Assembly of enveloped viruses in Madin-Darby canine kidney cells: polarized budding from single attached cells and from clusters of cells in suspension

In confluent monolayers of the dog kidney epithelial cell line Madin-Darby canine kidney (MDCK) assembly of RNA enveloped viruses reflects the functional polarization of the cells. Thus, influenza, Sendai, and Simian virus 5 bud from the apical (free) surface, while vesicular stomatitis virions (VSV) are assembled at basolateral plasma membrane domains (Rodriguez-Boulan, E., and D.D. Sabatini, 1978, Proc. Natl. Acad. Sci. U.S.A., 75:5071-5075). MDCK cells derived from confluent monolayers by dissociation with trypsin-EDTA and maintained as single cells in spinner medium for 12-20 h before infection, lose their characteristic structural polarity. Furthermore, when these cells were infected with influenza or VSV, virions assembled in a nonpolarized fashion over most of the cell surface. However, when dissociated MDCK cells infected in suspension were sparsely plated on collagen gels to prevent intercellular contact and the formation of junctions, the characteristic polarity of viral budding observed in confluent monolayers was again manifested; i.e., VSV budded preferentially from adherent surfaces and influenza almost exclusively from free surface regions. Similar polarization was observed in cells which became aggregated during incubation in spinner medium: influenza budded from the free surface, while VSV was produced at regions of cell-cell contact. It therefore appears that in isolated epithelial cells attachment to a substrate or to another cell is sufficient to trigger the expression of plasma membrane polarity which is manifested in the asymmetric budding of viruses.     

Epithelial cell-cell junctions and plasma membrane domains

Epithelial cells form a barrier against the environment, but are also required for the regulated exchange of molecules between an organism and its surroundings. Epithelial cells are characterised by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally, compositionally, and functionally distinct surface domains. Here we consider the (in)dependence of epithelial cell polarisation and the function of smaller plasma membrane domains (e.g. adherens junctions, gap junctions, tight junctions, apical lipid rafts, caveolae, and clathrin-coated pits) in the development and maintenance of cell surface polarity. Recent evidence of cross-talk and/or overlap between the different cell-cell junction components and alternate functions of junction components, including gene expression regulation, are discussed in the context of cell surface polarity.     

Polarity complex proteins

The formation of functional epithelial tissues involves the coordinated action of several protein complexes, which together produce a cell polarity axis and develop cell-cell junctions. During the last decade, the notion of polarity complexes emerged as the result of genetic studies in which a set of genes was discovered first in Caenorhabditis elegans and then in Drosophila melanogaster. In epithelial cells, these complexes are responsible for the development of the apico-basal axis and for the construction and maintenance of apical junctions. In this review, we focus on apical polarity complexes, namely the PAR3/PAR6/aPKC complex and the CRUMBS/PALS1/PATJ complex, which are conserved between species and along with a lateral complex, the SCRIBBLE/DLG/LGL complex, are crucial to the formation of apical junctions such as tight junctions in mammalian epithelial cells. The exact mechanisms underlying their tight junction construction and maintenance activities are poorly understood, and it is proposed to focus in this review on establishing how these apical polarity complexes might regulate epithelial cell morphogenesis and functions. In particular, we will present the latest findings on how these complexes regulate epithelial homeostasis.     

Polarity complex proteins

The formation of functional epithelial tissues involves the coordinated action of several protein complexes, which together produce a cell polarity axis and develop cell-cell junctions. During the last decade, the notion of polarity complexes emerged as the result of genetic studies in which a set of genes was discovered first in Caenorhabditis elegans and then in Drosophila melanogaster. In epithelial cells, these complexes are responsible for the development of the apico-basal axis and for the construction and maintenance of apical junctions. In this review, we focus on apical polarity complexes, namely the PAR3/PAR6/aPKC complex and the CRUMBS/PALS1/PATJ complex, which are conserved between species and along with a lateral complex, the SCRIBBLE/DLG/LGL complex, are crucial to the formation of apical junctions such as tight junctions in mammalian epithelial cells. The exact mechanisms underlying their tight junction construction and maintenance activities are poorly understood, and it is proposed to focus in this review on establishing how these apical polarity complexes might regulate epithelial cell morphogenesis and functions. In particular, we will present the latest findings on how these complexes regulate epithelial homeostasis.     

