Junctions as Organizing Centers in Epithelial Cells? A Fly Perspective

Junctions isolate the apical from the basal–lateral surfaces and are required for the polarization of epithelial cells. Genetics of junction formation in Drosophila has identified numerous transmembrane and scaffolding proteins that assemble in membrane-associated complexes. This review focuses on recent findings suggesting that junctions are autonomous organizing centers for the establishment and maintenance of apical-basal polarity in epithelial cells as well as other polarized processes such as the orientation of the division apparatus.     

Differential Sensitivity of Epithelial Cells to Extracellular Matrix in Polarity Establishment

Establishment of apical-basal polarity is crucial for epithelial sheets that form a compartment in the body, which function to maintain the environment in the compartment. Effects of impaired polarization are easily observed in three-dimensional (3-D) culture systems rather than in two-dimensional (2-D) culture systems. Although the mechanisms for establishing the polarity are not completely understood, signals from the extracellular matrix (ECM) are considered to be essential for determining the basal side and eventually generating polarity in the epithelial cells. To elucidate the common features and differences in polarity establishment among various epithelial cells, we analyzed the formation of epithelial apical-basal polarity using three cell lines of different origin: MDCK II cells (dog renal tubules), EpH4 cells (mouse mammary gland), and R2/7 cells (human colon) expressing wild-type α-catenin (R2/7 α-Cate cells). These cells showed clear apical-basal polarity in 2-D cultures. In 3-D cultures, however, each cell line displayed different responses to the same ECM. In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel. In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens. In EpH4 cells, the spheroids displayed an apical-basal polarity that was opposite to that seen in the other two cell types in both ECM gels, at least during the culture period. On the other hand, the three cell lines showed the same apical-basal polarity both in 2-D cultures and in 3-D cultures using the hanging drop method. The three lines also had similar cellular responses to ECM secreted by the cells themselves. Therefore, appropriate culture conditions should be carefully determined in advance when using various epithelial cells to analyze cell polarity or 3-D morphogenesis.     

The Drosophila PAR-1 spacer domain is required for lateral membrane association and for polarization of follicular epithelial cells

The Ser/Thr kinases of the PAR-1/MARK/Kin1 family are conserved regulators of polarity in epithelial and non-epithelial cells . Drosophila PAR-1 localizes laterally in the follicular epithelium of the ovary , where it has been shown to function at two distinct levels: It stabilizes the cytoskeleton and it regulates apical-basal polarity by directly inhibiting lateral assembly of the apical aPKC/Bazooka/PAR-6 complex . However, it has been unclear how lateral localization of Drosophila PAR-1 is achieved and whether this localization contributes to epithelial polarity in vivo. Here we show that, through its spacer domain, Drosophila PAR-1 accumulates on the lateral plasma membrane (PM) in cells of the follicular epithelium (FE). Rescue experiments indicate that in FE cells PAR-1 kinase activity is essential for all the described functions of PAR-1. In contrast, the spacer domain of PAR-1 is required for apical-basal polarity and growth control but is dispensable for microtubule (MT) stabilization. Our data indicate that the spacer domain of PAR-1 is required for lateral PM localization of PAR-1 kinase and for development of a polarized FE.     

Epithelial polarity and cell separation in the neoplastic l(1)dlg-1 mutant of Drosophila.

Lethal (1) discs-large-1 [l(1)dlg-1] is a non-epithelial overgrowth or neoplastic mutant of Drosophila, which results in tumor-like imaginal discs and enlarged larvae that never pupariate. In an ultrastructural analysis we found that the wing discs develop convoluted monolayers of epithelial cells characterized by well-defined apical-basal polarity and that these layered cells secrete large amounts of basement membrane material. Immuno-EM indicates that Drosophila laminin and collagen are components of this matrix. Late in development clusters or 'rosettes' of separated cells lacking cell-cell junctions and apical-basal polarity form. In in vitro culture experiments l(1)dlg-1 wing discs did not respond to a pulse of exogenous ecdysone by secreting cuticle or losing basement membrane as normal discs do. Our observations are consistent with the hypothesis that cell-cell interaction and communication is required for termination of disc cell proliferation, which must occur prior to a cellular response to ecdysone.     

