial stroma occurs during the late secretory phase. Maximum cadherin-11 mRNA levels were observed in the decidua of early pregnancy but were markedly reduced at term. In the placenta, cadherin-11 is expressed in the syncytial trophoblast and extravillous cytotrophoblast columns. However, cadherin-6 seems to be the predominant cadherin subtype present in highly invasive extravillous cytotrophoblasts.
Cadherin-11 and cadherin-6 may play a central role in the formation and organization of the human endometrium and placenta.
Extracellular Ca(2+) is essential for the development of stable epithelial tight junctions. We find that in the absence of extracellular Ca(2+), AMP-activated protein kinase (AMPK) activation and glycogen synthase kinase (GSK)-3β inhibition independently induce the localization of epithelial tight junction components to the plasma membrane. The Ca(2+)-independent deposition of junctional proteins induced by AMPK activation and GSK-3β inhibition is independent of E-cadherin. Furthermore, the nectin-afadin system is required for the deposition of tight junction components induced by AMPK activation, but it is not required for that induced by GSK-3β inhibition. Phosphorylation studies demonstrate that afadin is a substrate for AMPK. These data demonstrate that two kinases involved in regulating cell growth and metabolism act through distinct pathways to influence the deposition of the components of epithelial tight junctions.
Cells of the human retinal pigment epithelium (RPE) have a regular epithelial cell shape within the tissue in situ, but for reasons that remain elusive the RPE shows an incomplete and variable ability to re-develop an epithelial phenotype after propagation in vitro. In other epithelial cell cultures, formation of an adherens junction (AJ) composed of E-cadherin plays an important early inductive role in epithelial morphogenesis, but E-cadherin is largely absent from the RPE. In this review, the contribution of cadherins, both minor (E-cadherin) and major (N-cadherin), to RPE phenotype development is discussed. Emphasis is placed on the importance for future studies of actin cytoskeletal remodeling during assembly of the AJ, which in epithelial cells results in an actin organization that is characteristically zonular. Other markers of RPE phenotype that are used to gauge the maturation state of RPE cultures including tissue-specific protein expression, protein polarity, and pigmentation are described. An argument is made that RPE epithelial phenotype, cadherin-based cell–cell adhesion and melanization are linked by a common signaling pathway: the Wnt/β-catenin pathway. Analyzing this pathway and its intersecting signaling networks is suggested as a useful framework for dissecting the steps in RPE morphogenesis.
In this review, we define the two major tissue types, epithelium and mesenchyme, and we describe the transformations (transdifferentiations) of epithelium to mesenchyme (EMT) and mesenchyme to epithelium (MET) that occur during embryonic development. The differentiation of the metanephric blastema provides a striking example of MET. Differentiation of metanephric epithelium is promoted by matrix molecules and receptors (nidogen, laminins, alpha 6 integrins), hepatic growth factor/scatter factor, and products of the genes wnt-1, wnt-4, and Pax-2. Transformation of MDCK epithelium to mesenchyme-like cells is promoted in vitro by antibodies to E-cadherin, products of v-src, v-ras, and v-mos, and by manipulation of the epithelium on collagen gels. Suspension in collagen gel, transforming growth factors, and c-fos have also been shown to promote EMT in epithelia. We present studies from our laboratory showing that alpha 5 beta 1 integrin has a role in the EMT of lens epithelium that is brought about by suspension in collagen gel. Our laboratory has also shown that transfection with the E-cadherin gene induces embryonic corneal fibroblasts to undergo MET and that this MET is enhanced by interaction of the differentiating epithelium with living fibroblasts. This review calls attention to the roles that EMT and MET might have in kidney pathologies and urges further study of the involvement of these phenomena in renal development, renal injury, and renal malignancy.
Classic cadherins, which are known to be crucial for homotypic cell-cell adhesion, have been found to be present not only in vertebrate but also in invertebrate species. Their three-dimensional structures, novel functions, and novel expression patterns were reported recently. These have been important steps towards a deeper understanding of the morphogenetic roles of this family of molecules.
The retinal pigment epithelium was used to study the relationship between the cortical cytoskeleton and two plasma membrane proteins that associate with it. These proteins were the Na+,K+-ATPase, an ion pump, and the 5A11 antigen, a member of the immunoglobulin super-family of receptor proteins. The cytoskeleton was marked by two of its constituents, α-spectrin and ankyrin. Ankyrin links the Na+,K+-ATPase to spectrin in many cells. The RPE is of interest, because unlike most epithelia it distributes the Na+,K+-ATPase to the apical membrane. The development of polarity was studied during chick embryogenesis. On embryonic day 6 (E6), each of these proteins was observed in the apical and lateral plasma membranes. As development proceeded, only the Na+,K+-ATPase was removed from the lateral membranes. Beginning on E12, ankyrin, spectrin and 5A11 appeared together in patches along the basal plasma membrane. By E16, these patches coalesced into a uniform distribution along the basal membrane. At the apical pole, α-spectrin appeared near the base of the microvilli, but was undetected in the microvilli themselves. This distribution resembled the distribution of α-spectrin in the intestine and proximal kidney tubule. By contrast, a pool of ankyrin and 5A11 and nearly all the Na+,K+-ATPase appeared in the microvilli. Despite its segregation from α-spectrin, the Na+,K+-ATPase appeared to associate with a macromolecular complex, as judged by extraction with Triton X-100. Changes in spectrin distribution could not be related to changes in isoform expression, as only one isoform of β-spectrin was detected by co-immunoprecipitation with α-spectrin. By contrast, multiple ankyrin-like peptides could be identified by immunoblotting. These data illustrate some of the unique properties of RPE microvilli. These properties prevent the Na+,K+-ATPase from complexing with the α-spectrin-based cytoskeleton by sequestering the enzyme into the compartment where its activity is required.
