Abstract. Morphological and biochemical analyseshave identified a set of proteins which together form a structure known as the adherens junction. Elegant experiments in tissue culture support the idea that adherens junctions play a key role in cell--cell adhesion and in organizing cells into epithelia. During normal embryonic development, cells quickly organize epithelia; these epithelial cells participate in many of the key morphogenetic movements of gastrulation. This prompted the hypothesis that adherens junctions ought to be critical for normal embryonic development.Drosophila Armadillo, the homologue of vertebrate 13-catenin, is a core component of the adherens junction protein complex and has been hypothesized to be essential for adherens junction function in vivo. We have used an intermediate mutant allele of armadillo, armadillo xP33, to test these hypotheses in Drosophila embryos. Adherens junctions cannot assemble in the absence of Armadillo, leading to dramatic defects in cell-cell adhesion. The epithelial ceils of the embryo lose adhesion to each other, round up, and apparently become mesenchymal. Mutant cells also lose their normal cell polarity. These disruptions in the integrity of epithelia block the appropriate morphogenetic movements of gastrulation. These results provide the first demonstration of the effect of loss of adherens junctions on Drosophila embryonic development.

The tumor suppressor adenomatous polyposis coli (APC) negatively regulates Wingless (Wg)/Wnt signal transduction by helping target the Wnt effector β-catenin or its Drosophila homologue Armadillo (Arm) for destruction. In cultured mammalian cells, APC localizes to the cell cortex near the ends of microtubules. Drosophila APC (dAPC) negatively regulates Arm signaling, but only in a limited set of tissues. We describe a second fly APC, dAPC2, which binds Arm and is expressed in a broad spectrum of tissues. dAPC2's subcellular localization revealed colocalization with actin in many but not all cellular contexts, and also suggested a possible interaction with astral microtubules. For example, dAPC2 has a striking asymmetric distribution in neuroblasts, and dAPC2 colocalizes with assembling actin filaments at the base of developing larval denticles. We identified a dAPC2 mutation, revealing that dAPC2 is a negative regulator of Wg signaling in the embryonic epidermis. This allele acts genetically downstream of wg, and upstream of arm, dTCF, and, surprisingly, dishevelled. We discuss the implications of our results for Wg signaling, and suggest a role for dAPC2 as a mediator of Wg effects on the cytoskeleton. We also speculate on more general roles that APCs may play in cytoskeletal dynamics.

Wingless (Wg) is a morphogen required for the patterning of many Drosophila tissues. Several lines of evidence implicate heparan sulfate-modified proteoglycans (HSPGs) such as Dally-like protein (Dlp) in the control of Wg distribution and signaling. We show that dlp is required to limit Wg levels in the matrix, contrary to the expectation from overexpression studies. dlp mutants show ectopic activation of Wg signaling at the presumptive wing margin and a local increase in extracellular Wg levels. dlp somatic cell clones disrupt the gradient of extracellular Wg, producing ectopic activation of high threshold Wg targets but reducing the expression of lower threshold Wg targets where Wg is limiting. Notum encodes a secreted protein that also limits Wg distribution, and genetic interaction studies show that dlp and Notum cooperate to restrict Wg signaling. These findings suggest that modification of an HSPG by a secreted hydrolase can control morphogen levels in the matrix.

Adherens junctions are multiprotein complexes mediating cell-cell adhesion and communication. They are organized around a transmembrane cadherin, which binds a set of cytoplasmic proteins required for adhesion and to link the complex to the actin cytoskeleton. Three components of Drosophila adherens junctions, analogous to those in vertebrates, have been identified: Armadillo (homolog of ␤-catenin), Drosophila E-cadherin (DE-cadherin), and ␣-catenin. We carried out the first analysis of the interactions between these proteins using in vitro binding assays, the yeast two-hybrid system, and in vivo assays. We identified a 76-amino acid region of Armadillo that is necessary and sufficient for binding ␣ Adherens junctions consist of transmembrane cadherins and a set of cytoplasmic proteins associated with cadherin cytoplasmic domains (reviewed in Refs. 1 and 3). The extracellular domains of cadherins interact homotypically with cadherins of neighboring cells. The cytoplasmic proteins ␣-catenin, ␤-catenin, and plakoglobin (or ␥-catenin) are required for cadherin adhesive function and anchor the actin cytoskeleton. The Src tyrosine kinase substrate p120cas is also present in adherens junctions (4, 5); its function remains unknown. Changes in tyrosine phosphorylation of ␤-catenin (reviewed in Ref.2) and p120cas (6) correlate with transformation and associated changes in cell adhesion.To understand the cell biological function of adherens junctions, we must determine how interactions among different adherens junction proteins mediate assembly. ␤-Catenin and plakoglobin bind directly to the E-cadherin cytoplasmic domain in a mutually exclusive fashion (7,8). ␤-Catenin and plakoglobin are 70% identical in amino acid sequence; their central regions, containing ϳ13 copies of the 42-amino acid Arm 1 repeat (9), are particularly well conserved (ϳ80% amino acid identity). These highly conserved Arm repeats mediate interaction with cadherin (10 -12), suggesting that ␤-catenin and plakoglobin compete for the same binding site. The N-terminal regions of both ␤-catenin and plakoglobin bind to ␣-catenin; ␣-catenin does not bind cadherin directly (11,(13)(14)(15). ␣-Catenin, in turn, links adherens junctions to actin, directly (16) or via ␣-actinin (17). p120 cas also binds directly to E-cadherin (18), but likely to a site distinct from that bound by ␤-catenin/ plakoglobin (4, 5). p120cas does not interact with ␣-catenin (18), however, and thus does not appear to mediate interaction with actin. The core cadherin-catenin complex forms higher order assemblies such as the zonula adherens. Both E-and N-cadherins dimerize (19,20), and association with the cytoskeleton may help form larger assemblies.Adherens junctions were first described in vertebrates, but precisely analogous structures exist in Drosophila. The Drosophila homolog of ␤-catenin is Armadillo, first discovered because of its role in transducing the Wingless cell-cell signal (reviewed in Ref. 3). Arm is structurally similar to ␤-catenin and plakoglobin (it is 73% identical ...

