Protein Interactions

Dishevelled acts in both wingless and frizzled signaling. It is interesting to note that many frizzled proteins, including Frizzled2, the putative Wingless receptor, contain a Ser/Thr-X-Val motif at their C-terminal end: this motif has been shown to interact with PDZ (or DHR) domains in a variety of proteins (Gomperts, 1996). DSH contains a PDZ domain, suggesting a direct interaction of DSH with Frizzled2 (Klingensmith, 1994). Unlike DFZ2, there is no canonical PDZ consensus binding site in the C-terminus of Frizzled.

Wingless signaling generates a hyperphosphorylated form of DSH, which is associated with a membrane fraction. Overexpressed DSH becomes hyperphosphorylated in the absence of extracellular WG and increases levels of the Armadillo protein, thereby mimicking the wg signal (Yanagawa, 1995). DSH functions upstream, prior to the action of shaggy/ zw3, a serine-thronine kinase (Siegfried, 1994). armadillo is downstream of shaggy.

Dishevelled is phosphorylated by shaggy/zeste signaling in response to wingless. Overexpressed dsh results in an increase in cytoplasmic Armadillo. Deletion of the C-terminal basic domain of DSH protein has no effect on ARM accumulation. Mutation of the GLGF/DHR motif modifies DSH affect on ARM. (Yanagawa, 1995).

shotgun (DE-cadherin] transcription level is regulated through the Wingless pathway. Drosophila genetic studies suggest that in the Wingless (Wg) signaling pathway, the segment polarity gene products, Dishevelled (Dsh), Zeste-white 3 (Zw-3), and Armadillo (Arm), work sequentially; wg and dsh negatively regulate Zw-3, which in turn down-regulates Arm. To biochemically analyze interactions between the Wg pathway and Shotgun, which binds to Arm, three proteins (Dsh, Zw-3, and Arm) were overexpressed in the Drosophila wing disc cell line (clone 8), which responds to Wg signal. Dsh overexpression leads to accumulation of Arm primarily in the cytosol and elevation of Shotgun at cell junctions. Overexpression of wild-type and dominant-negative forms of Zw-3 decreases and increases Arm levels, respectively, indicating that modulation in Zw-3 activity negatively regulates Arm levels. Overexpression of an Arm mutant with an amino-terminal deletion elevates Shotgun protein levels, suggesting that Dsh-induced Shotgun elevation is caused by the Arm accumulation induced by Dsh. Moreover, the Dsh-, dominant-negative Zw-3-, and truncated Arm-induced accumulation of Shotgun protein is accompanied by a marked increase in the steady-state levels of Shotgun mRNA, suggesting that transcription of shotgun is activated by Wg signaling. In addition, overexpression of shotgun elevates Arm levels by stabilizing Arm at cell-cell junctions (Yanagawa, 1997).

Dishevelled protein is targeted by Casein kinase II. Immunoprecipitated Dsh protein is associated with Casein Kinase II. Tryptic phosphopeptide mapping indicates that identical peptides are phosphorylated by CkII in vitro and in vivo, suggesting that CkII is at least one of the kinases that phosphorylates Dsh. Overexpression of frizzled2, a Wingless receptor, also stimulates phosphorylation of Dsh, Dsh-associated kinase activity, and association of CkII with Dsh. It is not known whether association of CkII with unphosphorylated Dsh occurs first, or whether phosphorylation of Dsh promotes association with CkII. Unphosphorylated Dsh clearly has some affinity for CkII, however, in vivo phosphorylated Dsh is associated with more CkII than is underphosphorylated Dsh. This suggests a model in which CkII can bind with low affinity to underphosphorylated Dsh and effect its phosphorylation. The phosphorylated Dsh then has a higher affinity for CkII, leading to an increase in the amount of Dsh-CkII complex. Phosphorylation of Dsh in response to the Wg signal, leads to the phosphorylation of Dsh but this is insufficient for the transduction of the Wg signal to Armadillo. Thus the function of the phosphorylation of Dsh by CkII is unknown (Willert, 1997).

Wingless (Wg) treatment of the Drosophila wing disc clone 8 cells leads to Armadillo (Arm) protein elevation, and this effect has been used as the basis of in vitro assays for Wg protein. Previously analyzed stocks of Drosophila Schneider S2 cells could not respond to added Wg, because they lack the Wg receptor, Frizzled2. However, a line of S2 cells obtained from another source express both Frizzled-2 and Frizzled. Thus, this cell line was designated as S2R+ (S2 receptor plus). S2R+ cells respond to addition of extracellular Wg by elevating Arm and Shotgun protein levels and by hyperphosphorylating Dsh, just as clone 8 cells do. Moreover, overexpression of Wg in S2R+, but not in S2 cells, induces the same changes in Dsh, Arm, and DE-cadherin proteins as induced in clone 8 cells, indicating that these events are common effects of Wg signaling, which occurs in cells expressing functional Wg receptors. In addition, unphosphorylated Dsh protein in S2 cells is phosphorylated as a consequence of expression of Frizzled-2 or mouse Frizzled-6, suggesting that basal structures common to various frizzled family proteins trigger this phosphorylation of Dsh. S2R+ cells are as sensitive to Wg as are clone 8 cells, but theycan grow in simpler medium. Therefore, the S2R+ cell line is likely to prove highly useful for in vitro analyses of Wg signaling (Yanagawa, 1998).

Thus expression of Dfz2 or Mfz6 induces phosphorylation of Dsh in S2 cells and a small proportion of Dsh protein is phosphorylated in S2R+ and clone 8 cells. These results suggest that expression of frizzled family proteins induces the basal phosphorylation of Dsh. In this regard, Casein kinase 2 (CK2), which binds to the PDZ domain of Dsh, is known to be the major kinase responsible for phosphorylation of Dsh upon Dfz2 overexpression in S2 cells. Therefore, CK2 may take part in the basal phosphorylation of Dsh in Dfz2/S2, Mfz6/S2, clone 8 and S2R+ cells not stimulated with soluble Wg. In addition, Frizzled overexpression leads to translocation of Dsh from cytoplasm to plasma membrane. Overexpression of rat frizzled-1 has been shown to result in recruitment of Xwnt-8 and XDsh to the plasma membrane in Xenopus embryos. Thus, it is possible that Dfz2 or Mfz6 expression induces translocation of at least a part of Dsh to the plasma membrane in S2 cells and that this Dsh translocation in some way stimulates Dsh phosphorylation by CK2. However, it is not clear whether CK2 also participates in Wg-induced hyperphosphorylation of Dsh or whether other kinase(s) are activated by the binding of Wg to Dfz2 in clone 8, S2R+, and Dfz2/S2 cells and that these other kinases induce the hyperphosphorylation. In view of the reports indicating association (probably indirect) between frizzled family proteins and Dsh and the binding of Dsh to CK2, it is attractive to speculate that Wg binding induces aggregation of Fz2 receptors, which, in turn, brings the Dsh-CK2 or other kinase complexes close together, and this aggregation stimulates the Dsh phosphorylation by CK2 or other kinases in these Dsh-kinase complexes. This could explain how Wg induces Dsh hyperphosphorylation in clone 8, S2R+, and Dfz2/S2 cells. However, it is noteworthy that Dfz2 overexpression leads to marked phosphorylation of Dsh, but not to elevation of Arm, in S2 cells, indicating that phosphorylation of Dsh, at least by Dfz2 overexpression, cannot activate the Wg signaling pathway by itself. Clearly, further detailed experiments are necessary to evaluate the function of Dsh phosphorylation in Wg signaling (Yanagawa, 1998 and references).

naked cuticle targets dishevelled to antagonize Wnt signal transduction

In Drosophila embryos the protein Naked cuticle (Nkd) limits the effects of the Wnt signal Wingless (Wg) during early segmentation. nkd loss of function results in segment polarity defects and embryonic death, but how nkd affects Wnt signaling is unknown. Using ectopic expression, it has been found that Nkd affects, in a cell-autonomous manner, a transduction step between the Wnt signaling components Dishevelled (Dsh) and Zeste-white 3 kinase (Zw3). Zw3 is essential for repressing Wg target-gene transcription in the absence of a Wg signal, and the role of Wg is to relieve this inhibition. Double-mutant analysis shows that, in contrast to Zw3, Nkd acts to restrain signal transduction when the Wg pathway is active . Yeast two hybrid and in vitro experiments indicate that Nkd directly binds to the basic-PDZ region of Dsh. Specially timed Nkd overexpression is capable of abolishing Dsh function in a distinct signaling pathway that controls planar-cell polarity. These results suggest that Nkd acts directly through Dsh to limit Wg activity and thus determines how efficiently Wnt signals stabilize Armadillo (Arm)/ß-catenin and activate downstream genes (Rousset, 2001).

The Drosophila eye is composed of mechanosensory bristles present at vertices of ommatidia. Bristle formation is suppressed near the circumferential margin of the eye, and the degree of suppression is least at the extreme dorsum of the head, typically 0-2 ommatidial diameters in width. Wg signaling, active at the circumference of the developing eye where wg is expressed, is responsible for this suppression of peripheral bristle formation. To assay the function of nkd in eye bristle formation, the EGUF/hid method was used to make homozygous mutant nkd eyes in nkd/+ animals. In this technique, Flp-mediated recombination between a chromosome mutant for nkd and a chromosome harboring both recessive and dominant cell-lethal mutations is specifically induced in the eye using the eyeless promoter. During eye development, the only cells surviving are those that have lost the cell-lethal chromosome through recombination, producing an eye homozygous mutant for nkd. Examination of eyes mutant for the strong allele nkd7E89 reveals, at the dorsum of the eye, consistent eye bristle suppression 3-5 ommatidial diameters away from the margin, with occasional closer bristles. This result suggests that endogenous nkd regulates interommatidial bristle suppression by antagonizing the effects of endogenous Wg in cells farther than one cell diameter away from the Wg source (Rousset, 2001).

To determine how Nkd impinges on the Wg pathway, the ability of Nkd to block the action of the positive regulators Wg, Dsh, and Arm was tested. To do so, advantage was taken of a Drosophila eye misexpression system. Production of Wg in a subset of photoreceptor cells throughout the eye using a sevenless promoter transgene (P[sev-wg]) prevents formation of interommatidial bristles in a paracrine fashion; otherwise, the eye is normal. Previous Nkd misexpression experiments did not indicate whether Nkd blocks Wg synthesis, Wg distribution, or cellular responses to received Wg. To distinguish between these possibilities, the GAL4/UAS binary expression system was used to evaluate the effect of Nkd (UAS-nkd) on Wg-mediated eye bristle suppression. Misexpression of Nkd alone using multiple repeats of the eye-specific glass (gl) enhancer (GMR) to drive the yeast transcription factor GAL4 (P[GMR-GAL4]) has no visible effect on eye development. However, the combination of sev-wg with nkd misexpression results in nearly complete suppression of the P[sev-wg]-induced bristle-loss phenotype. Nkd misexpression did not alter the levels or distribution of Wg antigen, indicating that Nkd is probably blocking signaling events downstream from Wg (Rousset, 2001).

The effect of Nkd on the downstream Wg pathway components Dsh and Arm was also tested using the GMR-GAL4 system. Dsh misexpression (UAS-dsh) produces small, bristle-less eyes devoid of ommatidia. Nkd strongly suppresses the Dsh misexpression eye phenotype, restoring numerous bristles and ommatidia. If the Dsh misexpression eye phenotype is Wg-dependent, its suppression by Nkd could be due to Nkd acting on Wg rather than on Dsh or other downstream components. Previous work suggests that the Dsh misexpression eye phenotype is Wg-independent. To confirm the Wg-independence of the Dsh phenotype, a dominant-negative form of Dfz2 (UAS-GPI-Dfz2) was coexpressed with either sev-wg or UAS-dsh. UAS-GPI-Dfz2 effectively suppresses sev-wg-induced bristle loss in the eye. Coexpression of UAS-GPI-Dfz2 and UAS-Dsh results in some eye necrosis, but it has negligible effects on the UAS-dsh eye phenotype. These results confirm that the Dsh misexpression effect in the eye is Wg-independent. Therefore, rescue of the UAS-dsh phenotype by Nkd is not an indirect effect due to suppression of Wg activity (Rousset, 2001).

GMR-driven expression of UAS-armS10, a constitutively activated form of arm, also produces bristle loss and failure of proper ommatidial development. Nkd coexpression had no effect on the Arm misexpression phenotype. Dsh and Arm misexpression phenotypes are not affected by simultaneous expression of UAS-lacZ, indicating that suppression of the dominant eye phenotypes by Nkd was not due to GAL4 titration. The ability of Nkd to block effects of Wg and Dsh but not Arm suggests that Nkd is acting at the level of, or downstream from, Dsh but not downstream of Arm (Rousset, 2001).

The relationship between Nkd and Zw3 could not be determined by a similar suppression test because both proteins are negative regulators of Wg. In addition, the subtlety of the nkd phenotype in the eye made this tissue unsuitable for analyzing the epistasis between nkd and zw3. Instead, Zw3/Gsk3ß was overproduced in nkd mutant embryos using genetic and mRNA injection methods: Heat shock promoter (hsp70)-controlled GAL4 was used to drive Zw3 production, or injections with Xenopus gsk3ß mRNA. nkd mutants lack ventral denticle belts and are considerably smaller than wild-type embryos. Overproduction of Gsk3ß or Zw3 in nkd mutants results in partial to almost complete restoration of denticle belts and restoration of more normal embryo size. Because Zw3 restores denticles to nkd mutants, Zw3 cannot act genetically upstream of the defect in nkd mutants (i.e., by stimulating nkd function) in the linear Wg pathway. Nkd therefore is likely to act upstream of, or in a pathway parallel to, Zw3 and downstream from, or at the level of, Dsh (Rousset, 2001).

The eye misexpression results suggest that Nkd antagonizes Wg signaling at the level of, or downstream from, Dsh. Loss-of-function dsh clones reveal that Dsh acts autonomously in Wg-responsive cells, suggesting that Nkd must also act in Wg-responsive cells. Indeed, previous observations in fly embryos suggest an initial requirement for nkd in cells receiving the Wg signal. Because eye development allows fairly easy production of sharply bounded clones, the eye was chosen to assess the cell autonomy of Nkd action. Marked clones of Nkd-misexpressing cells were produced in developing eyes and the range of Nkd action on sev-wg was monitored (Rousset, 2001).

The flip-on GAL4 system was used to make random clones of cells misexpressing both Nkd and a cell-autonomous marker, green fluorescent protein (GFP), in eyes with excess Wg (sev-wg eyes). All clones examined showed suppression of bristle loss, with the suppression consistently within or immediately adjacent to GFP misexpression clones. No bristles were present outside the clones, indicating a local action of Nkd. To address whether Nkd was acting only in bristle precursor cells, and hence cell-autonomously, those cells were specifically marked with antisera against the Cut nuclear protein in pupal eye discs. In the vicinity of Nkd misexpression clones, there was a perfect correlation between GFP and Cut-labeled cells: all Cut-positive bristle precursor cells expressed GFP and hence Nkd; no GFP-negative/Cut-positive cells were found. These results suggest that Nkd acts within Cut-positive bristle precursor cells to antagonize the inhibitory effects of Wg on bristle cell differentiation (Rousset, 2001).

Cuticles derived from embryos lacking wg activity (wg, dsh, or arm) have nearly continuous fields of denticles, whereas HS-wg embryos, or those mutant for the negative regulator zw3, secrete naked cuticle. Wg misexpression and double-mutant analyses show that Wg acts sequentially through Dsh, Zw3, and Arm. Embryos doubly mutant for wg and zw3 (zw3;wg), as well as zw3;dsh embryos, resemble zw3 embryos, whereas zw3;arm embryos resemble arm embryos, indicating that zw3 acts downstream from dsh and upstream of arm. Mutations in either nkd or zw3 give rise to a naked cuticle phenotype, with posterior expansion of en expression and ectopic wg expression in the developing embryo. However, in contrast to the naked cuticle phenotype of the zw3; wg embryo, the wg;nkd embryo has a wg-like phenotype, indicating a dependence on Wg for the naked cuticle phenotype of nkd mutants (Rousset, 2001 and references therein).

To clarify the relationship between Nkd and other Wg pathway components in the embryo, embryos were made doubly mutant for nkd and dsh or arm, using both genetic means and RNA interference (RNAi). Whereas the nkd gene is strictly zygotic, dsh has a maternal contribution that must be removed via germ-line clones to obtain the embryonic dsh phenotype. Females heterozygous for nkd and carrying dsh germ-line clones were crossed to males heterozygous for nkd. Embryos derived from crosses using different combinations of nkd and dsh alleles were counted and grouped according to their cuticle phenotypes. The expected Mendelian ratios are 37.5% wild type (3/8), 37.5% dsh (3/8), 12.5% nkd (1/8), and 12.5% dsh;nkd (1/8). Only three phenotypes could be detected -- wild type, dsh, and nkd -- indicating that the dsh;nkd mutants exhibit one of these phenotypes or die before secreting cuticle. Whereas the observed percentages for the wild-type and nkd categories are very close to the expected percentages (38.2% and 14.3%, respectively), the percentage of dsh embryos is significantly higher (47.5%), suggesting that the dsh;nkdmutant resembles the dsh mutant (Rousset, 2001).

To confirm the dsh;nkd phenotype, RNAi experiments were performed. Injection of nkd double-stranded RNA (dsRNA) into wild-type embryos efficiently mimics nkd loss of function: 76% of the injected embryos develop with greatly reduced denticles compared to wild-type embryos. The majority of these mutant embryos (69%) show an intermediate to strong nkd cuticle phenotype, the others showing a weak expressivity characterized by a loss of only a few denticles. RNAi was also attempted with dsh, but, in contrast to nkd, both the penetrance and the expressivity of the dsh phenotype are very weak. Increasing the dsh dsRNA concentration has little effect, producing only a fusion between belts A4 and A5 in <5% of the injected embryos and ruling out the utility of nkd and dsh double injections. Instead nkd dsRNA was injected into dsh embryos derived from germ-line clones. Half of the collected embryos are wild type due to rescue by the paternal X-chromosome. To score only nonrescued dsh mutant embryos, females carrying germ-line clones were crossed to males carrying an X-chromosome GFP balancer and GFP-negative embryos were scored after eliminating GFP embryos. Injection of nkd dsRNA into dsh embryos had no effect on the dsh phenotype, confirming that dsh;nkd double mutants resemble dsh embryos (Rousset, 2001).

The null allele armYD35 was used to generate arm;nkd double-mutant embryos. Embryos homozygous for this allele have a strong arm phenotype, even without making germ-line clones. Male heterozygotes for the strong alleles nkd7H16 or nkd7E89 were crossed to females heterozygous for armYD35 and nkd7H16. Since these crosses generate a majority of wild-type embryos (a ratio of nine wild type to seven mutants), only cuticles from unhatched embryos were counted; these unhatched embryos are expected to be mutant for arm (ratio 3:7, 42.9%), nkd (3:7, 42.9%), and arm;nkd (1:7, 14.3%). Like the dsh; nkd embryos, the arm;nkd mutants do not exhibit a distinct phenotype. The results show that the nkd phenotype is found at the expected frequency (42.6%), but the arm phenotype is over-represented (57.4% instead of 42.9%), indicating that this category also contains the arm;nkd embryos. Therefore, the arm;nkd mutant is covered with denticles and resembles arm embryos (Rousset, 2001).

The double-mutant analysis indicates that the nkd phenotype occurs only if wg, dsh, and arm genes are active, confirming the requirement for Wg signaling to generate the nkd phenotype. Zw3 constitutively represses Wg target-gene transcription, and the role of Wg is to overcome this inhibition. These results indicate that Nkd, in contrast, is required to oppose Wg signal. Removal of nkd in the absence of wg, dsh, or arm has little effect on cuticle phenotype. Accordingly, increased levels of Nkd do not modify the wg mutant cuticle. The negative influence of Nkd could be mediated by inhibition of Dsh activity, stimulation of Zw3 activity, or by interactions with unknown pathway components. To test whether Nkd can directly interact with known Wg signaling components, yeast two-hybrid and in vitro binding assays were used (Rousset, 2001).

Expression in yeast of full-length Nkd protein fused to the GAL4 DNA-binding domain (GB-Nkd) does not activate transcription by itself. When GB-Nkd was coexpressed with Dsh fused to the activation domain of GAL4 (GAD-Dsh), strong ß-galactosidase activity was detected, indicating an interaction between Nkd and Dsh. The interaction between Dsh and Nkd was confirmed using coimmunoprecipitation and glutathione-S-transferase (GST) pull-down assays. Three protein association assays indicate that Nkd and Dsh can directly interact, in keeping with the epistasis results that suggested a role for Nkd at the level of Dsh or Zw3 (Rousset, 2001).

Production of Nkd-GFP fusion protein in larval salivary glands reveals striking colocalization with endogenous Dsh, stained with an anti-Dsh antibody, indicating that the two proteins may also interact in vivo. However, an association between the two proteins was not detected using coimmunoprecipitation experiments with embryo extracts or lysates from Drosophila cell lines. The negative results may be due to protein complex dynamics, accessibility to antibodies, low levels of the complex in fly cells, as well as possible modes of regulation of the interaction, which are currently being investigated (Rousset, 2001).

The Dsh protein contains three defined domains: DIX, PDZ, and DEP. The DIX (Dishevelled, Axin) domain shares homology with the C-terminal part of D-Axin, the PDZ (PSD-95, Dlg, Zo-1) domain is a modular region involved in protein-protein interactions, and the DEP (Dsh, Egl-10, Pleckstrin) domain is usually found in signaling proteins, although its role remains unclear. In addition, a stretch of basic residues is present between the DIX and PDZ domains. Using both the yeast two-hybrid system and GST pull-down experiments, the region in Dsh that binds Nkd was defined. These results indicated that the central region of Dsh containing the basic sequence and the PDZ domain is sufficient for binding Nkd. The PDZ domain of Dsh is necessary for the interaction with Nkd but, in contrast to proteins such as casein kinase I or Frat1, it cannot efficiently bind Nkd by itself (Rousset, 2001).