In epithelial cells interactions between the actin cytoskeleton and cell-cell junctions regulate paracellular permeability and participate in morphogenesis. We have studied the relationship between supracellular morphology and actin-junction interactions using primary cultures of porcine thyroid cells grown either as three-dimensional follicles or as open monolayers. Regardless of morphology, thyroid cells assembled occluding and adhesive junctions containing ZO-1 and E-cadherin, respectively, and showed F-actin staining in apical microvilli and a perijunctional ring. In monolayers, actin stress fibers were also observed in the apical and basal poles of cells, where they terminated in the vinculin-rich zonula adherens and in cell-substrate focal adhesions, respectively. Surprisingly, we were unable to detect vinculin localization in follicular cells, which also did not form stress fibers. Immunoblotting confirmed significantly greater vinculin in triton-insoluble fractions from monolayer cells compared with follicular cells. Incubation of monolayers with 8 chloro(phenylthio)-cyclic AMP decreased the level of immunodetectable vinculin in the zonula adherens, indicating that junctional incorporation of vinculin was regulated by cyclic AMP. In monolayer cultures, cytochalasin D (1 microM) cause actin filaments to aggregate associated with retraction of cells from one another and the disruption of cell junctions. Despite morphologically similar perturbations of actin organization in follicular cultures treated with cytochalasin D, junctional staining of ZO-1 and E-cadherin was preserved and cells remained adherent to one another. We conclude that in cultured thyroid cells structural and functional associations between actin filaments and cellular junctions differ depending upon the supracellular morphology in which cells are grown. One important underlying mechanism appears to be regulation of vinculin incorporation into adhesive junctions by cyclic AMP.
Restriction of sodium, potassium adenosine triphosphatase (Na+,K(+)-ATPase) to either the apical or basal-lateral membrane
domain of polarized epithelial cells is fundamental to vectorial ion and solute transport in many tissues and organs. A restricted
membrane distribution of Na+,K(+)-ATPase in Madin-Darby canine kidney (MDCK) epithelial cells was found experimentally to
be generated by preferential retention of active enzyme in the basal-lateral membrane domain and selective inactivation and
loss from the apical membrane domain, rather than by vectorial targeting of newly synthesized protein from the Golgi complex
to the basal-lateral membrane domain. These results show how different distributions of the same subunits of Na+,K(+)-ATPase
may be generated in normal polarized epithelial and in disease states.
The basement membrane stimulates the differentiation and polarity of simple transporting epithelia. We demonstrated for the retinal pigment epithelium (RPE) of chicken embryos that polarity develops gradually. Although the RPE and an immature basement membrane are established on embryonic day 4 (E4), the distribution of the Na,K-ATPase and a family of basement membrane receptors containing the beta 1 subunit of integrin is nonpolarized. The percentage of polarized cells increases gradually until cells in all regions of the epithelium are polarized on E11. During this time, the basement membrane increases in size and complexity to form Bruch's membrane. To study the ability of the basement membrane to stimulate the polarized distribution of the beta 1 integrins or the Na,K-ATPase, RPE was harvested from E7, E9, or E14 embryos and cultured on Bruch's membrane isolated (in association with the choroid) from E14 embryos. As a control, the RPE was plated on the side of the choroid lacking a Bruch's membrane. The distribution of the beta 1 integrins and the Na,K-ATPase was determined by indirect immunofluorescence. Bruch's membrane stimulated the polarized distribution of the beta 1 integrins regardless of the developmental age of the RPE even though E7 RPE is nonpolarized in vivo. To examine the role of individual matrix components, RPE was plated on matrix-coated filters. The polarized distribution of the beta 1 integrins was stimulated by laminin, collagen IV, and Matrigel but not by fibronectin. Interestingly, laminin and collagen IV are present in the basement membrane on E4 when RPE is not polarized in vivo. Under no circumstances was the distribution of the Na,K-ATPase polarized. These data indicate that the basement membrane influences the distribution of a subset of plasma membrane proteins but that other factors are required for full polarity.
In striking contrast to most other transporting epithelia (e.g., urinary or digestive systems), where Na,K-ATPase is expressed basolaterally, the retinal pigment epithelium (RPE) cells display Na,K-ATPase pumps on the apical membrane. We report here studies aimed to identify the mechanisms underlying this polarity "reversal" of the RPE Na,K-ATPase. By immunofluorescence on thin frozen sections, both alpha and beta subunits were localized on the apical surface of both freshly isolated rat RPE monolayers and RPE monolayers grown in culture. The polarity of the RPE cell is not completely reversed, however, since aminopeptidase, an apically located protein in kidney epithelia, was also found on the apical surface of RPE cells. We used subunit- and isoform-specific cDNA probes to determine that RPE Na,K-ATPase has the same isoform (alpha 1) as the one found in kidney. Ankyrin and fodrin, proteins of the basolateral membrane cytoskeleton of kidney epithelial cells known to be associated with the Na,K-ATPase (Nelson, W. J., and R. W. Hammerton. 1989. J. Cell Biol. 110:349-357) also displayed a reversed apical localization in RPE and were intimately associated to Na,K-ATPase, as revealed by cross-linking experiments. These results indicate that an entire membrane-cytoskeleton complex is assembled with opposite polarity in RPE cells. We discuss our observations in the context of current knowledge on protein sorting mechanisms in epithelial cells.
Calcium-dependent cell-cell adhesion is mediated in large part by a set of homologous integral membrane glycoproteins termed cadherins. In this report, antibodies to conserved domains in previously described cadherins have been used to isolate cDNAs encoding a novel chick cadherin. The deduced primary structure of this novel molecule, assigned the name B-cadherin, contains 726 amino acid residues which include five extracellular domains characteristic of this class of adhesion molecules, a single putative transmembrane spanning region, and a cytoplasmic tail. In each domain, B-cadherin shares extensive homologies with other cadherins, but is more closely related to E-cadherin, P-cadherin, and L-CAM than to N-cadherin. It is expressed in a wide variety of chick tissues at embryonic day 13. In particular, immunohistochemical staining and in situ hybridization localize B-cadherin protein and mRNA to the epithelial lining of the choroid plexus and to cells in specific layers of the optic tectum in chick brain. Levels of the protein and RNA transcript change dramatically as development proceeds in chick brain. These results suggest that B-cadherin has important functions in neurogenesis, in at least some epithelia, and in embryogenesis.
