Remodeling the zonula adherens in response to tension and the role of afadin in this response

Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions.     

Identification of a putative cell adhesion domain of uvomorulin.

A rat monoclonal antibody (DECMA-1) selected against the murine cell adhesion molecule uvomorulin blocks both the aggregation of mouse embryonal carcinoma cells and the compaction of pre-implantation embryos. However, decompacted embryos eventually become recompacted in the presence of DECMA-1 and form blastocysts composed of both trophectoderm and inner cell mass. DECMA-1 also disrupts confluent monolayers of Madin-Darby canine kidney (MDCK) epithelial cells. DECMA-1 recognizes uvomorulin in extracts from mouse and dog tissues. Protease digestion of mouse and dog uvomorulin generated core fragments including one of 26 kd which reacted with DECMA-1. The same 26-kd fragment is recognized by anti-uvomorulin monoclonal antibodies which have been obtained from other laboratories and which dissociate MDCK cell monolayers and block the formation of the epithelial occluding barrier. This 26-kd fragment therefore seems to be involved in the adhesive function of uvomorulin.     

Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin, and fodrin in Madin- Darby canine kidney epithelial cells

Cell-cell contact is an important determinant in the formation of functionally distinct plasma membrane domains during the development of epithelial cell polarity. In cultures of Madin-Darby canine kidney (MDCK) epithelial cells, cell-cell contact induces the assembly and accumulation of the Na+,K+-ATPase and elements of the membrane-cytoskeleton (ankyrin and fodrin) at the regions of cell-cell contact. Epithelial cell-cell contact appears to be regulated by the cell adhesion molecule uvomorulin (E-cadherin) which also becomes localized at the lateral plasma membrane of polarized cells. We have sought to determine whether the colocalization of these proteins reflects direct molecular interactions which may play roles in coordinating cell-cell contact and the assembly of the basal-lateral domain of the plasma membrane. Recently, we identified a complex of proteins containing the Na+,K+-ATPase, ankyrin, and fodrin in extracts of whole MDCK cells (Nelson, W.J., and R. W. Hammerton. 1989. J. Cell Biol. 108:893-902). We have now examined cell extracts for protein complexes containing the cell adhesion molecule uvomorulin. Proteins were solubilized from whole MDCK cells and fractionated in sucrose gradients. The sedimentation profile of solubilized uvomorulin is well separated from the majority of cell surface proteins, suggesting that uvomorulin occurs in a protein complex. A distinct portion of uvomorulin (30%) cosediments with ankyrin and fodrin (approximately 10.5S). Further fractionation of cosedimenting proteins in nondenaturing polyacrylamide gels reveals a discrete band of proteins that binds antibodies specific for uvomorulin, Na+,K+-ATPase, ankyrin, and fodrin. Significantly, ankyrin and fodrin, but not Na+K+-ATPase, coimmunoprecipitate in a complex with uvomorulin using uvomorulin antibodies. This result indicates that separate complexes exist containing ankyrin and fodrin with either uvomorulin or Na+,K+-ATPase. These results are discussed in the context of the possible roles of uvomorulin-induced cell-cell contact in the assembly of the membrane-cytoskeleton and associated membrane proteins (e.g., Na+,K+-ATPase) at the contact zone and in the development of cell polarity.     

ZO-1- and ZO-2-dependent integration of myosin-2 to epithelial zonula adherens

For the zonula adherens (ZA) to be established by linear arrangement of adherens junctions (AJs) in epithelial sheet cells, critical for the epithelial cell sheet formation and intercellular barrier function, myosin-2 is supposedly integrated into the ZA with the result of overlapping localization of E-cadherin/actin/myosin-2. Here, we immunofluorescently showed that myosin-2 failed to be integrated into the ZA in cultured epithelial-type ZO1(ko)/2(kd) Eph4 cells lacking ZO-1 and -2 (zonula occludens-1 and -2) by knockout and knockdown, respectively. Instead, a linearized but fragmented arrangement of AJs was formed in the way that it was positive for E-cadherin/actin, but negative for myosin-2 (designated prezonula-AJ). Transfection of full-length ZO-1 or ZO-2, or ZO-1 lacking its PDZ (PSD-95/discs large/zonula occludens-1)-1/2 domains (but not one lacking PDZ-1/2/3) into ZO1(ko)/2(kd) Eph4 cells restored the junctional integration of myosin-2 with prezonula-AJ to establish the ZA. Transfection of dominant-active RhoA or Rho-kinase (ROCK), as well as administration of lysophosphatidic acid or Y27632, which activates RhoA or inhibits ROCK, respectively, suggested that RhoA regulated the junctional integration of myosin-2 into ZA in a manner such that ROCK played a necessary but not-sufficient role. Fluorescence resonance energy transfer analyses revealed that spatiotemporal Rho-activation occurred in a ZO-1/2–dependent way to establish ZA from primordial forms in epithelial cells.     

