M.Umbhauer and A.Djiane contributed equally to this workFrizzled receptors are components of the Wnt signalling pathway, but how they activate the canonical Wnt/b-catenin pathway is not clear. Here we use three distinct vertebrate frizzled receptors (Xfz3, Xfz4 and Xfz7) and describe whether and how their C-terminal cytoplasmic regions transduce the Wnt/b-catenin signal. We show that Xfz3 activates this pathway in the absence of exogenous ligands, while Xfz4 and Xfz7 interact with Xwnt5A to activate this pathway. Analysis using chimeric receptors reveals that their C-terminal cytoplasmic regions are functionally equivalent in Wnt/b-catenin signalling. Furthermore, a conserved motif (Lys-Thr-X-X-X-Trp) located two amino acids after the seventh transmembrane domain is required for activation of the Wnt/b-catenin pathway and for membrane relocalization and phosphorylation of Dishevelled. Frizzled receptors with point mutations affecting either of the three conserved residues are defective in Wnt/b-catenin signalling. These ®ndings provide functional evidence supporting a role of this conserved motif in the modulation of Wnt signalling. They are consistent with the genetic features exhibited by Drosophila Dfz3 and Caenorhabditis elegans mom-5 in which the tryptophan is substituted by a tyrosine.

Formins are involved in a wide range of cellular processes that require the remodeling of the actin cytoskeleton. Here, we have analyzed a novel Drosophila formin, belonging to the recently described DAAM subfamily. In contrast to previous assumptions, we show that DAAM plays no essential role in planar cell polarity signaling, but it has striking requirements in organizing apical actin cables that define the taenidial fold pattern of the tracheal cuticle. These observations provide evidence the first time that the function of the taenidial organization is to prevent the collapse of the tracheal tubes. Our results indicate that although DAAM is regulated by RhoA, it functions upstream or parallel to the non-receptor tyrosine kinases Src42A and Tec29 to organize the actin cytoskeleton and to determine the cuticle pattern of the Drosophila respiratory system.

Planar cell polarity (PCP) is a common feature of many vertebrate and invertebrate epithelia and is perpendicular to their apical/basal (A/B) polarity axis. While apical localization of PCP determinants such as Frizzled (Fz1) is critical for their function, the link between A/B polarity and PCP is poorly understood. Here, we describe a direct molecular link between A/B determinants and Fz1-mediated PCP establishment in the Drosophila eye. We demonstrate that dPatj binds the cytoplasmic tail of Fz1 and propose that it recruits aPKC, which in turn phosphorylates and inhibits Fz1. Accordingly, components of the aPKC complex and dPatj produce PCP defects in the eye. We also show that during PCP signaling, aPKC and dPatj are downregulated, while Bazooka is upregulated, suggesting an antagonistic effect of Bazooka on dPatj/aPKC. We propose a model whereby the dPatj/aPKC complex regulates PCP by inhibiting Fz1 in cells where it should not be active.

The outcome of the Notch pathway on proliferation depends on cellular context, being growth promotion in some, including several cancers, and growth inhibition in others. Such disparate outcomes are evident in Drosophila wing discs, where Notch overactivation causes hyperplasia despite having localized inhibitory effects on proliferation. To understand the underlying mechanisms, we have used genomic strategies to identify the Notch-CSL target genes directly activated during wing disc hyperplasia. Among them were genes involved in both autonomous and non-autonomous regulation of proliferation, growth and cell death, providing molecular explanations for many characteristics of Notch induced wing disc hyperplasia previously reported. The Notch targets exhibit different response patterns, which are shaped by both positive and negative feed-forward regulation between the Notch targets themselves. We propose, therefore, that both the characteristics of the direct Notch targets and their cross-regulatory relationships are important in coordinating the pattern of hyperplasia.

The related Wnt-Frizzled(Fz)/beta-catenin and Fz/planar cell polarity (PCP) pathways are essential for the regulation of numerous developmental processes and are deregulated in many human diseases. Both pathways require members of the Dishevelled (Dsh or Dvl) family of cytoplasmic factors for signal transduction downstream of the Fz receptors. Dsh family members have been studied extensively, but their activation and regulation remains largely unknown. In particular, very little is known about how Dsh differentially signals to the two pathways. Recent work in cell culture has suggested that phosphorylation of Dsh by Casein Kinase I epsilon (CKIepsilon) may act as a molecular "switch," promoting Wnt/beta-catenin while inhibiting Fz/PCP signaling. Here, we demonstrate in vivo in Drosophila through a series of loss-of-function and coexpression assays that CKIepsilon acts positively for signaling in both pathways, rather than as a switch. Our data suggest that the kinase activity of CKIepsilon is required for peak levels of Wnt/beta-catenin signaling. In contrast, CKIepsilon is a mandatory signaling factor in the Fz/PCP pathway, possibly through a kinase-independent mechanism. Furthermore, we have identified the primary kinase target residue of CKIepsilon on Dsh. Thus, our data suggest that CKIepsilon modulates Wnt/beta-catenin and Fz/PCP signaling pathways via kinase-dependent and -independent mechanisms.