To examine the diversity of the cadherin family, we isolated cDNAs from brain and retina cDNA preparations with the aid of polymerase chain reaction. The products obtained included cDNAs for two of three known cadherins as well as eight distinct cDNAs, of which deduced amino acid sequences show significant similarity with the known cadherin sequences. Larger cDNA clones were isolated from human cDNA libraries for six of the eight new molecules. The deduced amino acid sequences show that the overall structure of these molecules is very similar to that of the known cadherins, indicating that these molecules are new members of the cadherin family. We have tentatively designated these cadherins as cadherin-4 through -11. The new molecules, with the exception of cadherin-4, exhibit features that distinguish them as a group from previously cloned cadherins; they may belong to a new subfamily of cadherins. Northern blot analysis showed that most of these cadherins are expressed mainly in brain, although some are expressed in other tissues as well. These findings show that the cadherin family of adhesion molecules is much larger than previously thought, and suggest that the new cadherins may play an important role in cell-cell interactions within the central nervous system.
This report demonstrates that the high affinity binding of ankyrin to two well characterized ankyrin-binding proteins, the erythrocyte anion exchanger and kidney Na+K(+)-ATPase, requires interaction of these proteins with unique sites on the ankyrin molecule. Binding of 125I-labeled erythrocyte ankyrin and ankyrin proteolytic domains was measured to the anion exchanger and Na+K(+)-ATPase incorporated into phosphatidylcholine liposomes. 125I-Labeled ankyrin associated with both anion exchanger and Na+K(+)-ATPase liposomes with a high affinity (KD ranging from 10 to 25 nM), and a capacity approaching 1 mol of ankyrin/2 mol of ATPase and 1 mol of ankyrin/8 mol of anion exchanger. The 43 kDa cytoplasmic domain of the erythrocyte anion exchanger inhibited binding of ankyrin to both the anion exchanger and Na+K(+)-ATPase liposomes with a 50% reduction at approximately 90 nM for both proteins. Further binding experiments using proteolytic domains derived from ankyrin demonstrated the following differences between the anion exchanger and Na+K(+)-ATPase in interactions with ankyrin: 1) 125I-Labeled Na+K(+)-ATPase associated with both the 89-kDa domain as well as the spectrin binding domain of ankyrin, while the anion exchanger only associated with the 89-kDa domain. 2) The 125I-labeled 89-kDa domain of ankyrin associated with Na+K(+)-ATPase liposomes with at least a 20-fold lower affinity compared with intact ankyrin while this domain associated with the anion exchanger with a 2-3-fold increase in affinity compared with intact ankyrin. 3) The 125I-labeled spectrin-binding domain of ankyrin associated with the Na+K(+)-ATPase liposomes to at least an 8-fold greater extent than to anion exchanger liposomes. The data are consistent with an independent acquisition of high affinity ankyrin binding activity for the anion exchanger and Na+K(+)-ATPase proteins through a convergent evolutionary process.
The polarity of retinal pigmented epithelia (RPE) from chicken embryos was studied in primary cell culture. Since cultured RPE approximates the morphological polarity of RPE in vivo, we investigated whether this polarity extends to the distribution of plasma membrane proteins that are peculiar to RPE. In contrast to other epithelia, the Na+,K(+)-ATPase of RPE is located in the apical rather than basolateral plasma membrane. To examine this property, we cultured RPE on extracellular matrix-coated filters. Primary cultures were compared to embryonic RPE in situ using electron microscopy and indirect immunofluorescence of frozen sections. The viability and morphology of RPE was improved by using a serum-free medium containing a bovine pituitary extract in conjunction with an extracellular matrix coating derived from Engelbreth-Holm-Swarm tumors. Cultured RPE mimicked the morphology of RPE in vivo with microvilli and junctional complexes on the apical pole and infoldings along the basolateral plasma membrane. Functional tight junctions formed as demonstrated by an EDTA-sensitive, transepithelial electrical resistance, and by the retention of [3H]inulin added to the apical chamber. In 2 hr, only 4-6% of the [3H]inulin crossed the monolayer, compared to 24% in control filters. Despite these features of polarity, the Na+,K(+)-ATPase was detected in both apical and basolateral membranes by immunofluorescence. In embryonic eyes in which the neural retina was removed, the Na+,K(+)-ATPase was confined to the apical membrane. In addition, the polarity of cultured RPE was probed with vesicular stomatitis virus. In contrast to other epithelia, budding virus particles were observed emerging from the apical, as well as basolateral, domain further suggesting the cultured cells were only partially polarized. These data indicate that structural criteria are inadequate to determine if cultured RPE have become polarized in the same manner as the epithelium in vivo.
Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.
In nonerythroid cells the distribution of the cortical membrane skeleton composed of fodrin (spectrin), actin, and other proteins varies both temporally with cell development and spatially within the cell and on the membrane. In monolayers of Madin-Darby canine kidney (MDCK) cells, it has previously been shown that fodrin and Na,K-ATPase are codistributed asymmetrically at the basolateral margins of the cell, and that the distribution of fodrin appears to be regulated posttranslationally when confluence is achieved (Nelson, W. J., and P. I. Veshnock. 1987. J. Cell Biol. 104:1527-1537). The molecular mechanisms underlying these changes are poorly understood. We find that (a) in confluent MDCK cells and intact kidney proximal tubule cells, Na,K-ATPase, fodrin, and analogues of human erythrocyte ankyrin are precisely colocalized in the basolateral domain at the ultrastructural level. (b) This colocalization is only achieved in MDCK cells after confluence is attained. (c) Erythrocyte ankyrin binds saturably to Na,K-ATPase in a molar ratio of approximately 1 ankyrin to 4 Na,K-ATPase's, with a kD of 2.6 microM. (d) The binding of ankyrin to Na,K-ATPase is inhibited by the 43-kD cytoplasmic domain of erythrocyte band 3. (e) 125I-labeled ankyrin binds to the alpha subunit of Na,K-ATPase in vitro. There also appears to be a second minor membrane protein of approximately 240 kD that is associated with both erythrocyte and kidney membranes that binds 125I-labeled ankyrin avidly. The precise identity of this component is unknown. These results identify a molecular mechanism in the renal epithelial cell that may account for the polarized distribution of the fodrin-based cortical cytoskeleton.