上皮细胞极性的建立和维持

细胞极性是生物中广泛存在的一个特征。上皮细胞是构成表皮、腺体、气管和消化道等组织的一类特化细胞。上皮细胞通常沿顶端-基底端轴向发生极化,形成紧密连接、粘附连接等胞间结构,同时细胞膜、细胞骨架和中心体、内膜系统、细胞核等也发生不对称分布,使细胞能行使分泌、吸收和屏障等多种重要的生理功能。有许多分子参与上皮细胞极性的建立和维持,其中最主要的是3个极性复合物,即Par-aPKC复合物,Scribble(Lg1-Dlg-Scrib)复合物和Crb(Crb-Pals-PATJ)复合物,三者共同配合发挥功能。     

Depletion of E-cadherin disrupts establishment but not maintenance of cell junctions in Madin-Darby canine kidney epithelial cells

E-cadherin forms calcium-dependent homophilic intercellular adhesions between epithelial cells. These contacts regulate multiple aspects of cell behavior, including the organization of intercellular tight junctions (TJs). To distinguish between the roles of E-cadherin in formation versus maintenance of junctions, Madin-Darby canine kidney (MDCK) cells were depleted of E-cadherin by RNA interference. Surprisingly, reducing E-cadherin expression had little effect on the protein levels or localization of adherens junction (AJ) or TJ markers. The cells underwent morphological changes, as the normally flat apical surface swelled into a dome. However, apical-basal polarity was not compromised, transmembrane resistance was normal, and zonula occludin protein 1 dynamics at the TJs were unchanged. Additionally, an E-cadherin/Cadherin-6 double knockdown also failed to disrupt established TJs, although beta-catenin was lost from the cell cortex. Nevertheless, cells depleted of E-cadherin failed to properly reestablish cell polarity after junction disassembly. Recovery of cell-cell adhesion, transepithelial resistance, and the localization of TJ and AJ markers were all delayed. In contrast, depletion of alpha-catenin caused long-term disruption of junctions. These results indicate that E-cadherin and Cadherin-6 function as a scaffold for the construction of polarized structures, and they become largely dispensable in mature junctions, whereas alpha-catenin is essential for the maintenance of functional junctions.     

A cell polarity protein aPKCλ is required for eye lens formation and growth

The organisation of individual cells into a functional three-dimensional tissue is still a major question in developmental biology. Modulation of epithelial cell shape is a critical driving force in forming tissues. This is well illustrated in the eye lens where epithelial cells elongate extensively during their differentiation into fibre cells. It is at the lens equator that epithelial cells elongate along their apical-basal axis. During this process the elongating epithelial cells and their earliest fibre cell derivatives remain anchored at their apical tips, forming a discrete region or modiolus, which we term the lens fulcrum. How this is achieved has received scant attention and is little understood. Here, we show that conditional depletion of aPKCλ, a central effector of the PAR polarity complex, disrupts the apical junctions in elongating epithelial cells so that the lens fulcrum fails to form. This results in disorganised fibre cell alignment that then causes cataract. Interestingly, aPKCλ depletion also promotes epithelial-mesenchymal transition of the lens epithelial cells, reducing their proliferation, leading ultimately to a small lens and microphthalmia. These observations indicate that aPKCλ, a regulator of polarity and apical junctions, is required for development of a lens that is the correct size and shape.     

Epithelial cell polarity: what flies can teach us about cancer

Epithelial cells are polarized along their apical-basal axis. Much of the cellular machinery that goes into establishing and maintaining epithelial cell polarity is evolutionarily conserved. Model organisms, including the fruit fly, Drosophila melanogaster, are thus particularly useful for the study of cell polarity. Work in Drosophila has identified several important components of the polarity machinery and has also established the surprising existence of a secondary cell polarity pathway required only under conditions of energetic stress. This work has important implications for the understanding of human cancer. Most cancers are epithelial in origin, and the loss of cell polarity is a critical step towards malignancy. Thus a better understanding of how polarity is established and maintained in epithelial cells will help us to understand the process of malignant transformation and may lead to improved therapies. In the present chapter we discuss the current understanding of how epithelial cell polarity is regulated and the known associations between polarity factors and cancer.     

