shotgun
Vertebrate E-cadherin, adherens junction proteins, cell polarity and polarized membrane growth Polarization of cells during mouse preimplantation development first occurs within blastomeres at the
eight-cell stage, as part of a process called compaction. Cell-cell contact mediated by the cell adhesion
molecule uvomorulin (E-cadherin) and the activity of the microfilament cytoskeleton are important in
the development of compaction, which is crucial for establishment of trophoblast and pluriblast (inner
cell mass) lineages and for subsequent development. Members of the Rho family of p21 GTPases have
been shown to regulate the organization of the actin cytoskeleton and adhesion in other cell types. The
potential role of these proteins in compaction was investigated. Inhibition of Rho with Clostridium
botulinum C3-transferase disturbs intercellular flattening at compaction and prevents cytocortical
microfilament polarization of eight-cell blastomeres, in contrast to cytochalasin D, which inhibits only
adhesion. Microinjection of a constitutively activated recombinant Rho protein into four-cell
blastomeres induces cortical microfilament disruption and apical displacement of nuclei associated with
polarized clustering of microtubules. Interblastomere adhesion is reduced and E-cadherin is
aberrently clustered at remaining cell-cell contacts. Similarly, activated Cdc42 protein induces nuclear
displacement with additional cytoplasmic actin bundle formation between nucleus and cell-cell contacts.
The effects produced by both of the activated GTPase proteins are indicative of prematurely induced
but aberrently organized polarity. These results suggest that Rho family GTPases are involved in the
polarization of early mouse blastomeres (Clayton, 1999).
Beta-Catenin (Drosophila homolog: Armadillo) is involved in the formation of adherens junctions of mammalian epithelia. It interacts with the cell adhesion molecule E-cadherin and also with the tumor suppressor gene product APC, and Armadillo, the Drosophila homolog of beta-Catenin that mediates morphogenetic signals. E-Cadherin and APC directly compete for binding to the internal, Armadillo-like repeats of beta-Catenin; the NH2-terminal domain of beta-Catenin mediates the interaction of the alternative E-Cadherin and APC complexes to the cytoskeleton by binding to alpha-Catenin. Plakoglobin (gamma-Catenin), structurally related to beta-Catenin, mediates identical interactions. The APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions. This suggests that beta-Catenin and Plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression (Hulsken, 1994).
The classical adherens junction that holds epithelial cells together consists of a protein complex in which members of the cadherin family linked to various catenins are the principal components. delta-catenin is a mammalian brain protein in the Armadillo repeat superfamily with sequence similarity
to the adherens junction protein p120(ctn). Delta-catenin can be immunoprecipitated as a complex with other components of the adherens junction, including cadherin and beta-catenin, from
transfected cells and brain. The interaction with cadherin involves direct contact within the highly conserved juxtamembrane region of the COOH terminus, where p120(ctn) also binds. In developing
mouse brain, staining with delta-catenin antibodies is prominent toward the apical boundary of the neuroepithelial cells, in the ventricular zone. When transfected into Madin-Darby canine kidney
(MDCK) epithelial cells delta-catenin colocalizes with cadherin, p120(ctn), and beta-catenin. The Arm domain alone is sufficient for achieving localization and coimmunoprecipitation with cadherin. The ectopic expression of delta-catenin in MDCK cells alters their morphology, induces the elaboration of lamellipodia, interfers with monolayer formation, and increases scattering in response to hepatocyte growth factor treatment. It is proposed that delta-catenin can regulate adhesion molecules to implement the organization of large cellular arrays necessary for tissue morphogenesis (Lu, 1999).
Organization of proteins into structurally and functionally distinct plasma membrane domains is an essential characteristic of polarized epithelial cells. Based
on studies with cultured kidney cells, it has been hypothesized that a mechanism for restricting Na/K-ATPase to the basal-lateral membrane involves
E-cadherin-mediated cell-cell adhesion and integration of Na/K-ATPase into the Triton X-100-insoluble ankyrin- and spectrin-based membrane
cytoskeleton. In this study, the relevance of these in vitro observations to the generation of epithelial cell polarity in vivo was examined during mouse kidney
development. Using differential detergent extraction, immunoblotting, and immunofluorescence histochemistry, the following have been demonstrated: (1)
expression of the 220-kDa splice variant of ankyrin-3 correlates with the development of resistance to Triton X-100 extraction for Na/K-ATPase,
E-cadherin, and catenins and precedes maximal accumulation of Na/K-ATPase; (2) expression of the 190-kDa slice variant of ankyrin-3 correlates
with maximal accumulation of Na/K-ATPase; (3) Na/K-ATPase, ankyrin-3, and fodrin specifically colocalize at the basal-lateral plasma membrane of all
epithelial cells in which they are expressed and during all stages of nephrogenesis; (4) the relative immunofluorescence staining intensities of
Na/K-ATPase, ankyrin-3, and fodrin become more similar during development until they are essentially identical in adult kidney. Thus, renal epithelial cells
in vivo regulate the accumulation of E-cadherin-mediated adherens junctions, the membrane cytoskeleton, and Na/K-ATPase through sequential protein
expression and assembly on the basal-lateral membrane. These results are consistent with a mechanism in which generation and maintenance of polarized
distributions of these proteins in vivo and in vitro involve cell-cell adhesion, assembly of the membrane cytoskeleton complex, and concomitant integration
and retention of Na/K-ATPase in this complex (Piepenhagen, 1998).