In polarized Madin-Darby canine kidney (MDCK) epithelial cells, ankyrin, and the alpha- and beta-subunits of fodrin are components of the basolateral membrane-cytoskeleton and are colocalized with the Na+,K+-ATPase, a marker protein of the basolateral plasma membrane. Recently, we showed with purified proteins that the Na+,K+-ATPase is competent to bind ankyrin with high affinity and specificity (Nelson, W. J., and P. J. Veshnock. 1987. Nature (Lond.). 328:533-536). In the present study we have sought biochemical evidence for interactions between these proteins in MDCK cells. Proteins were solubilized from MDCK cells with an isotonic buffer containing Triton X-100 and fractionated rapidly in sucrose density gradients. Complexes of cosedimenting proteins were detected by analysis of sucrose gradient fractions in nondenaturing polyacrylamide gels. The results showed that ankyrin and fodrin cosedimented in sucrose gradient. Analysis of the proteins from the sucrose gradient in nondenaturing polyacrylamide gels revealed two distinct ankyrin:fodrin complexes that differed in their relative electrophoretic mobilities; both complexes had electrophoretic mobilities slower than that of purified spectrin heterotetramers. Parallel analysis of the distribution of solubilized Na+,K+-ATPase in sucrose gradients showed that there was a significant overlap with the distribution of ankyrin and fodrin. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the alpha- and beta-subunits of the Na+,K+-ATPase colocalized with the slower migrating of the two ankyrin:fodrin complexes. The faster migrating ankyrin:fodrin complex did not contain Na+,K+-ATPase. These results indicate strongly that the Na+,K+-ATPase, ankyrin, and fodrin are coextracted from whole MDCK cells as a protein complex. We suggest that the solubilized complex containing these proteins reflects the interaction of the Na+,K+-ATPase, ankyrin, and fodrin in the cell. This interaction may play an important role in the spatial organization of the Na+,K+-ATPase to the basolateral plasma membrane in polarized epithelial cells.
Cell adhesion molecules (CAMs) are cell surface glycoproteins that may play a variety of roles in morphogenesis and histogenesis, particularly in defining borders of discrete cell populations. To examine the influence of CAM expression on such cell segregation events in vitro, we have transfected cells with cDNAs coding for two calcium-dependent CAMs of different specificity, the liver CAM (L-CAM) and the structurally related molecule N-cadherin. The cDNAs were introduced separately or together into murine sarcoma S180 cells, which normally do not express these molecules, to produce cell lines denoted S180L, S180cadN, and S180L/cadN, respectively. A number of cell lines of each type were produced that differed in their levels of CAM expression. In adhesion assays, S180L and S180cadN cells aggregated specifically via their respective CAMs, and S180L cells did not appear to adhere to S180cadN cells. Cells expressing high levels of each CAM aggregated more rapidly than cells expressing low levels. Segregation between two cell types occurred when they expressed CAMs of different specificity or different levels of the same CAM. S180L and S180cadN cells both sorted out from untransfected cells, and cells expressing high levels of either L-CAM or N-cadherin segregated from cells expressing low levels of the same CAM; in all cases segregation was inhibited by antibodies specific for the transfected CAM. S180L cells sorted out from S180cadN cells, but this segregation was inhibited only when antibodies to both CAMs were applied together. Doubly transfected S180L/cadN cells also sorted out from S180L cells and from S180cadN cells, and the process was inhibited by antibodies to the unshared CAM (N-cadherin or L-CAM, respectively). Cytochalasin D and nocodazole inhibited sorting-out, consistent with the probable role of microfilaments and microtubules in cell movement and in accord with evidence that the action of these CAMs depends on interactions with cortical cytoplasmic components. Using cDNAs for only two CAMs in these studies, we could distinguish at least eight cell lines by their behavior in sorting-out assays. This suggests that qualitative and quantitative differences in the expression in vivo of a relatively small number of CAMs can lead to a large variety of patterns among cell collectives and their borders during tissue formation.
Madin-Darby canine kidney (MDCK) epithelial cells exhibit a polarized distribution of membrane proteins between the apical and basolateral domains of the plasma membrane. We have initiated studies to investigate whether the spectrin-based membrane skeleton plays a role in the establishment and maintenance of these membrane domains. MDCK cells express an isoform of spectrin composed of two subunits, Mr 240,000 (alpha-subunit) and Mr 235,000 (gamma-subunit). This isoform is immunologically and structurally related to fodrin in lens and brain cells, which is a functional and structural analog of alpha beta-spectrin, the major component of the erythrocyte membrane skeleton. Analysis of fodrin in MDCK cells by immunoblotting, immunofluorescence, and metabolic labeling revealed significant changes in the biophysical properties, subcellular distribution, steady-state levels, and turnover of the protein during development of a continuous monolayer of cells. The changes in the cellular organization of fodrin did not appear to coincide with the distributions of microfilaments, microtubules, or intermediate filaments. These changes result in the formation of a highly insoluble, relatively dense and stable layer of fodrin which appears to be localized to the cell periphery and predominantly in the region of the basolateral plasma membrane of MDCK cells in continuous monolayers. The formation of this structure coincides temporally and spatially with extensive cell-cell contact, and with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral plasma membrane.
Cadherins are a family of glycoproteins involved in the Ca2+-dependent cell-cell adhesion mechanism which is detected in most kinds of tissues. Inhibition of the cadherin activity with antibodies induces dissociation of cell layers, indicating a fundamental importance of these molecules in maintaining the multicellular structure. Cadherins are divided into subclasses, including E-, N- and P-cadherins. While all subclasses are similar in molecular weight, Ca2+- and proteasesensitivity, each subclass is characterized by a unique tissue distribution pattern and immunological specificity. Analysis of amino acid sequences deduced from cDNA encoding these molecules showed that they are integral membrane proteins of 723–748 amino acids long and share common sequences; similarity in the sequences between subclasses is in a range of 50–60 % when compared within a single animal species.