Accumulation of a microtubule-binding protein, pp170, at desmosomal plaques

The establishment of epithelial cell polarity correlates with the formation of specialized cell-cell junctions and striking changes in the organization of microtubules. A significant fraction of the microtubules in MDCK cells become stabilized, noncentrosomally organized, and arranged in longitudinal bundles in the apical-basal axis. This correlation suggests a functional link between cell-cell junction formation and control of microtubule organization. We have followed the distribution of pp170, a recently described microtubule-binding protein, during establishment of epithelial cell polarity. This protein shows the typical patchy distribution along microtubules in subconfluent fibroblasts and epithelial cells, often associated with the peripheral ends of a subpopulation of microtubules. In contrast to its localization in confluent fibroblasts (A72) and HeLa cells, however, pp170 accumulates in patches delineating the regions of cell-cell contacts in confluent polarizing epithelial cells (MDCK and Caco-2). Double immunolocalization with antibodies specific for cell-cell junction proteins, confocal microscopy, and immunoelectron microscopy on polarized MDCK cells suggest that pp170 accumulates at desmosomal plaques. Furthermore, microtubules and desmosomes are found in close contact. Maintenance of the desmosomal association of pp170 is dependent on intact microtubules in 3-d-old, but not in 1-d-old MDCK cell cultures. This suggests a regulated interaction between microtubules and desmosomes and a role for pp170 in the control of changes in the properties of microtubules induced by epithelial cell-cell junction formation.     

The signaling adaptor gab1 regulates cell polarity by acting as a par protein scaffold

Cell polarity plays a key role in development and is disrupted in tumors, yet the molecules and mechanisms that regulate polarity remain poorly defined. We found that the scaffolding adaptor GAB1 interacts with two polarity proteins, PAR1 and PAR3. GAB1 binds PAR1 and enhances its kinase activity. GAB1 brings PAR1 and PAR3 into a transient complex, stimulating PAR3 phosphorylation by PAR1. GAB1 and PAR6 bind the PAR3 PDZ1 domain and thereby compete for PAR3 binding. Consequently, GAB1 depletion causes PAR3 hypophosphorylation and increases PAR3/PAR6 complex formation, resulting in accelerated and enhanced tight junction formation, increased transepithelial resistance, and lateral domain shortening. Conversely, GAB1 overexpression, in a PAR1/PAR3-dependent manner, disrupts epithelial apical-basal polarity, promotes multilumen cyst formation, and enhances growth factor-induced epithelial cell scattering. Our results identify GAB1 as a negative regulator of epithelial?cell polarity that functions as a scaffold for modulating PAR protein complexes on the lateral membrane.     

Ultrastructure of intermediate stages in polarity reversal of thyroid epithelium in follicles in suspension culture

Separated thyroid follicles can be maintained in suspension culture in Coon's modified F-12 medium in 0.5% calf serum. If the serum concentration is raised to 5%, the follicles undergo inversion in 3-5 d. During the process of inversion, epithelial cells can be observed in intermediate stages of polarity reversal. The earliest ultrastructural changes recognized are surface changes in which tight junctions and microvilli appear at the lateral margins of the cell near the medium. Later, changes in the distribution of intracellular organelles occur. The Golgi apparatus shifts towards the end of the cell facing the medium, and lysosomes shift toward the luminal end of the cell. The right junctions and microvilli at the luminal end of the cell disappear sometime after the cytoplasmic organelles rearrange. The luminal colloid disappears only after the surface changes (loss of tight junctions and microvilli) occur at the luminal end of the cell. There appears to be some regulation of the order in which changes occur during polarity reversal of the thyroid epithelial cell.     

A Highlights from MBoC Selection: Cdc42 Regulates Apical Junction Formation in Human Bronchial Epithelial Cells through PAK4 and Par6B

Cdc42 has been implicated in numerous biochemical pathways during epithelial morphogenesis, including the control of spindle orientation during mitosis, the establishment of apical-basal polarity, the formation of apical cell–cell junctions, and polarized secretion. To investigate the signaling pathways through which Cdc42 mediates these diverse effects, we have screened an siRNA library corresponding to the 36 known Cdc42 target proteins, in a human bronchial epithelial cell line. Two targets, PAK4 and Par6B, were identified as necessary for the formation of apical junctions. PAK4 is recruited to nascent cell–cell contacts in a Cdc42-dependent manner, where it is required for the maturation of primordial junctions into apical junctions. PAK4 kinase activity is essential for junction maturation, but overexpression of an activated PAK4 mutant disrupts this process. Par6B, together with its binding partner aPKC, is necessary both for junction maturation and for the retention of PAK4 at sites of cell–cell contact. This study demonstrates that controlled regulation of PAK4 is required for apical junction formation in lung epithelial cells and highlights potential cross-talk between two Cdc42 targets, PAK4 and Par6B.     