A functional assay for proteins involved in establishing an epithelial occluding barrier: identification of a uvomorulin-like polypeptide

A functional assay has been developed to identify cell surface proteins involved in the formation of epithelial tight junctions. Transepithelial electrical resistance was used to measure the presence of intact tight junctions in monolayers of Madin-Darby canine kidney (MDCK) cells cultured on nitrocellulose filters. The strain I MDCK cells used have a transmonolayer resistance greater than 2,000 ohm . cm2. When the monolayers were incubated at 37 degrees C without Ca2+, the intercellular junctions opened and the transmonolayer resistance dropped to the value of a bare filter, i.e., less than 40 ohm . cm2. When Ca2+ was restored, the cell junctions resealed and the resistance recovered rapidly. Polyclonal antibodies raised against intact MDCK cells inhibited the Ca2+-dependent recovery of electrical resistance when applied to monolayers that had been opened by Ca2+ removal. Cross-linking of cell surface molecules was not required because monovalent Fab' fragments also inhibited. In contrast, a variety of other antibodies that recognize specific proteins on the MDCK cell surface failed to inhibit the recovery of resistance. Monoclonal antibodies have been raised and screened for their ability to inhibit resistance recovery. One such monoclonal antibody has been obtained that stained the lateral surface of MDCK cells. This antibody, rr1, recognized a 118-kD polypeptide in MDCK cell extracts and an 81-kD fragment released from the cell surface by trypsinization in the presence of Ca2+. Sequential immunoprecipitation with antibody rr1 and a monoclonal antibody to uvomorulin showed that this polypeptide is related to uvomorulin. The role of uvomorulin-like and liver cell adhesion molecule (L-CAM)-like polypeptides in the establishment of the epithelial occluding barrier is discussed.     

Nonmitogenic morphoregulatory action of pp60v-src on multicellular epithelial structures.

Madin-Darby canine kidney (MDCK) cells form polarized, multicellular epithelial structures in vitro. Low-level expression of pp60v-src in MDCK cells elicits plasticity in these multicellular structures. Plasticity was revealed by the displacement of cells from mechanically stressed regions of the epithelial monolayers; however, the two-dimensional relationship between the cells in the remainder of the monolayer was maintained. Electron microscopy of multicellular structures revealed abnormal separation of the lateral membranes of adjacent cells and selective uncoupling of the junctional complex; the zonula adherens was disrupted, but the zonula occludens and desmosomes were retained. Significantly, this result was not accompanied by transformation of the cells, as judged by the absence of anchorage-independent growth potential. These results demonstrate a nonmitogenic biological activity of pp60v-src which is experimentally dissociable from transformation. This morphoregulatory action on higher-order epithelial structures may reflect a function of related cellular tyrosine kinases.     

Dissociation of Madin-Darby canine kidney epithelial cells by the monoclonal antibody anti-arc-1: mechanistic aspects and identification of the antigen as a component related to uvomorulin

It has previously been shown that the monoclonal antibody anti-Arc-1 dissociates Madin-Darby canine kidney (MDCK) epithelial cells and changes their morphology in vitro (Imhof, B.A., H.P. Vollmers, S.L. Goodman, and W. Birchmeier, 1983, Cell, 35:667-675). In this article we demonstrate that the anti-Arc-1 antibody recognizes an uvomorulin-like molecule on MDCK cells, i.e., it immunoprecipitates an 84-kD protein fragment from a tryptic digest of cell surfaces in the presence of Ca2+ (as does anti-uvomorulin antiserum). Furthermore, anti-uvomorulin antiserum prevents the binding of anti-Arc-1 to MDCK cells. The distribution of the Arc-1 antigen is also quite similar to that of uvomorulin: it is enriched at the cell-cell contacts both of MDCK cells and of cells in various canine tissues. In the intestinal epithelium the antigen could be further localized in the region of the junctional complex. To study the mechanism of action of the dissociating antibody, MDCK cells grown on Nuclepore filters in Boyden chambers were exposed to anti-Arc-1 from either the upper or lower compartment. It could be shown that the antibody interfered with cell adhesion only from the basolateral but not from the apical cell surface. Antibody action was inhibited in the presence of colchicine but not cytochalasin B. Furthermore, cell dissociation was prevented when the cellular cAMP level was raised. These findings indicate that the anti-Arc-1 antibody acts on a target below the tight junctions (possibly on the antigen located in the junctional complex), and they confirm that cytoskeleton and metabolic factors are actively involved in the maintenance of junctional integrity.     