Drosophila alpha-Catenin is homologous to vertebrate Vinculin and vertebrate beta-Catenin is homologous to human Plakoglobin and Drosophila Armadillo. Plakoglobin is a component of the Cadherin-Catenin complex. Plakoglobin binds directly to E-Cadherin. It seems that two distinct and separable E-Cadherin-Catenin complexes exist in the same cell. One complex is composed of E-Cadherin, alpha- and beta-Catenin, the other of E-Cadherin, alpha-Catenin and Plakoglobin. A similar distinct association with Catenins is also found for other Cadherins. Comparison of different cell lines reveals that the relative amounts of the two
complexes vary depending on cell types (Butz, 1994).
Fascin (the mammalian homolog of Drosophila Singed) binds to ß-catenin's Armadillo repeat domain (ß-catenin in the vertebrate of Drosophila Armadillo). In vitro competition and domain-mapping experiments demonstrate that Fascin and E-cadherin utilize a similar binding site within ß-catenin, such that they form mutually exclusive complexes with ß-catenin. Fascin and ß-catenin colocalize at cell-cell borders and dynamic cell leading edges of epithelial and endothelial cells. In addition to cell-cell borders, cadherins colocalize with Fascin and ß-catenin at cell leading edges. It is likely that ß-catenin participates in modulating cytoskeletal dynamics in association with Fascin, perhaps in a coordinate manner with its functions in Cadherin and APC (adenomatous polyposis coli) complexes. Whatever the biological role of the Fascin-ß-catenin complex, Fascin itself does not appear to be required for the function of all bundled filaments. For example, Singed mutants evidence a variety of apparently normal cell activities even in embryos harboring strong singed alleles, leaving open the possibility that other bundling proteins effectively assume the role of Fascin in various contexts (Tao, 1996).
Human LAR, the homolog of DLAR, a Drosophila transmembrane protein tyrosine phosphatase, associates with the cadherin-catenin complex. This association requires the amino-terminal domain of ß-catenin but does not require the armadillo repeats, which mediate association with cadherins. The association is not mediated by alpha-catenin or by cadherins. LAR-protein tyrosine phosphatases are phosphorylated on tyrosine in a TrkA-dependent manner, and their association with the cadherin-catenin complex is reduced in cells treated with NGF. It is proposed that changes in tyrosine phosphorylation of ß-catenin, mediated by TrkA and LAR-PTPs control cadherin adhesive function during processes such as neurite outgrowth (Kypta, 1996).
The small guanosine triphosphatases (GTPases) Cdc42 and Rac1 regulate E-cadherin-mediated cell-cell adhesion. IQGAP1, a
target of Cdc42 and Rac1, is localized with E-cadherin and beta-catenin at sites of cell-cell contact in mouse L fibroblasts
expressing E-cadherin (EL cells), and interacts with E-cadherin and beta-catenin both in vivo and in vitro. IQGAP1 induces
the dissociation of alpha-catenin from a cadherin-catenin complex in vitro and in vivo. Overexpression of IQGAP1 in EL cells,
but not in L cells expressing an E-cadherin-alpha-catenin chimeric protein, results in a decrease in E-cadherin-mediated
cell-cell adhesive activity. Thus, IQGAP1, acting downstream of Cdc42 and Rac1, appears to regulate cell-cell adhesion
through the cadherin-catenin pathway (Kuroda, 1998).
The E-cadherin-based adherens junction (AJ) is essential for organogenesis of epithelial tissues including the liver, although the regulatory
mechanism of AJ formation during development remains unknown. Using a primary culture system of fetal hepatocytes in which
oncostatin M (OSM) induces differentiation, it has been shown that OSM induces AJ formation by altering the subcellular localization of AJ
components, including E-cadherin and catenins. By retroviral expression of dominant-negative forms of signaling molecules, Ras was
shown to be required for the OSM-induced AJ formation. Fetal hepatocytes derived from K-Ras knockout (K-Ras-/-) mice fail to
form AJs in response to OSM, whereas AJ formation is induced normally by OSM in mutant hepatocytes lacking both H-Ras and
N-Ras. Moreover, the defective phenotype of K-Ras-/- hepatocytes is restored by expression of K-Ras, but not by H-Ras and N-Ras. Finally, pull-down assays
using the Ras-binding domain of Raf1 demonstrate that OSM directly activates K-Ras in fetal hepatocytes. These results indicate that K-Ras specifically mediates
cytokine signaling for formation of AJs during liver development (Matsui, 2002).
While these results indicate that among the three Ras proteins, K-Ras specifically mediates OSM signaling to induce the formation of E-cadherin-based adhesion, the
molecular basis for the specificity of K-Ras currently is unknown. A structural difference in the C-terminal short stretches may provide a hint. H-Ras and N-Ras have
homologous C-terminal stretches, by which both are palmitoylated. This modification enables them to be recruited to a particular
subdomain of the plasma membrane, called the caveola. In contrast, K-Ras is not palmitoylated and is anchored to
the membrane through the basic domain near the C-terminus. There is no evidence so far that K-Ras is concentrated in a certain subdomain
of the plasma membrane. Based on this difference, it is tempting to speculate that K-Ras stimulates a distinct array of effector molecules and thereby elicits cellular
responses unique to K-Ras. It is thus possible that OSM induces the localization of E-cadherin through K-Ras by activating unique effector proteins that are not
activated by H- or N-Ras (Matsui, 2002).