CAS/CSE 1 stimulates E-cadhrin-dependent cell polarity in HT-29 human colon epithelial cells

The establishment and maintenance of epithelial polarity are crucial for tissue organization and function in mammals. Epithelial cadherin (E-cadherin) is expressed in epithelial cell membrane and is important for cell-cell adhesion, intercellular junctions formation, as well as epithelial cell polarization. We report herein that CAS (CAS/CSE 1), the human cellular apoptosis susceptibility protein, interacts with E-cadherin and stimulates polarization of HT-29 human colon epithelial cells. CAS binds with E-cadherin but not with beta-catenin in the immunoprecipitation assays. Interaction of CAS with E-cadherin enhances the formation of E-cadherin/beta-catenin cell-cell adhesive complex. Electron microscopic study demonstrated that CAS overexpression in cells stimulates intercellular junction complex formation. The disorganization of cellular cytoskeleton by cytochalasin D, colchicine, or acrylamide treatment disrupts CAS-stimulated HT-29 cell polarization. CAS-mediated HT-29 cell polarity is also inhibited by antisense E-cadherin DNA expression. Our results indicate that CAS cooperates with E-cadherin and plays a role in the establishment of epithelial cell polarity.     

Interactions between the crumbs,lethal giant larvae and bazooka pathways in epithelial polarization

Several protein complexes that are involved in epithelial apicobasal polarity have been identified. However, the mechanism by which these complexes interact to form an integrated polarized cell morphology remains unclear. Crumbs (Crb) and Lethal giant larvae (Lgl) are components of distinct complexes that regulate epithelial polarization in Drosophila melanogaster, but may not interact directly as they localize to the apical and basolateral membrane, respectively. Nevertheless, a genetic screen identifies marked functional interactions between crb and lgl. These interactions extend to other genes within the crb (stardust, sdt) and lgl (discs large, dlg; scribble, scrib) pathways. Our findings suggest that the crb and lgl pathways function competitively to define apical and basolateral surfaces. They also suggest that in the absence of lgl pathway activity, the crb pathway is not required to maintain epithelial polarity. Moreover, we show that crb and lgl cooperate in zonula adherens formation early in development. At later stages, epithelial cells in these mutants acquire normal polarity, indicating the presence of compensatory mechanisms. We find that bazooka (baz) functions redundantly with crb/sdt to support apical polarity at mid- to late-embryogenesis. Despite regaining cell polarity, however, epithelial cells in crb and lgl pathway mutants fail to re-establish normal overall tissue architecture, indicating that the timely acquisition of polarized cell structure is essential for normal tissue organization.     

Hydrocortisone induces changes in gene expression and differentiation in immature human enterocytes

It is known that functional maturation of the small intestine occurring during the weaning period is facilitated by glucocorticoids (such as hydrocortisone, HC), including an increased expression of digestive hydrolases. However, the molecular mechanisms are not well understood, particularly in the human gut. Here we report a microarray analysis of HC-induced changes in gene expression in H4 cells (a well-characterized human fetal small intestinal epithelial cell line). This study identified a large number of HC-regulated genes, some involved in metabolism, cell cycle regulation, cell-cell or cell-extracellular matrix communication. HC also regulates the expression of genes important for cell maturation such as development of cell polarity, tight junction formation, and interactions with extracellular matrices. Using human small intestinal xenografts, we also show that HC can regulate the expression of genes important for intestinal epithelial cell maturation. Our dataset may serve as a useful resource for understanding and dissecting the molecular mechanisms of intestinal epithelial cell maturation.     

Complete polarization of single intestinal epithelial cells upon activation of LKB1 by STRAD

The LKB1 gene encodes a serine/threonine kinase that is mutated in the Peutz-Jeghers cancer syndrome. LKB1 is homologous to the Par-4 polarity genes in C. elegans and D. melanogaster. We have previously reported the identification and characterization of an LKB1-specific adaptor protein, STRAD, which activates LKB1 and translocates it from nucleus to cytoplasm. We have now constructed intestinal epithelial cell lines in which inducible STRAD activates LKB1. Upon LKB1 activation, single cells rapidly remodel their actin cytoskeleton to form an apical brush border. The junctional proteins ZO-1 and p120 redistribute in a dotted circle peripheral to the brush border, in the absence of cell-cell contacts. Apical and basolateral markers sort to their respective membrane domains. We conclude that LKB1 can induce complete polarity in intestinal epithelial cells. In contrast to current thinking on polarization of simple epithelia, these cells can fully polarize in the absence of junctional cell-cell contacts.     

