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.     

Sec3-containing Exocyst Complex Is Required for Desmosome Assembly in Mammalian Epithelial Cells

The Exocyst is a conserved multisubunit complex involved in the docking of post-Golgi transport vesicles to sites of membrane remodeling during cellular processes such as polarization, migration, and division. In mammalian epithelial cells, Exocyst complexes are recruited to nascent sites of cell–cell contact in response to E-cadherin–mediated adhesive interactions, and this event is an important early step in the assembly of intercellular junctions. Sec3 has been hypothesized to function as a spatial landmark for the development of polarity in budding yeast, but its role in epithelial cells has not been investigated. Here, we provide evidence in support of a function for a Sec3-containing Exocyst complex in the assembly or maintenance of desmosomes, adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. We show that Sec3 associates with a subset of Exocyst complexes that are enriched at desmosomes. Moreover, we found that membrane recruitment of Sec3 is dependent on cadherin-mediated adhesion but occurs later than that of the known Exocyst components Sec6 and Sec8 that are recruited to adherens junctions. RNA interference-mediated suppression of Sec3 expression led to specific impairment of both the morphology and function of desmosomes, without noticeable effect on adherens junctions. These results suggest that two different exocyst complexes may function in basal–lateral membrane trafficking and will enable us to better understand how exocytosis is spatially organized during development of epithelial plasma membrane domains.     

Failure of epithelial tube maintenance causes hydrocephalus and renal cysts in Dlg5-/- mice

Epithelial tubes represent fundamental building blocks of metazoan organisms; however, the mechanisms responsible for their formation and maintenance are not well understood. Here, we show that the evolutionarily conserved coiled-coil MAGUK protein Dlg5 is required for epithelial tube maintenance in mammalian brain and kidneys. We demonstrate that Dlg5(-/-) mice develop fully penetrant hydrocephalus and kidney cysts caused by a deficiency in membrane delivery of cadherin-catenin adhesion complexes and loss of cell polarity. Dlg5 travels with cadherin-containing vesicles and binds to syntaxin 4, a t-SNARE protein that regulates fusion of transport vesicles with the lateral membrane domain. We propose that Dlg5 functions in plasma membrane delivery of cadherins by linking cadherin-containing transport vesicles with the t-SNARE targeting complex. These findings show that Dlg5 is causally involved in hydrocephalus and renal cysts and reveal that targeted membrane delivery of cadherin-catenin adhesion complexes is critical for cell polarity and epithelial tube maintenance.     

Viral interactions with receptors in cell junctions and effects on junctional stability

Several viruses can use, as entry receptors, cell adhesion molecules that localize to junctional complexes of epithelial cells and other cell types. A recent publication in Cell describes how adenovirus can disrupt cell junctions, thereby effecting its release from basal surfaces of an infected epithelium to the apical or external environment.     

MYC-induced apoptosis in mammary epithelial cells is associated with repression of lineage-specific gene signatures

Apoptosis caused by deregulated MYC expression is a prototype example of intrinsic tumor suppression. However, it is still unclear how supraphysiological MYC expression levels engage specific sets of target genes to promote apoptosis. Recently, we demonstrated that repression of SRF target genes by MYC/MIZ1 complexes limits AKT-dependent survival signaling and contributes to apoptosis induction. Here we report that supraphysiological levels of MYC repress gene sets that include markers of basal-like breast cancer cells, but not luminal cancer cells, in a MIZ1-dependent manner. Furthermore, repressed genes are part of a conserved gene signature characterizing the basal subpopulation of both murine and human mammary gland. These repressed genes play a role in epithelium and mammary gland development and overlap with genes mediating cell adhesion and extracellular matrix organization. Strikingly, acute activation of oncogenic MYC in basal mammary epithelial cells is sufficient to induce luminal cell identity markers. We propose that supraphysiological MYC expression impacts on mammary epithelial cell identity by repressing lineage-specific target genes. Such abrupt cell identity switch could interfere with adhesion-dependent survival signaling and thus promote apoptosis in pre-malignant epithelial tissue.     

Emerging role for ERM proteins in cell adhesion and migration

The highly related ERM (Ezrin, Radixin, Moesin) proteins provide a regulated linkage between the membrane and the underlying actin cytoskeleton. They also provide a platform for the transmission of signals in responses to extracellular cues. Studies in different model organisms and in cultured cells have highlighted the importance of ERM proteins in the generation and maintenance of specific domains of the plasma membrane. A central question is how do ERM proteins coordinate actin filament organization and membrane protein transport/stability with signal transduction pathways to build up complex structures? Through their interaction with numerous partners including membrane proteins, actin cytoskeleton and signaling molecules, ERM proteins have the ability to organize multiprotein complexes in specific cellular compartments. Likewise, ERM proteins participate in diverse functions including cell morphogenesis, endocytosis/exocytosis, adhesion and migration. This review focuses on aspects still poorly understood related to the function of ERM proteins in epithelial cell adhesion and migration.Key words: epithelial cells, membrane-cytoskeleton interface, morphogenesis, ERM proteins, cell adhesion     

Setting up a selective barrier at the apical junction complex

Across the animal kingdom the apical junction complex of epithelial cells creates both a permeability barrier and cell polarity. Although based on overlapping and evolutionarily conserved proteins, the cell-cell contacts of nematodes, flies and mammals appear to differ in morphology and functional organization. Emerging evidence shows that the selective pore-like properties of vertebrate and invertebrate barriers are created by the claudin family. Similarly, assembly of the barriers requires a conserved set of polarity-generating protein complexes, particularly the PAR protein complexes.     