L cells, with very little endogenous cadherin activity, transfected with the cadherin cDNA acquired high cadherin-mediated aggregating activity. Their colony morphology was altered by the ectopic expression of cadherins from the dispersed type to the compact type, providing direct evidence for a key role of cadherins in cell-cell adhesion. It has been suggested that cadherins bind cells by their homophilic interactions at the extracellular domain and are associated with actin bundles at the cytoplasmic domain.
It appears that each cadherin subclass has binding specificity and this molecular family is involved in selective cell-cell adhesion. In development, the expression of each cadherin subclass is spatiotemporally regulated and associated with a variety of morphogenetic events; e.g. the termination or initiation of expression of a cadherin subclass in a given cell collective is correlated with its segregation from or connection with other cell collectives. Antibodies to cadherins were shown to perturb the morphogenesis of some embryonic organs in vitro. These observations suggest that cadherins play a crucial role in construction of tissues and the whole animal body.
This report describes a combined biochemical and autoradiographic approach to localization of ouabain binding sites in the frog choroid plexus. This tissue is known to secrete sodium by a ouabain sensitive pump, which is thought to reside on the apical surface of the epithelium (Wright, 1972). Since ouabain is only effective in the solution bathing the apical surface of the epithelium, the cerebrospinal fluid side, ouabain should bind to the apical (brush border) membrane.
In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution-binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.
Treatment of Xenopus animal pole tissue with activin results in the induction of mesodermal cell types and a dramatic elongation of the tissue. The morphogenetic movements involved in the elongation appear similar to those in normal gastrulation, which is driven by cell rearrangement and cell intercalations. We have used this system to explore the potential regulation of cell-cell adhesion and cadherin function during morphogenesis. Quantitative blastomere aggregation assays revealed that activin induction reduced the calcium-dependent adhesion between blastomeres. Activin-induced blastomeres formed smaller aggregates, and a greater proportion of the population remained as single cells compared to uninduced blastomeres. The aggregation was mediated by C-cadherin because C-cadherin was present in the blastomeres during the aggregation assay, and monoclonal antibodies against C-cadherin inhibited the calcium-dependent aggregation of blastomeres. E-cadherin was not detectable until after the completion of the assay and, therefore, does not explain the adhesive differences between induced and uninduced blastomeres. L cells stably expressing C-cadherin (LC cells) were used to demonstrate that C-cadherin activity was specifically altered after activin induction. Blastomeres induced with activin bound fewer LC cells than uninduced blastomers. L cells not expressing C-cadherin did not adhere to blastomeres. The changes in C-cadherin-mediated adhesion occurred without detectable changes in the steady-state levels of C-cadherin or the amount of C-cadherin present on the surface of the cell. Immunoprecipitation of C-cadherin and its associated catenins revealed that the ratio of C-cadherin and the catenins was not altered by activin induction. These results demonstrate that activin decreases the adhesive function of existing C-cadherin molecules on the surface of blastomeres and suggest that decreased cadherin mediated cell-cell adhesion is associated with increased morphogenetic movement.
The sorting-out of embryonic cells from a cell mixture and the selective spreading of one cell population over the surface of another have been attributed to various causes. These include differentials in chemotaxis, in cellular adhesiveness, in cell surface contractility, in speed of cell movement, and in the timing of postulated changes in cellular adhesive and motile properties. One of us earlier predicted on mathematical grounds that two motile cell types differing only in the level of expression of a single cell adhesion system should not only segregate from one another but also arrange themselves with the less cohesive cells enveloping a core of the more cohesive ones. To test these predictions, we combined two populations of L cells transfected with P-cadherin cDNA and expressing this homophilic adhesion molecule in substantially differing amounts. When the two cell populations were intermixed, they segregated to approach a sphere-within-a-sphere configuration, the cell population expressing more P-cadherin forming islands which fused to become an internal "medulla." When the two cell populations were first formed into separate aggregates which were subsequently allowed to fuse, the cell population expressing more P-cadherin was enveloped by its partner, which formed an external "cortex." These observations confirm the early prediction and support the conclusion that both morphogenetic movements and the specific anatomical configurations to which they lead can be determined by particular sets of intercellular adhesive intensities, regardless of how these are generated and in the absence of differentials in other parameters.
Polarized epithelial cells play fundamental roles in the ontogeny and function of a variety of tissues and organs in mammals. The morphogenesis of a sheet of polarized epithelial cells (the trophectoderm) is the first overt sign of cellular differentiation in early embryonic development. In the adult, polarized epithelial cells line all body cavities and occur in tissues that carry out specialized vectorial transport functions of absorption and secretion. The generation of this phenotype is a multistage process requiring extracellular cues and the reorganization of proteins in the cytoplasm and on the plasma membrane; once established, the phenotype is maintained by the segregation and retention of specific proteins and lipids in distinct apical and basal-lateral plasma membrane domains.
In striking contrast to most other transporting epithelia (e.g., urinary or digestive systems), where Na,K-ATPase is expressed basolaterally, the retinal pigment epithelium (RPE) cells display Na,K-ATPase pumps on the apical membrane. We report here studies aimed to identify the mechanisms underlying this polarity "reversal" of the RPE Na,K-ATPase. By immunofluorescence on thin frozen sections, both alpha and beta subunits were localized on the apical surface of both freshly isolated rat RPE monolayers and RPE monolayers grown in culture. The polarity of the RPE cell is not completely reversed, however, since aminopeptidase, an apically located protein in kidney epithelia, was also found on the apical surface of RPE cells. We used subunit- and isoform-specific cDNA probes to determine that RPE Na,K-ATPase has the same isoform (alpha 1) as the one found in kidney. Ankyrin and fodrin, proteins of the basolateral membrane cytoskeleton of kidney epithelial cells known to be associated with the Na,K-ATPase (Nelson, W. J., and R. W. Hammerton. 1989. J. Cell Biol. 110:349-357) also displayed a reversed apical localization in RPE and were intimately associated to Na,K-ATPase, as revealed by cross-linking experiments. These results indicate that an entire membrane-cytoskeleton complex is assembled with opposite polarity in RPE cells. We discuss our observations in the context of current knowledge on protein sorting mechanisms in epithelial cells.