Dsh is a branchpoint connecting two distinct signaling pathways in Drosophila development: the Wg pathway and the planar cell polarity pathway (PCP). nkd mutant clones have normal planar cell polarity; so there is no detectable normal role for nkd in the PCP pathway. If Nkd affects Dsh during Wg signaling, as the data suggest, then appropriately timed overexpression of Nkd might be able to specifically alter Dsh function in PCP signaling. Nkd was tested for its ability to interfere with PCP signaling during a time when Fz and Dsh are not appreciably participating in Wg signaling. Timed overexpression of Nkd at 24 h after puparium formation (APF) produces adult flies with wing hair polarity defects that are indistinguishable from those seen in dsh1 mutant adults. dsh1 is an adult viable allele of dsh that harbors a missense mutation in the C-terminal DEP domain. Genetic tests have shown dsh1 to be a null allele for PCP signaling. The Nkd overexpression polarity pattern is reproducible and qualitatively distinct from that produced by complete loss of function of other known PCP mutants, including fz or prickle (pk). The Nkd overexpression defect is also different from those associated with Fz or Dsh overexpression (Rousset, 2001).

The PCP phenotype associated with Fz overexpression is sensitive to the dose of dsh. To determine whether Nkd could similarly titrate Dsh from PCP signaling induced by Fz overexpression, Fz and Nkd were simultaneously expressed. Indeed, overexpressed Nkd suppresses the effects of excess Fz. Neither excess Nkd nor decreased nkd dosage modifies the wing bristle polarity of dsh1 mutant flies. The results suggest that Nkd can specifically interfere with Dsh function in planar cell polarity and that this effect requires wild-type Dsh protein (Rousset, 2001).

That Nkd can act through Dsh has important implications for the dynamic control of Wg/Wnt signaling. By acting upstream of the ß-catenin degradation machinery, Nkd may determine how effectively a given dose of Wnt causes ß-catenin accumulation and target-gene activation and thereby influences the sensitivity of a cell to a given amount or type of Wnt ligand. The kinetic and dynamic parameters of the feedback loop involving Wg, Dsh, and Nkd may play key roles in controlling the duration and extent of signaling activity. Tight regulation of this feedback loop is clearly important for normal Drosophila embryonic development, and in various animals it may be subject to spatial and temporal adjustments during evolution or during disease progression. Future experiments will test how the interaction between Nkd and Dsh affects responses to Wnt signals during development and may provide insight into Wnt-associated tumor progression (Rousset, 2001).

PAR-1 is a Dishevelled-associated kinase and a positive regulator of Wnt signalling

Wnt signaling regulates ß-catenin-dependent developmental processes through the Dishevelled protein (Dsh). Dsh regulates two distinct pathways, one mediated by ß-catenin and the other by Jun kinase (JNK). A Dsh-associated kinase has been purified from Drosophila that encodes a homologue of Caenorhabditis elegans PAR-1, a known determinant of polarity during asymmetric cell divisions. Treating cells with Wnt increases endogenous PAR-1 activity coincident with Dsh phosphorylation. PAR-1 potentiates Wnt activation of the ß-catenin pathway but blocks the JNK pathway. Suppressing endogenous PAR-1 function inhibits Wnt signaling through ß-catenin in mammalian cells, and Xenopus and Drosophila embryos. PAR-1 seems to be a positive regulator of the ß-catenin pathway and an inhibitor of the JNK pathway. These findings show that PAR-1, a regulator of polarity, is also a modulator of Wnt-ß-catenin signaling, indicating a link between two important developmental pathways (Sun, 2001).

Dsh is progressively phosphorylated in Drosophila during embryonic development. To identify the kinase that phosphorylates Dsh, various domains of Dsh were expressed as glutathione S-transferase (GST)-fusion proteins and tested for their ability to bind the kinase from Drosophila embryos. Only the GST fusion protein containing the middle domain, DM, but not the N-terminal or C-terminal domain of Dsh, has a high affinity for the kinase. The Dsh-associated kinase activity that is precipitated with GST-DM increases as Dsh becomes progressively phosphorylated during development. Similarly, there is a kinase activity present in Dsh immunoprecipitates. An in-gel kinase experiment has shown that the kinase activity, eluted from Dsh immunoprecipitates, is associated with two major bands and a minor band on a polyacrylamide gel. These bands of 110 kDa, 64 kDa and 130 kDa (minor band) are the kinase and its major fragment. The region on Dsh that interacts with the kinase was mapped more precisely to a 36 amino acid segment, DM5, which is N-terminal to the PDZ domain. Importantly, this region in Dsh is well conserved among Drosophila, C. elegans, Xenopus and mammals (Sun, 2001).

Using GST-DM5 precipitation and in vitro phosphorylation as an assay to monitor the Dsh-associated kinase activity, the kinase was purified 60,000-fold from Drosophila embryos and several peptide sequences were then used to clone the cDNA of this kinase. The cDNA clones encoded a protein kinase that is highly homologous to the C. elegans protein PAR-1 (85% identity in the kinase domain and 42% identity overall), which is known to regulate embryo polarity but whose function in Wnt signaling has not been explored (Sun, 2001).

To determine whether dPAR-1 and Dsh form a complex in vivo, endogenous Dsh was immunoprecipitated from Drosophila cells with an affinity purified anti-Dsh antibody and dPAR-1 was detected in the immunocomplex by Western blot using anti-dPAR-1 antibody. These data indicate that dPAR-1 is the kinase responsible for the activity that co-purified with Dsh in the GST pull-down experiment. The PAR-1 protein, prepared by in vitro translation, strongly phosphorylates the middle region of Dsh in vitro. Co-expression of Dsh with wild-type dPAR-1, but not kinase-negative dPAR-1 (dPAR-1 KN), in NIH3T3 cells results in a reduction in Dsh mobility on SDS-PAGE. These data indicate that dPAR-1 directly phosphorylates Dsh in vitro and in vivo (Sun, 2001).

If PAR-1 acts in Wnt signaling, PAR-1 activity might be expected to change in response to Wnt. Drosophila clone-8 cells were stimulated with conditioned medium containing Wingless and it was confirmed that Dsh becomes phosphorylated, and Armadillo is stabilized. The kinase activity of dPAR-1 was also measured under the same conditions. Several experiments have shown that soluble Wg stimulates dPAR-1-specific activity. Wg treatment does not increase the amount of dPAR-1 protein that interacts with GST-DM5. The increased dPAR-1 activity correlates with enhanced Dsh phosphorylation and elevation of Arm levels in Clone-8 cells (Sun, 2001).

To examine whether PAR-1 is required in the Wnt pathway, endogenous PAR-1 activity was suppressed by expressing a kinase-negative PAR-1 (hPAR-1Balpha KN). Chinese hamster ovary (CHO) cells were used because good expression from transfected DNA can be achieved and these cells have a well-characterized response to Wnt. Three hallmarks of Wnt activity were measured: Dsh phosphorylation, ß-catenin stabilization and transcriptional activation. Wnt treatment of CHO cells retards the mobility of Dvl proteins (mammalian homologs of Dsh) on SDS-PAGE, and phosphatase treatment increases the mobility of the Dvl band, thereby confirming that Dvl is phosphorylated in response to Wnt. The hPAR-1Balpha KN suppresses Wnt-mediated phosphorylation of endogenous Dvl proteins, as shown by the reduced amount of a retarded Dvl band. This result is consistent with the data that PAR-1 phosphorylates Dsh in vitro and in cells. Furthermore, both human and Drosophila PAR-1 KN strongly suppress Wnt-induced ß-catenin stabilization. The kinase-negative forms of hPAR-1A, hPAR-1B and hPAR-1C all strongly suppress Wnt-mediated transcriptional activation (measured by LEF1/TCF reporters) in a dose-dependent manner. Importantly, co-expression of wild-type hPAR1 can override the suppression mediated by hPAR-1 KN, indicating that hPAR-1 KN affects Wnt signaling by specifically blocking the effects of endogenous PAR-1 in cells. However, hPAR-1 KN is unable to inhibit the transcriptional activation induced by overexpression of ß-catenin, consistent with PAR-1's role in regulation of Dsh function upstream of ß-catenin (Sun, 2001).

All three human PAR-1 homologs strongly potentiated the responses to Wnt or Dvl3 in CHO cells. The hPAR-1 proteins alone do not activate the signaling pathway but require co-expression of either Wnt or Dvl, indicating that there is synergy between hPAR-1 and other components of the Wnt pathway. The specificity of these responses was verified by their dependency on the co-expression of the LEF1 transcription factor, which is required for Wnt signaling. Furthermore, the effects of hPAR-1 were suppressed by Axin, a negative regulator of the Wnt pathway that acts downstream of Dsh. As predicted, hPAR-1 overexpression does not alter the gene response induced by overexpression of ß-catenin, consistent with the idea that PAR-1 regulates Wnt signaling at a step upstream of Axin and ß-catenin (Sun, 2001).

Dsh also activates an alternative pathway that culminates in the stimulation of JNK in mammalian cells and controls of cell polarity in Drosophila and in vertebrates. Expression of Dvl in NIH3T3 cells induces JNK activation. Interestingly, hPAR-1 strongly suppresses the ability of Dvl3 to activate JNK in NIH3T3 cells. This inhibitory effect depends on the kinase activity of hPAR-1, because a mutation in hPAR-1, which is expected to inactivate its kinase activity (hPAR-1 KN), leads to a loss of its inhibitory effect on JNK activation. Together, these data indicate that PAR-1 promotes the participation of Dsh in the Wnt-ß-catenin pathway but suppresses its function in the JNK pathway, thereby acting as a switch for the downstream responses mediated by Dsh protein (Sun, 2001).

Several studies have shown that Wnt signaling controls axis specification during Xenopus embryogenesis. Ectopic ventral expression of Wnt before the onset of zygotic transcription results in duplication of the dorsal axis: this duplication can be blocked by dominant-negative forms of Frizzled, Dsh and CKIepsilon. Likewise, interference with negative regulators of the Wnt pathway, such as GSK3ß or Axin, by ventral expression of dominant negatives also induces axis duplication. In order to further the understanding of the function of PAR-1 in vertebrates, whether PAR-1 KN can alter Wnt signaling was investigated by injecting PAR-1 RNA into Xenopus embryos. Ventral blastomere injection of XWnt8 RNA results in significant axis duplication, as expected. Co-injection of human or Drosophila kinase-negative PAR-1 RNA significantly inhibits XWnt8-induced axis duplication in injected embryos. Both human and Drosophila kinase-negative PAR-1 can exert functional effects on the Wnt pathway in Xenopus axis duplication experiments (Sun, 2001).

To examine the function of dPAR-1 in Wg signaling in Drosophila embryos, double-stranded RNA-mediated interference (RNAi) was used to generate a dpar-1 loss-of-function phenotype. The ventral epidermis of a wild-type embryo is covered with alternating smooth cuticle and denticle belts. The Wg pathway is required to specify the fate of the epidermal cells that secrete smooth cuticle. RNAi of dpar-1 causes localized ectopic denticle formation and fusion of denticle belts, resembling the wg RNAi phenotype previously described. A similar wg phenotype is also generated with RNAi using double-stranded RNAs derived from different regions of the dpar-1 transcript (Sun, 2001).

Whether Dsh itself or perhaps other proteins bound to Dsh are the most important substrates of PAR-1 remains to be determined. Previous reports have shown that CKIepsilon, a positive regulator of the Wnt pathway, also interacts with Dsh. However, CKIpsilon seems to exert its major effect on more downstream components of the pathway. GBP/Frat (GSK3-binding protein) also interacts with Dsh and has a positive role in the Wnt pathway. Detailed biochemical studies will be required to elucidate the precise function(s) of PAR-1 and its functional interactions with Dsh and other regulators of Dsh in Wnt signaling (Sun, 2001).

Prickle mediates feedback amplification to generate asymmetric planar cell polarity signaling

Planar cell polarity signaling in Drosophila requires the receptor Frizzled and the cytoplasmic proteins Dishevelled and Prickle. From initial, symmetric subcellular distributions in pupal wing cells, Frizzled and Dishevelled become highly enriched at the distal portion of the cell cortex. A Prickle-dependent intercellular feedback loop is described that generates asymmetric Frizzled and Dishevelled localization. In the absence of Prickle, Frizzled and Dishevelled remain symmetrically distributed. Prickle localizes to the proximal side of pupal wing cells and binds the Dishevelled DEP domain, inhibiting Dishevelled membrane localization and antagonizing Frizzled accumulation. This activity is linked to Frizzled activity on the adjacent cell surface. Prickle therefore functions in a feedback loop that amplifies differences between Frizzled levels on adjacent cell surfaces (Tree, 2002).

Using a glutathione-S-transferase (GST) pull-down assay, it was found that full-length, in vitro translated PkSple (one of the three alternatively spliced forms of Prickle) binds to a GST-Dsh fusion protein. To determine the domain dependency of this interaction, domains of Pk were tested for binding to individual Dsh domains. A Pk construct containing just the conserved PET and LIM domains that are common to all Pk isoforms (PkPETLIM), but not either domain individually, is sufficient for binding to full-length Dsh. Furthermore, of the three defined Dsh domains, DIX, PDZ, and DEP, it was found that PkPETLIM binds a construct containing the DEP domain but not other Dsh domains. The binding between Pk and Dsh was confirmed, and the domain specificity was verified, using a yeast two-hybrid assay. PkPETLIM fused to a DNA binding domain interacts specifically with the Dsh DEP domain but not with the other domains of Dsh. It is concluded that Pk, via its conserved PET and LIM domains, binds the Dsh DEP domain (Tree, 2002).

Since the Dsh DEP domain is required for membrane localization, it was hypothesized that Pk antagonizes Fz activity by interfering with Dsh membrane localization. To test this, it was necessary to find conditions in which Pk activity is uncoupled from Fz/Dsh activity in the adjacent cell. Fz-dependent Dsh localization was therefore reconstituted in cultured cells. In U20S cells, transfected GFP-tagged Dsh (Dsh::GFP) can be seen in punctate patches in the cytoplasm. On cotransfection with Fz, Dsh::GFP is translocated to the cell membrane, as was seen in a similar frog animal cap assay. To test whether the physical interaction between Pk and Dsh affects Dsh membrane localization, Fz, Dsh::GFP, and Pk were cotransfected. Dsh is localized in the cytoplasm in 90% of the cotransfected cells, resembling cells transfected with Dsh::GFP alone. In contrast, Pk lacking the PET domain is severely impaired in its ability to block Dsh membrane localization. Pk, therefore, interferes with Dsh membrane association in this heterologous system and by extension may interfere with Dsh membrane localization at the proximal boundary of pupal wing cells. Since Dsh is required to generate asymmetric localization of Fz, it is suggested that blocking Dsh membrane localization also inhibits the accumulation of Fz at the proximal boundary (Tree, 2002).

Fz activity on the distal side of the cell is linked to the accumulation of Pk on the proximal side of the adjacent cell and that accumulation of Pk suppresses Fz/Dsh localization at the proximal cell cortex. In this way, Fz and Pk appear to function in a feedback loop rather than a linear signaling pathway. The ordering of these members of the PCP pathway was analyzed through epistasis analysis. The loss-of-function phenotypes of pkpk-sple, dsh, and fz are very similar, suggesting that they activate the same signaling mechanism. However, these similarities make classical genetic epistasis tests difficult to interpret. In previous work, Fz overexpression phenotypes have been used to show that Fz signaling requires Dsh. These results were interpreted as implying that the PCP genes form a linear pathway with Dsh acting downstream of Fz. Consistent with this is the finding that Dsh localization requires Fz protein. In contrast, other observations do not fit this model. For example, localization of Fz requires Dsh, a presumed 'downstream' element of the pathway. Fz localization also requires the other presumed downstream elements Pk, the 7 cadherin Flamingo (Fmi), and novel TM protein Van gogh/Strabismis (Vang/Stbm) (Tree, 2002).

To clarify these results, several further epistasis tests were performed. Dsh was found to be required to generate a Fz overexpression phenotype and Fz is required to produce a Dsh overexpression phenotype. In addition, Pk is required for the Dsh and Fz overexpression phenotypes. These results argue against a simple linear pathway but are consistent with Fz, Dsh, and Pk participating in a feedback loop (Tree, 2002).

Evidence is provided that Fmi acts with Pk on the proximal side of the cell. Like Pk, overexpression of fmi using ptc-Gal4 causes hairs to point toward the midline of the wing. fmi overexpression clones show distal-domineering nonautonomy, phenocopying fz loss-of-function and pk overexpression clones. Thus, it is possible that Fmi could be acting similarly to Pk in feedback amplification of PCP signaling. Fmi is likely to be present at both sides of the cell, but it may act with Pk at the proximal side to suppress Fz signaling. However, Fmi must have an additional function since, within and surrounding fmi clones, very little Pk and Dsh localize to cell boundaries. In addition to functioning with Pk to suppress Fz/Dsh activity, it is suggested that Fmi could have a role in stabilizing the Fz complex at the membrane, without which no signaling occurs (Tree, 2002).

Whether differences in Pk activity between adjacent cells affect the localization of Dsh and Fz was examined. pk was overexpressed in the posterior of the wing (using UAS-pk, engrailed-Gal4), and Dsh and Fz localization were assessed near the edge of the pk overexpression domain. Dsh and Fz were relocalized to the antero-posterior cell boundaries, indicating that accumulation of Dsh and Fz occurs at interfaces between cells expressing high and low levels of Pk. Furthermore, within the posterior domain, Dsh and Fz are seen to accumulate to higher levels than in the anterior, consistent with a role for Pk in promoting PCP signaling. Two conclusions are drawn from these observations: (1) Dsh and Fz localization occur preferentially at boundaries between cells where one cell expresses high levels and the other expresses lower levels of Pk; (2) providing high levels of Pk amplifies Fz signaling, as measured by the increased accumulation of both Fz and Dsh at cell peripheries. In the wild-type, therefore, Pk at the proximal side of the cell drives Fz and Dsh accumulation at the distal side of the adjacent cell. Conversely, providing high levels of Fz induces higher levels of Pk accumulation (Tree, 2002).

Since Pk accumulates at boundaries between cells expressing different levels of Fz, and Dsh accumulates at boundaries between cells expressing different levels of Pk, it is suggested that Fz and Dsh on one side and Pk on the opposite side of an intercellular boundary form a self-organizing complex. Indeed, evidence is found for this in the posterior, pk-overexpressing domain of the same wings. In the posterior, Dsh and Fz accumulate in discrete patches around the cell peripheries. Simultaneous staining for Pk, Dsh, and Fmi, or Fz and Fmi, reveals that all four proteins colocalize to these patches. Thus, Pk, Dsh, Fmi, and Fz form self-organizing complexes bridging adjacent cells (Tree, 2002).

Various models have been proposed for the distribution of PCP information within Drosophila epithelia. Regardless of which model is correct, it is likely that this cue induces only a slight asymmetry within each cell. In the Drosophila wing, this initial small asymmetry may result in slightly higher levels of Fz accumulation or activation on the distal than on the proximal side of cells. However, this initial level of asymmetry is insufficient to polarize the cytoskeletal architecture of the cell and is undetectable as assessed by Dsh localization. Fz and Dsh localization subsequently become highly polarized through the action of a feedback amplification loop. Although the mechanism for such a feedback loop has not been demonstrated, it has previously been speculated that Fz activity on distal cell surfaces might promote expression of an inhibitory Wnt molecule that would suppress Fz activity on the adjacent cell surface. This study demonstrates suppression of Fz activity on adjacent cell surfaces but finds that it is mediated through asymmetric localization of Pk, which antagonizes Fz/Dsh accumulation by blocking cortical Dsh localization (Tree, 2002).

A model is proposed for a Pk-dependent feedback mechanism that functions across each P-D cell interface to amplify the difference between the initial levels of Fz/Dsh activity. Pk and Dsh are initially distributed nearly symmetrically, but in largely nonoverlapping distributions around the cells' apices. As a result of an initial, slight asymmetry in one or more components, Pk suppresses Fz accumulation on the proximal side of the cell by antagonizing Dsh cortical localization. Dsh association with the cortex is required for asymmetric Fz accumulation. Reduced proximal Fz accumulation then decreases Pk activity on the adjacent, distal side of the neighboring cell, allowing even greater Dsh cortical localization and Fz accumulation. Conversely, Pk localization is induced by Fz localization on the neighboring cell. The result is the highly asymmetric distribution of Fz, Dsh, and Pk observed at 30 hr APF. This mechanism is bidirectional, constituting a negative feedback loop that is predicted to be unstable; once any asymmetry is initiated, high levels of Fz/Dsh are promoted on one side of the boundary and low levels on the other. Fmi is proposed to play two roles. The localization of all components to the apical cell cortex depends on Fmi, which is thought to be on both sides of the boundary. Since Fmi overexpression mimics Pk overexpression, it is proposed that Fmi is activated selectively on the proximal side of the cell, where it works with Pk to suppress Fz/Dsh activity. In essence, the function of this regulatory loop depends on the balance of forces regulating Dsh membrane association on either side of the boundary. Fz recruits Dsh to the cell cortex, and Pk blocks this recruitment. Once an initial asymmetry is induced, these forces become different on either side of the boundary, and the feedback mechanism amplifies the differences (Tree, 2002).