Planar cell polarity in Drosophila

In all multicellular organisms, epithelial cells are not only polarized along the apical-basal axis, but also within the epithelial plane, giving cells a sense of direction. Planar cell polarity (PCP) signaling regulates establishment of polarity within the plane of an epithelium. The outcomes of PCP signaling are diverse and include the determination of cell fates, the generation of asymmetric but highly aligned structures, such as the stereocilia in the human inner ear or the hairs on a fly wing, or the directional migration of cells during convergence and extension during vertebrate gastrulation. In humans, aberrant PCP signaling can result in severe developmental defects, such as open neural tubes (spina bifida), and can cause cystic kidneys. In this review, we discuss the basic mechanism and more recent findings of PCP signaling focusing on Drosophila melanogaster, the model organism in which most key PCP components were initially identified.     

Cell polarity from cell division

Apical-basal polarity of epithelial cells is critical for their symmetric versus asymmetric division and commonly thought to be established in interphase. In a novel type of cell division termed "mirror-symmetric", apical cell constituents accumulate during M-phase at the cleavage furrow, resulting in epithelial daughter cells with opposite apical-basal polarity.     

Lateral membrane biogenesis in human bronchial epithelial cells requires 190-kDa ankyrin-G

Ankyrin-G polypeptides are required for restriction of voltage-gated sodium channels, L1 cell adhesion molecules, and beta IV spectrin to axon initial segments and are believed to couple the Na/K-ATPase to the spectrin-actin network at the lateral membrane in epithelial cells. We report here that depletion of 190-kDa ankyrin-G in human bronchial epithelial cells by small interfering RNA results in nearly complete loss of lateral plasma membrane in interphase cells, and also blocks de novo lateral membrane biogenesis following mitosis. Loss of the lateral membrane domain is accompanied by an expansion of apical and basal plasma membranes and preservation of apical-basal polarity. Expression of rat 190-kDa ankyrin-G, which is resistant to human small interfering RNA, prevents loss of the lateral membrane following depletion of human 190-kDa ankyrin-G. Human 220-kDa ankyrin-B, a closely related ankyrin isoform, is incapable of preserving the lateral membrane following 190-kDa ankyrin-G depletion. Moreover, analysis of rat 190-kDa ankyrin G/ankyrin B chimeras shows that all three domains of 190-kDa ankyrin-G are required for preservation of the lateral membrane. These results demonstrate that 190-kDa ankyrin-G plays a pleiotropic role in assembly of lateral membranes of bronchial epithelial cells.     

Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells

This study of epithelial-mesenchymal transformation and epithelial cell polarity in vitro reveals that environmental conditions can have a profound effect on the epithelial phenotype, cell shape, and polarity as expressed by the presence of apical and basal surfaces. A number of different adult and embryonic epithelia were suspended within native collagen gels. Under these conditions, cells elongate, detach from the explants, and migrate as individual cells within the three-dimensional lattice, a previously unknown property of well-differentiated epithelia. Epithelial cells from adult and embryonic anterior lens were studied in detail. Elongated cells derived from the apical surface develop pseudopodia and filopodia characteristic of migratory cells and acquire a morphology and ultrastructure virtually indistinguishable from that of mesenchymal cells in vivo. It is concluded from these experiments that the three-dimensional collagen gel can promote dissociation, migration, and acquisition of secretory organelles by differentiated epithelial cells, and can abolish the apical-basal cell polarity characteristic of the original epithelium.     

glaikit is essential for the formation of epithelial polarity and neuronal development

Epithelial cells have a distinctive polarity based on the restricted distribution of proteins and junctional complexes along an apical-basal axis. Studying the formation of the polarized ectoderm of the Drosophila embryo has identified a number of the molecules that establish this polarity. The Crumbs (Crb) complex is one of three separate complexes that cooperate to control epithelial polarity and the formation of zonula adherens. Here we show that glaikit (gkt), a member of the phospholipase D superfamily, is essential for the formation of epithelial polarity and for neuronal development during Drosophila embryogenesis. In epithelial cells, gkt acts to localize the Crb complex of proteins to the apical lateral membrane. Loss of gkt during neuronal development leads to a severe CNS architecture disruption that is not dependent on the Crb pathway but probably results from the disrupted localization of other membrane proteins. A mutation in the human homolog of gkt causes the neurodegenerative disease spinocerebellar ataxia with neuropathy (SCAN1), making it possible that a failure of membrane protein localization is a cause of this disease.     