Na,K-ATPase beta-subunit is required for epithelial polarization, suppression of invasion, and cell motility

The cell adhesion molecule E-cadherin has been implicated in maintaining the polarized phenotype of epithelial cells and suppression of invasiveness and motility of carcinoma cells. Na,K-ATPase, consisting of an alpha- and beta-subunit, maintains the sodium gradient across the plasma membrane. A functional relationship between E-cadherin and Na,K-ATPase has not previously been described. We present evidence that the Na,K-ATPase plays a crucial role in E-cadherin-mediated development of epithelial polarity, and suppression of invasiveness and motility of carcinoma cells. Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK) have highly reduced levels of E-cadherin and beta(1)-subunit of Na,K-ATPase. Forced expression of E-cadherin in MSV-MDCK cells did not reestablish epithelial polarity or inhibit the invasiveness and motility of these cells. In contrast, expression of E-cadherin and Na,K-ATPase beta(1)-subunit induced epithelial polarization, including the formation of tight junctions and desmosomes, abolished invasiveness, and reduced cell motility in MSV-MDCK cells. Our results suggest that E-cadherin-mediated cell-cell adhesion requires the Na,K-ATPase beta-subunit's function to induce epithelial polarization and suppress invasiveness and motility of carcinoma cells. Involvement of the beta(1)-subunit of Na,K-ATPase in the polarized phenotype of epithelial cells reveals a novel link between the structural organization and vectorial ion transport function of epithelial cells.     

Integrins contribute to the establishment and maintenance of cell polarity in the follicular epithelium of the Drosophila ovary

The generation of epithelial cell polarity is a key process during development. Although the induction and orientation of cell polarity by cell-cell and cell-extracellular matrix (ECM) interactions is well established, the molecular mechanisms by which signals from the ECM control cell polarity in developing epithelial tissues remain poorly understood. Here, we have used the follicular epithelium of the Drosophila ovary to investigate the role that integrins, the main cell-ECM receptors, play in the establishment of apicobasal polarity. Mature follicle cells have an apical side facing the germ line and a basal side in contact with a basement membrane. Our results show that integrins - presumably via interactions with the basement membrane - play a reinforcing role in follicle cell polarization, as they are required to establish and/or maintain follicle cell membrane asymmetry only when contact with the germ line is prevented. We suggest that the primary cue for polarization of the follicular epithelium is contact with the germline cells. In addition, while interfering with apical and lateral polarization cues leads to apoptosis, we show here that inhibition of contact with the basement membrane mediated by integrins does not affect cell survival. Finally, we provide evidence to suggest that integrins are required to orientate epithelial polarity in vivo.     

Differentiation of an epithelium: factors affecting the polarized distribution of Na+,K(+)-ATPase in mouse trophectoderm

Na+,K(+)-ATPase is a marker of the basolateral plasma membrane domain of polarized epithelial cells, including the mural trophectoderm of the mammalian blastocyst (Watson and Kidder (1988). Dev. Biol. 126, 80-90). We have used this marker to explore the factors governing the establishment and maintenance of apical/basolateral polarity during differentiation of trophectoderm. A polyclonal antiserum (anti-GP80) against human cell-CAM 120/80, a homolog of the mouse cell-cell adhesion protein, uvomorulin, was used to prevent cell flattening (compaction) and formation of the epithelial junctional complex. The majority of treated embryos failed to develop a blastocoel; instead their blastomeres developed fluid-filled cavities that expanded while untreated control embryos were cavitating. Immunocytochemistry revealed that the catalytic subunit of Na+,K(+)-ATPase was contained within the membranes lining these cavities, as well as within numerous punctate foci in the cytoplasm. The down-regulation of expression of the enzyme that normally occurs in the ICM and polar trophectoderm did not take place, since the immunoreactivity remained equally strong in all blastomeres. The enzyme could not be detected in plasma membranes. We conclude that uvomorulin-mediated cell adhesion is involved in spatially restricting the expression of the catalytic subunit and is a prerequisite for the insertion of enzyme-laden vesicles into plasma membranes, but not for expression of the catalytic subunit gene. When fully developed blastocysts were treated with cytochalasins to disrupt the epithelial junctional complex, the catalytic subunit shifted from the basolateral to the apical plasma membrane. This finding suggests a primary role for the apical plasma membrane in the process of polarization, and implies that tight junctions are a manifestation of polarity that serve to maintain the separation between apical and basolateral markers.     