Because fmi overexpression produces polarity phenotypes very similar to those seen with pk overexpression, it is likely that Fmi activity is also polarized across cell boundaries and participates in blocking Fz/Dsh accumulation on the distal side of the interface. However, Fmi has been proposed to exist on both sides of the boundary, and Fmi is required for the efficient localization of Fz, Dsh, and Pk to boundaries. Fmi, therefore, appears to have an additional, complex-stabilizing function. Diego (Dgo), an ankyrin repeat protein, is required for PCP signaling and localizes to P-D cell boundaries, though it has not been possible to resolve on which side of the boundary Diego is found. Dgo may function as a scaffold for assembly of PCP-signaling components (Tree, 2002).

The core PCP protein Van Gogh (Vang, also known as Strabismus [Stbm], a transmembrane protein, is likely to be involved in the feedback amplification mechanism. In a vang/stbm mutant background, Fz is localized symmetrically to the membrane in the same manner as in a pk null background, and vang/stbm has been proposed to function downstream of pk. Interestingly, in Xenopus and zebrafish, Stbm binds Dsh and seems to toggle its activity between the canonical Wnt and a PCP-like signaling pathway. Stbm antagonizes canonical Wnt signaling and activates a PCP-like pathway that regulates convergent extension and induces JNK signaling. It is proposed that both fly and vertebrate Vang/Stbm may function together with Pk, facilitating PCP signaling by antagonizing Dsh activity (Tree, 2002).

The feedback amplification mechanism described here provides a clue to understanding the long-standing problem of domineering nonautonomy. Loss-of-function clones of fz, vang/stbm, and to a lesser extent pk induce polarity phenotypes in neighboring wild-type tissue. The feedback loop model predicts that loss of Fz will disrupt the localization of these components in the neighboring distal cells, causing their polarity to reverse. Furthermore, if the immediate clone neighbors have reversed polarity, this could then cause the reversal to propagate over a distance. This phenomenon is observed near the lateral borders of a fz clone, where Pk accumulation is seen to run parallel to the clone, even at a distance from the mutant cells. Thus, the reversal in Pk localization may underlie, in part, the domineering nonauontomy observed within wild-type tissue distal to fz clones (Tree, 2002).

Once asymmetrically localized, the Fz/Dsh complex must direct reorganization of the cytoskeleton by both determining the location for prehair initiation and by limiting the number of prehairs to one. Rho and Rho-associated kinase directly regulate myosins to limit the number of prehairs. The recently described Formin homology protein, Daam1, links Dsh to Rho during vertebrate PCP-like signaling, and a Drosophila homolog may function similarly. However, further studies will be required to understand how localized Fz and Dsh orient prehair initiation (Tree, 2002).

Strabismus is asymmetrically localized and binds to Prickle and Dishevelled during Drosophila planar polarity patterning

Planar polarity decisions in the wing of Drosophila involve the assembly of asymmetric protein complexes containing the conserved receptor Frizzled. This study analyses the role of the Van Gogh/strabismus gene in the formation of these complexes and in determination of cell polarization. The Strabismus protein becomes asymmetrically localized to the proximal edge of cells. In the absence of strabismus activity, the planar polarity proteins Dishevelled and Prickle are mislocalized in the cell. Strabismus binds directly to Dishevelled and Prickle and is able to recruit them to membranes. Furthermore, the putative PDZ-binding motif at the C terminus of Strabismus is not required for its function. A two-step model is proposed for assembly of Frizzled-containing asymmetric protein complexes at cell boundaries. First, Strabismus acts together with Frizzled and the atypical cadherin Flamingo to mediate apicolateral recruitment of planar polarity proteins including Dishevelled and Prickle. In the second phase, Dishevelled and Prickle are required for these proteins to become asymmetrically distributed on the proximodistal axis (Bastock, 2003).

The subcellular localiaation of Stbm protein was investigated during wing morphogenesis using both a Stbm-YFP (Stbm yellow fluorescent protein) expressing transgene and using specific antibodies raised against Stbm. During the third instar stage, Stbm-YFP in the wing pouch localizes unevenly around apicolateral cell boundaries. Based on its molecular homology as a multi-pass transmembrane protein, it is assumed that Stbm is present in the outer cell membrane. At 18 hours of pupal life, a similar pattern is seen, Stbm-YFP still being distributed patchily in an apicolateral ring. By 24 hours, there is preferential distribution of Stbm-YFP to proximodistal cell boundaries; this distribution is clearly present at 28 hours and persists until at least 32 hours, which corresponds to the time of trichome initiation. The pattern seen with Stbm antibodies confirms that Stbm-YFP is a faithful reporter of Stbm protein distribution (Bastock, 2003).

The timecourse and distribution of Stbm broadly fits that described for other planar polarity proteins such as Fmi, Fz, Dsh and Pk-Sple. Consistent with this, good colocalization is found between Stbm-YFP and other polarity proteins. The localization of Stbm-YFP to the adherens junction zone was confirmed by costaining for Armadillo distribution. Conversely, Stbm-YFP shows no overlap with the distribution of Discs-Large, which is localized in the septate junction region. Mosaic analysis revealed that Stbm-YFP becomes preferentially distributed to the proximal edges of cells with no appreciable accumulation at distal edges (Bastock, 2003).

The three putative multipass transmembrane proteins Fmi, Fz and Stbm all play important roles in the first step of localizing planar polarity proteins to the apicolateral adherens junction zone. It is thought that Fmi acts at the top of the hierarchy in this process, since, in its absence, negligible amounts of any planar polarity proteins become apicolaterally localized. Stbm is also key, because, in its absence, both Fz and Fmi recruitment are reduced. Additionally, Stbm is also required for Dsh apicolateral recruitment and for efficient localization of Pk to membranes. Fz is not significantly required for apicolateral recruitment of Fmi, but is partly needed for apicolateral localization of Stbm and is absolutely required for apicolateral localization of Dsh. Hence, in the absence of Fmi, Fz or Stbm, one or more planar polarity proteins do not become apicolaterally localized and the process of asymmetric localization on the proximodistal axis does not occur (Bastock, 2003).

An important question is which of these factors are directly binding together, in the process of apicolateral recruitment. So far no direct protein interactions have been reported for Fmi, although it is tempting to speculate that Fmi might bind directly to Fz and Stbm in the process of apicolateral recruitment. However, Fz is able to recruit Dsh to membranes in a heterologous cell type, suggesting that these factors directly interact. In addition, vertebrate Stbm and Dsh homologs have been shown to directly interact. Direct interactions are shown between Drosophila Stbm and Dsh, and Stbm and Pk. This suggests a model in which Dsh and Pk both become apicolaterally localized as a result of direct interactions with Fz and Stbm. Notably, in the absence of Stbm, Pk accumulates in the cytoplasm, suggesting that its interaction with Stbm is important for regulating its level in the cell in addition to its subcellular localization (Bastock, 2003).

At the stage when the planar polarity proteins are apicolaterally localized, but prior to the stage when they are asymmetrically localized on the proximodistal axis of the wing, it is possible that they are present in either 'symmetric' or 'asymmetric' complexes assembled across cell-cell boundaries. If the complexes were symmetric, then Fmi, Fz, Stbm, Pk and Dsh would all be present in a complex together on the same side of the cell-cell boundary. Such symmetric complexes would then subsequently evolve into asymmetric complexes, with Fz/Dsh at distal cell edges and Stbm/Pk at proximal cell edges and Fmi on both sides. Alternatively, the initial apicolateral complexes formed could be asymmetric, with Fz/Dsh always on the opposite side of the cell-cell boundary from Stbm/Pk. These asymmetric complexes would initially be randomly oriented relative to the axes of the wing, but would gradually become aligned to the proximodistal axis. The possibility is favored that planar polarity protein complexes are initially symmetric, since Stbm directly interacts with Dsh and these molecules colocalize during earlier stages of wing development. However, it has been reported that Pk and Dsh-GFP do not precisely colocalize in early pupal wings: this observation supports the early presence of asymmetric complexes (Bastock, 2003).

After the apicolateral recruitment of planar polarity proteins, over a number of hours their localization alters such that they become asymmetrically distributed on the proximodistal axis of the wing. Although Dsh and Pk play negligible roles in the apicolateral recruitment of proteins, both are required for this subsequent proximodistal redistribution. Since overexpression of both factors leads to the accumulation of polarity proteins at apicolateral cell boundaries, it is suggested that they both function to promote the assembly and/or stabilization of protein complexes. Removal of the function of the planar polarity gene dgo also blocks asymmetric proximodistal localization but not apicolateral localization of other polarity proteins. Furthermore, overexpression of Dgo causes an accumulation of other polarity proteins at cell boundaries similar to that seen when Dsh and Pk are overexpressed. Therefore, it is proposed that Dsh and Pk act together with Dgo in the assembly of asymmetric complexes (Bastock, 2003).

Recently, it has been proposed that the function of Pk in asymmetric complex assembly is to antagonize Dsh localization to membranes. This model is mechanistically attractive, in providing an explanation for the formation of asymmetric complexes in which Dsh and Pk are found on opposite sides of cell-cell boundaries. However, it is found that in the presence of Stbm, Dsh and Pk will colocalize at the same membranes. Furthermore, it was not possible to show an effect of overexpressing Pk on the association of Fz and Dsh at membranes. In addition, high level Pk expression in vivo does not cause Dsh to lose its membrane localization but instead appears to increase levels of Dsh at the membrane. Resolution of these issues will require a more detailed understanding of the composition and properties of the protein complexes involved (Bastock, 2003).

A role of Dishevelled in relocating Axin to the plasma membrane during Wingless signaling

Wnt signaling causes changes in gene transcription that are pivotal for normal and malignant development. A key effector of the canonical Wnt pathway is ß-catenin, or Drosophila Armadillo. In the absence of Wnt ligand, ß-catenin is phosphorylated by the Axin complex, which earmarks it for rapid degradation by the ubiquitin system. Axin acts as a scaffold in this complex, to assemble ß-catenin substrate and kinases (casein kinase I [CKI] and glycogen synthase kinase 3ß [GSK3]). The Adenomatous polyposis coli (APC) tumor suppressor also binds to the Axin complex, thereby promoting the degradation of ß-catenin. In Wnt signaling, this complex is inhibited; as a consequence, ß-catenin accumulates and binds to TCF proteins to stimulate the transcription of Wnt target genes. Wnt-induced inhibition of the Axin complex depends on Dishevelled (Dsh), a cytoplasmic protein that can bind to Axin, but the mechanism of this inhibition is not understood. This study shows that Wingless signaling causes a striking relocation of Drosophila Axin from the cytoplasm to the plasma membrane. This relocation depends on Dsh. It may permit the subsequent inactivation of the Axin complex by Wingless signaling (Cliffe, 2003).

To identify further components of the Wingless pathway that are required for Axin relocation to the membrane, Axin-GFP was examined in various mutants. In sgg mutants, there are no significant changes in the subcellular distribution of the Axin-GFP dots, and their relocation to the plasma membrane in +Wg cells appears normal. Likewise, the few residual GFP-Axin in +Wg cells of APC double mutants are associated with the plasma membrane. Thus, neither GSK3 nor APC are required for relocation of Axin-GFP to the plasma membrane. Interestingly however, none of the Axin-GFP dots are associated with the plasma membrane in dsh mutants; Wingless is still expressed in these mutants at this stage). This is the case even if Wingless is coexpressed with Axin-GFP in these mutants. Thus, Dsh is the most downstream-acting component of the Wnt pathway that is required for the relocation of Axin-GFP to the plasma membrane (Cliffe, 2003).

It was asked whether overexpressed Dsh may mediate additional relocation. In wing discs, GFP-Dsh is associated with apicolateral regions of the plasma membrane whether or not Wingless signals. In the embryo, GFP-Dsh is expressed very weakly, and it can only be detected in late stages when Wingless has ceased to signal in the epidermis. In these -Wg cells, GFP-Dsh is detectable in the cytoplasm throughout the embryo and forms occasional dots; it is also weakly associated with the plasma membrane. Notably, overexpressed Dsh causes additional relocation of GFP-Axin from the cytoplasm to the plasma membrane. Most embryos show wide zones of membrane-associated Axin-GFP spanning +Wg cells that alternate with narrow zones of cytoplasmic Axin-GFP dots coincinding with -Wg cells, but, occasionally, the membrane relocation is seen throughout the embryo. This suggests that the additional Dsh-mediated relocation depends on low levels of Wingless signaling (Cliffe, 2003).

Overexpressed Dsh results in a partially naked cuticle, with only small denticles remaining. Thus, Dsh may inhibit endogenous Axin by relocating it to the plasma membrane. Consistent with this, limited restoration of naked cuticle is seen if Axin-GFP is coexpressed with Dsh, and an abundance of small denticles are seen that are sparse in wg mutants or in cuticles expressing Axin-GFP alone. As in the case of Wingless, this suppression of the activity of Axin-GFP is mild, suggesting that the putative limiting component is upstream of Dsh. This component may be Arrow, given that Arrow can bind to Axin and that an interaction between these two components in mammalian cells is induced by Wnt signaling (Cliffe, 2003).

Therefore, a possible model is that the Axin complex and Dsh are associated with the same vesicles, which may be recycling endocytic vesicles. Dsh may target these vesicles constitutively to the plasma membrane, where the Axin complex can interact potentially with Wnt receptors. This complex may be retained at the plasma membrane as a result of a Wnt-induced interaction between Axin and LRP/Arrow, and this retention may allow its subsequent inactivation. It is noted that LRPs are thought to recycle to the plasma membrane through endocytic vesicles, like their rapidly recycling LDL receptor relative. Recycling vesicles may thus provide a platform for APC-mediated assembly of the Axin complex and may convey this complex to the plasma membrane for inactivation by Wnt receptors (Cliffe, 2003).

Diego and Prickle regulate Frizzled planar cell polarity signalling by competing for Dishevelled binding

Epithelial planar cell polarity (PCP) is evident in the cellular organization of many tissues in vertebrates and invertebrates. In mammals, PCP signalling governs convergent extension during gastrulation and the organization of a wide variety of structures, including the orientation of body hair and sensory hair cells of the inner ear. In Drosophila melanogaster, PCP is manifest in adult tissues, including ommatidial arrangement in the compound eye and hair orientation in wing cells. PCP establishment requires the conserved Frizzled/Dishevelled PCP pathway. Mutations in PCP-pathway-associated genes cause aberrant orientation of body hair or inner-ear sensory cells in mice, or misorientation of ommatidia and wing hair in Drosophila. This study provides mechanistic insight into Frizzled/Dishevelled signalling regulation. The ankyrin-repeat protein Diego binds directly to Dishevelled and promotes Frizzled signalling. Dishevelled can also be bound by the Frizzled PCP antagonist Prickle. Strikingly, Diego and Prickle compete with one another for Dishevelled binding, thereby modulating Frizzled/Dishevelled activity and ensuring tight control over Frizzled PCP signalling (Jenny, 2005).

Mathematical modeling of planar cell polarity to understand domineering nonautonomy

Planar cell polarity (PCP) signaling generates subcellular asymmetry along an axis orthogonal to the epithelial apical-basal axis. Through a poorly understood mechanism, cell clones that have mutations in some PCP signaling components, including some, but not all, alleles of the receptor frizzled, cause polarity disruptions of neighboring wild-type cells, a phenomenon referred to as domineering nonautonomy. A contact-dependent signaling hypothesis, derived from experimental results, is shown by reaction-diffusion, partial differential equation modeling and simulation to fully reproduce PCP phenotypes, including domineering nonautonomy, in the Drosophila wing. This work suggests that Fz does not require a Wnt ligand in PCP signaling but that its activity is regulated by interactions between neighboring cells and differential levels of the cytoplasmic mediators Pk and Dsh. The sufficiency of this model and the experimental validation of model predictions reveal how specific protein-protein interactions produce autonomy or domineering nonautonomy (Amonlirdviman, 2005).

As the understanding of cellular regulatory networks grows, system behaviors resulting from feedback effects have proven sufficiently complex so as to preclude intuitive understanding. The challenge now is to show that enough of a network is understood to explain such behaviors. Using mathematical modeling, the sufficiency of a proposed biological model is shown and its properties studied, to demonstrate that it can explain complex PCP phenotypes and provide insight into the system dynamics that govern them (Amonlirdviman, 2005).

Many epithelia are polarized along an axis orthogonal to the apical-basal axis. On the Drosophila adult cuticle, each hexagonally packed cell elaborates an actin-rich hair that develops from the distal vertex and points distally. Genetic analyses have identified a group of PCP proteins whose activities are required to correctly polarize these arrays. The domineering nonautonomy adjacent to cell clones mutant for some, but not other, PCP genes has not yet been adequately explained. For example, in the Drosophila wing, Van Gogh/strabismus (Vang; encoding a four-pass transmembrane protein) clones disrupt polarity proximal to the mutant tissue, whereas null frizzled (fz; encoding a seven-pass transmembrane protein) alleles disrupt polarity distal to the clone. Models to explain this phenomenon have often invoked diffusible factors, referred to as factor X or Z, because they have not yet been identified. It is proposed instead that the observed behaviors of known PCP proteins are sufficient to explain domineering nonautonomy (Amonlirdviman, 2005).

Fz and other PCP signaling components accumulate selectively on the distal or proximal side of wing cells. Evidence has been provided that these proteins function in a feedback loop that amplifies an asymmetry cue, which converts uniform distributions of PCP proteins into highly polarized distributions. The proposed feedback mechanism depends on several functional relationships. Fz recruits Dishevelled (Dsh; a cytoplasmic protein) to the cell membrane. In addition, Fz promotes the recruitment of Prickle-spinylegs (Pk; a LIM domain protein) and Vang to the cell membrane of the adjacent cell. Feedback is provided by the ability of Pk (and Vang) to cell-autonomously block Fz-dependent recruitment of Dsh. This feedback loop functions strictly locally, between adjacent cells. Global directionality is imposed through the agency of the novel transmembrane protein Four-jointed and the cadherins Dachsous and Fat (Ft). Widerborst, a regulatory subunit of protein phosphatase 2A, accumulates asymmetrically within each cell and is required to bias the feedback loop. Although the mechanism by which Ft biases the direction of the feedback loop is unknown, one possibility is that Ft may direct Widerborst distribution (Amonlirdviman, 2005).

However, it is not readily apparent that this biological model does not readily explain the complex patterns observed in fields of cells containing mutant clones, and it has been argued that it cannot account for some of the observed phenotypes. Indeed, progress in understanding PCP signaling has been hampered by an inability to deduce, given a particular signaling network hypothesis, definitive links between molecular genetic interventions and tissue patterning effects. For example, although it is apparent that removing Dsh or Fz would disrupt the feedback loop, it is not obvious how the feedback loop in adjacent wild-type cells responds, such that dsh mutant clones behave autonomously, whereas for most fz alleles, mutant clones behave nonautonomously. Interestingly, though, for some fz alleles, mutant clones produce an almost cell-autonomous phenotype. As another example, Pk overexpression promotes the asymmetric accumulation of Dsh and Fz, despite the role of Pk in the feedback loop as an inhibitor of Dsh membrane recruitment (Amonlirdviman, 2005).

A mathematical model has been developed based on the described feedback loop and an initial asymmetry input representing the global directional cue. Although mathematical modeling cannot prove the correctness of the underlying biological model, the ability of the mathematical model to capture the known behaviors of the system proves the feasibility of the biological model, provides testable hypotheses, and yields insight into the factors contributing to autonomy and nonautonomy (Amonlirdviman, 2005).

The features of the biological feedback loop model have been represented as a mathematical reaction-diffusion model that describes the concentrations of Dsh, Fz, Vang, and Pk throughout a network of cells. Although the mechanisms that underlie the local feedback loop are not fully understood, the essential logic of this feedback loop is preserved by representing these interactions as binding to form protein complexes (Amonlirdviman, 2005).

Inhibition of Dsh membrane recruitment by Pk and Vang is represented in the mathematical model as an increase in the backward reaction rate of reactions in which Dsh binds Fz (or Fz complexes) by a factor dependent on the local concentration of Pk and Vang. The specific mechanism for the introduction of the directional bias into the feedback loop network is not known. Two forms of a global biasing signal were therefore implemented, and the results using either of these models were similar. The resulting mathematical model consists of a system of 10 nonlinear partial differential equations. With a given set of model parameters, an array of cells could then be simulated, and the resulting hair pattern assigned on the basis of the final distribution of Dsh (Amonlirdviman, 2005).

The model parameters, including the initial protein concentrations, reaction rates, and diffusion constants, were not known, and so these parameters were identified by constraining them to result in specific qualitative features of the hair pattern phenotypes. A sensitivity analysis showed that the model results are not highly sensitive to the precise parameter values and suggests that the conclusions regarding the feasibility of the model are valid for considerable ranges of parameters (Amonlirdviman, 2005).

In simulated wild-type cells, Dsh and Fz localize to the distal membrane, and Vang and Pk localize to the proximal membrane, as is seen in vivo. Simulated clones of cells lacking fz function disrupt polarity in wild-type cells distal to the clones, whereas simulated clones lacking Vang function disrupt polarity on the proximal side of the clones. Simulated clones lacking dsh function result in the disruption of polarity within the mutant cells, but only show a mild effect outside of the clones. The nearly, though not fully cell autonomous, phenotype is similar to that which is observed experimentally. Clones lacking all pk function show only a subtle phenotype. Examination of protein distributions shows that the results are highly concordant with published observations. Similarly, simulated overexpression clones produce results closely mimicking observed experimental results. In simulations and in wings, relatively small clones lacking a global biasing signal show no phenotype, demonstrating that not all cells need to respond to the global directional signal for the feedback loop to cooperatively align all of the cells (Amonlirdviman, 2005).