Sec6/8 (exocyst) complex (see Drosophila Sec5) regulates vesicle delivery and polarized membrane growth in a variety of cells, but mechanisms regulating Sec6/8 localization are unknown. In epithelial cells, Sec6/8 complex is recruited to cell-cell contacts with a mixture of junctional proteins, but then sorts out to the apex of the lateral membrane with components of tight junction and nectin complexes. Sec6/8 complex fractionates in a high molecular mass complex with tight junction proteins and a portion of E-cadherin, and co-immunoprecipitates with cell surface-labeled E-cadherin and nectin-2alpha. Recruitment of Sec6/8 complex to cell-cell contacts can be achieved in fibroblasts when E-cadherin and nectin-2alpha are co-expressed. These results support a model in which localized recruitment of Sec6/8 complex to the plasma membrane by specific cell-cell adhesion complexes defines a site for vesicle delivery and polarized membrane growth during development of epithelial cell polarity (Yeaman, 2004).
Phosphatidylinositol 3-kinase (PI3K) is recruited to and activated by E-cadherin engagement. This PI3K activation is essential for adherens junction integrity and intestinal epithelial cell differentiation. Evidence is provided that hDlg, the homolog of disc-large tumor suppressor, is another key regulator of adherens junction integrity and differentiation in mammalian epithelial cells. This study reports the following: (1) hDlg co-localizes with E-cadherin, but not with ZO-1, at the sites of cell-cell contact in intestinal epithelial cells; (2) reduction of hDlg expression levels by RNAi in intestinal cells not only severely alters adherens junction integrity but also prevents the recruitment of p85/PI3K to E-cadherin-mediated cell-cell contact and inhibits sucrase-isomaltase gene expression; (3) PI3K and hDlg are associated with E-cadherin in a common macromolecular complex in living differentiating intestinal cells; (4) this interaction requires the association of hDlg with E-cadherin and with Src homology domain 2 domains of the p85/PI3K subunit; (5) phosphorylation of hDlg on serine and threonine residues prevents its interaction with the p85 Src homology domain 2 in subconfluent cells, whereas phosphorylation of hDlg on tyrosine residues is essential. It is concluded that hDlg may be a determinant in E-cadherin-mediated adhesion and signaling in mammalian epithelial cells (Laprise, 2004).
Cadherin adhesion molecules are key determinants of morphogenesis and tissue
architecture. Nevertheless, the molecular mechanisms responsible for the
morphogenetic contributions of cadherins remain poorly understood in vivo.
Besides supporting cell-cell adhesion, cadherins can affect a wide range of
cellular functions that include activation of cell signalling pathways,
regulation of the cytoskeleton and control of cell polarity. To determine the
role of E-cadherin in stratified epithelium of the epidermis, its gene was conditionally inactivated in
mice. Loss of E-cadherin
in the epidermis in vivo results in perinatal death of mice due to the inability
to retain a functional epidermal water barrier. Absence of E-cadherin leads to
improper localization of key tight junctional proteins, resulting in permeable
tight junctions and thus altered epidermal resistance. In addition, both Rac and
activated atypical PKC, crucial for tight junction formation, are mislocalized.
Surprisingly, the results indicate that E-cadherin is specifically required for
tight junction (but not desmosome) formation and this appears to involve
signalling rather than cell contact formation (Tunggal, 2005).
Scribble (Scrib) is a conserved polarity protein required in Drosophila for synaptic function, neuroblast differentiation, and epithelial polarization. It is also a tumor suppressor. In rodents, Scrib has been implicated in receptor recycling and planar polarity but not in apical/basal polarity. Knockdown of Scrib disrupts adhesion between Madin-Darby canine kidney epithelial cells. As a consequence, the cells acquire a mesenchymal appearance, migrate more rapidly, and lose directionality. Although tight junction assembly is delayed, confluent monolayers remain polarized. These effects are independent of Rac activation or Scrib binding to ßPIX. Rather, Scrib depletion disrupts E-cadherin-mediated cell-cell adhesion. The changes in morphology and migration are phenocopied by E-cadherin knockdown. Adhesion is partially rescued by expression of an E-cadherin-alpha-catenin fusion protein but not by E-cadherin-green fluorescent protein. These results suggest that Scrib stabilizes the coupling between E-cadherin and the catenins and are consistent with the idea that mammalian Scrib could behave as a tumor suppressor by regulating epithelial cell adhesion and migration (Qin, 2005).
Desmosomes are cadherin-based adhesive intercellular junctions, which are present in tissues such as heart and skin. Despite considerable efforts, the molecular interfaces that mediate adhesion remain obscure. This study applied cryo-electron tomography of vitreous sections from human epidermis to visualize the three-dimensional molecular architecture of desmosomal cadherins at close-to-native conditions. The three-dimensional reconstructions show a regular array of densities at ~70 Å intervals along the midline, with a curved shape resembling the X-ray structure of C-cadherin, a representative 'classical' cadherin. Model-independent three-dimensional image processing of extracted sub-tomograms reveals the cadherin organization. After fitting the C-cadherin atomic structure into the averaged sub-tomograms, a periodic arrangement is seen of a trans W-like and a cis V-like interaction corresponding to molecules from opposing membranes and the same cell membrane, respectively. The resulting model of cadherin organization explains existing two-dimensional data and yields insights into a possible mechanism of cadherin-based cell adhesion (Al-Amoudi, 2007).
In contrast to previous tomographic studies with conventional preparation techniques, in which a stochastic arrangement and interaction of cadherins was found, in this study the cadherins are quasi-periodically arranged and adopt a specific organization with alternating trans and cis interactions. The differences from the previous tomographic study can be explained by the dehydration procedure, which is shown to cause aggregation. In the current study, the molecules interact by their tips in a zipper-like arrangement similar to the N-cadherin crystal, but in a different manner from the one interpreted when the crystal structure was solved. Despite the quasi-periodicity of the cadherin arrangement, the cadherins retain a significant flexibility without losing their alternating interaction pattern (Al-Amoudi, 2007).