Cell-cell adhesion is at the top of a molecular cascade of protein interactions that leads to the remodeling of epithelial cell structure and function. The earliest events that initiate this cascade are poorly understood. Using high resolution differential interference contrast microscopy and retrospective immunohistochemistry, we observed that cell-cell contact in MDCK epithelial cells consists of distinct stages that correlate with specific changes in the interaction of E-cadherin with the cytoskeleton. We show that formation of a stable contact is preceded by numerous, transient contacts. During this time and immediately following formation of a stable contact, there are no detectable changes in the distribution, relative amount, or Triton X-100 insolubility of E-cadherin at the contact. After a lag period of approximately 10 min, there is a rapid acquisition of Triton X-100 insolubility of E-cadherin localized to the stable contact. Significantly, the total amount of E-cadherin at the contact remains unchanged during this time. The increase in the Triton X-100 insoluble pool of E-cadherin does not correlate with changes in the distribution of actin or fodrin, suggesting that the acquisition of the Triton X-100 insolubility is due to changes in E-cadherin itself, or closely associated proteins such as the catenins. The 10 minute lag period, and subsequent prompt and localized nature of E-cadherin reorganization indicate a form of signaling is occurring.
The specificity of adhesion between embryonal carcinoma cells and fibroblastic cells of various origins was studied. Embryonal carcinoma cells have intercellular adhesion sites requiring Ca2+ (CDS). These sites were found to be sensitive to proteases but resistant to them in the presence of Ca2+. CDS with a similar protease sensitivity is present in fibroblastic cells. When embryonal carcinoma cells of different lines were mixed, they adhered to each other nonselectively by CDS. Nonselective adhesion by CDS occurred also between fibroblastic cells of various lines. When embryonal carcinoma and fibroblastic cells were mixed, they preferentially adhered to homotypic cells. Fab fragments of antibodies raised against F9 cells (a nullipotent line of embryonal carcinoma) inhibited the adhesion between embryonal carcinoma cells but not between fibroblastic cells. This inhibitory activity of Fab was absorbed with embryonal carcinoma cells with CDS, but not with fibroblastic cells with CDS or embryonal carcinoma cells from which CDS was experimentally removed. SDS-polyacrylamide gel electrophoresis of radioiodinated cell surface proteins showed that the presence of a 140K-dalton component correlated with the presence of CDS in embryonal carcinoma cells, while the presence of a 150K-dalton component correlated with the presence of CDS in fibroblastic cells. These results suggest that CDS in embryonal carcinoma and fibroblastic cells comprise distinct molecules.
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Recent developments in several areas of cell and developmental biology support the concept that adhesion molecules serve receptor and signal transduction functions in addition to more classically defined structural roles (Takeichi 1988; Albelda and Buck 1990; Hynes 1992). Adhesion receptors, including integrins, cadherins, and members of the immunoglobulin superfamily, influence many aspects of cell behavior, including growth control, differentiation, and motility (Edelman 1986; Buck and Horwitz 1987; Takeichi 1991; Schwartz et al. 1991). The integrin family of adhesion receptors mediates the interaction of cells with many different extracellular matrix (ECM) proteins. Specificity is imparted to this adhesive interaction by the combination of particular α and β chains to form integrin heterodimers that may recognize single or several ECM proteins (Albelda and Buck 1990; Hemler 1990; Hynes 1992). Integrin subunits span the plasma membrane and, in general, have relatively short and highly conserved cytoplasmic domains. Information initiated by the interactions of...
Congenital polycystic kidney disease is characterized by the formation of large fluid-filled cysts in kidney tubules. It has been postulated that increased epithelial cell proliferation and altered transtubular fluid transport are necessary for cyst formation. To address the latter problem, we have studied the plasma membrane distribution of the al and beta-1 subunits of Na+/K+-ATPase during progressive stages of proximal and collecting tubular cyst formation in the CPK mouse, a murine model of autosomal recessive polycystic kidney disease. In both control and cystic proximal tubules, Na+/K+-ATPase distribution was restricted to the basal-lateral membrane of cells. However, in newborn through day 5 kidney tissue, 16% of control vs. 47% of cystic outer cortical, 6% of control vs. 46% of cystic inner cortical, and 2% of control vs. 63% of cystic medullary collecting tubules demonstrated apical and lateral membrane distribution of Na+/K+-ATPase. In all nephrogenic zones, the percentage of control or cystic collecting tubules demonstrating apical membrane distribution of Na+/K+-ATPase decreased over time, but the percentage of cystic collecting tubules with apical membrane Na+/K+-ATPase remained significantly greater than in developmentally matched controls. No alterations in the normal distributions of other apical or basal-lateral membrane marker proteins were noted at any stage of control or cystic proximal or collecting tubule development. We conclude that apical-lateral membrane Na+/K+-ATPase expression is a normal transient feature of early collecting tubule development. However, apical membrane Na+/K+-ATPase persists in cystic kidneys, suggesting that such expression may be a manifestation of the relatively undifferentiated phenotype of epithelial cells lining collecting tubule cysts. The persistence of apical membrane Na+/K+-ATPase, if the enzyme is functional, may have pathogenic import in abnormal transtubular fluid transport in polycystic kidney disease.