Previously, it was found that Pk overexpression in the posterior wing domain enhances the accumulation of Fz and Dsh at cell boundaries, despite the observed ability of Pk and Vang to block Dsh recruitment. Consistent with these results, Dsh and Fz are seen in a simulation of this experiment to accumulate to higher levels in the region overexpressing pk than in the wild-type region, and they accumulate perpendicular to the wildtype orientation near the anterior-posterior boundary (Amonlirdviman, 2005).

The results suggested a mechanistic explanation for the difference between autonomous and nonautonomous fz alleles. Because the nearly autonomous fz alleles (fzJ22 and fzF31) have phenotypes similar to dsh clones, it is hypothesized that these alleles may be selectively deficient in complexing with Dsh, but normal in their ability to complex with Vang. Simulations of clones in which the interaction was disrupted between Dsh and Fz by reducing the corresponding forward reaction rates produced nearly cell autonomous polarity phenotypes (Amonlirdviman, 2005).

This hypothesis makes two easily testable predictions. (1) Fz autonomous proteins should be present in the membrane and should recruit Vang to the adjacent membrane, whereas Fz nonautonomous protein should not recruit Vang. It has previously been shown that GFP-tagged FzJ22, expressed in a wild-type background, is present at the apical cell cortex, but remains symmetrically distributed, a distribution in accordance with the simulation of this condition. Examining this further, it was found that in cells adjoining clones of the autonomous fzF31 allele, Vang is recruited to the boundary between wild-type and mutant cells, whereas substantially less Vang is recruited to those boundaries in cells adjoining clones of the nonautonomous fzR52 allele. Thus, Fzautonomous proteins recruit Vang to the opposing cell surface, whereas nonautonomous alleles do not. (2) Autonomous Fz proteins should fail to recruit Dsh. Indeed, it was found that both are substantially impaired in Dsh recruitment, though somewhat less impaired than the very strong, nonautonomous fzR52 allele. Thus, strong fz alleles, many of which fail to accumulate Fz protein, display no or severely impaired interaction with Dsh and Vang, whereas autonomous alleles have impaired interaction with Dsh, but retain substantial ability to recruit Vang to the adjacent membrane. Notably, simulated overexpression of Fz with impaired Dsh interaction also produced the correct polarity disruption in cells proximal to the clones (Amonlirdviman, 2005).

The Dsh1 protein produces nearly autonomous clones, and it carries a mutation in its DEP domain, which is required for membrane localization; autonomous fz alleles bear point mutations in the first cytoplasmic loop, suggesting these mutations may affect the same interaction. A low affinity interaction between the Dsh PDZ domain and a sequence in the cytoplasmic tail of Fz has been demonstrated. These data suggest that sequences in the Dsh DEP domain, and in the Fz first intracellular loop, are also important for Dsh membrane association. Thus, a regulated, bipartite, high affinity association of Dsh with Fz may be selectively disrupted in fzautonomous alleles (Amonlirdviman, 2005).

The ability of the mathematical model to simultaneously reproduce all of the most characteristic PCP phenotypes demonstrates the feasibility of the underlying biological model as a PCP signaling mechanism. Further, the mathematical model demonstrates how the overall scheme of the model -- a local feedback loop between adjacent cells amplifying an initial asymmetry -- can explain the autonomous and nonautonomous behavior of PCP mutant clones. Alternative models invoking diffusible factors have not been supported by the identification of such factors, and the contact-dependent intercellular signaling model more readily accounts for the slight nonautonomy of dsh and fzautonomous clones than do the diffusible factor models (Amonlirdviman, 2005).

The ability of the mathematical model to make predictions and provide a detailed picture of PCP signaling is limited by the lack of complete biological understanding. Although the validity of quantitative model predictions is subject to its assumptions and the set of features used in parameter identification, the model has allowed a direct connection of a biological model to the complex behaviors it is hypothesized to explain and to explore the implications of variations in the model (Amonlirdviman, 2005).

An unconventional nuclear localization motif is crucial for function of the Drosophila Wnt/wingless antagonist Naked cuticle; Nkd targets Dsh-dependent signal transduction steps in both cytoplasmic and nuclear compartments of cells receiving the Wnt signal

Wnt/ß-catenin signals orchestrate cell fate and behavior throughout the animal kingdom. Aberrant Wnt signaling impacts nearly the entire spectrum of human disease, including birth defects, cancer, and osteoporosis. If Wnt signaling is to be effectively manipulated for therapeutic advantage, how Wnt signals are normally controlled must first be understood. Naked cuticle (Nkd) is a novel and evolutionarily conserved inducible antagonist of Wnt/ß-catenin signaling that is crucial for segmentation in Drosophila. Nkd can bind and inhibit the Wnt signal transducer Dishevelled (Dsh), but the mechanism by which Nkd limits Wnt signaling in the fly embryo is not understood. This study shows that nkd mutants exhibit elevated levels of the ß-catenin homolog Armadillo but no alteration in Dsh abundance or distribution. In the fly embryo, Nkd and Dsh are predominantly cytoplasmic, although a recent report suggests that vertebrate Dsh requires nuclear localization for activity in gain-of-function assays. While Dsh-binding regions of Nkd contribute to its activity, a conserved 30-amino-acid motif, separable from Dsh-binding regions, was identified that is essential for Nkd function and nuclear localization. Replacement of the 30-aa motif with a conventional nuclear localization sequence rescued a small fraction of nkd mutant animals to adulthood. This studies suggest that Nkd targets Dsh-dependent signal transduction steps in both cytoplasmic and nuclear compartments of cells receiving the Wnt signal (Waldrop, 2006; full text of article).

This study reports a structure–function analysis of Drosophila Nkd. The finding that nkd mutants have elevated Arm/ß-catenin levels concomitant with broadened domains of Wg target gene expression is consistent with prior reports of Nkd targeting Dsh, an enigmatic Wnt signal transducer that acts upstream of ß-catenin degradation. Although Wnt-signal-induced Dsh accumulation has been observed in cultured cells, transgenic mice, and some cancers, and recent studies indicate that Dsh, like ß-catenin, can be degraded by the ubiquitin–proteasome pathway, the current data show that Nkd does not attenuate Wnt signaling in the embryo by significantly altering steady-state Dsh levels or distribution. If Nkd promotes Dsh degradation in the fly embryo, as has recently been proposed on the basis of overexpression of mammalian Nkd in cultured cells, it must act only on a subset of Dsh, perhaps the fraction engaged in signaling. Consistent with this idea, rare, punctate Nkd/Dsh colocalization was observed in embryonic ectodermal cells (Waldrop, 2006).

Several Nkd constructs with mutant or deleted Dsh-binding regions possessed a reduced but still substantial nkd rescue activity. Perhaps NkdΔR1S/GFPC, lacking both Dsh-binding regions, is able to target Dsh in vivo (and hence rescue a nkd mutant) by virtue of overexpression, through other low-affinity Nkd/Dsh-binding regions, or by as yet uncharacterized proteins that bridge Nkd to Dsh. Consistent with these possibilities, some NkdΔR1S/GFPC/Dsh colocalization was also observed (Waldrop, 2006).

Three independent lines of investigation—evolutionary sequence comparisons, sequencing of lethal nkd alleles, and transgenic nkd rescue assays—pinpointed a 30-aa motif, separable from Dsh-binding regions, that is crucial for fly Nkd activity and nuclear localization. The comparable positions, identical sequence length, and similar predicted structure of insect and mammalian 30-aa motifs suggests that the family of Nkd proteins may inhibit Wnt signaling through a common mechanism. Given the small size and presumably simple α-helical structure of the 30-aa motif, it is unlikely to possess intrinsic catalytic activity but, in addition to its weak NLS activity, it could serve as a protein-docking motif (Waldrop, 2006).

In addition to several reports that have documented nucleo-cytoplasmic shuttling of ß-catenin, Axin, and APC, it is noteworthy that two recent reports revealed a potential role for Fz and Dsh in the nucleus. In response to Wg signaling at the fly neuromuscular synapse, the Fz2 C terminus was detected in puncta of postsynaptic muscle nuclei although not in ectodermal nuclei, so this report's significance to Nkd's action in ectoderm is unclear. Xenopus Dsh has a separable NLS and nuclear export sequence (NES), with the former required for 'signaling activity' in gain-of-function assays. However, a vertebrate Dsh construct with a mutant NES exhibited increased nuclear accumulation but no activity increase relative to that of wild-type Dsh, arguing against nuclear Dsh concentration—at least when it is overexpressed—being rate limiting for activity. Intriguingly, Dsh NES and NLS motifs seem to be conserved in D. melanogaster Dsh, but their significance remains to be investigated (Waldrop, 2006).

These data extend the still rudimentary knowledge of Nkd action in the fly embryo. The epistatic relationship between wg and nkd suggests that, in the absence of Wg ligand, the low levels of Nkd in a wg mutant (because Wg normally upregulates nkd transcription) inhibit spontaneous ligand-independent signaling through the Wnt receptor complex. Wg exposure promotes Arm accumulation and induction of target genes, including en, hh, and nkd. Nkd, synthesized in the cytoplasm, accumulates and targets an uncharacterized fraction of cytoplasmic Dsh. However, Nkd/Dsh binding alone is apparently insufficient to limit Wg signaling during stages 10–11, since Nkd uses its 30-aa motif to inhibit Arm accumulation, restrict Wg-dependent gene expression, and access the nucleus. Although it is possible that the 30-aa motif is required in the cytoplasm, and that the ability of the 30-aa motif to confer nuclear access is a consequence rather than a cause of activity, three lines of evidence support a nuclear role for Nkd: (1) a subpool of Nkd normally accumulates in the embryonic nuclei after stage 10; (2) the 30-aa motif, distinct from the Dsh-binding sequence, is necessary for both nuclear localization and activity and is sufficient to increase the activity of mouse Nkd1 when expressed in the fly; and (3) a heterologous NLS increased nuclear localization and nkd rescue activity of NkdΔ30aa (Waldrop, 2006).

While these experiments strongly suggest a role for Nkd in the nucleus, they do not reveal the nature of that role. Likewise, lacking insight into how Dsh transmits Wnt signals into the nucleus, the experiments thus far reveal neither the relevant subcellular location(s) of Nkd action nor a molecular mechanism by which Nkd inhibits Dsh activity. The punctate Nkd/Dsh colocalization that was observed in embryonic cytoplasm, and rarely, in nuclei, is consistent with Nkd either affecting Dsh nucleo-cytoplasmic transport or impinging directly on the chromatin of Wnt-responsive genes. The inability to observe increased nuclear Nkd or Dsh after treatment with a nuclear export inhibitor suggests that nuclear export of Nkd (and possibly Dsh) in the fly embryo (1) does not occur (e.g., if each protein were degraded in the nucleus following import); (2) occurs over a longer time period relative to proteins such as Lines that can rapidly shuttle between nucleus and cytoplasm; (3) is independent of CRM-1; or (4) like the presumed Nkd/Dsh interaction, involves only a fraction of the total pool of each protein. Future experiments will be required to distinguish among these possibilities (Waldrop, 2006).

The four-domain structure of both insect and vertebrate Nkd's argues that there once existed an ancient 'core' mechanism by which Nkd engaged Dsh to inhibit Wnt signaling. However, given the sequence divergence between insect and mammalian Nkds, their current mechanisms may share little similarity beyond Dsh binding. Recently, PR72 and PR130, two alternatively spliced B'' subunits of the multi-subunit enzyme protein phosphatase 2A (PP2A), were shown to associate with mammalian Nkd and to modulate its inhibitory effect on ectopic Wnt signaling. Since Dsh is phosphorylated by kinases such as CK1, CK2, and Par1 following Wnt stimulation, recruitment of phosphatases to Dsh by Nkd represents an attractive hypothesis to explain the inhibitory action of Nkd on Wnt signaling via Dsh. Consistent with this possibility, Nkd, PP2A, and Dsh kinases co-immunoprecipitated with vertebrate Dsh. However, unlike the vertebrate Nkd/PR72 interaction, thus no direct interactions by Y2H were detected between the fly PR72 homolog (CG4733) and full-length fly Nkd or any of the regions in Nkd, in particular the 30-aa motif, that are crucial for activity. Thus, regulation of Nkd activity by PR72/PR130 may be a derived, vertebrate-specific phenomenon—analogous in some ways to the effect of mammalian Nkd2 but not Nkd1 on intracellular TGF-α trafficking that may be distinct from the mechanism by which Nkd regulates Wg signaling in Drosophila (Waldrop, 2006).

In Drosophila, nkd is crucial for shaping gradients of Wnt activity, but is this role conserved in vertebrates? Mouse nkd genes are expressed during embryogenesis in dynamic patterns reminiscent of known Wnt gradients. A recent report described nkd1 mutant mice with a targeted deletion of exons 6 and 7 (encoding the EFX domain) but allowing in-frame splicing between exons 5 and 8, resulting in expression of a residual Nkd1 protein very much analogous to the NkdΔR1S/GFPC construct that lacks Dsh-binding sequences but retains three conserved motifs. Given that nkd1 is more broadly expressed than nkd2 during mouse development, it was surprising that nkd1–/– mice are viable and fertile, even though mutant mouse embryo fibroblasts show elevated Wnt reporter activity and homozygous male mice exhibit a sperm maturation defect. Although genetic redundancy between nkd1 and nkd2 could account for these observations, the results suggest an alternative hypothesis, namely that the residual protein produced in the reported nkd1 mutant mice, like EFX-deleted NkdΔEFX/GFPC and NkdΔR1S/GFPC constructs, has significant activity in vivo, despite the observation that a mutant mNkd1 protein lacking the EF hand is defective at blocking Wnt signaling in cultured cells. Resolution of this quandary awaits an investigation of strong loss-of-function mutations in each mammalian nkd gene. Given the broad involvement of Wnt/ß-catenin signaling in mammalian development and cancer, coupled with the similar loss-of-function phenotypes of fly nkd, axin, and apc homologs, it is hoped that these studies guide future investigations of vertebrate Nkd proteins as regulators of Wnt signaling and candidate tumor suppressor genes (Waldrop, 2006).

Frizzled-Dishevelled signaling specificity outcome can be modulated by Diego in Drosophila

Members of the Frizzled (Fz) family of seven-pass transmembrane receptors are required for the transduction of both Wnt-Fz/β-catenin and Fz/planar cell polarity (PCP) signals. Although both pathways transduce signals via interactions between Fz and the cytoplasmic protein Dishevelled (Dsh), each pathway has specific and distinct effectors. One explanation for the pathway specificity is that signal-induced conformational changes result in unique Fz-Dsh interactions. Mutational analyses of Fz-Dsh activities in vivo do however not support this model, since both pathways are affected by all mutations tested. Alternatively, the interaction of Fz or Dsh with other proteins could modulate the signaling outcome. The role of a Dsh-binding PCP molecule, Diego (Dgo), was studied in both Wnt-Fz/β-catenin and Fz/PCP signaling. Both loss-of-function and gain-of-function results suggest that Dgo promotes Fz-Dsh/PCP signaling at the expense of Wnt-Fz/β-catenin signaling. The data suggest that Dgo sequesters Dsh to a functionally distinct Fz/PCP signaling compartment within the cell (Wu, 2008).

It has suggested that the KTXXXW motif in Fz C-tails is important for the activation of Wnt-Fz/β-cat signaling targets, but conversely other data implied that this motif was dispensable for Wnt-Fz/β-cat signaling. This issue was addressed in a physiological context. Expressing Fz under control of the tubulin (tub)-promoter fully rescues Fz-activity in fz, fz2 double mutant flies with respect to both Wnt-Fz/β-cat and Fz–PCP signaling. Importantly no dominant phenotypes result from tub-fz expression and thus the tubulin promoter presumably drives the Fz transgenes close to endogenous levels. All Fz C-tail isoforms defective in the Dsh interaction motifs in the C-tail and third cytoplasmic loop (M469R) failed to rescue Wnt-Fz/β-cat signaling, indicating that the Dsh interacting sites are important for Wnt-Fz/β-cat signaling in vivo (Wu, 2008).

The function of the KTXXXW C-tail motif in PCP signaling has previously not been determined. All KTXXXW mutations tested in vivo in this study failed to rescue the fz PCP mutant phenotypes, indicating that the Dsh interacting sites are required for both signaling pathways. These data suggest that there are no obvious differences in how Fz and Dsh interact with each other in the context of either pathway, and therefore that additional factors are likely involved to modulate the signaling outcome and to provide specificity (Wu, 2008).

Dgo is a core Fz/PCP signaling factor (Feiguin, 2001: Jenny, 2005). During the interactions of the core PCP factors, Fz, Dsh, and Dgo become localized to the distal end of pupal wing cells (or the R3-side of the R3/R4 border in the eye), suggesting that they form a functional complex (Axelrod, 2001; Das, 2004; Strutt, 2001). The localization of Dsh and Dgo depends on Fz (Axelrod, 2001; Das, 2004). Dgo localization also partially depends on Dsh and Dgo and Dsh interact physically (Jenny, 2005). Taken together, these data are consistent with the notion that Fz, Dsh, and Dgo are forming a functional complex during PCP signaling that promotes Dsh PCP-activity (Wu, 2008).

Co-expression of Dgo enhances Fz-mediated inhibition of Wnt-Fz/β-cat signaling in the wing. Furthermore, overexpressed Dgo sequesters more Dsh into the subapical junctional region (where PCP signaling takes place) in a Fz dependent manner, suggesting that a Dgo-Dsh association sequesters Dsh away from canonical Wnt-signaling. Thus, a Fz–Dsh–Dgo complex selectively acts in Fz/PCP signaling and is likely not active for the Wnt-Fz/β-cat pathway (Wu, 2008).

Does Dgo affect the levels of Wnt-Fz/β-cat signaling in a loss-of-function (LOF) scenario? Although dgo LOF alleles show only minor effects on Wnt-Fz/β-cat associated phenotypes, double mutant LOF combinations of dgo and nmo, a mild inhibitor of Wnt-Fz/β-cat signaling, show more robust defects (manifest in the observation that the nmoP allele, which does not show Wg GOF defects, in combination with dgo LOF alleles frequently displayed ectopic margin bristles). This indicates that in vivo Dgo can affect the levels of Wnt-Fz/β-cat signaling, but redundantly with other Wg-signaling inhibitors. It is interesting to note that while Dgo presumably acts at the level of Dsh, Nmo phosphorylates the nuclear transcription factors of the TCF family and thus inhibits their association with β-cat and/or the DNA (Wu, 2008).

How does Dgo negatively affect Dsh in Wnt-Fz/β-cat signaling? in vivo data suggest that Dgo acts mainly by sequestering Dsh away from the cytoplasmic and/or basolateral cell regions where Wnt-Fz/β-cat signaling is thought to take place. Thus, a Dgo influenced shift in Dsh subcellular localization, caused either by loss or excess of Dgo, makes the pathway sensitive to additional changes. When Dsh itself or other Wnt-Fz/β-cat signaling factors become more limiting, alteration of Dgo levels can have effects on Wnt-Fz/β-cat signaling strength (Wu, 2008).

Does Dgo affect overall Dsh levels? Studies with the vertebrate Dgo homologue Inversin have suggested that Inversin, the vertebrate Dgo homologue, can affect Dsh levels through ubiquitination and associated degradation in HEK 293T cells (Simons, 2005). It seemed thus possible that Dgo affected the overall Dsh levels: Dgo could stabilize Dsh at the subapical membrane but cause its destabilization in the cytoplasm. However, no evidence was seen for a destabilization mechanism in vivo or in HEK 293T cells. Thus, it seems that Dgo and Inversin do not share this biochemical property (Wu, 2008).

Diversin is a second Dgo-related vertebrate factor that can act as a repressor of Wnt-Fz/β-cat signaling (Schwarz-Romond, 2002; Simons, 2005). The Diversin and Dgo sequences C-terminal to the Ankyrin repeats do not share homologous domains, although clusters of high homology are present. Diversin is thought to inhibit Wnt-Fz/β-cat signaling through its interaction with Axin and CKIε (Schwarz-Romond, 2002). Dgo does not interact with Axin. Thus, it appears that both Diversin and Inversin can inhibit Wnt-Fz/β-cat signaling by (at least partially) different mechanisms from Dgo, suggesting that these features have diverged evolutionarily (Wu, 2008).

Taken together, the in vivo and cell culture data suggest that Dgo can negatively affect Wnt-Fz/β-cat signaling by trapping Dsh in a Fz/PCP specific complex that is inactive for canonical Wnt-Fz/β-cat signaling. Comparative analyses with Dgo, Inversin, and Diversin will be interesting to shed light on conserved mechanisms of action for these three related proteins (Wu, 2008).

Dishevelled interactor Naked cuticle engages the nuclear import adaptor Importin-α3 to antagonize Wnt/β-catenin signaling

Precise control of Wnt/β-catenin signaling is critical for animal development, stem cell renewal, and prevention of disease. In Drosophila, the naked cuticle (nkd) gene limits signaling by the Wnt ligand Wingless (Wg) during embryo segmentation. Nkd is an intracellular protein that is composed of separable membrane- and nuclear-localization sequences (NLS) as well as a conserved EF-hand motif that binds the Wnt receptor-associated scaffold protein Dishevelled (Dsh), but the mechanism by which Nkd inhibits Wnt signaling remains a mystery. This study identified a second NLS in Nkd that is required for full activity and that binds to the canonical nuclear import adaptor Importin-α3. The Nkd NLS is similar to the Importin-α3-binding NLS in the Drosophila heat-shock transcription factor (dHSF), and each Importin-α3-binding NLS required intact basic residues in similar positions for nuclear import and protein function. These results provide further support for the hypothesis that Nkd inhibits nuclear step(s) in Wnt/β-catenin signaling and broaden the understanding of signaling pathways that engage the nuclear import machinery (Chan, 2008).