Involvement of the membrane-cytoskeleton in development of epithelial cell polarity

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.     

CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation

CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized human mammary epithelial cells (HMEC) caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The primary sequence of the CLCA2 TMS was found to be conserved in CLCA2 orthologs throughout mammals, suggesting that its interaction with EVA1 co-evolved with the mammary gland. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines.     

Par6 is phosphorylated by aPKC to facilitate EMT

The conserved polarity proteins Par6 and aPKC regulate cell polarization processes. However, increasing evidence also suggests that they play a role in oncogenic progression. During tumor progression, epithelial to mesenchymal transition (EMT) delineates an evolutionary conserved process that converts stationary epithelial cells into mesenchymal cells, which have an acquired ability for independent migration and invasion. In addition to signaling pathways that alter genetic programes that trigger the loss of cell-cell adhesion, alternative pathways can alter cell plasticity to regulate cell-cell cohesion and increase invasive potential. One such pathway involves TGFβ-induced phosphorylation of Par6. In epithelial cells, Par6 phosphorylation results in the dissolution of junctional complexes, cytoskeletal remodelling, and increased metastatic potential. Recently, we found that aPKC can also phosphorylate Par6 to drive EMT and increase the migratory potential of non-small cell lung cancer cells. This result has implications with respect to homeostatic and developmental processes involving polarization, and also with respect to cancer progression—particularly since aPKC has been reported to be an oncogenic regulator in various tumor cells.     

Prostaglandin E2 enhances mitogen-activated protein kinase/Erk pathway in human cholangiocarcinoma cells: involvement of EP1 receptor, calcium and EGF receptors signaling

The cross-signaling between (cell) adhesion molecules is nowadays a well-accepted phenomenon and includes orchestrated cellular changes and changes in the microenvironment. For example, Ep-CAM is an epithelial adhesion molecule that prevails in active proliferating tissue and is suppressed in a more differentiated state of the cell. E-cadherin adhesion complexes are typical for the advanced and terminal differentiated cell status. During normal proliferation, E-cadherin is not suppressed. We have demonstrated the effect of overexpression of Ep-CAM on E-cadherin, which probably affects the connection of cadherins and F-actin. Phosphatidylinositol 3-kinase (Pi3K) participates in various regulating mechanisms, for example in signaling to nuclei, vesicle transport, and cytoskeletal rearrangements. The effect of Ep-CAM on E-cadherin mediated junctions as well as the involvement of Pi3K in regulating adherens junctions, led us to investigate the potential interaction between Pi3K and Ep-CAM. Introduction of Ep-CAM in the epithelial cells caused abrogation of N-cadherin mediated cell–cell adhesion, which could be inhibited by Pi3K inhibitor LY294002. Moreover, the Pi3K subunit p85 was precipitated with Ep-CAM from cell lysates, and this complex showed kinase activity. The Pi3K activity shuttled from N-cadherin to Ep-CAM. From our results, we conclude that Ep-CAM cross signaling with N-cadherin involves Pi3K, resulting in the abrogation of the cadherin adhesion complexes in epithelial cells.     

Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts

In MDCK cells, presenilin-1 (PS1) accumulates at intercellular contacts where it colocalizes with components of the cadherin-based adherens junctions. PS1 fragments form complexes with E-cadherin, beta-catenin, and alpha-catenin, all components of adherens junctions. In confluent MDCK cells, PS1 forms complexes with cell surface E-cadherin; disruption of Ca(2+)-dependent cell-cell contacts reduces surface PS1 and the levels of PS1-E-cadherin complexes. PS1 overexpression in human kidney cells enhances cell-cell adhesion. Together, these data show that PS1 incorporates into the cadherin/catenin adhesion system and regulates cell-cell adhesion. PS1 concentrates at intercellular contacts in epithelial tissue; in brain, it forms complexes with both E- and N-cadherin and concentrates at synaptic adhesions. That PS1 is a constituent of the cadherin/catenin complex makes that complex a potential target for PS1 FAD mutations.     

alpha3beta1 integrin-CD151, a component of the cadherin-catenin complex, regulates PTPmu expression and cell-cell adhesion

The beta1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for alpha3beta1 integrin in association with the tetraspanin CD151 as a component of a cell-cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin-tetraspanin complex affects epithelial cell-cell adhesion at the level of gene expression both by regulating expression of PTPmu and by organizing a multimolecular complex containing PKCbetaII, RACK1, PTPmu, beta-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.     