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During development, axons grow along highly stereospecific pathways to innervate and synapse with target tissue. In recent years, much effort has been devoted to attempting to understand these processes in molecular terms. A variety of studies (for review, see Doherty and Walsh 1989; Bixby and Harris 1991; Lumsden and Cohen 1991) have implicated a number of cellular components in regulating axonal growth and guidance. These are, first, soluble components such as neurotransmitters (Kater and Guthrie 1990) and chemotropic or trophic factors (Lumsden and Davies 1986; Heffner et al. 1990); second, components of the extracellular matrix such as fibronectin and a variety of laminin isoforms (Reichardt et al. 1990; Sanes et al. 1990); and third, an increasing number of membrane-associated glycoproteins that are members of different cell and substrate adhesion molecule families (Reichardt et al. 1990; Ranscht 1991; Rathjen and Jessell 1991; Walsh and Doherty 1991). In addition to positive cues...
The generation of unique domains on the cell, cell surface polarity, is critical for differentiation into the diversity of cell structures and functions found in a wide variety of organisms and cells, including the bacterium Caulobacter crescentus, the budding yeast Saccharomyces cerevisiae, and mammalian polarized epithelial cells. Comparison of the mechanisms for establishing polarity in these cells indicates that restricted membrane protein distributions are generated by selective protein targeting to, and selective protein retention at, the cell surface. Initiation of these mechanisms involves reorientation of components of the cytoskeleton and protein transport pathways toward restricted sites at the cell surface and formation of a targeting patch at those sites for selective recruitment and retention of proteins.
The identification of protein factors, such as epimorphin, scatter factor, and activin, that induce epithelial branching and convergent extension-like movements in embryonic tissues are important breakthroughs in our understanding of the role of mesenchyme in epithelial morphogenesis. Moreover, the development of simple in vitro epithelial cell systems that undergo morphogenesis in response to these factors should provide a means to investigate the cellular and molecular bases of the morphogenetic movements themselves. Although many different cellular processes are involved in such morphogenetic behaviors, cell rearrangement is a particularly intriguing one that will be important to study further. Several considerations lead to the prediction that a dynamic regulation of cell-cell adhesion is likely to play a central role in cell rearrangements and epithelial morphogenesis. Ultimately, a greater issue to be addressed is how the different cellular mechanisms participating in epithelial morphogenesis are coordinated and regulated, so as to generate the diverse patterns found in various epithelia.
Cadherins represent a gene family of Ca(2+)-dependent cell adhesion molecules (CAMs) identified during development and in adult organs. They generally mediate cell-cell adhesion by homotypic interaction, although heterotypic binding between different cadherin molecules is possible. Molecular cloning and sequence comparison has led to the characterization of a highly homologous group of 'classical' cadherins and more distantly related members, together composing a gene superfamily. The classical cadherins are transmembrane glycoproteins which exhibit, in addition to the structural homologies, a very similar overall protein topology. Protein sequence comparison has led to the identification of domains of common functional importance. The cytoplasmic domains of cadherins associate with peripheral cytoplasmic proteins termed catenin alpha, beta and gamma with molecular weights of 102, 88 and 80 kDa respectively. This complex formation seems to regulate the adhesive function of cadherins, most likely by connecting cadherins with actin microfilaments. Possible implications of catenins for cadherin function are discussed.
We present evidence that the morphoregulatory activities of neural cell adhesion molecule (NCAM) and N-cadherin involve activation of intracellular second messenger pathways. PC12 cells were cultured on monolayers of control 3T3 cells or 3T3 cells expressing transfected N-cadherin or NCAM. NCAM and N-cadherin directly induced a transcription-independent change in the morphology of PC12 cells from an adrenal to neuronal phenotype and also specifically increased Thy-1, but not L1/NILE or low affinity NGF receptor, immunoreactivity. The morphological response was more rapid and, in the case of N-cadherin, more substantial than that induced by NGF. It could be fully inhibited by pertussis toxin and a combination of L- and N-type Ca2+ channel antagonists, but not by broad-specificity kinase inhibitors. It was blocked, however, by the kinase inhibitor K-252b. These studies suggest that cell adhesion molecules directly alter cell phenotype and provide direct evidence for transmembrane signaling mediating both the morphological and biochemical responses induced by NCAM and N-cadherin.
The generation of cell surface polarity in transporting epithelial cells occurs in three distinct stages that involve cell-cell recognition and adhesion, cell surface remodelling to form biochemically and functionally distinct cell surface domains, and development of vectorial function. A widely used model system to study mechanisms involved in these stages is the Madin-Darby canine kidney (MDCK) cell line. Under appropriate growth conditions, MDCK cells develop in similar stages into polarized, multicellular epithelial structures. Analysis of membrane-cytoskeletal proteins ankyrin and fodrin during development of MDCK cell surface polarity shows that they gradually assemble into an insoluble protein complex on the basal-lateral membrane domain upon cell-cell adhesion, concomitantly with the redistribution of Na+,K(+)-ATPase, a marker protein of the basal-lateral membrane. Biochemical analysis shows that ankyrin, fodrin occur in a complex with Na+,K(+)-ATPase and the cell adhesion molecule uvomorulin in MDCK cells. A model is presented in which assembly of membrane-cytoskeletal complexes at sites of uvomorulin-induced cell-cell contact causes a remodelling of the cell surface distribution of specific membrane proteins which, in turn, contributes to the generation of epithelial cell surface polarity.
Cadherins homophilically bind cells. Thus, cells expressing identical cadherins adhere selectively to each other, and they do not randomly intermix with the cells expressing other types of cadherins in vitro. Neural tissues express multiple types of cadherins, and the expression of each cadherin type is spatiotemporally regulated within a tissue during development. This molecular family therefore could operate for the sorting of different cell types in the nervous system. The regulation of N-cadherin expression is also important for the early development of the neural tube. The ectopic expression of N-cadherin in Xenopus embryos, which was induced by mRNA injection, led to the disorganization of neural tube structures or the fusion of the neural tube to the epidermis. These results suggest that the precise regulation of cadherin expression at the quantitative as well as at the qualitative level si crucial for neural morphogenesis.