A growing body of evidence indicates that the traditionally 'cytoplasmic' Wnt signal transducers Axin, Apc, and Dsh also act in the nucleus. In the cytoplasm, Apc promotes β-catenin degradation and regulates the cytoskeleton, but nuclear Apc can recruit transcriptional corepressors to Wnt target genes and, like Axin, escort β-catenin from the nucleus. In light of recent evidence that Axin/Dsh oligomers cross-link Wnt-bound receptors at the plasma membrane during signal activation, it remains unclear whether the nuclear roles for Axin or Dsh are similar to their cytoplasmic functions or whether they, like Apc, have novel nuclear functions (Chan, 2008).

Nkd is also a conserved Wnt signal regulator whose subcellular localization initially suggested a cytoplasmic site of action. These studies have shown that fly Nkd is composed of discrete motifs that confer membrane localization and binding to Dsh, as well as two NLSs. In addition to Nkd targeting an uncharacterized fraction of Dsh in the cytoplasm and/or at the plasma membrane, these data strongly support a nuclear role for Nkd, but whether Nkd inhibits Wnt signaling by altering nucleo-cytoplasmic transport of critical signaling components, such as Dsh or Arm, or by acting on the chromatin of Wnt target genes remains to be elucidated (Chan, 2008).

Genetic epistasis can be a powerful method to infer the regulatory logic of signal transduction cascades. Because Nkd is a 'side-regulator' whose loss-of-function phenotype is dependent on intact Wg signaling, double-mutants between nkd and dsh or arm did not help discern at which level Nkd inhibits the linear Wg signaling pathway. However, Nkd overexpression suppressed the gain-of-Wg signaling phenotype caused by overexpression of Dsh but not that caused by overexpression of an N-terminally deleted and hence degradation-resistant Arm/β-catenin; taken together with the observation that Nkd binds Dsh, epistasis experiments have led to the conclusion that Nkd acted at the level of Dsh and not 'downstream' of Arm/β-catenin in Wg signaling. However, in view of the present data, Nkd might also act in the nucleus at or above the level of Arm/β-catenin. Unfortunately, overproduction of wild type Arm is without phenotypic consequence, presumably because of an excess capacity of the β-catenin 'destruction complex' to degrade ectopic Arm, thus preventing the making further conclusions at present about the epistatic relationship between Nkd and endogenous, degradation-sensitive Arm/β-catenin (Chan, 2008).

Despite the deletion of Dsh-binding sequences in otherwise wild type Nkd having only a minor effect on cuticle rescue activity, the present experiments further support the hypothesis that the Nkd/Dsh interaction is important for Nkd to inhibit Wg signaling. However, the experiments thus far do not clarify how the interaction is regulated in vivo or whether it must occur in the cytoplasm, nucleus, or both locations. Nevertheless, several lines of evidence indicate that a Nkd/Dsh interaction in the cytoplasm and/or near the plasma membrane is important for Nkd function: First, both proteins are predominantly cytoplasmic and/or membrane-associated. Second, punctate cytoplasmic Nkd/Dsh colocalization can be observed in embryos and in salivary gland. Third, the Dsh-binding EFX motif fused to GFP was predominantly cytoplasmic. Fourth, deletion of Dsh-binding sequences in Nkd promoted nuclear localization, consistent with Dsh anchoring Nkd in the cytoplasm. Fifth, deletion of both Nkd NLSs eliminated nuclear localization whether or not Dsh-binding sequences were present, but Dsh-binding sequences were required for Nkd activity (Chan, 2008).

How Nkd acts on Dsh in the cytoplasm to inhibit Wg signaling is not known. One possibility is that Nkd sequesters Dsh away from Fz and/or Axin during signal activation, freeing Axin to regenerate β-catenin destruction complexes. Alternatively, Nkd might target 'activated' Dsh, possibly the pool of Dsh bound to the Wnt receptor complex, for degradation; consistent with Nkd targeting only a fraction of Dsh is the minimal colocalization of the two proteins in embryos as well as the lack of any obvious changes in Dsh levels in nkd mutants. Since Nkd can block the gain-of-Wg signaling phenotypes caused by overexpression of the Dsh kinase CK1, a third possibility is that Nkd blocks CK1-dependent phosphorylation of Dsh via a steric mechanism, although the relationship between Dsh phosphorylation status and activity remains unclear. Future experiments should clarify this issue, because each of these hypotheses makes distinct predictions about phosphorylation status and associated proteins in a native Nkd/Dsh complex (Chan, 2008).

These studies also provide several lines of evidence that the Nkd/Dsh interaction is not sufficient for Nkd to inhibit Wg signaling, and that binding in the nucleus might also be required to fully antagonize Wg signaling. First, the Dsh-binding regions of Nkd when overexpressed blocked phenotypes induced by Dsh overexpression but had no nkd rescue activity. Second, (fly) Nkd and (vertebrate) Dsh have NLSs, although it is not yet known whether fly Dsh acts in the nucleus. Third, rare punctate Nkd/Dsh nuclear colocalization can be observed by confocal microscopy in fly embryos. Fourth, the SV40-NLS increased NkdΔ30aa/GFPC activity when Dsh-binding sequences were intact but reduced activity when Dsh-binding sequences were deleted. The possibility cannot be ruled out that the activity of NkdΔ30aaNLS/GFPC, some of which remains outside the nucleus despite the strong heterologous NLS, is due to cytoplasmic Nkd/Dsh interactions. Similarly, NkdΔR1SΔ30aaNLS/GFPC, which was exclusively nuclear in embryos, might lack activity because of its inability to bind and be retained by Dsh in the cytoplasm. While one must be cautious when inferring site(s) of protein action from subcellular localizations, these studies collectively suggest that fly Nkd is required at multiple locations in Wg-receiving cells (Chan, 2008).

The Nkd-D6 motif has been subject to intense selection pressure, as it is identical in Nkd from D. pseudoobscura, a fly species that diverged from D. melanogaster approximately one billion generations ago. Similarly, the 30 aa NLS is part of a 58 aa motif, and the EFX is part of a 91 aa motif, that are also identical in the two Drosophila species. Using yeast two hybrid, Nkd-EFX residues have been identified that are either dispensable or critical for NkdEFX/DshbPDZ interactions, suggesting that interactions between the EFX motif and proteins other than Dsh might enforce strict motif conservation. Although each NLS contributes to Nkd activity and nuclear localization, heterologous NLSs did not fully replace the function of each Nkd NLS in rescue assays, and in both cases in this work, a heterologous NLS was deleterious to protein function. Absolute conservation of each of these motifs implies that both the tertiary structure and every square angstrom of each motif's surface are necessary for species survival. These experiments suggest that each Nkd motif is required for distinct thresholds and/or duration of Wg signal inhibition: the N-terminal and 30 aa motifs were required for reduction of Arm levels by stage 10, whereas the Dsh-binding EFX and Importin-α3-binding D6 motifs were dispensable to reduce Arm levels but were required, either directly or indirectly, to fully repress en and/or wg transcription by stage 11. Since the deletion of two highly conserved motifs (EFX and D6) preserved the mutant Nkd protein's ability to reduce Arm levels during stage 10, it seems unlikely that these motifs will be shown to possess an intrinsic catalytic activity. The hypothesis is therefore favored that Nkd acts as an inducible protein scaffold, with each of the conserved motifs able to bind additional protein(s). Perhaps there exist distinct Nkd-complexes depending on the subcellular compartment, state of signal activation, or time following signal initiation (Chan, 2008).

Alignment of the Importin-α3-binding NLSs in Nkd and dHSF revealed several conserved residues. Interestingly, the dHSF-NLS has been shown to be bifunctional, suppressing dHSF trimerization in the absence of heat-shock, and in response to heat or other stress conferring Importin-α3-dependent dHSF nuclear translocation and transcriptional induction of heat-responsive genes such as hsp70. These data suggest that the Nkd D6-NLS is also bifunctional, conferring Importin-α3-dependent nuclear localization as well as possibly binding nuclear protein(s) that repress Wg target gene transcription in some cells through stages 10-11. While non-import (presumably scaffolding) functions for Importin-αs have been inferred from phenotypes observed with importin-α deficiency in flies and worms, all of the current experiments support the hypothesis that the Nkd/Importin-α3 interaction promotes nuclear localization. The central region of Importin-α consists of 10 alpha-helical 'Arm' repeats (so named because they were first identified in the Drosophila Arm protein) stacked to form a banana-shaped molecule, the concave side of which harbors a groove that binds basic residues within NLSs. At present, it is not possible based on primary sequence to predict which Importin-α a given NLS will bind, although both the NLS and its three-dimensional context (i.e., adjacent sequence) have been demonstrated to contribute to NLS/Importin-α specificity. Future experiments will investigate whether the residues conserved between Nkd and dHSF represent a consensus Importin-α3-specific binding motif (Chan, 2008).

Vertebrate Nkds have a conserved 30 aa motif between the EFX and C-terminal histidine-rich regions, but whether the vertebrate proteins act in the nucleus like fly Nkd is not known. In this regard, no obvious difference was observed between the subcellular localizations of mouse Nkd1 fused to C-terminal GFP (mNkd1GFPC) vs. a similar construct that lacks the 30 aa motif (mNkd1Δ30aa/GFPC) when the proteins were produced in cultured mammalian cells. However, no obvious difference was observed between fly Nkd and NkdΔ30aa localizations in Drosophila S2 cells, but the differences in localization and function of these two constructs when produced in nkd mutant embryos were dramatic. These findings illustrate the importance of investigating the subcellular localizations of mutant proteins in a native environment that lacks the endogenous wild type protein. It might therefore be interesting to examine the subcellular localization of vertebrate Nkd constructs in nkd-mutant mice or zebra fish just as has been done in Drosophila. More importantly, future experiments must address the critical question of how Nkd antagonizes Wnt/β-catenin signaling in each of the subcellular compartments to which it localizes (Chan, 2008).

Differential stability of flamingo protein complexes underlies the establishment of planar polarity

The planar polarization of developing tissues is controlled by a conserved set of core planar polarity proteins. In the Drosophila pupal wing, these proteins adopt distinct proximal and distal localizations in apicolateral junctions that act as subcellular polarity cues to control morphological events. The core polarity protein Flamingo (Fmi) localizes to both proximal and distal cell boundaries and is known to have asymmetric activity, but the molecular basis of this asymmetric activity is unknown. This study examined the role of Fmi in controlling asymmetric localization of polarity proteins in pupal wing cells. Fmi was found to interact preferentially with distal-complex components, rather than with proximal components, and evidence is presented that there are different domain requirements for Fmi to associate with distal and proximal components. Distally and proximally localized proteins cooperate to allow stable accumulation of Fmi at apicolateral junctions, and evidence is presented that the rates of endocytic trafficking of Fmi are increased when Fmi is not in a stable asymmetric complex. Finally, evidence is provided that Fmi is trafficked from junctions via both Dishevelled-dependent and Dishevelled-independent mechanisms. A model is presented in which the primary function of Fmi is to participate in the formation of inherently stable asymmetric junctional complexes: Removal from junctions of Fmi that is not in stable complexes, combined with directional trafficking of Frizzled and Fmi to the distal cell edge, drives the establishment of cellular asymmetry (Strutt, 2009).

The differing ability of overexpressed Fmi to modulate Fz:Dsh and Stbm:Pk levels at junctions could be explained by a number of mechanisms. One likely hypothesis is that Fmi may require a cofactor for a robust interaction with Stbm, and that this cofactor is limiting when Fmi is overexpressed. Alternatively, Fmi may require posttranslational modification or a conformational change to interact with Stbm, and a factor needed for this modification is limiting. The cytoplasmic C-terminal tail of Fmi is a likely region to mediate an interaction with Fz:Dsh or Stbm:Pk; therefore, a truncated form of Fmi was constructed, in which this region is either absent or replaced with GFP (Strutt, 2009).

When overexpressed in pupal wing cells, FmideltaIntra is much more efficient at recruiting Fz and Dsh to junctions than full-length Fmi, an effect similar to that caused by removal of stbm or pk. Stbm is still reduced at junctions, although less than when full-length Fmi is overexpressed. This suggests that the C-terminal intracellular domain of Fmi is dispensible for the interaction of Fmi with Fz:Dsh and, importantly, that Fz:Dsh no longer have to compete with Stbm:Pk for access to Fmi (Strutt, 2009).

Interestingly, two isoforms of Fmi have been identified, one of which contains a PDZ binding motif (PBM) at its C terminus. It is possible that loss of the PBM alone could account for the failure of overexpressed Fmi or FmideltaIntra to associate with Stbm:Pk. However, this is unlikely, because Fmi that lacks the PBM can rescue the planar polarity phenotype of fmi mutants (Strutt, 2009).

Endogenous Fmi is thought to be localized on both proximal and distal cell boundaries. This was confirmed by expressing CFP-tagged Fmi at physiological levels in clones in pupal wings, and it was observed that levels of staining appear similar at each end of the cell, consistent with the homophilic-interaction model. Notably, expression of a GFP-tagged form of FmideltaIntra results in its preferential localization to distal cell edges, where Fz and Dsh also localize (Strutt, 2009).

Interestingly, junctional localization of FmideltaIntra-EGFP is not dependent on endogenous, full-length Fmi, suggesting that this molecule is still able to participate in homophilic interactions. Hence, the ability of FmideltaIntra-EGFP to functionally rescue the polarity phenotype of fmi null mutant clones was investigated. If FmideltaIntra-EGFP interacts preferentially with the distal Fz:Dsh complex, then Stbm recruitment to junctions inside clones would be compromised. Consequently, FmideltaIntra-EGFP:Fz complexes inside the clone would preferentially interact with Fmi:Stbm outside the clone, leading to a reversal in polarity on proximal clone edges. Importantly, this prediction is upheld, and fmi clones rescued with FmideltaIntra-EGFP exhibit weak proximal polarity inversions, such that trichomes point away from the clone, and polarity proteins are recruited to the clone boundary (Strutt, 2009).

Nevertheless, Stbm localizes asymmetrically inside the clone, although not always at the correct site, whereas in a fmi null mutant it lacks any asymmetric localization. Thus, FmideltaIntra-EGFP must retain some ability to interact with Stbm. To confirm this, the ability of full-length Fmi or FmideltaIntra-EGFP to interact with Fz and Stbm in Drosophila S2 cells was analyzed. In this assay, Fmi and FmideltaIntra-EGFP are recruited to sites of cell contact, as a result of homophilic interactions between their extracellular domains. Cotransfection of Fz or Stbm with either full-length Fmi or FmideltaIntra-EGFP in Drosophila S2 cells results in the recruitment of both to sites of cell contact (Strutt, 2009).

Interestingly, if S2 cells were transfected with either Fz or Stbm and then mixed, weak recruitment is also observed to sites of cell contact, arguing that their extracellular domains can interact independently of Fmi. Nevertheless, recruitment was weaker and less frequent than when Fmi was cotransfected, suggesting that Fmi:Fmi interactions are more important than Fz:Stbm interactions in stabilizing complexes between adjacent cells (Strutt, 2009).

The data suggest that Fz:Dsh and Stbm:Pk complexes differ in their ability to associate with Fmi. Whereas endogenous levels of Fmi result in the formation of asymmetric complexes with Fz:Dsh on one side of the boundary and Stbm:Pk on the other, overexpressing Fmi favors Fz:Dsh recruitment. Furthermore, a C-terminally deleted form of Fmi preferentially localizes distally with Fz, and overexpression of this form has an even greater preference for Fz:Dsh recruitment. Thus, the C terminus of Fmi is important in promoting the interaction with Stbm:Pk. The Fmi truncation data could be explained simply by the possibility that the C terminus of Fmi contains a direct binding site for Stbm; however, this fails to explain why overexpressed full-length Fmi prefers to recruit Fz:Dsh. It is therefore proposed that the association of Fmi with Stbm:Pk requires a limiting factor that is saturated by Fmi overexpression. The most plausible hypothesis is a requirement for a cofactor for Stbm:Pk binding, but other possibilities include saturation of the machinery for a posttranslational modification or a conformational change in Fmi (Strutt, 2009).

The data also suggest that Fmi itself needs to associate with both proximal and distal components in order to be stably localized to apicolateral junctions. Although it can form homophilic dimers between adjacent cell membranes in tissue culture, in pupal wings Fmi does not localize strongly to apical junctions and presumably fails to form stable homodimers in trans. Fz on one side of the junction and Stbm:Pk on the opposite side stabilize Fmi at junctions, most likely by promoting homophilic interactions or preventing internalization. However, Fmi appears to be capable of forming complexes with either distal or proximal components alone, but these complexes (particularly the proximal complex) are apparently less stable at junctions. Taken together with overexpression experiments, this would suggest that the most stable configuration is Fz:Fmi on one side of the boundary and Fmi*:Stbm:Pk on the other (where Fmi* denotes the modified form able to preferentially associate with Stbm:Pk) (Strutt, 2009).

In order for an asymmetric complex to be stabilized across junctions, the extracellular domains must somehow 'look' different. One possibility is that the Fz and Stbm extracellular loops interact - a view supported by S2 cell data. Alternatively, the Fmi extracellular domain, when associated with either Fz or Stbm:Pk, could undergo a conformational change that promotes homophilic Fmi interactions (Strutt, 2009).

An intriguing question is why clones of cells that overexpress Fmi behave like fz loss-of-function clones. It is suggested that within the clones, excess Fmi associates with the entire available pools of both Fz and Stbm. However, there is still a pool of uncomplexed Fmi that can associate with Fmi:Fz in adjacent wild-type cells, forming the relatively stable Fmi-Fmi:Fz configuration, thus causing polarity to be reversed on distal clone boundaries. In support of this model, an identical nonautonomous effect is seen when FmideltaIntra is overexpressed, which itself interacts only poorly with Stbm but presumably can interact with Fmi:Fz in adjacent cells outside the clone (Strutt, 2009).

Interestingly, Fmi accumulates in excess at junctions in a dsh, stbm double mutant, whereas Fz does not. Thus, although Fz acts to stabilize Fmi at junctions, Fmi does not always need to associate with Fz in a stoichiometric fashion in order to be stabilized. Perhaps as long as there is some Fz associated with Fmi, this may permit local stabilization of other Fmi molecules in cis. Alternatively, this excess accumulation of Fmi might simply represent 'unstable' Fmi homodimers that are no longer being removed from junctions by the actions of Dsh and Stbm (Strutt, 2009).

The composition of the complex with which Fmi is associated appears to be critical for determining the frequency and manner by which Fmi is turned over from the plasma membrane. Most compellingly, Fmi accumulates more strongly in an enlarged endosomal compartment in Rab7TN mutant tissue when stbm and fz are absent than when they are present. Thus, it is suggested that more Fmi is resident in the endocytic pathway when it is unable to form stable asymmetric complexes. Fmi:Fz puncta have been observed that are selectively trafficked to distal cell edges. In the current experiments, these puncta colocalize with YFP-Rab4, suggesting that Fmi and Fz are recycled back to the plasma membrane by a Rab4-dependent mechanism. Furthermore, the increased intracellular and junctional levels of Fz and Fmi in dor mutant clones suggests that in addition to being recycled to the plasma membrane, a significant fraction of internalized Fmi and Fz is also sent for degradation. It is formally possible that the intracellular accumulation of Fmi and Fz seen when lysosomal trafficking is blocked by loss of Rab7 or in dor clones is due to their being sent for degradation immediately after synthesis (e.g., if damaged or misfolded); however this is unlikely because newly synthesized Fmi-ECFP appears first at junctions before been seen in puncta (Strutt, 2009).

Stbm has not been observed in large intracellular puncta, but it seems likely that it is also internalized and recycled, possibly together with Fmi, although it must do so by alternative pathways involving smaller or more rapidly recycling particles that are not visible by confocal microscopy. Indeed, the data suggest a potential role for Dsh and Stbm in regulating junctional levels of Fmi. A stbm mutant alone results in a loss of Fmi from junctions, consistent with a need for Stbm in stabilizing Fmi in asymmetric complexes. In contrast, loss of Dsh and Stbm together increases Fmi levels at junctions, suggesting a role for Stbm in internalization. It is suggested that the outcome of any interaction of Stbm with Fmi is dependent upon whether Fmi is able to form stable homodimers with Fz on the opposite cell membrane. In a wild-type situation, one could envisage that Fmi forms stable homodimers in a Fz:Fmi-Fmi*:Stbm configuration, and that both Dsh and Stbm promote internalization of any Fmi that is not in this configuration, the majority of which is subsequently recycled back to the plasma membrane. In dsh mutants, there is reduced internalization, but the effect on Fmi levels is subtle; Fz and Stbm are still present to promote Fmi homodimer formation, and Stbm still promotes internalization of any unstable Fmi. In contrast, in stbm mutants, the number of less stable Fmi complexes (associating only with Fz) is greatly increased, favoring internalization by Dsh. Finally in dsh, stbm double mutants, Fmi is again less stable (associating only with Fz), but there is no Dsh- or Stbm-mediated internalization, leading to an overall increase of Fmi at junctions (Strutt, 2009).

How do Dsh and Stbm regulate Fmi levels at junctions? Stbm contains potential interaction motifs for the endocytic adaptor AP2, but their role has not been functionally tested. In addition, in vertebrate Wnt signaling, there is evidence that Dsh interacts with the endocytic adaptor protein β-arrestin and mu2 subunit of AP2 to mediate Wnt/Fz endocytosis and downregulation of Wnt signaling. Interestingly, in planar polarity this is no evidence that Dsh directly mediates internalization of Fz, but the data rather point to Dsh promoting Fmi internalization when it is not associated with Fz. Instead, the trafficking of Fmi together with Fz into the lysosomal pathway is Dsh independent (Strutt, 2009).