PATJ regulates tight junction formation and polarity in mammalian epithelial cells

Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. Here, we find that PATJ is targeted to the apical region and tight junctions once cell polarization is initiated. We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs-PALS1-PATJ complex function in epithelial polarity.     

The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium

Studies of the developing trophectoderm in the mouse embryo have shown that extensive cellular remodeling occurs during epithelial formation. In this investigation, confocal immunofluorescence microscopy is used to examine the three-dimensional changes in cellular architecture that take place during the polarization of a terminally differentiated epithelial cell line. Madin-Darby canine kidney cells were plated at a low density on permeable filter supports. Antibodies that specifically recognize components of the tight junction, adherens junction, microtubules, centrosomes, and the Golgi complex were used to study the spatial remodeling of the cytoarchitecture during the formation of the polarized cell layer. The immunofluorescence data were correlated with establishment of functional tight junctions as measured by transepithelial resistance and back-exchange of the cell surface, labeled with metabolites of the fluorescent lipid analogue N-(7-[4- nitrobenzo-2-oxa-1,3-diazole]) aminocaproyl sphingosine. 1 d after plating, single cells had microtubules, radiating from a broad region, that contained the centrosomes and the Golgi complex. 2 d after plating, the cells had grown to confluence and had formed functional tight junctions close to the substratum. The centrioles had split and no longer organized the microtubules which were running above and below the nucleus. The Golgi complex had spread around the nucleus. By the fifth day after plating, the final polarized state had been achieved. The junctional complex had moved greater than 10 microns upward from its basal location. The centrioles were together below the apical membrane, and the Golgi complex formed a ribbon-like convoluted structure located in the apical region above the nucleus. The microtubules were organized in an apical web and in longitudinal microtubule bundles in the apical-basal axis of the columnar cell. The longitudinal microtubules were arranged with their minus ends spread over the apical region of the cell and their plus ends toward the basal region. These findings show that there is an extensive remodeling of epithelial cytoarchitecture after formation of cell-cell contacts. Reorganization of the microtubule network results in functional polarization of the cytoplasm.     

Self-association of PAR-3-mediated by the conserved N-terminal domain contributes to the development of epithelial tight junctions

PAR-3 is a scaffold-like PDZ-containing protein that forms a complex with PAR-6 and atypical protein kinase C (PAR-3-atypical protein kinase C-PAR-6 complex) and contributes to the establishment of cell polarity in a wide variety of biological contexts. In mammalian epithelial cells, it localizes to tight junctions, the most apical end of epithelial cell-cell junctions, and contributes to the formation of functional tight junctions. However, the mechanism by which PAR-3 localizes to tight junctions and contributes to their formation remains to be clarified. Here we show that the N-terminal conserved region, CR1-(1-86), and the sequence 937-1,024 are required for its recruitment to the most apical side of the cell-cell contact region in epithelial Madin-Darby canine kidney cells. We also show that CR1 self-associates to form an oligomeric complex in vivo and in vitro. Further, overexpression of CR1 in Madin-Darby canine kidney cells disturbs the distribution of atypical protein kinase C and PAR-6 as well as PAR-3 and delays the formation of functional tight junctions. These results support the notion that the CR1-mediated self-association of the PAR-3-containing protein complex plays a role during the formation of functional tight junctions.     

PAR3 is essential for cyst-mediated epicardial development by establishing apical cortical domains

Epithelial cysts are one of the fundamental architectures for mammalian organogenesis. Although in vitro studies using cultured epithelial cells have revealed proteins required for cyst formation, the mechanisms that orchestrate the functions of these proteins in vivo remain to be clarified. We show that the targeted disruption of the mouse Par3 gene results in midgestational embryonic lethality with defective epicardial development. The epicardium is mainly derived from epicardial cysts and essential for cardiomyocyte proliferation during cardiac morphogenesis. PAR3-deficient epicardial progenitor (EPP) cells do not form cell cysts and show defects in the establishment of apical cortical domains, but not in basolateral domains. In PAR3-deficient EPP cells, the localizations of aPKC, PAR6beta and ezrin to the apical cortical domains are disturbed. By contrast, ZO1 and alpha4/beta1 integrins normally localize to cell-cell junctions and basal domains, respectively. Our observations indicate that EPP cell cyst formation requires PAR3 to interpret the polarity cues from cell-cell and cell-extracellular matrix interactions so that each EPP cell establishes apical cortical domains. These results also provide a clear example of the proper organization of epithelial tissues through the regulation of individual cell polarity.     