Regulation of mitotic spindle orientation: an integrated view

Mitotic spindle orientation is essential for cell fate decisions, epithelial maintenance, and tissue morphogenesis. In most animal cell types, the dynein motor complex is anchored at the cell cortex and exerts pulling forces on astral microtubules to position the spindle. Early studies identified the evolutionarily conserved Gαi/LGN/NuMA complex as a key regulator that polarizes cortical force generators. In recent years, a combination of genetics, biochemistry, modeling, and live imaging has contributed to decipher the mechanisms of spindle orientation. Here, we highlight the dynamic nature of the assembly of this complex and discuss the molecular regulation of its localization. Remarkably, a number of LGN‐independent mechanisms were described recently, whereas NuMA remains central in most pathways involved in recruiting force generators at the cell cortex. We also describe the emerging role of the actin cortex in spindle orientation and discuss how dynamic astral microtubule formation is involved. We further give an overview on instructive external signals that control spindle orientation in tissues. Finally, we discuss the influence of cell geometry and mechanical forces on spindle orientation.     

Apico-basal polarity complex and cancer

Apico-basal polarity is a cardinal molecular feature of adult eukaryotic epithelial cells and appears to be involved in several key cellular processes including polarized cell migration and maintenance of tissue architecture. Epithelial cell polarity is maintained by three well-conserved polarity complexes, namely, PAR, Crumbs and SCRIB. The location and interaction between the components of these complexes defines distinct structural domains of epithelial cells. Establishment and maintenance of apico-basal polarity is regulated through various conserved cell signalling pathways including TGFβ, Integrin and WNT signalling. Loss of cell polarity is a hallmark for carcinoma, and its underlying molecular mechanism is beginning to emerge from studies on model organisms and cancer cell lines. Moreover, deregulated expression of apico-basal polarity complex components has been reported in human tumours. In this review, we provide an overview of the apico-basal polarity complexes and their regulation, their role in cell migration, and finally their involvement in carcinogenesis.     

Identification of a novel protein, LYRIC, localized to tight junctions of polarized epithelial cells

Tight junctions (TJ) are multiprotein complexes that function to regulate paracellular transport of molecules through epithelial and endothelial cell layers. Many new tight junction-associated proteins have been identified in the past few years, and their functional roles and interactions have just begun to be elucidated. In this paper, we describe a novel protein LYsine-RIch CEACAM1 co-isolated (LYRIC) that is widely expressed and highly conserved between species. LYRIC has no conserved domains that would indicate function and does not appear to be a member of a larger protein family. Data from analysis of rat and human tissue sections and cell lines show that LYRIC colocalizes with tight junction proteins ZO-1 and occludin in polarized epithelial cells, suggesting that LYRIC is part of the tight junction complex. LYRIC dissociates from ZO-1 when junctional complexes are disrupted, and as tight junctions reform, ZO-1 relocalizes before LYRIC. These results suggest that LYRIC is most likely not a structural component required for TJ formation, but rather is recruited during the maturation of the tight junction complex.     

Molecular mechanism for orienting membrane and actin dynamics to nascent cell-cell contacts in epithelial cells

The small GTPase Rac1 has been implicated in regulation of cell migration and cell-cell adhesion in epithelial cells. Little is known, however, about the spatial and temporal coordination of Rac1 activity required to balance these competing processes. We fractionated endogenous Rac1-containing protein complexes from membranes of Madin-Darby canine kidney cells and identified three major complexes comprising a Rac1.PAK (p21-activated kinase) complex, and 11 S and 16 S Rac1 complexes. Significantly, Rac1 shifts from the 11 S to a 16 S particle during initiation of cell-cell adhesion. This shift may reflect a diffusion trapping mechanism by which these Rac1 complexes are localized to cadherin-mediated cell-cell contacts through an interaction with annexin II.     

Identification of a novel intracellular interaction domain essential for Bves function

While Blood vessel epicardial substance (Bves) confers adhesive properties, the molecular mechanism of regulating this activity is unknown. No predicted functional motifs in this highly conserved integral membrane protein, other than the transmembrane domain, have been identified. Here, we report for the first time that Bves interacts with itself through an intracellular interaction domain that is essential for its intercellular adhesion activity. Glutathione-S-transferase (GST) pull-down and SPOTs analyses mapped this domain to amino acids 268-274 in the intracellular C-terminus. Site-directed mutagenesis revealed that lysines 272 and 273 are essential for homodimerization and cell adhesion. Human corneal cells transfected with wild-type Bves trafficked the protein to the cell surface, assembled junction complexes and formed epithelial sheets. In contrast, cells expressing Bves mutated at these positions did not form continuous epithelial sheets or maintain junctional proteins such as ZO-1 and E-cadherin at the membrane. A dramatic reduction in transepithelial electrical resistance was also observed indicating a functional loss of tight junctions. Importantly, expression of mutated Bves in epithelial cells promoted the transformation of cells from an epithelial to a mesenchymal phenotype. This study is the first to demonstrate the essential nature of any domain within Bves for maintenance of epithelial phenotype and function.