In summary, it is proposed that a number of mechanisms exist by which Fmi contributes to the generation of asymmetry at the molecular level. First, the characterization of the previously inferred asymmetry in Fmi activity indicates that Fmi normally prefers to bind to Fz and requires a limiting factor for association with Stbm:Pk. Second, Fmi stability at junctions is dependent on both Fz and Stbm:Pk, with the most stable form being Fz:Fmi bound to Fmi*:Stbm. Finally, it is proposed that entry of Fmi into the endocytic trafficking pathway is decreased if it is in a stable complex, and this is regulated either by Dsh and Stbm or independently of Dsh and Stbm, depending on whether it is associated with Fz (Strutt, 2009).

An outstanding question is how these mechanisms translate into cellular asymmetry, such that in any particular cell, heterophilic polarity complexes preferentially form with Fz:Dsh at the distal junctions, rather than having heterophilic complexes in both orientations. It is thought that the acquisition of cellular asymmetry is likely to be driven by directional trafficking of Fmi:Fz, although other models, such as a mechanism for preferential stabilization of Fmi:Fz interactions at the distal cell edge, are also possible. In addition, it seems likely that an amplification mechanism would be required, although the molecular mechanisms remain to be elucidated (Strutt, 2009).

While this manuscript was in preparation, another manuscript was published, in which Fmi was proposed to mediate an asymmetric and instructive signal between proximal and distal complexes to generate asymmetry, and thus does not act merely as a scaffold for Fz:Stbm interactions across membranes. It is argued that the current data do not provide evidence for a specific signaling function of Fmi. Instead, the hypothesis is favored that the composition of the proximal and distal complexes is distinct, and that heterophilic complexes are inherently more stable than homophilic complexes. Together, removal of unstable nonasymmetric complexes through increased endocytic turnover, in concert with directional trafficking and an unknown amplification mechanism, may be sufficient to generate asymmetry without the need to invoke a specific signaling function for any components of the complexes (Strutt, 2009).

The trimeric G protein Go inflicts a double impact on axin in the Wnt/frizzled signaling pathway

The Wnt/Frizzled signaling pathway plays crucial roles in animal development and is deregulated in many cases of carcinogenesis. Frizzled proteins initiating the intracellular signaling are typical G protein-coupled receptors and rely on the trimeric G protein Go for Wnt transduction in Drosophila. However, the mode of action of Go and its interplay with other transducers of the pathway such as Dishevelled and Axin remained unclear. This study shows that the alpha-subunit of Go directly acts on Axin, the multidomain protein playing a negative role in the Wnt signaling. G alpha o physically binds Axin and re-localizes it to the plasma membrane. Furthermore, G alpha o suppresses Axin's inhibitory action on the Wnt pathway in Drosophila wing development. The interaction of G alpha o with Axin critically depends on the RGS domain of the latter. Additionally, the betagamma-component of Go (see Gβ13F) can directly bind and recruit Dishevelled from cytoplasm to the plasma membrane, where activated Dishevelled can act on the DIX domain of Axin. Thus, the two components of the trimeric Go protein mediate a double-direct and indirect-impact on different regions of Axin, which likely serves to ensure a robust inhibition of this protein and transduction of the Wnt signal (Egger-Adam, 2009).

This study has demonstrated that Gαo can physically bind the RGS domain of Axin and recruit it to the plasma membrane, the action likely leading to the destabilization of the Axin-based β-catenin destruction complex and propagation of the Wnt signal inside the cell. In support of this idea, this study has shown that Gαo can suppress the Wnt loss-of-function phenotypes induced by Axin over-expression in wing imaginal discs. This rescue critically depends on the presence of the RGS domain, reiterating the crucial role of this domain for the interaction with Gαo. While the GTP-bound form of Gαo is unable to change the phenotypes of the AxinΔRGS expression, the GDP-bound forms of Gαo even dramatically enhance these phenotypes. It is hypothesized that this enhancement is due to sequestration of the Gβγ heterodimer by the GDP-forms of Gαo. It was also shown that Gβγ can directly bind and recruit Dsh from the cytoplasm to the plasma membrane, thus possibly contributing to the propagation of the Wnt signal (Egger-Adam, 2009).

The RGS domain of Axin, responsible for the interaction with Gαo, is important for the full range of Axin activity in wing imaginal discs. Indeed, over-expression of the ΔRGS form of Axin only partially suppresses Wnt signaling in this tissue. The RGS domain of Axin is known to bind APC, another component of the β-catenin-destruction complex. The inability of AxinΔRGS to directly interact with APC is the likely reason for the reduced activity of this construct in Drosophila wings and in vertebrates. The Gαo and Gαq proteins were shown to dissociate the Axin-based destruction complexes in mammalian cells. It is proposed that in Drosophila, Gαo leads to a similar dissociation of the destruction complex through direct binding to the RGS domain of Axin, which recruits Axin to the plasma membrane and probably displaces APC from Axin (Egger-Adam, 2009).

In vitro, the purified RGS domain of Axin binds equally well both the GDP- and the GTP-loaded forms of Gαo. It also lacks the GTPase-activating protein (GAP) activity towards Gαo, typical for other RGS domains. These data agree with the absence of some of the conserved residues required for the GAP action in Axin RGS. Thus, biochemically Axin binds Gαo regardless of its nucleotide form. However, in vivo the GDP- and the GTP-loaded forms of Gαo behave differently towards Axin. Only Gαo[GTP] is capable of recruiting Axin-GFP to the plasma membrane in the salivary glands. Similarly, Gαo[GTP] is much more potent in rescuing the Axin full-length over-expression effects in wing imaginal discs and adult wings. This seeming contradiction is explained by the fact that in vivo the GDP-loaded forms of Gαo bind the βγ-subunits, recreating the trimeric Go complexes. Indeed, over-expressed, the wild-type Gαo was shown to compete with other Gα proteins for the βγ-subunits. Only the Gαo[GTP] form can stay free and thus exert its activities on Axin in full (Egger-Adam, 2009).

In contrast, the wild-type Gαo also possesses a capacity of over-activating the Wnt pathway in wing imaginal discs, and can to a certain degree rescue the phenotypes of Axin over-expression in this tissue. This contrasts with its inability to recruit Axin-GFP to the plasma membrane in salivary glands. These differences between the two tissues correlate with the degree of Wnt signal transduction. Indeed, the Wnt pathway is highly active in the wing imaginal discs, and Gαo can further enhance the pathway relying on the activity of Fz receptors. In contrast, in larval salivary glands the Wnt pathway is silent, which is illustrated by the cytoplasmic localization of Dsh in this tissue, expected to be plasma membrane localized when the pathway is on. It thus seems probable that in the salivary glands Gαo, forming trimeric Go complexes with Gβγ, fails to be further converted into the monomeric form due to the absence of the Wnt/Fz activity. In contrast, wing imaginal discs provide enough Wnt/Fz activity to activate endogenous as well as exogenous Go, which can then recruit Axin and thus propagate the signal (Egger-Adam, 2009).

The ability of the GDP-bound forms of Gαo to bind to the βγ-subunits is the likely reason for the aggravation of the AxinΔRGS phenotype induced by Gαo. This form, even upon conversion to the GTP-bound state by the action of the Wnt/Fz complexes, can no longer bind the RGS-lacking Axin and suppress Axin's negative action on the Wnt signal transduction. However, it can bind Gβγ. It is proposed that Gβγ plays, in addition to Gαo-GTP, a positive role in the Wnt signal transduction through its ability to bind and recruit Dsh to the plasma membrane. Over-expression of Gαo reduces the amounts of free Gβγ, reducing the efficiency of Dsh re-localization. It is proposed that when the endogenous full-length Axin is present, over-expression of Gαo has the overall stimulating effect on the Wnt signaling in wing discs due to increased generation of Gαo-GTP, which binds and antagonizes Axin. It is only in the artificial situation of over-expression of AxinΔRGS that the other, negative, effect of Gαo can be revealed. To prove that Gαo aggravates the AxinΔRGS phenotypes due to sequestration of Gβγ, the mutant Gαo[GDP] protein unable to charge with GTP but still capable to bind Gβγ was ested, and this form was found to be similar to Gαo in enhancing the AxinΔRGS phenotypes (Egger-Adam, 2009).

Direct experiments were performed testing the involvement of Gβγ in Wnt signaling. In accordance with predictions, down-regulation of Gβγ results in a clear reduction of the Wnt signaling in Drosophila wings and wing discs, affecting the short-range target genes of the Wnt pathway. As over-expression of Gβ alone leads to trapping Dsh in the cytoplasm, such over-expression also produces drastic dominant effects on Wnt signaling in wing discs. Unfortunately, it was not possible to confirm that Dsh was trapped in the cytoplasm of the epithelial cells of such discs due to the low resolution of the Dsh staining obtained in these thin columnar cells. Additionally, not only localization but also abundance of the components of the Wnt pathway are known to change in cells with high levels of Fz activation as part of the feedback regulation. Thus, interpretation of Dsh localization in wing imaginal discs upon perturbations of the Wnt pathway will be difficult. Instead, analysis of a tissue where the Wnt pathway is endogenously silent, such as salivary glands, allows analysis of the direct influence of the subunits of the trimeric Go complex on cellular localization of the components of the Wnt pathway. This analysis led to the identification of the plasma membrane re-localization of Axin by Gαo and of Dsh by Gβγ as such direct cellular responses. These primary responses are probably then utilized in the physiological context as the basis to build positive and negative feedbacks for the final outcome of Wnt signal propagation (Egger-Adam, 2009).

While the numerous data indicate that Gβγ is necessary for the proper activation of the Wnt pathway, probably through plasma membrane re-localization of Dsh, it was not possible to over-activate the Wnt pathway by over-expression of Gβ and Gγ together. Instead, the pathway was down-regulated, although to a weaker extent than that seen by over-expression of Gβ alone. This observation is not easy to reconcile with the other data. One possible explanation is that in the wing discs, unlike the salivary glands, co-overexpression of Gγ might be insufficient to attract the complete pool of Gβ to the plasma membrane, and significant amounts of Gβ may still remain cytoplasmic and retain Dsh. Along these lines, co-overexpression of Gγ shows a partial 'rescue' of the phenotypes induced by Gβ over-expression. Another possible explanation involves the notion of the negative feedback regulation in the Wnt cascade. Proteosomal degradation of Dsh during Wnt signal transduction has been demonstrated. A recent work has shown that targeted plasma membrane localization of Dsh by the Wnt activation or by the Gβγ subunits also destines it for the lysosomal degradation in vertebrate cells. Thus, the activity of Gβγ in the Wnt signaling may be multistep: the initial recruitment of Dsh from the cytosol may serve to activate the pathway, but the persistent membrane localization will lead to Dsh degradation. While Gβ RNAi targeting shows that the Gβγ complex is necessary for the proper Wnt signaling, activation of such a negative feedback loop may underlie the phenotypes observed upon the persistent over-expression of Gβγ. In this scenario, Gβγ will be added to the growing list of regulators of the Wnt pathway, which have both positive and negative activities in this signaling (Egger-Adam, 2009).

A model ia favored whereby Gβγ-induced plasma membrane re-localization of Dsh serves as an initial positive impact to activate the Wnt signal propagation. If this is correct, what may be the immediate consequences of the Gβγ-induced plasma membrane recruitment of Dsh? This scaffolding protein is known to become hyper-phosphorylated upon plasma membrane localization, which correlates with its activity in the Wnt signal transduction. Dsh is known to directly bind Axin through the DIX domain heterodimerization. Although a direct interaction of Gβγ with Axin's protein phosphatase 2A-binding region (N-terminal to the DIX domain) has recently been demonstrated in mammalian cells, no ability was found of Gβγ to re-localize or directly bind Drosophila Axin. Overall, the data and the above considerations lead to the proposal of the following model of the action of the trimeric Go protein in the Drosophila Wnt/Fz pathway (Egger-Adam, 2009).

The trimeric Go protein is a direct target of the activated Fz receptors. Wnt ligand binding to Fz activates the guanine nucleotide exchange activity of Fz towards Go. This in turn dissociates the trimeric Go complex into Gαo-GTP and Gβγ. It is proposed that both these components of the trimeric complex have the initial positive activity in Wnt signal propagation. Gαo-GTP directly binds to the RGS domain of Axin, recruiting Axin to the plasma membrane and dissociating the Axin-based β-catenin destruction complex. In contrast, Gβγ recruits and contributes to activation of Dsh, which then can bind the DIX domain of Axin and thus also promote dissociation of the destruction complex. These two branches of G protein–mediated signal propagation converge on the Axin complex to cooperatively ensure its efficient inhibition. Such a double effect on Axin emanating from the trimeric Go complex may serve to ensure a robust activation of the Wnt signaling (Egger-Adam, 2009).

Abelson family kinases regulate Frizzled planar cell polarity signaling via Dsh phosphorylation

Abelson (Abl) family tyrosine kinases have been implicated in cell morphogenesis, adhesion, motility, and oncogenesis. Using a candidate approach for genes involved in planar cell polarity (PCP) signaling, Drosophila Abl (dAbl) was identified as a modulator of Frizzled(Fz)/PCP signaling. dAbl positively regulates the Fz/Dishevelled (Dsh) PCP pathway without affecting canonical Wnt/Wg-Fz signaling. Genetic dissection suggests that Abl functions via Fz/Dsh signaling in photoreceptor R3 specification, a well-established Fz-PCP signaling readout. Molecular analysis shows that dAbl binds and phosphorylates Dsh on Tyr473 within the DEP domain. This phosphorylation event on Dsh is functionally critical, as the equivalent DshY473F mutant is nonfunctional in PCP signaling and stable membrane association, although it rescues canonical Wnt signaling. Strikingly, mouse embryonic fibroblasts (MEFs) deficient for Abl1 and Abl2/Arg genes also show reduced Dvl2 phosphorylation as compared with control MEFs, and this correlates with a change in subcellular localization of endogenous Dvl2. As in Drosophila, such Abl-deficient MEFs show no change in canonical Wnt signaling. Taken together, these results argue for a conserved role of Abl family members in the positive regulation of Dsh activity toward Fz-Dsh/PCP signaling by Dsh phosphorylation (Singh, 2010).

Evidence is provided for a specific role of tyrosine phosphorylation of Dsh by Abl family kinases in Fz/Dsh-PCP signaling. dAbl is required for R3/R4 fate specification. dAbl interacts with fz and dsh genetically in PCP signaling. Biochemical experiments indicate that dAbl binds Dsh and phosphorylates it on Tyr473 within the DEP domain, which has been specifically implicated in PCP signaling and is largely dispensable for canonical Wnt/Wg signaling. The data further show that Abl kinases do not affect canonical Wnt signaling in either Drosophila or MEFs. Taken together, these data indicate that Abl family kinases positively regulate PCP signaling by affecting Dsh/Dvl family members via phosphorylation of Tyr473. Abl family kinases appear to provide a molecular gating mechanism to increase the capability of Dsh/Dvl proteins to signal via the Fz/Dsh-PCP pathway. These data suggest the possibility that this function of Abl family kinases might be conserved from flies to mammals, as similar effects were observed with mammalian Abl and Dvl family members (Singh, 2010).

Most Abl studies in mammalian cell culture have focused on their role in tumor formation. Little is known about Abl’s normal physiological roles during development, except in the context of junctional stability and cytoskeletal events. Previous studies in the Drosophila eye established that dAbl is expressed dynamically in all photoreceptors, and dAbl mutant flies have a rough eye phenotype with significant photoreceptor loss. Its potential role in cell fate specification has not been addressed. A detailed analysis of the dAbl eye phenotype was performed in the context of PCP establishment and R3/R4 specification. It was demonstrated that dAbl is required for specification of both R3-R4 cells. Differential activation of Fz/PCP signaling specifies R3 and leads to the activation of Notch signaling in the neighboring R4 to induce its proper fate. The data suggest that dAbl is required in R3 for fate specification via its interaction with Dsh and positive input into Fz/PCP signaling. This Abl function appears to be common to Fz/PCP signaling in general, as Fz and dAbl also synergize in PCP establishment in the wing. Moreover, dAbl phosphorylation of DshTyr473 is essential for Dsh PCP function in general. In vertebrates, Abl kinases affect Dvl localization in MEFs, and Abl1-/-,Abl2-/- mice display similar phenotypes as dvl1-/-,dvl2-/- mice, with severe open neural tube defects in 9.5-day embryos. The requirement of dAbl in R4 specification remains obscure, as Fz/PCP signaling is not required in R4 (Singh, 2010).

A likely explanation derives from studies of dAbl in noncanonical Notch signaling, where dAbl has been suggested to act downstream from Notch. Although the role of Notch in the developing eye has focused on canonical Su(H)-dependent Notch activity, it is quite likely that Abl could modulate Notch signaling activity in R4. Further work will be needed to explain the role of dAbl in R4 fate specification and associated Notch signaling (Singh, 2010).

Dsh contains three highly conserved domains and a stretch of basic residues and several serine/threonine-rich regions between the DIX and PDZ domains, as well as a proline-rich region downstream from the PDZ domain, which encodes a class I consensus sequence for an SH3-binding protein. Many proteins that have been shown to bind Dsh/Dvl bind to Dsh in the PDZ domain. Abl binding, however, maps to the proline-rich region of Dsh just C-terminal to the PDZ domain, while the PDZ domain alone showed no binding. Different Dsh domain requirements are known for canonical and PCP signaling. While the DIX domain functions exclusively in canonical Wnt signaling, the DEP domain is required for PCP signaling and, in particular, stable membrane association. The results indicate that dAbl phosphorylates Dsh at Tyr473 (and possibly other Tyr residues in the DEP/C-term region). Phosphorylation of Dsh-Tyr473 is unique, as dAbl is a tyrosine kinase and all previously analyzed Dsh kinases have been serine/threonine kinases (Singh, 2010).

Wnt signaling is important in diverse physiological processes and, when deregulated, often leads to disease states. Studies in model organisms have unraveled two conserved pathways, now referred to as canonical Wnt/Wg signaling and Wnt-Fz/PCP signaling. The canonical Wnt signal is transduced via Fz family receptors (along with the LRP5/6 coreceptors), leading to Dsh-Axin complex formation, which in turn causes the stabilization of cytoplasmic β-catenin and allows gene transcription. In Fz/PCP signaling, Dsh is recruited to the membrane in an Axin- and LRP5/6-independent manner and acts on different downstream effectors, depending on the cellular context. The mechanism of pathway-specific Dsh 'activation' is poorly understood, and, similarly, the question as to how the individual pathways are specifically activated at the level of either Fz or Dsh remains unresolved. This study highlights the importance of Abl in the context of Fz/PCP signaling at the level of Dsh/Dvl. At the level of both Abl mutants as well as the DshY473F phosphorylation mutant, canonical Wnt/Wg signaling remains unaffected, while PCP signaling is defective. As the signal that activates dAbl in this context is not known, it is possible that Abl acts in a permissive manner in PCP signaling. In conclusion, this study provides evidence for Dsh tyrosine phosphorylation and a role of Abl in PCP signaling; further studies will be needed to establish a full framework for the regulation of Abl in PCP signaling and in the biology of Dsh (Singh, 2010).

dachsous and frizzled contribute separately to planar polarity in the Drosophila ventral epidermis.

Cells that comprise tissues often need to coordinate cytoskeletal events to execute morphogenesis properly. For epithelial tissues, some of that coordination is accomplished by polarization of the cells within the plane of the epithelium. Two groups of genes--the Dachsous (Ds) and Frizzled (Fz) systems--play key roles in the establishment and maintenance of such polarity. There has been great progress in uncovering the how these genes work together to produce planar polarity, yet fundamental questions remain unanswered. The Drosophila larval ventral epidermis has been studied to begin to address several of these questions. ds and fz are shown to contribute independently to polarity, and they do so over spatially distinct domains. Furthermore, it was found that the requirement for the Ds system changes as field size increases. Lastly, it was found that Ds and its putative receptor Fat (Ft) are enriched in distinct patterns in the epithelium during embryonic development (Donoughe, 2011).

In early embryos, the body axis is subdivided into parasegments, each of which is further subdivided into two domains. One half of the epithelial cells will secrete smooth cuticle and the other half will form cuticular protrusions called denticles (the denticle field). The denticle field pattern is the product of a series of distinct polarized events. First, cells align into columns as a consequence of the reorganization of select cell interfaces. Second, one to three F-actin bundles protrude from the posterior edge of each cell. Third, the F-actin bundles guide the secretion of extracellular matrix (cuticle) such that denticles take on their final tapered orientation and hooked shapes. The result is that each column of denticles corresponds to a single column of underlying cells. This study has taken advantage of this polarized pattern to investigate the roles of ds, ft and fz in establishing this planar polarity (Donoughe, 2011).

With each molt, a growing larva secretes a new cuticle that is patterned on the underlying epidermis. Since there are no major cell rearrangements nor any increase in cell number during larval growth, cells of this epithelium maintain their specific fates and relative positions. Thus, the denticle pattern is resynthesized for each successive cuticle, where the columns of protruding denticles remain intact until the next molt, enabling the crawling larvae to grip the substrate during locomotion (Donoughe, 2011).

This study addresses long-standing questions in the planar cell polarity (PCP) field: (1) how do Fz and the members of the Ds system each contribute to planar polarity in an epithelium and (2) how do Ds and Ft influence the polarized placement of F-actin protrusions (Donoughe, 2011)?