Even though the images are static, these results support the hypothesis that desmosomal cadherins on the cell surface are first clustered into small groups interacting through specific residues in the EC1 domains to form cis homodimers. The opposing cell membranes are then brought in close proximity to enable the formation of the trans homodimers, which relies on Trp 2 and the hydrophobic pocket together with residues involved in molecular specificity. Once the initial recognition is established, more molecules are brought to the contact zone, thus compacting the junction. This compaction process is regularized by building blocks of alternate cis and trans dimers so that the strength of cell-cell contact is homogeneous. These processes are repeated to extend the junction and finally form the fully mature desmosome (Al-Amoudi, 2007).
This study asked if the mouse homolog of Drosophila Scribbled is required for establishment and/or maintenance of epithelial identity in vivo. To do so, Scrib was conditionally deleted in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting epithelial-mesenchymal transition (EMT) was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of alphaSMA, Smad3 and Smad4, TGFβ signaling intermediates, accumulated in the nucleus and Snail, a TGFβ target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFβ signaling (Yamben, 2013).
Snail and Zeb (see Drosophila Snail and Zinc finger homeodomain 1) transcription factors induce epithelial to mesenchymal transition (EMT) in embryonic and adult tissues by direct repression of E-Cadherin (see Drosophila Shotgun) transcription. The repression of E-Cadherin transcription by the EMT inducers Snail1 and Zeb2 plays a fundamental role in defining embryonic territories in the mouse, as E-Cadherin needs to be downregulated in the primitive streak and in the epiblast concomitant with the formation of mesendodermal precursors and the neural plate, respectively. This study shows that in the chick embryo, E-Cadherin is weakly expressed in the epiblast at pre-primitive streak stages where it is substituted by P-Cadherin. Snail2 and Zeb2 were shown to repress P-Cadherin transcription in the primitive streak and the neural plate, respectively. This indicates that E- and P-Cadherin expression patterns evolved differently between chick and mouse. As such, the Snail1/E-Cadherin axis described in the early mouse embryo corresponds to Snail2/P-Cadherin in the chick, but both Snail factors and Zeb2 fulfill a similar role in chick and mouse in directly repressing ectodermal Cadherins to promote the delamination of mesendodermal precursors at gastrulation and the proper specification of the neural ectoderm during neural induction (Acloque, 2017).
The integrity and homeostasis of the vertebrate epidermis depend on various cellular junctions. How these junctions are assembled during development and how their number is regulated remain largely unclear. This study addressed these issues by analysing the function of Lgl2, E-cadherin and atypical Protein kinase C (aPKC) in the formation of hemidesmosomes in the developing basal epidermis of zebrafish larvae. It has been shown that a mutation in lgl2 (penner) prevents the formation of hemidesmosomes. This study shows that Lgl2 function is essential for mediating the targeting of Integrin alpha 6 (Itga6), a hemidesmosomal component, to the plasma membrane of basal epidermal cells. In addition, it was shown that whereas aPKClambda seems dispensable for the localisation of Itga6 during hemidesmosome formation, knockdown of E-cadherin function leads to an Lgl2-dependent increase in the localisation of Itga6. Thus, Lgl2 and E-cadherin act antagonistically to control the localisation of Itga6 during the formation of hemidesmosomes in the developing epidermis (Sonawane, 2009).
How do Lgl2 and E-cadherin, localised at the lateral domain, regulate the
formation of hemidesmosomes formed at the basal domain in epidermal cells? It was shown at the lateral domain, Itga6 localises with Lgl2
as well as with E-cadherin. This observation indicates that after its
synthesis, a fraction of Itga6 is first targeted to the lateral domain. This
lateral Itga6 fraction diminishes by 5 days of development, indicating that Itga6 localisation at the lateral domain is dynamic. In early lgl2 mutant larvae (3.75 days), there is a selective loss of Itga6 localisation at the lateral membrane domain. Moreover, in lgl2 mutant larvae, Itga6 vesicles accumulate in the cytoplasm, especially near the lateral and apical domains. Thus, it is plausible that beyond 3.5 days, a fraction of the Itga6 synthesised is targeted to the lateral membrane domain first and that Lgl2 mediates this targeting. This fraction at the lateral domain then translocates to the basal domain, where it joins the existing Itga6 fraction (localised prior to 3.5 days)
clustered at the intermediate filaments, and becomes assembled into functional
hemidesmosomes. The translocation of the lateral Itga6 fraction to the basal domain may occur by passive diffusion or by transcytosis. In the latter case, a likely mechanism might be Rab21/Rab5-mediated endocytosis and trafficking of Itga6 from the lateral domain and Rab11-mediated delivery to the basal domain
through recycling endosomes. Since, in lgl2 mutant larvae, the Itga6 fraction targeted beyond 3.5 dpf fails to reach the lateral membrane domain and thus also the basal domain, the existing levels of Itga6 at the basal domain (localised before 3.5 dpf) remain insufficient to form functional hemidesmosomes (Sonawane, 2009).
Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that α-catenin links the adhesion protein E-cadherin through ß-catenin to the actin cytoskeleton, it has been shown that α-catenin does not bind simultaneously to both E-cadherin-ß-catenin and actin filaments (Yamada, 2005). This study demonstrates that α-catenin exists as a monomer or a homodimer with different binding properties. Monomeric α-catenin binds more strongly to E-cadherin-ß-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that α-catenin is an allosteric protein. Significantly, α-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for α-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion (Drees, 2005).