E-Cadherin-based Adherens Junctions (AJs) are a defining feature of all epithelial sheets. Through the homophilic association of E-Cadherin molecules expressed on neighboring cells, they ensure intercellular adhesion amongst epithelial cells, and regulate many key aspects of epithelial biology. While their adhesive role requires these structures to remain stable, AJs are also extremely plastic. This plasticity allows for the adaptation of the cell to its changing environment: changes in neighbors after cell division, cell death, or cell movement, and changes in cell shape during differentiation. In this review we focus on the recent advances highlighting the critical role of the apico-basal polarity machinery, and in particular of the Par3/Bazooka scaffold, in the regulation and remodeling of AJs. We propose that by regulating key phosphorylation events on the core E-Cadherin complex components, Par3 and epithelial polarity promote meta-stable protein complexes governing the correct formation, localization, and functioning of AJ.

Wnt signaling has an important role in cell-fate determination, tissue patterning and tumorigenesis. Wnt proteins signal through seven-pass transmembrane receptors of the frizzled family to activate β-catenindependent transcription of target genes. Using early Xenopus embryos, we show that frizzled receptors can dimerize and that dimerization is correlated with activation of the Wnt/β-catenin pathway. Co-immunoprecipitation studies revealed that the receptor Xfz3 exists as a dimer when expressed in Xenopus embryos, and it has been shown to activate the Wnt/β-catenin pathway as revealed by expression of the target gene siamois. Xfz3 dimerization requires intramolecular and/or intermolecular disulfide linkages, and the N-terminal extracellular region of the receptor, including the cysteine-rich domain (CRD), is sufficient for dimerization. The receptor Xfz7 behaves differently from Xfz3 when overexpressed in the embryo as Xfz7 is monomeric and is unable to directly activate the Wnt/β-catenin pathway. However, activation of this pathway can be achieved by artificially forcing Xfz7 dimerization. These results provide the first direct evidence for the dimerization of frizzled receptors and suggest that dimerization contributes to transducing the Wnt/β-catenin signal. Research Article 2542 the cytoplasm and nuclei of dorsal blastomeres during early cleavage stages (Larabell et al., 1997). Moreover, overepression of GSK3 and Axin or depletion of maternal β-catenin RNA causes deficiencies in dorsal structures (He et al., 1995; Heasman et al., 1994;Yost et al., 1998;Zeng et al., 1997).The biochemical mechanisms by which the binding of the Wnt ligand to its frizzled receptor elicits signal transduction within the cell are poorly characterized. Numerous studies have suggested that several G-protein-coupled receptor (GPCR) families exist as dimers or even higher structures (Devi, 2001; Gouldson et al., 2000; Hebert and Bouvier, 1998;. Recently, biophysical methods based on luminescence and fluorescence energy transfer have confirmed the existence of such oligomeric complexes in living cells (Angers et al., 2000;Angers et al., 2001; Kroeger et al., 2001;Overton and Blumer, 2000). However, whether dimerization is a general property of this class of receptors and whether this is functionally relevant for signal transduction remains controversial (Cvejic and Devi, 1997; George et al., 1998; Gouldson et al., 1998; Hebert et al., 1998;Marshall et al., 1999). In several cases, receptors appear to fold as constitutive dimers shortly after biosynthesis, whereas ligand-promoted dimerization at the cell surface has been proposed for others (Jones et al., 1998; Kaupmann et al., 1998; Kuner et al., 1999;White et al., 1998). Dimerization is required for normal functioning of β-adrenergic receptors and has been shown to rescue the function of mutant forms of β-adrenergic and angiotensin type I receptors (Hebert et al., 1998).In this report, we address the question of the potential dimerization property of two Xenopus frizzled receptors...

Vertebrate oocytes arrest in the second metaphase of meiosis (metaphase II [MII]) by an activity called cytostatic factor (CSF), with aligned chromosomes and stable spindles. Segregation of chromosomes occurs after fertilization. The Mos/…/MAPK (mitogen-activated protein kinases) pathway mediates this MII arrest. Using a two-hybrid screen, we identified a new MAPK partner from a mouse oocyte cDNA library. This protein is unstable during the first meiotic division and accumulates only in MII, where it localizes to the spindle. It is a substrate of the Mos/…/MAPK pathway. The depletion of endogenous RNA coding for this protein by three different means (antisense RNA, double-stranded [ds] RNA, or morpholino oligonucleotides) induces severe spindle defects specific to MII oocytes. Overexpressing the protein from an RNA not targeted by the morpholino rescues spindle destabilization. However, dsRNA has no effect on the first two mitotic divisions. We therefore have discovered a new MAPK substrate involved in maintaining spindle integrity during the CSF arrest of mouse oocytes, called MISS (for MAP kinase–interacting and spindle-stabilizing protein).