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Division abnormally delayed (Dally) is one of two glycosylphosphatidylinositol (GPI)-linked heparan sulfate proteoglycans in Drosophila. Numerous studies have shown that it influences Decapentaplegic (Dpp) and Wingless signaling. It has been generally assumed that Dally affects signaling by directly interacting with these growth factors, primarily through its heparan sulfate (HS) chains. To understand the functional contributions of HS chains and protein core we have (1) assessed the growth factor binding properties of purified Dally using surface plasmon resonance, (2) generated a form of Dally that is not HS modified and evaluated its signaling capacity in vivo. Purified Dally binds directly to FGF2, FGF10, and the functional Dpp homolog BMP4. FGF binding is abolished by preincubation with HS, but BMP4 association is partially HS-resistant, suggesting the Dally protein core contributes to binding. Cell binding and co-immunoprecipitation studies suggest that non-HS-modified Dally retains some ability to bind Dpp or BMP4. Expression of HS-deficient Dally in vivo showed it does not promote signaling as well as wild-type Dally, yet it can rescue several dally mutant phenotypes. These data reveal that heparan sulfate modification of Dally is not required for all in vivo activities and that significant functional capacity resides in the protein core.

The adenomatous polyposis coli (APC) tumour-suppressor protein controls the Wnt signalling pathway by forming a complex with glycogen synthase kinase 3beta (GSK-3beta), axin/conductin and betacatenin. Complex formation induces the rapid degradation of betacatenin. In colon carcinoma cells, loss of APC leads to the accumulation of betacatenin in the nucleus, where it binds to and activates the Tcf-4 transcription factor (reviewed in [1] [2]). Here, we report the identification and genomic structure of APC homologues. Mammalian APC2, which closely resembles APC in overall domain structure, was functionally analyzed and shown to contain two SAMP domains, both of which are required for binding to conductin. Like APC, APC2 regulates the formation of active betacatenin-Tcf complexes, as demonstrated using transient transcriptional activation assays in APC -/- colon carcinoma cells. Human APC2 maps to chromosome 19p13.3. APC and APC2 may therefore have comparable functions in development and cancer.

Drosophila Armadillo and its mammalian homologue ␤-catenin are scaffolding proteins involved in the assembly of multiprotein complexes with diverse biological roles. They mediate adherens junction assembly, thus determining tissue architecture, and also transduce Wnt/Wingless intercellular signals, which regulate embryonic cell fates and, if inappropriately activated, contribute to tumorigenesis. To learn more about Armadillo/␤-catenin's scaffolding function, we examined in detail its interaction with one of its protein targets, cadherin. We utilized two assay systems: the yeast two-hybrid system to study cadherin binding in the absence of Armadillo/␤-catenin's other protein partners, and mammalian cells where interactions were assessed in their presence. We found that segments of the cadherin cytoplasmic tail as small as 23 amino acids bind Armadillo or ␤-catenin in yeast, whereas a slightly longer region is required for binding in mammalian cells. We used mutagenesis to identify critical amino acids required for cadherin interaction with Armadillo/␤-catenin. Expression of such short cadherin sequences in mammalian cells did not affect adherens junctions but effectively inhibited ␤-catenin-mediated signaling. This suggests that the interaction between ␤-catenin and T cell factor family transcription factors is a sensitive target for disruption, making the use of analogues of these cadherin derivatives a potentially useful means to suppress tumor progression.