Epithelial cell-cell junctions are organized by adhesion proteins and the underlying actin cytoskeleton. They provide adaptable interfaces that can respond to signals for cell movement during convergent extension in gastrulation or changes in cell shape during tube formation but also provide a constant permeability barrier between different biological compartments in the body. Analysis of how migrating cells initiate cell-cell adhesion has revealed dramatic changes in membrane dynamics and organization of the actin cytoskeleton. Migrating cells have characteristic forward-moving lamellipodia produced by rapid Arp2/3-nucleated assembly of a branched actin network perpendicular to the leading edge. Upon cell-cell adhesion, lamellipodial activity is reduced over the contacting area, and there is a concomitant reorganization of actin filaments; electron microscopy of simple epithelial cells indicates the formation of bundles of actin filaments parallel to the contacting membranes, an organization very different from that of branched actin in lamellipodia. It is unknown how engagement of cadherins between migrating cells causes these dramatic changes in actin-filament assembly and organization. The intracellular domain of cadherins binds cytoplasmic proteins that are thought to recruit and organize actin filaments. These molecular linkages include high-affinity binding of ß-catenin to the cadherin cytoplasmic domain and a lower-affinity interaction between ß-catenin and α-catenin (Pokutta, 2000). Since α-catenin can also bind actin filaments in vitro (Pokutta, 2002; Rimm, 1995), it is widely accepted that α-catenin bound to the cadherin-ß-catenin complex bridges these components to actin. In addition, many actin binding proteins have been reported to bind α-catenin, including vinculin and α-actinin, suggesting that they could also link the cadherin-catenin complex to the actin cytoskeleton. Furthermore, the Rho family of small GTPases and the actin nucleators formin and the Arp2/3 complex are involved in cell-cell adhesion and may regulate actin dynamics around the cadherin-catenin complex (Drees, 2005 and references therein).
In an accompanying paper (Yamada, 2005), experiments with purified proteins demonstrated that the ternary complex of cadherin-ß-catenin-αcatenin does not bind directly to actin filaments or indirectly through vinculin or α-actinin. Moreover, live-cell imaging showed that the cadherin-catenin complex has dynamics that are very different from those of actin, consistent with the lack of a stable linkage between the cadherin-catenin complex and actin in cells (Drees, 2005).
The inability of α-catenin to bind simultaneously to the cadherin-ß-catenin complex and actin indicates that it may function as a molecular switch, whereby binding to one partner changes the ability to interact with the other. Evidence is provided in this study that these changes are associated with distinct conformational states of α-catenin and that dimerization of α-catenin influences its ability to selectively bind to ß-catenin or actin. Given the highly dynamic properties of the actin network at cell-cell contacts, the role of α-catenin in regulating actin assembly was further examined. α-catenin is shown to suppress actin polymerization by the Arp2/3 complex, suggesting that assembly and clustering of the cadherin-catenin complex at cell-cell contacts may provide a pool of α-catenin that can locally regulate actin dynamics and organization (Drees, 2005).
Cell-cell contacts are considered to be stable structures that maintain the structural integrity of tissues and are thought to be formed by clustering of cell-adhesion proteins through binding between opposed extracellular domains and linkage through the cytoplasmic domain to the underlying actin cytoskeleton. The cadherin cytoplasmic domain binds with high affinity to ß-catenin, which in turn binds with weaker affinity to α-catenin (Pokutta, 2000). Given that α-catenin binds to actin filaments (Pokutta, 2002; Rimm, 1995) and to other actin binding proteins such as vinculin and α-actinin (Hazan, 1997; Knudsen, 1995; Watabe-Uchida, 1998; Weiss, 1998), it was reasonable to assume that α-catenin bound to the cadherin-ß-catenin complex also binds directly or indirectly to actin filaments. However, direct tests of this model failed. In the accompanying paper, it is shown that α-catenin does not bind simultaneously to the cadherin-catenin complex and actin filaments (Yamada, 2005). These results predict that interactions between the cadherin-catenin complex and underlying actin cytoskeleton in cells might be very dynamic rather than being static as has been assumed. Although direct interactions between the cadherin complex and actin filaments were not verified experimentally, there is a considerable body of work concluding that some sort of interaction of actin filaments and the cadherin-catenin complex is important in cell-cell adhesion. Cytochalasin D and latrunculin A, which change the dynamic organization of the actin cytoskeleton, reduce adhesion and weaken cell-cell contacts. However, these drugs have global effects on actin organization that are not restricted to effects on only cell-cell contacts. Genetic deletion of α-catenin potentially provides a more direct approach to disrupt the putative cadherin-actin linkage. Cell-cell adhesion in α-catenin null cells is reduced and can be rescued by re-expression of α-catenin. However, it is noted that cell-cell adhesion occurs in some α-catenin null cells, presumably because there is sufficient cadherin on the cell surface to initiate cell-cell adhesion. αcatenin null cells have also been used to express chimeras between α-catenin-E-cadherin, α-catenin-vinculin and α-catenin-formin-1, all of which partially rescue cell-cell adhesion. However, no direct evidence is presented in those studies that these chimeric proteins bind actin filaments. Moreover, the current findings that the molecular and functional properties of α-catenin are altered upon binding to ß-catenin - in particular, that the αcatenin/ ß-catenin complex cannot bind actin - demonstrate that the use of E-cadherin-α-catenin chimeras cannot recapitulate the behavior of the cadherin-catenin complex at the membrane. It is possible that expression of some of these chimeric proteins could locally change actin dynamics or simply increase the amount of cadherin at the cell surface to a level that can partially rescue cell-cell adhesion (Drees, 2005).