aPKC acts upstream of PAR-1b in both the establishment and maintenance of mammalian epithelial polarity

BACKGROUND: aPKC and PAR-1 are required for cell polarity in various contexts. In mammalian epithelial cells, aPKC localizes at tight junctions (TJs) and plays an indispensable role in the development of asymmetric intercellular junctions essential for the establishment and maintenance of apicobasal polarity. On the other hand, one of the mammalian PAR-1 kinases, PAR-1b/EMK1/MARK2, localizes to the lateral membrane in a complimentary manner with aPKC, but little is known about its role in apicobasal polarity of epithelial cells as well as its functional relationship with aPKC. RESULTS: We demonstrate that PAR-1b is essential for the asymmetric development of membrane domains of polarized MDCK cells. Nonetheless, it is not required for the junctional localization of aPKC nor the formation of TJs, suggesting that PAR-1b works downstream of aPKC during epithelial cell polarization. On the other hand, aPKC phosphorylates threonine 595 of PAR-1b and enhances its binding with 14-3-3/PAR-5. In polarized MDCK cells, T595 phosphorylation and 14-3-3 binding are observed only in the soluble form of PAR-1b, and okadaic acid treatment induces T595-dependent dissociation of PAR-1b from the lateral membrane. Furthermore, T595A mutation induces not only PAR-1b leakage into the apical membrane, but also abnormal development of membrane domains. These results suggest that in polarized epithelial cells, aPKC phosphorylates PAR-1b at TJs, and in cooperation with 14-3-3, promotes the dissociation of PAR-1b from the lateral membrane to regulate PAR-1b activity for the membrane domain development. CONCLUSIONS: These results suggest that mammalian aPKC functions upstream of PAR-1b in both the establishment and maintenance of epithelial cell polarity.     

Planar cell polarity in Drosophila

In all multicellular organisms, epithelial cells are not only polarized along the apical-basal axis, but also within the epithelial plane, giving cells a sense of direction. Planar cell polarity (PCP) signaling regulates establishment of polarity within the plane of an epithelium. The outcomes of PCP signaling are diverse and include the determination of cell fates, the generation of asymmetric but highly aligned structures, such as the stereocilia in the human inner ear or the hairs on a fly wing, or the directional migration of cells during convergence and extension during vertebrate gastrulation. In humans, aberrant PCP signaling can result in severe developmental defects, such as open neural tubes (spina bifida), and can cause cystic kidneys. In this review, we discuss the basic mechanism and more recent findings of PCP signaling focusing on Drosophila melanogaster, the model organism in which most key PCP components were initially identified.     

Peptidase allergen Der p 1 initiates apoptosis of epithelial cells independently of tight junction proteolysis

Loss of epithelial cell polarity, which can arise following disruption of tight junctions (TJs), is a precursor to the carefully orchestrated removal of moribund cells from epithelia in apoptosis. Ordinarily, this cycle of events has minimally disruptive effects on the function of the epithelial barrier, but some agents have been identified that induce apoptosis and promote epithelial leakiness. The allergen Der p 1 is a cysteine peptidase that cleaves TJ adhesion proteins and induces apoptosis in epithelial cells. This suggests the possibility that, at least for some inducers of apoptosis, these events might be causally linked. We report here that Der p 1 induces epithelial apoptosis before outright cell detachment and that apoptosis occurs within the same time span as increased paracellular permeability in polarized epithelial monolayers. Whilst TJ-deficient BEAS-2B cells were resistant to Der p 1-induced apoptosis, the cell line 1HAEo-, which was also TJ deficient, was sensitive to Der p 1, providing evidence against TJ proteolysis as a cause of apoptosis. To provide direct evidence, we propagated cells that normally express TJs in low calcium medium that prevented intercellular junction assembly. These cells retained full susceptibility to Der p 1, indicating that Der p 1-induced apoptosis is independent from TJ proteolysis.