This study has elucidated the contributions of several key polarity genes in the larval ventral epidermis. The genes in the Ds system are essential for proper polarity in this tissue. Notably, the Ds extracellular domain is able to reorient adjacent cells even when they are null for ds. The Fz protein operates largely redundantly and in parallel to the Ds system, and appears to contribute more in some columns than others. As field size increases, it is likely that Fz is less able to polarize the tissue on its own. By contrast, the Ds system is able to polarize the tissue equally well at small and large field sizes. Finally, it was found that in embryos, Ds and Ft are enriched in the posterior half of each denticle field. This correlates with the domain of the embryonic denticle field where actin protrusion placement defects appear in ds M-Z- embryos (Donoughe, 2011).

Several observations are in line with what is understood from other tissues. First, the polarity disruptions in ds- or ft- single mutants are comparable in severity to those observed in ds- ft- double mutants. This confirms that Ds and Ft act within the same process to polarize tissues. Second, in the adult abdomen, an experimentally induced high point of Ds extracellular domain expression causes an adjacent cell to reorient its polarity toward that high point (see Ds extracellular domain can reorient neighboring denticle columns). Likewise, overexpression of the Ds extracellular domain in one cell column of an otherwise wild-type larva causes the flanking cell columns to reorient toward this (presumed) enhanced source of Ds. By repeating this experiment in the ds- mutant, any potentially confounding contributions from the superimposed distribution of endogenous Ds were avoided. Therefore, it can be concluded that cells polarize toward high levels of Ds. Whether this is the case during normal patterning is more difficult to address (Donoughe, 2011).

Finally, it was found that gain-of-function effects are propagated farther than just the adjacent cell. Thus, in a wild-type background, excess Ds in column 1 caused reorientation in columns 2 and 3. This implies that the signal was received in column 2 (resulting in altered polarity there), and then a polarizing effect was propagated to column 3. When such overexpression was repeated in a ds- background, however, column 2 reoriented whereas column 3 largely did not. This demonstrates that Ds is not required for a cell to respond to a Ds polarity signal, but it is important in propagating that signal onward. Altogether, these findings support the hypothesis that Ds and Ft work together to send, implement and propagate a polarity signal (Donoughe, 2011).

A central focus of ongoing research is to determine how the Fz and Ds systems each contribute to the establishment and maintenance of planar polarity. In both the Drosophila eye and wing it appears that the Ds system provides a directional cue that is amplified and implemented by the Fz system. In the abdomen, by contrast, the Ds system can polarize in the absence of Fz and Stan, both of which are essential for the non-cell-autonomous effects of the Fz system. The current findings make it clear that for the larval denticle field, the Fz protein acts in a way that is inconsistent with its proposed role downstream of the Ds system. However, this observation still leaves room for the possibility that Ds-Ft engages downstream components within the Fz system (Donoughe, 2011).

The larval epidermis is unique in that the relative requirements for the Ds and Fz systems differ in different domains. The most obvious example of this is that when the Ds system is removed, polarity is completely removed in some columns (e.g., columns 0 and 4) but at least some polarity is still present in others (e.g., columns 1, 2, 3, 5, 6). Thus, it appears that the Fz system (still intact) is acting in those columns to impart polarity, suggesting that the two systems have independent and redundant inputs to polarity (Donoughe, 2011).

It was also demonstrated that Ds extracellular domain overexpression is able to reorient adjacent columns in an fz null background, and this signal is propagated onward. This shows that the Ds system can send, receive and propagate polarity information without contribution from the Fz protein. It remains possible that even when Fz-dependent intercellular signaling is absent, intracellular components of the Fz-system, such as Dsh, act in implementing the Ds signal. This function of Dsh would have to be unaffected in dsh[1] MZ mutants, as the polarity of dsh[1] MZ and dsh[1] MZ ds- larvae appear similar to that of fz- and ds- fz- larvae, respectively. Testing for Ds-mediated polarity in dsh null cells would be the true test of this hypothesis, but is precluded by the essential role of dsh in canonical Wnt signaling (Donoughe, 2011).

If, however, the Ds system operates independently of the Fz system, this would have significant ramifications for understanding of the molecular mechanisms that must be engaged downstream of each polarity system. The two systems must eventually converge at the point when cells create the oriented read-out (in this case, denticle formation). It is possible that the common polarity effectors might be far downstream of the initial effects in signaled cells. Given that the Fz and Ft receptors are so dissimilar from a molecular standpoint, their immediate effectors are likely to be distinct. Only by identifying the proteins that interact with Ft to implement Ds system polarity will it be possible to determine whether these effectors intersect downstream components of the Fz system or act independently on the polarity read-out (Donoughe, 2011).

Another observation that requires explanation is that the Ds and Fz systems seem to operate serially in some contexts (e.g. in the eye or wing) but in parallel in others (e.g. in the abdomen or the larval epidermis). Ds system-mediated microtubule (MT) orientation has been suggested as one mechanism by which the Ds system could feed into the Fz system. When MTs are oriented along the axis of polarity of wing cells, MT-mediated polarized transport brings Fz to the cell membrane, and it was recently shown that the maintenance of the correct MT orientation is Ds dependent. In the embryonic ventral epidermis, however, MTs are oriented perpendicular to the axis of planar polarity, at least at steady state. Therefore, unless careful imaging uncovers a minor, posteriorly polarized and Ds-dependent MT track, it seems unlikely that the Ds system is operating in the ventral epidermis in the manner proposed for wing polarity. This could explain why the Ds system only functions independently of Fz protein in the denticle field (Donoughe, 2011).

In ds- fz- larvae, all columns were largely disordered, but the flanking cell columns exhibited a slight, yet statistically significant, tendency toward reversed polarity. It is difficult to explain why there is residual polarity rather than randomization. Although the ds and fz alleles that were used are nulls, fz2 cannot be additionally removed owing to its essential role in canonical Wnt signaling. Thus, it is possible that Fz2-dependent polarization makes some contribution in the larval epidermis, although Fz2 has not as yet been implicated in PCP in any tissue. Alternatively, even if fz were the only Fz system receptor active for PCP, some latent activation of downstream components of the Fz system could, in principle, be responsible for imparting this subtle but polarized output (Donoughe, 2011).

An alternative explanation for the residual polarization in ds- fz- mutants is that there is an underlying bias in the tissue that is ordinarily masked in the presence of Ds or Fz proteins, but uncovered when both are removed. Since the residual orientation in double mutants tends to be directed away from the smooth field, perhaps that domain is somehow responsible for the latent polarity. Alternatively, the orientation signal might derive from within the denticle field. For instance, the 4-5 column interface is a boundary for Notch and EGFR signaling. Perhaps a low-level orientation signal emanates from that position (Donoughe, 2011).

This work also suggests that the Ds and Fz systems have different capacities to adjust to changes in field size. Current models for creating planar polarity begin with a gradient across the field of unpolarized tissue. A subtle bias is presumably then established within each cell across the field, as cells compare the level of the polarizing gradient they detect with that detected by their neighbors. This bias is then reinforced in each cell through a feedback mechanism, converting it into a sharp intracellular gradient of effector protein distribution (Axelrod, 2009). At those initial stages, when a given cell compares the level it perceives with that of adjacent cells, the magnitude of the difference under comparison should be influenced by the size of the field: as field size increases, the contrast perceived by adjacent cells decreases. Correspondingly, any comparison mechanism will be challenged as field size increases (Donoughe, 2011).

The larval epidermis presents such a challenge to the polarizing systems as tremendous growth occurs across the field between each larval molt. This study succeeded in analyzing the effects on the Fz system as field size increased by examining ds null animals at each molt. At small field size (i.e. first instar), polarity defects are rare; however, at large field size (i.e., third instar, five times larger), the disruption to polarity is dramatic. This suggests that the Fz system loses potency as field size increases. By contrast, the Ds system did not appear to be affected, as there are only rare defects in fz null animals at first or third instar. Since the change in field size through the larval instars occurs in the absence of cell division, it will be of interest to explore what other parameters of cell growth affect the Fz system in this tissue (Donoughe, 2011).

Note also that this work demonstrates that denticle field polarity can change over the course of larval growth. This supports the recent finding that third instar polarity is not determined at the embryonic stage. Together, these findings strongly imply that planar polarity in the larval epidermis is not permanently set, but rather requires input throughout larval growth (Donoughe, 2011).

The ventral epidermis also provides the opportunity to study how the two polarity systems influence distinct polarized outputs from the same tissue. Cell alignment and denticle orientation were largely unaffected in ds M-Z- embryos/first instar larvae, but there were F-actin protrusion placement defects in cell columns 3 through 5. This result is compelling, as the domain affected matched the region of peak Ds and Ft accumulation. In fz M-Z- and dsh[1] MZ backgrounds, there are subtle column 1 and 2 defects in F-actin protrusion placement. It is intriguing that the embryonic protrusion placement defects appear in complementary patterns for the Fz system as compared with the Ds system; this suggests that in embryos, as in larvae, the two systems function mainly in spatially distinct domains (Donoughe, 2011).

In several tissues, protein distributions have provided a window into the mechanism of polarization. However, in the embryonic epidermis, this analysis so far has not been suggestive. As neither Ds nor Ft showed an obvious bias toward particular interfaces around a given cell, it is not immediately apparent how these accumulation patterns might be related to proposed Ds-Ft dimer distributions or to the polarity of the tissue. It is of course possible that the protein accumulations would be more suggestive if one could analyze them during the larval molts, but this cannot presently be done (Donoughe, 2011).

In this context, it is worth noting that the endogenous distributions of Fz system components have not yet been determined in the ventral epidermis. Staining for Fz-GFP and Dsh-GFP, however, reveals a difference in their enrichments as compared with Ds and Ft: both Fz system members are strongly enriched along cell interfaces that separate cell columns and are depleted from interfaces between cells within the same column. Whether these putative enrichments are necessary for polarity in this tissue remains to be tested (Donoughe, 2011).

The Adenomatous polyposis coli tumour suppressor is essential for Axin complex assembly and function and opposes Axin's interaction with Dishevelled

Most cases of colorectal cancer are linked to mutational inactivation of the Adenomatous polyposis coli (APC) tumour suppressor. APC downregulates Wnt signalling by enabling Axin to promote the degradation of the Wnt signalling effector β-catenin (Armadillo in flies). This depends on Axin's DIX domain whose polymerization allows it to form dynamic protein assemblies ('degradasomes'). Axin is inactivated upon Wnt signalling, by heteropolymerization with the DIX domain of Dishevelled, which recruits it into membrane-associated 'signalosomes'. How APC promotes Axin's function is unclear, especially as it has been reported that APC's function can be bypassed by overexpression of Axin. Examining apc null mutant Drosophila tissues, it was discovered that APC is required for Axin degradasome assembly, itself essential for Armadillo downregulation. Degradasome assembly is also attenuated in APC mutant cancer cells. Notably, Axin becomes prone to Dishevelled-dependent plasma membrane recruitment in the absence of APC, indicating a crucial role of APC in opposing the interaction of Axin with Dishevelled. Indeed, co-expression experiments reveal that APC displaces Dishevelled from Axin assemblies, promoting degradasome over signalosome formation in the absence of Wnts. APC thus empowers Axin to function in two ways-by enabling its DIX-dependent self-assembly, and by opposing its DIX-dependent copolymerization with Dishevelled and consequent inactivation (Mendoza-Topaz, 2011).

Drosophila apc null mutants wer used for stringent in vivo function tests to show that APC is indispensable in Drosophila tissues for Axin's activity in assembling functional degradasomes that destabilize Armadillo. The evidence suggests that the same is also true for APC mutant colorectal cancer cells in which Axin–GFP, if expressed at low levels, shows a marked tendency to fail in assembling functional degradasomes. This provides a new insight into how APC promotes the destabilization of Armadillo/β-catenin—namely by enabling Axin to assemble degradasomes. The failure of this assembly step explains why Axin fails to destabilize Armadillo/β-catenin, given that this function of Axin crucially depends on its DAX-dependent polymerization. It is emphasized that previous studies have shown that APC loss-of-function can be bypassed by Axin overexpression, which indicated a non-obligatory role of APC in the destabilization of β-catenin/Armadillo. However, these studies underestimated APC's essential role in this process since Axin was assayed under conditions of residual APC function, and possibly also since high Axin overexpression levels were used for complementation (Mendoza-Topaz, 2011).

Why does Axin fail to assemble functional degradasomes in the absence of APC? This is believed to be true due to a combination of two different effects of APC loss on Axin. First, Axin is destabilized in the absence of APC, so its levels may fall below the minimal cellular concentration required for DAX-dependent polymerization: note that the DIX domain auto-affinity is in the micromolar range, and so the DAX-dependent polymerization may not occur spontaneously at low cellular Axin concentrations, but might require a co-factor capable of clustering Axin, increasing its local concentration and nucleating polymerization. APC is a candidate for such a co-factor, given its relatively high cellular abundance and affinity to Axin which allow it to associate efficiently with Axin at physiological concentrations. APC might cluster Axin directly, by binding simultaneously to multiple Axin molecules through its multiple Axin-binding sites, or indirectly through additional factors (such as CtBP, itself capable of clustering APC. Notably, Axin would become independent of this co-factor if overexpressed at high enough levels, as this would allow it to overcome its low auto-affinity and to polymerize spontaneously (Mendoza-Topaz, 2011).

Second, the absence of APC (or of binding to APC) renders Axin prone to Dsh-dependent relocation from the cytoplasm to the PM, into signalosome-like particles. Since the recruitment of Axin into signalosomes normally blocks its function in promoting the phosphorylation and destabilization of β-catenin/Armadillo, its relocation to the PM might also explain its inactivity in the absence of APC. The evidence indicates that APC shields Axin from interaction with Dsh in the absence of Wnt signalling, to ensure Axin's function in the cytoplasmic degradasomes. This shielding function of APC may be particularly important in cells experiencing non-canonical Wnt signalling (promoting PM-association of Dishevelled), such as in third-larval instar wing discs.The observations in Drosophila tissues suggested that APC may compete with Dishevelled for association with Axin, which is strongly supported by evidence from co-expression experiments in mammalian cells (carried out in the absence of Wnt stimulation): these indicate that Axin cannot interact simultaneously with APC and Dvl2, and that APC is capable of displacing Dvl2 from Axin protein assemblies. The notion of a competition between APC and Dishevelled for their association with Axin is consistent with previous evidence from epistasis experiments in Drosophila embryos, which indicated that APC acts at the same level as Dishevelled rather than below it (Mendoza-Topaz, 2011).

Evidently, the function of APC that shields Axin from its interaction with Dishevelled is somehow antagonized by Wnt stimulation, which enables Dishevelled to bind to Axin and recruit it to the PM into signalosomes. Indeed, Wnt signalling may overcome the competition between APC and Dishevelled for their binding to Axin, allowing simultaneous interaction of all three proteins. Consistent with this, Axin–GFP appears to co-localize with E-APC in the Wg signalling zones of Drosophila embryos within the PM-associated signalosomes that are likely to also contain Dsh (although this has not been confirmed directly owing to the lack of a suitable Dsh antibody), suggesting that Wg signalling allows all three proteins to coincide in signalosomes (Mendoza-Topaz, 2011).

It is noted that a previous study in Drosophila uncovered a positive role of APC in antagonizing Axin (rather than promoting its function), thereby stimulating signalling through Armadillo. The key evidence supporting this rather unexpected conclusion was that the levels of Axin were upregulated in apc null mutant wing disc clones, as shown by immunofluorescence. This contrasts with the current result from apc mutant embryos, which shows much reduced levels of Axin–GFP, as judged by Western blotting. Although this quantitative biochemical approach is difficult to apply to apc mutant wing disc clones (owing to insufficient apc mutant material), the dramatic PM relocation of Axin–GFP observed in these clones may have been misled by the high levels of apical Axin, and mistaken for a general upregulation rather than simply a relocation (as has been shown for Axin–GFP). The current evidence reaffirms the negative role of APC in Wg/Armadillo signalling, demonstrating an essential function of APC in keeping Axin in the cytoplasm, where it enables it to assemble functional degradasomes (Mendoza-Topaz, 2011).

The main corollary of these results from Drosophila tissues is that APC promotes the DAX-dependent homopolymerization of Axin (required for degradasome assembly), and that it antagonizes the heteropolymerization between Axin and Dishevelled (mediated by DIX–DAX interaction). The latter is further supported by the evidence from co-expression experiments in mammalian cells that APC displaces Dvl2 from Axin puncta. This creates a mechanistic conundrum: APC binds to the N-terminal RGS domain of Axin, but appears to control the DAX-dependent interactions at its C-terminus. Although it is conceivable that APC achieves this at 'long range', given its unusually large size it is more likely that APC relies on additional factors, perhaps even on enzymes, to promote Axin's self-assembly at the expense of its heteropolymerization with Dishevelled. Future work will be required to determine the precise molecular mechanism by which APC enables Axin to assemble functional degradasomes and opposes its recruitment by Dishevelled, and how this is overcome during Wnt signalling (Mendoza-Topaz, 2011).

Planar cell polarity controls directional Notch signaling in the Drosophila leg

The generation of functional structures during development requires tight spatial regulation of signaling pathways. Thus, in Drosophila legs, in which Notch pathway activity is required to specify joints, only cells distal to ligand-producing cells are capable of responding. This study shows that the asymmetric distribution of planar cell polarity (PCP) proteins correlates with this spatial restriction of Notch activation. Frizzled and Dishevelled are enriched at distal sides of each cell and hence localize at the interface with ligand-expressing cells in the non-responding cells. Elimination of PCP gene function in cells proximal to ligand-expressing cells is sufficient to alleviate the repression, resulting in ectopic Notch activity and ectopic joint formation. Mutations that compromise a direct interaction between Dishevelled and Notch reduce the efficacy of repression. Likewise, increased Rab5 levels or dominant-negative Deltex can suppress the ectopic joints. Together, these results suggest that PCP coordinates the spatial activity of the Notch pathway by regulating endocytic trafficking of the receptor (Capilla, 2012).

Spatially coordinated regulation of signaling pathways is essential to generate correct anatomical and functional structures, as exemplified by the Drosophila leg, in which activity of the N pathway is required to specify leg joints. In this case, only cells distal to the stripe of Ser expression appear to be capable of responding to the ligand. This study shows that activity of the core PCP pathway is required in those cells proximal to the domain of Ser expression to prevent them from responding to this N ligand. This regulation correlates with the asymmetric distribution of the core PCP proteins, since this study shows that Fz and Dsh are enriched at the distal side of each cell, which in the non-responding cells faces the neighboring Ser-expressing cells. Conversely, in those cells distal to Ser, Fz and Dsh are depleted from the proximal side, leaving N free to interact with its ligand to promote joint formation. It appears that elimination of core PCP gene function in cells proximal to the Ser-expressing cells is sufficient to alleviate the repression resulting in ectopic N activity and ectopic joint formation. Such regulation of the membrane availability of Notch could equally affect Dl-mediated activation, although Ser appears to be the major ligand responsible in the joints. Other factors are likely to influence proximal repression of N because ectopic joints are also observed in alterations of the EGFR pathway and mutants of defective proventriculus (Capilla, 2012).

It is also noted that the domains of N activation (both normal and ectopic) extend beyond the cells at the interface with Ser. This additional level of regulation has not been investigated, but the results indicate that it is unlikely to be due to a secondary signal emanating from the Ser-interfacing cells because the loss of function clones show complete autonomy, without any 'shadow' of activation adjacent to the clone. An alternative possibility is that the cells make more extensive contacts, as has been seen in other tissues (Capilla, 2012).

PCP regulation of N has been observed in other developmental processes, most notably in photoreceptor fate choice in the Drosophila eye. There, much of the regulation is via effects on levels and activity of the ligand. However, no change was detected in the pattern of N or Ser expression in PCP mutants. Instead, the evidence suggests that regulation involves direct interaction between Dsh and N and that this interaction has consequences on the endocytic trafficking of N, resulting in its inactivation. The interaction requires the amino-terminal portion of the Dsh DIX domain, which is also required for Axin binding in the canonical Wnt pathway, making it difficult to dissect its role in the PCP-mediated N inhibition. Nevertheless, one mutation was generated that reduced interactions with N with minor consequences on Axin binding. Rescue experiments with this mutant form of Dsh indicated that it was less effective in PCP function in the leg joints compared with others (e.g., polarity of leg bristles). These results support the model that a direct interaction between Dsh and N is relevant in the context of joint determination. However, the possibility cannot be fully ruled that the mutation has more generalized effects on Dsh, if the joints are particularly sensitive to the levels of Dsh activity (Capilla, 2012).

Several studies indicate that endocytic sorting of N is involved in its regulation, with either positive or negative effects depending on the particular context. The current findings suggest that regulation of N by PCP in the leg is mediated by interaction with Dsh, and probably involves the control of N endocytic trafficking. This suggests a model whereby the interaction between Dsh and N results in increased endocytosis of the N receptor, so reducing its capability to interact with ligands on neighboring cell. Removal of Fz or Dsh compromises this endocytic trafficking, allowing N to be activated. The interaction between Dsh and N is thus only likely to be relevant under circumstances in which there is a strong localization of Dsh co-incident with an interface between N and ligand-expressing cells (Capilla, 2012).

Previous studies have also suggested a role for Dsh in regulating N and on promoting its endocytosis (Axelrod, 1996; Munoz-Descalzo, 2010). In both instances, these effects were linked to Wg signaling, rather than to the core PCP pathway as in this study. Nevertheless several aspects are consistent with the current results, most notably the direct binding between Dsh and N. Additionally it has been argued that Dsh specifically antagonizes Dx-mediated effects of N, which is compatible with their complementary effects on joint formation. However, it is also evident that the ability of Dsh to inhibit N depends on the developmental context. For example, whereas overexpression of Dsh in the leg is sufficient to inhibit N activation at presumptive joints, overexpression of Dsh at the wing margin is not sufficient to repress N signaling: there are no nicks and cut expression is not inhibited. Interestingly, differences in Dx behavior are also evident in these two contexts. At the wing margin, Deltex mutation DxΔpro acts as a dominant-negative form of Dx, whereas DxΔNBS is inactive. By contrast, in the leg joints DxΔpro behaves as wild-type Dx, whereas DxΔNBS is a dominant negative. It is postulated, therefore, that the subcellular localization of Dsh and the availability of Dx are important for determining the regulation of N trafficking at joints (Capilla, 2012).