Na+,K(+)-ATPase has distinctly different distributions in mesenchymal cells, where it has an unrestricted distribution over the entire cell surface, compared with polarized epithelial cells, where it is restricted to the basal-lateral membrane domain. The generation of this restricted distribution is important in mesenchyme to epithelia conversion in development and the function of transporting epithelia, but the mechanisms involved are unknown. Here we show that expression of the epithelial CAM uvomorulin in transfected fibroblasts is sufficient to induce a redistribution of Na+,K(+)-ATPase to sites of uvomorulin-mediated cell-cell contacts, similar to that in polarized epithelial cells. This restricted distribution of Na+,K(+)-ATPase occurs in the absence of tight junctions but coincides with the reorganization of the membrane cytoskeleton. The results indicate a direct role for CAMs as inducers of cell surface polarity of selective cytoplasmic and membrane proteins.
We studied the surface delivery pathways followed by newly synthesized plasma membrane proteins in intestinal cells. To this end, we developed an assay and characterized an epithelial cell line (SK-CO-15) derived from human colon adenocarcinoma. Polarized confluent monolayers (2000 omega.cm2), grown on polycarbonate filter chambers, were pulsed with radioactive methionine/cysteine and, at different times of chase, the protein fraction reaching the apical or basolateral surface was recovered by domain-selective biotinylation, immunoprecipitation, and immobilized streptavidin precipitation. Both an apical and a basolateral marker were found to be delivered vectorially to the respective surface, with a sorting efficiency of 50:1 for the basolateral marker and 14:1 for the apical marker.
Selective biotinylation of the apical or basolateral domains of confluent MDCK monolayers grown on polycarbonate filters with a water soluble biotin analog, sulfo-NHS-biotin, was employed to reveal strikingly distinct patterns of endogenous "peripheral" and "integral" membrane proteins. "Peripheral" proteins were found to be approximately fivefold more abundant with this procedure than "integral" membrane proteins, both on the apical and on the basolateral surface. The distinct apical and basal patterns were shown to depend upon the integrity of the monolayer; when the tight junctions were disrupted by preincubation in calcium-depleted medium, the patterns appeared practically indistinguishable. Two-dimensional gel electrophoresis demonstrated that only a very small percentage of the biotinylated proteins were found in similar amounts on both apical and basolateral domains. These results indicate that the sorting mechanisms that segregate apical and basolateral epithelial proteins are very strict. The simple procedure described here has clear advantages over other methods available to label apical and basal epithelial surface domains, namely, higher accessibility of the biotin probe to the basolateral membrane, possibility of purifying biotinylated proteins via immobilized streptavidin and minimal exposure of the researcher to isotopes. It should be very useful in characterizing the apical and basolateral protein compositions of other epithelial cells and in studies on the development of epithelial cell polarity.
Interactions between integral proteins of the plasma membrane and the cytoskeleton may be important for localizing certain membrane proteins in a nonrandom fashion at specialized domains of the cell surface. Here, we show that ankyrin, the key protein for the linkage of the erythrocyte anion exchanger (band 3) to the spectrin-based membrane cytoskeleton, is also present in kidney distal tubular cells where ankyrin is precisely colocalized with Na+,K+-ATPase. Both proteins are confined to the basolateral plasma membrane and are absent from the apical membrane, the junctional complex and the membrane surface that contacts the basal lamina. Purified Na+,K+-ATPase of sheep and pig kidney contains a binding site for erythrocyte ankyrin as demonstrated by immunoprecipitation experiments. A band 3-like binding site for ankyrin is likely, since binding of ankyrin to Na+,K+-ATPase could be inhibited in a competitive fashion by the isolated cytoplasmic domain of erythrocyte band 3.
The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.
In vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.
Cadherins are cell-surface glycoproteins responsible for Ca2+-dependent cell-to-cell adhesion. E- or P-cadherin was transfected into L cells, which normally have little cadherin activity, and cellular aggregation of the resulting transfectants was observed to be a function of the cadherin molecule expressed. Transfected cells preferentially adhered to cells expressing the same cadherin subclass. Furthermore, in reconstituted embryonic lung tissue, E-cadherin-expressing L cells were associated with epithelial tubules expressing E-cadherin, while untransfected L cells associated with mesenchymal cells. These results provide the first direct evidence that the differential expression of cadherins can play a role in cell sorting in heterogeneous cell populations.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Desmosomes are junctions between epithelial, myocardiac, and certain other kinds of cells. They represent plasma membrane domains enriched in specific transmembrane glycoproteins, notably desmoglein (Dsg) and desmocollin (Dsc), both of which have recently been identified as members of the larger family of Ca(2+)-dependent cell adhesion molecules, the cadherins. Previously described forms of desmoglein have been isolated as proteins and cloned as cDNAs from epidermis and related stratified epithelia but have not been detected in the majority of other desmosome-containing tissues and cell culture lines. Here we present the complete cDNA-derived amino acid (aa) sequence of a different desmoglein polypeptide, termed Dsg2 (1069 aa, mol wt 116,760) and its precursor molecule (1117 aa, mol wt 122,384), which occurs in all human and bovine desmosome-producing tissues, tumors, and cell lines examined, epithelial as well as nonepithelial ones. We conclude that Dsg2, the largest molecule in this protein family, is the fundamental desmoglein common to all desmosome-possessing tissues, including simple epithelia and myocardium, and many cell cultures. Furthermore, in several tissues and cell lines Dsg2 is the only Dsg isoform detected so far. By contrast, the epidermal isoforms Dsg1 and Dsg3 are restricted to certain specialized epithelia, mostly stratified squamous ones. The importance of the junction-specific cadherin Dsg2 in tissue formation and carcinogenesis as well as in the development of autoimmune diseases of the Pemphigus type is discussed. In addition, we propose to use Dsg2 as a general marker common to all epithelial cells and tumors and to use the specific pattern of occurrence of Dsg and Dsc isoforms as an additional criterion for cell typing in tumor diagnosis.
The establishment and maintenance of epithelial-cell polarity are prerequisites for normal epithelial-cell and organ function. Knowledge of the processes involved in cell polarity has provided insight into the mechanisms of cell dysfunction and the pathogenesis of several diseases. These insights should lead to the development of specific strategies aimed at preventing or minimizing the progression of these diseases.