An RPTPα/Src family kinase/Rap1 signaling module recruits myosin IIB to support contractile tension at apical E-cadherin junctions

Cell–cell adhesion couples the contractile cortices of epithelial cells together, generating tension to support a range of morphogenetic processes. E-cadherin adhesion plays an active role in generating junctional tension by promoting actin assembly and cortical signaling pathways that regulate myosin II. Multiple myosin II paralogues accumulate at mammalian epithelial cell–cell junctions. Earlier, we found that myosin IIA responds to Rho-ROCK signaling to support junctional tension in MCF-7 cells. Although myosin IIB is also found at the zonula adherens (ZA) in these cells, its role in junctional contractility and its mode of regulation are less well understood. We now demonstrate that myosin IIB contributes to tension at the epithelial ZA. Further, we identify a receptor type-protein tyrosine phosphatase alpha–Src family kinase–Rap1 pathway as responsible for recruiting myosin IIB to the ZA and supporting contractile tension. Overall these findings reinforce the concept that orthogonal E-cadherin–based signaling pathways recruit distinct myosin II paralogues to generate the contractile apparatus at apical epithelial junctions.     

E-cadherin supports steady-state Rho signaling at the epithelial zonula adherens

In simple polarized epithelial cells, the Rho GTPase commonly localizes to E-cadherin-based cell–cell junctions, such as the zonula adherens (ZA), where it regulates the actomyosin cytoskeleton to support junctional integrity and tension. An important question is how E-cadherin contributes to Rho signaling, notably whether junctional Rho may depend on cadherin adhesion. We sought to investigate this by assessing Rho localization and activity in epithelial monolayers depleted of E-cadherin by RNAi. We report that E-cadherin depletion reduced both Rho and Rho-GTP at the ZA, an effect that was rescued by expressing a RNAi-resistant full-length E-cadherin transgene. This impact on Rho signaling was accompanied by reduced junctional localization of the Rho GEF ECT2 and the centralspindlin complex that recruits ECT2. Further, the Rho signaling pathway contributes to the selective stabilization of E-cadherin molecules in the apical zone of the cells compared with E-cadherin at the lateral surface, thereby creating a more defined and restricted pool of E-cadherin that forms the ZA. Thus, E-cadherin and Rho signaling cooperate to ensure proper ZA architecture and function.     

Differential regulation of junctional complex assembly in renal epithelial cell lines

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and -catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner. calpeptin; tight junctions; adherens junctions; Rho; cadherin; p120ctn     

Dissecting tumor cell invasion: epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cell-cell adhesion

The generation of invasiveness in transformed cells represents an essential step of tumor progression. We show here, first, that nontransformed Madin-Darby canine kidney (MDCK) epithelial cells acquire invasive properties when intercellular adhesion is specifically inhibited by the addition of antibodies against the cell adhesion molecule uvomorulin; the separated cells then invade collagen gels and embryonal heart tissue. Second, MDCK cells transformed with Harvey and Moloney sarcoma viruses are constitutively invasive, and they were found not to express uvomorulin at their cell surface. These data suggest that the loss of adhesive function of uvomorulin (which is identical to E-cadherin and homologous to L-CAM) is a critical step in the promotion of epithelial cells to a more malignant, i.e., invasive, phenotype. Similar modulation of intercellular adhesion might also occur during invasion of carcinoma cells in vivo.     

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.     

Assembly of the tight junction: the role of diacylglycerol

Extracellular Ca2+ triggers assembly and sealing of tight junctions (TJs) in MDCK cells. These events are modulated by G-proteins, phospholipase C, protein kinase C (PKC), and calmodulin. In the present work we observed that 1,2-dioctanoylglycerol (diC8) promotes the assembly of TJ in low extracellular Ca2+, as evidenced by translocation of the TJ-associated protein ZO-1 to the plasma membrane, formation of junctional fibrils observed in freeze-fracture replicas, decreased permeability of the intercellular space to [3H]mannitol, and reorganization of actin filaments to the cell periphery, visualized by fluorescence microscopy using rhodamine-phalloidin. In contrast, diC8 in low Ca2+ did not induce redistribution of the Ca-dependent adhesion protein E-cadherin (uvomorulin). Extracellular antibodies to E-cadherin block junction formation normally induced by adding Ca2+. diC8 counteracted this inhibition, suggesting that PKC may be in the signaling pathway activated by E-cadherin-mediated cell-cell adhesion. In addition, we found a novel phosphoprotein of 130 kD which coimmunoprecipitated with the ZO-1/ZO-2 complex. Although the assembly and sealing of TJs may involve the activation of PKC, the level of phosphorylation of ZO-1, ZO-2, and the 130-kD protein did not change after adding Ca2+ or a PKC agonist. The complex of these three proteins was present even in low extracellular Ca2+, suggesting that the addition of Ca2+ or diC8 triggers the translocation and assembly of preformed TJ subcomplexes.     

Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies

We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, connexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termii of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on approximately 50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation.     