The autonomous effect of core PCP mutants was clear when the E(spl)mβ1.5-CD2 N reporter and disco-lacZ was used. However, the consequences on bib-lacZ were more complex. Although larger clones of mutant cells always exhibited autonomous ectopic expression, similar to E(spl)mβ1.5-CD2, some narrow clones exhibited no ectopic expression. It is suggested that this might be due to bib-lacZ having a higher threshold of response, so it would need stronger N activation. The domain of bib-lacZ is narrower than that of the other known reporters, in agreement with this model. Furthermore, some cases were found in which there was a reduction of the normal bib-lacZ expression in the mutant cells, in addition to ectopic expression. This suggests that PCP-mediated distal localization of Dsh would be required not only for inhibition of N in proximal cells, but also for efficient activation of N in distal ones (Capilla, 2012).

The Drosophila homologue of the amyloid precursor protein is a conserved modulator of Wnt PCP signaling

Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. This study therefore investigated the role of Drosophila APPL during brain development. APPL was found to be involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, APPL was found to be a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover,APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together these data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth (Soldano, 2013).

AD is a neurodegenerative disorder characterized by progressive loss of neurons in specific regions of the brain that correlates with progressive impairment of higher cognitive functions. A growing body of evidence identifies the APP and its metabolite the Aβ peptide as main players in the pathogenesis of AD. In particular, the accumulation of Aβ peptides in the brain seems to be the trigger of the pathological cascade that eventually results in neuronal loss and degeneration. Despite efforts to characterize the molecular mechanisms underlying Aβ's toxic function, it is still not clear what triggers the accumulation of the peptide and how this is correlated with the pathogenesis of the disease and the dementia. In fact, most of the work done to unveil the pathogenesis of the disease has focused on the analysis of Aβ-peptide and the search for its receptors and downstream effectors. Even though the numerous in vitro studies performed in cell culture identified several molecules that interact with Aβ peptide, the in vivo biological relevance of these interactions remains to be clarified. The amyloid cascade hypothesis has also dominated the search for AD treatments, but the promising molecular candidates developed to modulate the Aβ peptide and reached clinical trials failed. Finally, over the last few years many studies indicated that there is no linear correlation between the accumulation of the peptide and the cognitive decline, leading to a revision of the amyloidogenic hypothesis. Taken together, these observations suggest that the accumulation of the peptide is not the only cause of the pathology and that other factors are involved. Interestingly, under physiological conditions APP is mainly found in its uncleaved or α-cleaved form, suggesting that the shift towards amyloidogenic processing not only increases the production of Aβ peptide but also depletes the pool of APP that undergoes non-amyloidogenic processing, with hitherto unknown consequences. It is therefore of paramount importance to understand the physiological role of APP and how perturbing this role could contribute to the pathogenesis of the disease. An important contribution to the study of the function of a protein comes from the analysis of the knock-out (KO) animals. In the case of APP, several KO models have been generated and analyzed in detail both from the morphological and behavioral point of view. Despite these efforts, the normal physiological function of APP in vivo in the nervous system remains largely elusive and highly controversial. This is due to the lack of consensus over the neuronal phenotypes in null mutant animals and the mechanism of action in vivo. The data collected by different labs confirmed the involvement of APPs in development and function of the nervous system, but these studies do not provide an in-depth analysis of the development of the brain during the pre-natal stages or the molecular mechanism underlying APPs' putative functions. Therefore this study took advantage of Drosophila melanogaster to further analyze the consequence of loss of APP Like (APPL) during brain development (Soldano, 2013).

The present study demonstrates that APPL is involved in brain development of Drosophila melanogaster, particularly in the Mushroom Body (MB) neurons. APPL is required for the development of αβ neurons. In Appl-/- flies, MB neurons fail to project the α lobe in 14% of the cases and the β-lobe in 12% of the cases. Further analysis of the phenotype reveals that APPL is required cell-autonomously for the development of the β lobe and non-cell autonomously for the development of the α lobe. In fact, single cell Appl-/- clones display only β-lobe loss and no α loss. The re-introduction of a full-length, membrane-tethered form of APPL, but not a soluble form, rescues β-lobe los. This is of particular interest because it confirms that, similar to mammalian APPs, the physiological role of APPL is mediated both by its full-length form, required in the neurons to achieve the correct β-lobe pattern, and by its soluble form (sAPPL) that regulates the extension of the α lobe. Moreover, the rescue data indicate that, at least in this context, the function of sAPPL is mediated not by homo-dimerization with the full-length form but by some other receptor, hitherto unknown. Further experiments are required to clarify the sAPPL non-cell autonomous effect, but it is hypothesized that it might be involved in modulating signaling mediated by the cells that surround the MB axons. Taken together, the analysis of the Appl-/- animals confirmed the important role of APPs during brain development but reinforced the idea that the phenotypes are present with incomplete penetrance and might be subtle. It would therefore be of interest to analyze the phenotype of the KO mice in greater detail and, in particular, to better characterize the APP's downstream pathway leading to these defects (Soldano, 2013).

Moreover, the results described clearly support a model of APPL as a novel, neuronal-specific positive modulator of the Wnt-PCP pathway. The PCP pathway was initially described because of its role in tissue polarity establishment and, in particular, of its regulation of cell orientation in plane of an epithelium. Among the different processes regulated by PCP signaling, axon growth and guidance is of particular interest. Mice null for Fzd3/Ceslr3-/- genes show severe defects in several major axon tracts like thalamocortical, corticothalamic, and nigrostriatal tracts, defects of the anterior commissure, and similarly to APP KO mice, the variable loss of the corpus callosum (Soldano, 2013).

The molecular mechanism underlying the function of PCP-signaling in regulating tissue polarity has been broadly studied. The current model suggests that, upon polarized expression of the different core proteins, Dsh is recruited to the membrane via Fz and leads to the activation of a cascade of small GTPases finally resulting in cytoskeleton rearrangements. In the case of regulation of axon growth and guidance, it is less clear how the signaling is regulated and transmitted to the cytoskeleton. A recent publication suggested that during axon growth the transmembrane PCP receptor-like Vang and Fzd are localized at the growth cone area on the tip of the fillopodia, thus suggesting that in this context the asymmetric localization is not needed (Soldano, 2013).

Furthermore, Dsh needs to relocalize from the cytoplasm to the membrane to ensure the proper activation of PCP signaling, and this is dependent on its phosphorylation status. Abelson has been shown to be one kinase responsible for this modification, but the receptor upstream of the kinase was not identified (Singh, 2010). Based on current evidence, it is proposed that APPL is a novel regulator of Wnt-PCP pathway involved in axon growth and guidance. This is of interest because while the PCP core proteins are ubiquitously expressed, APPL is restricted to the nervous system, suggesting that it could be the first described tissue-specific modulator of the pathway (Soldano, 2013).

Mechanistically, it is proposed that APPL-Abl complex modulates Dsh via dual protein-protein interactions. First, Abl might have an intrinsic affinity for its substrate Dsh (Singh, 2010). Secondly, this interaction is strengthened or stabilized by the inclusion of APPL in a PCP receptor complex. This dual affinity complex leads to increased PCP signaling efficiency at the developing growth cone. Both biochemical and physiological data show that this function is highly conserved in mammalian APP, suggesting that it may play a similar role in the mammalian brain. The canonical-Wnt signaling pathway has already been connected to AD pathogenesis because of its link to the tau-kinase GSK-3β. Interestingly, no clear link between the Wnt-PCP pathway and this neurodegenerative disorder has been made. Previous reports have show that, in flies and mice, Jun N-terminal Kinase (JNK) is the final effector of PCP in axon outgrowth and JNK was shown to be required for the effect of APP overexpression in the fly. Interestingly, JNK signaling has also been linked to the neuronal loss observed in AD. It is therefore worth investigating whether the physiological function of APP as a neuronal PCP modulator explains the JNK-AD connection (Soldano, 2013).

Kinesin-II recruits Armadillo and Dishevelled for Wingless signaling in Drosophila

Wingless (Wg)/Wnt signaling is fundamental in metazoan development. Armadillo (Arm)/beta-catenin and Dishevelled (Dsh) are key components of Wnt signal transduction. Recent studies suggest that intracellular trafficking of Wnt signaling components is important, but underlying mechanisms are not well known. This study shows that Klp64D, the Drosophila homolog of Kif3A kinesin II subunit, is required for Wg signaling by regulating Arm during wing development. Mutations in klp64D or RNAi cause wing notching and loss of Wg target gene expression. The wing notching phenotype by Klp64D knockdown is suppressed by activated Arm but not by Dsh, suggesting that Klp64D is required for Arm function. Furthermore, klp64D and arm mutants show synergistic genetic interaction. Consistent with this genetic interaction, Klp64D directly binds to the Arm repeat domain of Arm and can recruit Dsh in the presence of Arm. Overexpression of Klp64D mutated in the motor domain causes dominant wing notching, indicating the importance of the motor activity. Klp64D shows subcellular localization to intracellular vesicles overlapping with Arm and Dsh. In klp64D mutants, Arm is abnormally accumulated in vesicular structures including Golgi, suggesting that intracellular trafficking of Arm is affected. Human KIF3A can also bind β-catenin and rescue klp64D RNAi phenotypes. Taken together, it is proposed that Klp64D is essential for Wg signaling by trafficking of Arm via the formation of a conserved complex with Arm (Vuong, 2014).

Hipk2 and PP1c cooperate to maintain Dvl protein levels required for Wnt signal transduction

The phosphoprotein Dishevelled (Dvl) is a common essential component of Wnt/beta-catenin and Wnt/planar cell polarity (PCP) signaling pathways. However, the regulation and significance of Dvl phosphorylation are not fully understood. This study shows that homeodomain-interacting protein kinase 2 (Hipk2; see Drosophila Hipk) facilitates protein phosphatase 1 catalytic subunit (PP1c)-mediated dephosphorylation of Dvl via its C-terminal domain and that this dephosphorylation blocks ubiquitination and consequent degradation mediated by the E3 ubiquitin ligase Itch, which targets the phosphorylated form of Dvl proteins. Inhibition of Hipk2 or PP1c function reduces Dvl protein levels and suppresses Wnt/beta-catenin and Wnt/PCP pathway-dependent events in mammalian cells and zebrafish embryos, suggesting that Hipk2 and PP1c are essential for maintaining Dvl protein levels that are sufficient to activate Wnt signaling. It was also shown that Wnt-3a, a Wnt/beta-catenin ligand, induces dissociation of the Dvl-Hipk2-PP1c complex and Dvl degradation under high-cell-density conditions. This regulation may be a negative feedback mechanism that fine-tunes Wnt/beta-catenin signaling (Shimizu, 2014: 25159144).

Dishevelled binds the Discs large 'Hook' domain to activate GukHolder-dependent spindle positioning in Drosophila

Communication between cortical cell polarity cues and the mitotic spindle ensures proper orientation of cell divisions within complex tissues. Defects in mitotic spindle positioning have been linked to various developmental disorders and have recently emerged as a potential contributor to tumorigenesis. Despite the importance of this process to human health, the molecular mechanisms that regulate spindle orientation are not fully understood. Moreover, it remains unclear how diverse cortical polarity complexes might cooperate to influence spindle positioning. Spindle orientation roles have been identified for Dishevelled (Dsh), a key regulator of planar cell polarity, and Discs large (Dlg), a conserved apico-basal cell polarity regulator, effects which were previously thought to operate within distinct molecular pathways. This study identified a novel direct interaction between the Dsh-PDZ domain and the alternatively spliced 'I3-insert' of the Dlg-Hook domain, thus establishing a potential convergent Dsh/Dlg pathway. Furthermore, a Dlg sequence motif that is necessary for the Dsh interaction was identified that shares homology to the site of Dsh binding in the Frizzled receptor. Expression of Dsh enhanced Dlg-mediated spindle positioning similar to deletion of the Hook domain. This Dsh-mediated activation was dependent on the Dlg-binding partner, GukHolder (GukH). These results suggest that Dsh binding may regulate core interdomain conformational dynamics previously described for Dlg. Together, these results identify Dlg as an effector of Dsh signaling and demonstrate a Dsh-mediated mechanism for the activation of Dlg/GukH-dependent spindle positioning. Cooperation between these two evolutionarily-conserved cell polarity pathways could have important implications to both the development and maintenance of tissue homeostasis in animals (Garcia, 2014: PubMed).

The exon junction complex regulates the splicing of cell polarity gene dlg1 to control Wingless signaling in development

Wingless (Wg)/Wnt signaling is conserved in all metazoan animals and plays critical roles in development. The Wg/Wnt morphogen reception is essential for signal activation, whose activity is mediated through the receptor complex and a scaffold protein Dishevelled (Dsh). This study reports that the exon junction complex (EJC) activity is indispensable for Wg signaling by maintaining an appropriate level of Dsh protein for Wg ligand reception in Drosophila. Transcriptome analyses in Drosophila wing imaginal discs indicate that the EJC controls the splicing of the cell polarity gene discs large 1 (dlg1), whose coding protein directly interacts with Dsh. Genetic and biochemical experiments demonstrate that Dlg1 protein acts independently from its role in cell polarity to protect Dsh protein from lysosomal degradation. More importantly, human orthologous Dlg protein is sufficient to promote Dvl protein stabilization and Wnt signaling activity, thus revealing a conserved regulatory mechanism of Wg/Wnt signaling by Dlg and EJC (Liu, 2016).

The EJC is known to act in several aspects of posttranscriptional regulation, including mRNA localization, translation and degradation. After transcription, the pre-mRNA associated subunit eIF4AIII is loaded to nascent transcripts about 20-24 bases upstream of each exon junction, resulting in binding of Mago nashi (Mago)/Magoh and Tsunagi (Tsu)/Y14 proteins to form the pre-EJC core complex. The pre-EJC then recruits other proteins including Barentsz (Btz) to facilitate its diverse function). In vertebrates, the EJC is known to ensure translation efficiency as well as to activate nonsense-mediated mRNA decay (NMD). In Drosophila, however, the EJC does not contribute to NMD. It is instead required for the oskar mRNA localization to the posterior pole of the oocyte. Very recently, the pre-EJC has been shown to play an important role in alternative splicing of mRNA in Drosophila. Reduced EJC expression results in two forms of aberrant splicing. One is the exon skipping, which occurs in MAPK and transcripts that contain long introns or are located at heterochromatin (Ashton-Beaucage, 2010; Roignant, 2010). The other is the intron retention on piwi transcripts. Furthermore, transcriptome analyses in cultured cells indicates the role of EJC in alternative splicing is also conserved in vertebrates (Liu, 2016).

This study has utilized the developing Drosophila wing as an in vivo model system to investigate new mode of regulation of Wg signaling. The pre-EJC was found to positively regulate Wg signaling through its effect on facilitating Wg morphogen reception. Further studies reveal that the basolateral cell polarity gene discs large 1 (dlg1) is an in vivo target of the pre-EJC in Wg signaling. Dlg1 acts independently from its role on cell polarity to stabilize Dsh protein, thus allowing Wg protein internalization required for signaling activation. Furthermore, it was demonstrated that human Dlg2 exhibits a similar protective role on Dvl proteins to enhance Wnt signaling in cultured human cells. Taken together, this study unveils a conserved regulatory mechanism of the EJC and Dlg in Wg/Wnt signaling (Liu, 2016).

In summary, this study uncovers a specific role of the RNA binding protein complex EJC in the Drosophila wing morphogenesis. Genetic and biochemical analyses demonstrate that the pre-EJC is necessary for Wg morphogen reception to activate the signal transduction. The identification of the cell polarity determinant dlg1 as one of the pre-EJC targets provides mechanistic basis for the pre-EJC regulation of the Wg signaling. Dlg1 controls the stability of the scaffold protein Dsh, which is the hub of the Wg signaling cascade. Importantly, this mode of regulation of Dvl by Dlg is conserved from flies to vertebrates (Liu, 2016).

The EJC as well as other RNA binding protein complexes are thought to function in a pleiotropic manner. However, the current data together with several recent studies argue that RNA regulatory machineries can act specifically on developmental signaling for pattern formation and organogenesis. It has been increasingly recognized that the production, transport or the location of mRNA are subject to precise regulation in Wg/Wnt signaling. For example, apical localization of wg RNA is essential for signal activation in epithelial cells. The specific role of RNA machineries on cell signaling is not limited to Wg/Wnt signaling. It has been reported that RNA-binding protein Quaking specifically binds to the 3'UTR of transcription factor gli2a mRNA to modulate Hedgehog signaling in zebrafish muscle development. RNA binding protein RBM5/6 and 10 could differentially control alternative splicing of a negative Notch regulator gene NUMB, thus antagonistically regulating the Notch signaling activity for cancer cell proliferation. Therefore, generally believed pleotropic RNA regulatory machineries emerge as important regulatory means to specifically control cell signaling and related developmental processes (Liu, 2016).

The most studied function of the EJC in development is to localize oskar mRNA to the posterior pole of the oocyte for oocyte polarity establishment and germ cell formation in Drosophila. Further study suggests that the proper oskar RNA localization relies on its mRNA splicing. In light of the current study of the EJC activity on dlg1 mRNA as well as the roles of EJC on mapk and piwi splicing, it is suspected that EJC might regulate oskar mRNA splicing to mediate its mRNA localization. RNA-seq analyses identified several hundreds of candidate mRNAs whose expression may be directly or indirectly subjected to EJC regulation. Apart from defects in Wg and MAPK signaling, however, altered wing patterning associated with other developmental signaling systems was not seen in EJC defective flies, arguing that EJC may primarily regulate Wg and MAPK signaling in patterning the developing wing (Liu, 2016).

Wg/Wnt signaling plays a fundamental role in development and tissue homeostasis in both flies and vertebrates. Its activation and maintenance rely on appropriate activity of the ternary receptor complex including Fz family proteins. In Drosophila, polarized localization of Fz and Fz2 proteins is essential for activation of non-canonical and canonical Wg signaling, respectively. Dsh, which acts as a hub mediating both canonical and non-canonical Wg signaling, however, is found at both the apical cell boundary and in the basal side of the cytoplasm. Thus, the polarized activity of Dsh must require distinct regulatory mechanisms at different sub-membrane compartments. The results provide the in vivo evidence suggesting that the basolateral polarity determinant Dlg1 may play a dominant role to control the Dsh abundance/activity in canonical Wg signaling (Liu, 2016).

Altered Dvl production or activity has been linked with several forms of cancer. The stability of Dvl proteins can be controlled through regulated protein degradation both in vertebrates and in Drosophila as reported in this study. In HEK293T cells, Dapper1 induces whilst Myc-interacting zinc-finger protein 1 (MIZ1) antagonizes autophagic degradation of Dvl2 in lysosome. It is also reported that a tumor suppressor CYLD deubiquitinase inhibits the ubiquitination of Dvl. As Dlg1 prevents Dsh from degradation in Drosophila, it is important to investigate if Dlg1 participates in a posttranslational regulatory network of Dvl to integrate endocytosis and autophagy. Furthermore, upregulation of dvl2 and dlg2 expression has been found in various forms of cancer as shown in the COSMIC database. The study of the interaction between Dlg1 and Dsh may aid the development of novel approaches to prevent or treat relevant diseases. (Liu, 2016).

Dlg1 acts together with L(2)gl to form a basolateral complex in polarized epithelium. Dsh is known to interact with L(2)gl. On one hand, Dsh activity is required for correct localization of L(2)gl to establish apical-basal polarity in Xenopus ectoderm and Drosophila follicular epithelium. On the other hand, L(2)gl can regulate Dsh to maintain planar organization of the embryonic epidermis in Drosophila. Despite the complex interaction between L(2)gl and Dsh, not much is known about mutual regulation between Dlg1 and Dsh. A recent report suggests that Dsh binds to Dlg1 to activate Guk Holder-dependent spindle positioning in Drosophila. The current results unveil another side of the relationship in which Dlg1 controls the turnover of Dsh to ensure developmental signal propagation. Apart from its apical localization at the cell boundary, Dsh is also found in the basal side of the cytoplasm. It is likely that the interactions among Dsh, Dlg1 and L(2)gl may be dependent on their localization, and Dsh may serve as a bridge to connect cell signaling and polarity (Liu, 2016).

Developmental signaling and cell polarity intertwine to control a diverse array of cellular events. It is well known that Wg/Wnt signaling controls cell polarity in distinct manner. Non-canonical signaling acts through cytoskeletal regulators to establish planar cell polarity. Canonical signaling may also directly affect apical-basal cell polarity. On the other hand, disruption of epithelial cell polarity has a profound impact on protein endocytosis and recycling, both of which are essential regulatory steps for signal activation and maintenance. The current results add another layer of complexity by which polarity determinants could contribute to cell signaling independent of their conventional roles in polarity establishment and maintenance. Interestingly, this mode of regulation is also observed for other signaling processes. Loss of Dlg5 impairs Sonic hedgehog-induced Gli2 accumulation at the ciliary tip in mouse fibroblast cells that may not rely on cell polarity regulation. Similarly, L(2)gl regulates Notch signaling via endocytosis, independent of its role in cell polarity. It is believed that other cell polarity determinants may similarly participate in polarity-independent processes, however, the exact mechanism of how they cooperate to modulate developmental signaling awaits further investigation (Liu, 2016).

Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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