Although it is surprising that a stable linkage does not exist between cadherins and the underlying actin cytoskeleton, cell-cell adhesion is a dynamic process during embryonic development, wound healing and cancer cell metastasis. This may require a more dynamic interaction between cadherin and the actin cytoskeleton rather than the static, stable linkage proposed in previous models. In addition, it is noteworthy that, in most cell types, cadherins are not the only means of cell-cell adhesion. Many other adhesion proteins are expressed, including members of the nectin occludin/claudin, JAM, and desmosomal cadherin families, all of which are thought to interact directly or indirectly with the actin or intermediate filament cytoskeletons and thereby contribute to cell-cell adhesion (Drees, 2005).
There are dramatic changes in membrane and actin dynamics associated with the formation of cell-cell adhesions. Initial cell-cell contact formation is driven by overlapping membrane lamellipodia from contacting cells. These lamellipodia are regulated by actin polymerization and branching induced by the Arp2/3 complex and local activation of the Rho family of small GTPases. However, lamellipodial activity decays as the cadherin-catenin complex accumulates and the contacts mature into stable cell-cell junctions. It is not known what regulates this contact-dependent decrease of membrane activity, which presumably depends upon a decrease in Arp2/3-mediated actin polymerization. It is interesting to note, in this context, that decreased levels of α-catenin result in increased membrane activity in hippocampal neurons, while overexpression of α-catenin suppress membrane activity, indicating that α-catenin directly regulates membrane protrusive activity. Furthermore, keratinocytes from α-catenin knockout mice are characterized by loss of contact inhibition and increased migratory activity (Drees, 2005).
How might changes in both actin assembly and organization (from networks to bundles) that drive the transition from active lamellipodia to quiescent contacts be coordinated during the formation of cell-cell adhesions? The finding that α-catenin suppresses Arp2/3-mediates actin polymerization in a concentration-dependent manner, combined with the actin bundling activity of α-catenin (Rimm, 1995), may provide an explanation. The cytoplasmic α-catenin monomer concentration (0.6 mM) is too low to bind actin significantly and would need to be concentrated to bind actin and to dimerize. A significant increase in the local concentration of α-catenin at the membrane occurs upon accumulation of the cadherin-catenin complex at nascent contacts. This cadherin bound pool of α-catenin can exchange with the cytoplasmic pool; note that the amount of exchange was probably underestimated because it cannot be directly measure d locally at cell-cell contacts. Although the local concentrations of α-catenin and Arp2/3 immediately adjacent to contacting membranes are unknown, a 10-fold increase in local concentration of α-catenin would be sufficient for α-catenin to compete with the Arp2/3 complex for actin filaments. This would suppress formation of branched actin networks and inhibit lamellipodial activity and would also favor formation of α-catenin homodimers that bundle actin filaments (Rimm, 1995), resulting in a reorganization of actin filaments and a change in membrane dynamics underneath the junction. It has also been proposed that formins, which promote formation of linear actin cables, are recruited to the adherens junction by α-catenin. If so, α-catenin would serve as a switch that turns off Arp2/3-mediated branched-actin-network formation required for lamellipodial activity during the initiation of adhesion and turn on αcatenin- mediated bundling of actin filaments and formation of linear cables by formins during maturation of the adherens junction. While further work is needed to test specific tenets of this hypothesis, the results provide new mechanistic insights into many aspects of the local dynamics of actin and membranes associated with cell-cell contacts not accounted for in previous models (Drees, 2005).
Transmembrane cadherins are calcium-dependent intercellular adhesion molecules. Recently, they have also been shown to be sites of actin assembly during adhesive contact formation. However, the roles of actin assembly on transmembrane cadherins during development are not fully understood. This study shows, using the developing ectoderm of the Xenopus embryo as a model, that F-actin assembly is a primary function of both N-cadherin in the neural ectoderm and E-cadherin in the non-neural (epidermal) ectoderm, and that each cadherin is essential for the characteristic morphogenetic movements of these two tissues. However, depletion of N-cadherin and E-cadherin did not cause dissociation in these tissues at the neurula stage, probably owing to the expression of C-cadherin in each tissue. Depletion of each of these cadherins is not rescued by the other, nor by the expression of C-cadherin, which is expressed in both tissues. One possible reason for this is that each cadherin is expressed in a different domain of the cell membrane. These data indicate the combinatorial nature of cadherin function, the fact that N- and E-cadherin play primary roles in F-actin assembly in addition to roles in cell adhesion, and that this function is specific to individual cadherins. They also show how cell adhesion and motility can be combined in morphogenetic tissue movements that generate the form and shape of the embryonic organs (Nandadasa, 2009).
This study demonstrates that planar cell polarity signaling regulates morphogenesis in Xenopus embryos in part through the assembly of the fibronectin (FN) matrix. A regulatory pathway is outlined that includes cadherin adhesion and signaling through Rac and Pak, culminating in actin reorganization, myosin contractility, and tissue tension, which, in turn, directs the correct spatiotemporal localization of FN into a fibrillar matrix. Increased mechanical tension promotes FN fibril assembly in the blastocoel roof (BCR), while reduced BCR tension inhibits matrix assembly. These data support a model for matrix assembly in tissues where cell-cell adhesions play an analogous role to the focal adhesions of cultured cells by transferring to integrins the tension required to direct FN fibril formation at cell surfaces (Dzamba, 2009).