Polar delivery in plants; commonalities and differences to animal epithelial cells

Although plant and animal cells use a similar core mechanism to deliver proteins to the plasma membrane, their different lifestyle, body organization and specific cell structures resulted in the acquisition of regulatory mechanisms that vary in the two kingdoms. In particular, cell polarity regulators do not seem to be conserved, because genes encoding key components are absent in plant genomes. In plants, the broad knowledge on polarity derives from the study of auxin transporters, the PIN-FORMED proteins, in the model plant Arabidopsis thaliana. In animals, much information is provided from the study of polarity in epithelial cells that exhibit basolateral and luminal apical polarities, separated by tight junctions. In this review, we summarize the similarities and differences of the polarization mechanisms between plants and animals and survey the main genetic approaches that have been used to characterize new genes involved in polarity establishment in plants, including the frequently used forward and reverse genetics screens as well as a novel chemical genetics approach that is expected to overcome the limitation of classical genetics methods.     

Apicobasal polarity in the kidney

The apicobasal polarization of epithelia is critical for many aspects of kidney function. Over the last decade there have been major advances in our understanding of the mechanisms that underlie this polarity. Critical to this understanding has been the identification of protein complexes on the apical and basolateral sides of epithelial cells that act in a mutually antagonistic manner to define these domains. Concomitant with the creation of apical and basolateral domains is the formation of highly specialized cell-cell junctions including adherens junctions and tight junctions. Recent research points to variability in the polarity and junctional complexes amongst different species and between different cell types of the kidney. Defects in apicobasal polarity are prominent in several disorders including acute renal failure and polycystic kidney disease.     

Cell biology of planar polarity transmission in the Drosophila wing

The coordination of epithelial planar polarization is a critical step in the formation of well-ordered tissues. The process has been extensively studied in Drosophila, where genetic analysis has identified a set of "tissue polarity" genes that serve to coordinate planar polarity of cells in the developing wings, bristles and eyes. In the last several years, it has emerged that six of these genes encode junctional proteins. In the wing epithelium, these proteins undergo a polarized redistribution, forming separate proximal and distal cortical domains within each cell. The mechanisms that mediate cortical polarization and cue its direction have been the subject of intense investigation. Cuing the orientation of cortical polarization appears to depend on the atypical Cadherins Fat and Dachsous, although these proteins do not become polarized themselves, nor do they colocalize with components of polarized cortical domains. Interestingly, these Cadherins also act at earlier developmental stages to polarize tissue growth along the proximal-distal axis and it will be interesting to see whether these processes are mechanistically related. Once the axis of polarization is determined, cortical polarity seems to be propagated, at least locally, by a cascade of direct cell-cell interactions mediated by the proximal and distal domains. The cell biological mechanisms leading to polarization are still unclear, but the process depends on the control of Protein Phosphatase 2A activity by its regulatory subunit, Widerborst. Interestingly, Widerborst is found on a planar web of microtubules with connections to apical junctions, suggesting that these microtubules may have an important function in polarizing the cortex.     

The keratin-binding protein Albatross regulates polarization of epithelial cells

The keratin intermediate filament network is abundant in epithelial cells, but its function in the establishment and maintenance of cell polarity is unclear. Here, we show that Albatross complexes with Par3 to regulate formation of the apical junctional complex (AJC) and maintain lateral membrane identity. In nonpolarized epithelial cells, Albatross localizes with keratin filaments, whereas in polarized epithelial cells, Albatross is primarily localized in the vicinity of the AJC. Knockdown of Albatross in polarized cells causes a disappearance of key components of the AJC at cell–cell borders and keratin filament reorganization. Lateral proteins E-cadherin and desmoglein 2 were mislocalized even on the apical side. Although Albatross promotes localization of Par3 to the AJC, Par3 and ezrin are still retained at the apical surface in Albatross knockdown cells, which retain intact microvilli. Analysis of keratin-deficient epithelial cells revealed that keratins are required to stabilize the Albatross protein, thus promoting the formation of AJC. We propose that keratins and the keratin-binding protein Albatross are important for epithelial cell polarization.