Involvement of the Interaction of Afadin with ZO-1 in the Formation of Tight Junctions in Madin-Darby Canine Kidney Cells

Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with β- and α-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-ΔPR1–2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells.     

A WAVE2–Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens

The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2–Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2–Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2–Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin.     

Actomyosin tension is required for correct recruitment of adherens junction components and zonula occludens formation

The adherens junction (AJ) densely associated with actin filaments is a major cell-cell adhesion structure. To understand the importance of actin filament association in AJ formation, we first analyzed punctate AJs in NRK fibroblasts where one actin cable binds to one AJ structure unit. The accumulation of AJ components such as the cadherin/catenin complex and vinculin, as well as the formation of AJ-associated actin cables depended on Rho activity. Inhibitors for the Rho target, ROCK, which regulates myosin II activity, and for myosin II ATPase prevented the accumulation of AJ components, indicating that myosin II activity is more directly involved than Rho activity. Depletion of myosin II by RNAi showed similar results. The inhibition of myosin II activity in polarized epithelial MTD-1A cells affected the accumulation of vinculin to circumferential AJ (zonula adherens). Furthermore, correct zonula occludens (tight junction) formation along the apicobasal axis that requires cadherin activity was also impaired. Although MDCK cells which are often used as typical epithelial cells do not have a typical zonula adherens, punctate AJs formed dependently on myosin II activity by inducing wound closure in a MDCK cell sheet. These findings suggest that tension generated by actomyosin is essential for correct AJ assembly.     

Hepatocyte growth factor acutely perturbs actin filament anchorage at the epithelial zonula adherens

Cadherin adhesion molecules function in close cooperation with the actin cytoskeleton. At the zonula adherens (ZA) of polarized epithelial cells, E-cadherin adhesion induces the cortical recruitment of many key cytoskeletal regulators, which act in a dynamic integrated system to regulate junctional integrity and cell-cell interactions. This capacity for the cytoskeleton to support the ZA carries the implication that regulators of the junctional cytoskeleton might also be targeted to perturb junctional integrity. In this report, we now provide evidence for this hypothesis. We show that hepatocyte growth factor (HGF), which is well-known to disrupt cell-cell interactions, acutely perturbs ZA integrity much more rapidly than generally appreciated. This is accompanied by significant loss of junctional F-actin, a process that reflects loss of filament anchorage at the junctions. We demonstrate that this involves uncoupling of the unconventional motor myosin VI from junctional E-cadherin, a novel effect of HGF that is mediated by intracellular calcium. We conclude that regulators of the junctional cytoskeleton are likely to be major targets for cadherin junctions to be acutely modulated in development and perturbed in disease.     

Myelin-associated glycoprotein, a member of the L2/HNK-1 family of neural cell adhesion molecules, is involved in neuron-oligodendrocyte and oligodendrocyte-oligodendrocyte interaction

A monoclonal antibody to the myelin-associated glycoprotein (MAG) was prepared and characterized to probe for the involvement of MAG in cell surface interactions among neural cells in vitro. The antibody reacts specifically with oligodendrocyte cell surface and myelin-rich brain regions as expected from previous investigations. Not all O4 antigen- positive oligodendrocytes express MAG in vitro. Fab fragments of the antibody interfere with neuron to oligodendrocyte and oligodendrocyte to oligodendrocyte adhesion, but not with oligodendrocyte to astrocyte adhesion. MAG-containing liposomes bind to the cell surfaces of the appropriate target cells by a mechanism that is specifically inhibitable by Fab fragments of monoclonal MAG antibodies, demonstrating that MAG is a neural cell adhesion molecule.     

Role of the Crumbs complex in the regulation of junction formation in Drosophila and mammalian epithelial cells

The formation of a belt-like junctional complex separating the apical from the lateral domain is an essential step in the differentiation of epithelial cells. Thus protein complexes regulating this event are of first importance for the development of cell polarity and physiological functions of epithelial tissues. In Drosophila, the discovery of a gene, crb, controlling the coalescence of the spots of zonula adherens (ZA) into a adhesive ring around the cells was a major step. We know now that Crumbs, the product of crb is an apical transmembrane protein conserved in mammals and that it interacts by its cytoplasmic domain with two cortical modular proteins, Stardust (Sdt) and Discs lost (Dlt) that are also essential for the correct assembly of the ZA. These two proteins are also conserved in mammals and it is most likely that the Crumbs complex plays a similar role in very different species. Recently, we have shown that Crumbs interacts with the cortical cytoskeleton made of DMoesin and beta heavy-Spectrin and this connection could explain in part the role of Crumbs in building the ZA. Future work will help to understand several aspects of the Crumbs complex that are still unknown, like the role of the large extracellular domain or the precise function of Sdt and Dlt in the building of the ZA. Finding an answer to these questions will help to find new therapies for Retinitis pigmentosa and other retina degeneration in which CRB1, the human homologue of crb, has been involved.