Both PCP signaling and FN are required for gastrulation movements in Xenopus. The current study shows that normal assembly of FN matrix is inhibited following expression of dnWnt11. It is proposed that PCP signaling acts upstream to regulate FN fibrillogenesis by increasing cadherin adhesive activity and tension in BCR cells. Thus, one function of the PCP pathway in these embryos is to regulate FN matrix assembly in the marginal zone of the BCR. In both cultured mammalian cells and in the embryo, FN is first observed as diffuse punctae across cell surfaces. With time, both in cultured cells and on the BCR, fine fibrils are found initially at cell-cell junctions. These newly assembled FN fibrils are soluble in 2% DOC, but with time become detergent insoluble. The fibrils identified morphologically at gastrulation are DOC soluble, but, by neurula stages, they display DOC insolubility, suggesting that this progression is, in fact, similar to the progression of FN assembly and DOC solubility reported for cultured cells. Moreover, FN fibrils are required for radial intercalation and epiboly in the BCR, and nonfibrillar FN promotes high-speed migration of mesendodermal cells. Therefore, while early embryonic fibrillar and non-fibrillar FNs are indistinguishable in terms of DOC solubility, differences in biological functions supported by these two physical states of FN are evident in vivo (Dzamba, 2009).
The small GTPase, Rac, is a critical component of the pathway through which cadherins contribute to tissue tension. Both cadherin ligation and Wnt/PCP signaling can promote the activation of the small GTPases, Rac and Rho. While Rho has been shown to promote FN fibril assembly in cultured cells by promoting contractility through the phosphorylation of MLC, in the current system, Rac is the critical GTPase for FN assembly. Tension is generated via regulation of the actin cytoskeleton and MLC phosphorylation by Rac and its downstream effector, Pak. Inhibiton of either Rac or Pak abrogated cortical actin assembly in BCR cells. Activated Pak colocalized with FN fibrils. When Pak was inhibited, the phosphorylation of MLC at cell-cell junctions was reduced. Taken together, these data indicate that Pak is the key downstream effector of Rac in this system regulating cell tension and FN assembly (Dzamba, 2009).
Endocytosis of E-cadherin The establishment of cadherin-dependent cell-cell contacts in human epidermal keratinocytes are known to be regulated by the
Rac1 small GTP-binding protein, although the mechanisms by which Rac1 participates in the assembly or disruption of cell-cell
adhesion are not well understood. Green fluorescent protein (GFP)-tagged Rac1 expression vectors were used to examine the subcellular distribution of Rac1 and its effects on E-cadherin-mediated cell-cell adhesion. Microinjection of
keratinocytes with constitutively active Rac1 results in cell spreading and disruption of cell-cell contacts. The ability of active Rac1 to
disrupt cell-cell adhesion is dependent on colony size, with large established colonies being resistant to the effects of active Rac1. Disruption of cell-cell contacts in small preconfluent colonies is achieved through the selective recruitment of E-cadherin-catenin complexes to the perimeter of multiple large intracellular vesicles, which are bounded by GFP-tagged constitutively active Rac1. Similar vesicles were observed in noninjected keratinocytes when cell-cell adhesion is disrupted by removal of extracellular calcium or with the use of an E-cadherin blocking antibody. Moreover, formation of these structures in noninjected keratinocytes is dependent on endogenous Rac1 activity. Expression of GFP-tagged effector mutants of Rac1 in keratinocytes demonstrates that reorganization of the actin cytoskeleton is important for vesicle formation. Characterization of these Rac1-induced vesicles revealsthat they are endosomal in nature and tightly colocalized with the transferrin receptor, a marker for recycling endosomes. Expression of GFP-L61Rac1 inhibits uptake of transferrin-biotin, suggesting that the endocytosis of E-cadherin is a clathrin-independent mechanism. This is supported by the observation that caveolin, but not clathrin, localizes around these structures. Furthermore, an inhibitory form of dynamin, known to inhibit internalization of caveolae, inhibits formation of cadherin vesicles. These data suggest that Rac1 regulates adherens junctions via clathrin independent endocytosis of E-cadherin (Akhtar, 2001).
Regulation of E-cadherin transcription E-cadherin plays a pivotal role in the biogenesis of the first epithelium during development, and its down-regulation is associated with metastasis of carcinomas. Inactivation of RB family proteins by simian virus 40 large T antigen (LT) in MDCK epithelial cells results in a mesenchymal conversion associated with invasiveness and a down-regulation of c-Myc. Reexpression of RB or c-Myc in such cells allows the reexpression of epithelial markers, including E-cadherin. Both RB and c-Myc specifically activate transcription of the E-cadherin promoter in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated in both cases by the transcription factor AP-2. In vitro AP-2 and RB interaction involves the N-terminal domain of AP-2 and the oncoprotein binding domain and
C-terminal domain of RB. In vivo physical interaction between RB and AP-2 has been demonstrated in MDCK and HaCat cells. In LT-transformed MDCK cells, LT, RB, and AP-2 were all coimmunoprecipitated by each of the corresponding antibodies, and a mutation of the RB binding domain of the oncoprotein inhibits its binding to both RB and AP-2. Taken together, these results suggest that there is a tripartite complex between LT, RB, and AP-2 and that the physical and functional interactions between LT and AP-2 are mediated by RB. Moreover, they define RB and c-Myc as coactivators of AP-2 in epithelial cells and shed new light on the significance of the LT-RB complex, linking it to the dedifferentiation processes occurring during tumor progression. These data confirm the important role for RB and c-Myc in the maintenance of the epithelial phenotype and reveal a novel mechanism of gene activation by c-Myc (Batsche, 1998).
Transcriptional downregulation of E-cadherin appears to be an important event in the progression of various epithelial tumors. SIP1 (ZEB-2) is a Smad-interacting, multi-zinc finger protein that shows specific DNA binding activity. Expression of wild-type but not of mutated SIP1 downregulates mammalian E-cadherin transcription via binding to both conserved E2 boxes of the minimal E-cadherin promoter. SIP1 and Snail bind to partly overlapping promoter sequences and show similar silencing effects. SIP1 can be induced by TGF-beta treatment and shows high expression in several E-cadherin-negative human carcinoma cell lines. Conditional expression of SIP1 in E-cadherin-positive MDCK cells abrogates E-cadherin-mediated intercellular adhesion and simultaneously induces invasion. SIP1 therefore appears to be a promoter of invasion in malignant epithelial tumors (Comijn, 2001).
deltaEF1 and SIP1 (or Zfhx1a and Zfhx1b, respectively) are the only
known members of the vertebrate Zfh1 family of homeodomain/zinc
finger-containing proteins. Similar to other transcription factors, both
Smad-interacting protein-1 (SIP1) and deltaEF1 are capable of repressing
E-cadherin transcription through binding to the E2 boxes located in its
promoter. In the case of deltaEF1, this repression has been proposed to
occur via interaction with the corepressor C-terminal binding protein
(CtBP). In this study, it is shown by coimmunoprecipitation that SIP1
and CtBP interact in vivo and that an isolated CtBP-binding SIP1
fragment depends on CtBP for transcriptional repression. However, and
most importantly, full-length SIP1 and deltaEF1 proteins do not depend
on their interaction with CtBP to repress transcription from the
E-cadherin promoter. Furthermore, in E-cadherin-positive kidney
epithelial cells, the conditional synthesis of mutant SIP1 that cannot
bind to CtBP, abrogates endogenous E-cadherin expression in a similar way
as wild-type SIP1. These results indicate that full-length SIP1 can
repress E-cadherin in a CtBP-independent manner (van Grunsven, 2003).
Cadherin-mediated cell-cell adhesion plays important roles in mouse embryonic development, and changes in cadherin expression are often linked to morphogenetic events. For proper embryonic development and organ formation, the expression of E-cadherin must be tightly regulated. Dysregulated expression during tumorigenesis confers invasiveness and metastasis. Except for the E-box motifs in the E-cadherin promoter, little is known about the existence and location of cis-regulatory elements controlling E-cadherin gene expression. Examination of putative cis-regulatory elements in the murine E-cadherin gene shows a pivotal role for intron 2 in activating transcription. Upon deleting the genomic intron 2 entirely, the E-cadherin locus becomes completely inactive in embryonic stem cells and during early embryonic development. Later in development, from E11.5 onwards, the locus is activated only weakly in the absence of intron 2 sequences. In differentiated epithelia, intron 2 sequences are required both to initiate transcriptional activation and additionally to maintain E-cadherin expression. Detailed analysis also revealed that expression in the yolk sac is intron 2 independent, whereas expression in the lens and the salivary glands absolutely relies on cis-regulatory sequences of intron 2. Taken together, these findings reveal a complex mechanism of gene regulation, with a vital role for the large intron 2 (Stemmler, 2005).
The basic helix-loop-helix TAL-1/SCL essential for hematopoietic development is also required during vascular development for embryonic angiogenesis. TAL-1 acts positively on postnatal angiogenesis by stimulating endothelial morphogenesis. This study investigated the functional consequences of TAL-1 silencing in human primary endothelial cells. It was found that TAL-1 knockdown caused the inhibition of in vitro tubulomorphogenesis, which was associated with a dramatic reduction in vascular endothelial cadherin (VE-cadherin) at intercellular junctions. Consistently, silencing of TAL-1 as well as of its cofactors E47 and LMO2 down-regulated VE-cadherin at both the mRNA and the protein level. Endogenous VE-cadherin transcription could be activated in nonendothelial HEK-293 cells by the sole concomitant ectopic expression of TAL-1, E47, and LMO2. Transient transfections in human primary endothelial cells derived from umbilical vein (HUVECs) demonstrated that VE-cadherin promoter activity was dependent on the integrity of a specialized E-box associated with a GATA motif and was maximal with the coexpression of the different components of the TAL-1 complex. Finally, chromatin immunoprecipitation assays showed that TAL-1 and its cofactors occupied the VE-cadherin promoter in HUVECs. Together, these data identify VE-cadherin as a bona fide target gene of the TAL-1 complex in the endothelial lineage, providing a first clue to TAL-1 function in angiogenesis (Deleuze, 2007).
The class V POU domain transcription factor Oct4 (Pou5f1) is a pivotal regulator of embryonic stem cell (ESC) self-renewal and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. Oct4 is also an important evolutionarily conserved regulator of progenitor cell differentiation during embryonic development. This study examined the function of Oct4 homologs in Xenopus embryos and compared this to the role of Oct4 in maintaining mammalian embryo-derived stem cells. Based on a combination of expression profiling of Oct4/POUV-depleted Xenopus embryos and in silico analysis of existing mammalian Oct4 target data sets, a set of evolutionary-conserved Oct4/POUV targets was defined. Most of these targets, were regulators of cell adhesion. This is consistent with Oct4/POUV phenotypes observed in the adherens junctions in Xenopus ectoderm, mouse embryonic, and epiblast stem cells. A number of these targets, including E-cadherin, could rescue both Oct4/POUV phenotypes in cellular adhesion and multipotent progenitor cell maintenance, whereas expression of cadherins on their own could only transiently support adhesion and block differentiation in both ESC and Xenopus embryos. Currently, the list of Oct4 transcriptional targets contains thousands of genes. Using evolutionary conservation, a core set of functionally relevant factors were identified that linked the maintenance of adhesion to Oct4/POUV. It was found that the regulation of adhesion by the Oct4/POUV network occurred at both transcriptional and posttranslational levels and was required for pluripotency (Livigni, 2013).
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