Wnt pathway activation by ADP-ribosylation: Both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation

Wnt/β-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multi-protein 'destruction complex', assembled by the scaffold protein Axin, targets the key transcriptional activator β-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. This study used an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. For nearly all Wingless-driven developmental processes, a three- to four-fold increase in Axin was found to be insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, both the tumor suppressor Adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) were found to have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and separable domains were defined in Axin that are important for APC- or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation (Yang, 2016).

The Wnt/β-catenin signal transduction pathway directs fundamental processes during metazoan development and tissue homeostasis, whereas deregulation of Wnt signalling underlies numerous congenital disorders and carcinomas. Two multimeric protein complexes with opposing functions -- the cytoplasmic destruction complex and the plasma membrane-associated signalosome -- control the stability of the transcriptional co-factor β-catenin to coordinate the state of Wnt pathway activation. In the absence of Wnt stimulation, β-catenin is targeted for proteasomal degradation by the destruction complex, which includes the two tumour suppressors: Axin and Adenomatous polyposis coli (APC), and two kinases: casein kinase α (CK1α) and glycogen synthase kinase 3 (GSK3). Engagement of Wnt with its transmembrane receptors, Frizzled and low-density lipoprotein receptor-related protein 5/6 (herein LRP6), induces rapid LRP6 phosphorylation, recruitment of Axin to phospho-LRP6, and assembly of the signalosome, which includes two other Axin-associated components, GSK3 and Dishevelled (Dvl). Signalosome assembly results in the inhibition of β-catenin proteolysis; consequently stabilized β-catenin promotes the transcriptional regulation of Wnt pathway target genes (Yang, 2016).

As a key component in both the destruction complex and the signalosome, Axin is tightly regulated. Under basal conditions, Axin is maintained at very low levels, and serves as the concentration-limiting scaffold for assembly of the destruction complex. Following Wnt exposure, the rapid association of phospho-Axin with phospho-LRP6 triggers Axin dephosphorylation, inducing a conformational change that inhibits Axin's interaction with both the destruction and signalosome complexes. Axin is subsequently degraded; however, Axin proteolysis occurs several hours after Wnt exposure, and thus does not regulate Axin's essential role during the initial activation of the Wnt pathway (Yang, 2016).

The mechanisms that rapidly reprogram Axin from inhibitory to stimulatory roles following Wnt exposure remain uncertain. In current models, Wnt stimulation induces Axin's dissociation from the destruction complex, thereby promoting its interaction with the signalosome. As Wnt stimulation induces Axin dephosphorylation, decreased phosphorylation was postulated to facilitate the dissociation of Axin from the destruction complex; however, recent work revealed that the interaction of Axin with LRP6 precedes Axin dephosphorylation, and that dephosphorylation serves to inhibit, rather than enhance this interaction (Kim, 2013) Furthermore, some findings have challenged prevailing models, providing evidence that Axin's interaction with the destruction complex is not diminished upon Wnt stimulation. Thus, whereas the rapid switch in Axin function following Wnt stimulation is essential for the activation of signalling, the underlying mechanisms remain uncertain (Yang, 2016).

During investigation of this critical process, an unanticipated role was discovered for the ADP-ribose polymerase Tankyrase (Tnks) in the reprogramming of Axin activity following Wnt exposure. As Tnks-mediated ADP-ribosylation is known to target Axin for proteolysis, small molecule Tnks inhibitors have become lead candidates for development in the therapeutic targeting of Wnt-driven cancers. This study identified a novel mechanism through which Tnks regulates Axin: by promoting Axin's central role in rapid Wnt pathway activation. Wnt stimulation was found to modulate Axin levels biphasically in both Drosophila and human cells. Unexpectedly, Axin is rapidly stabilized following Wnt stimulation, before its ultimate proteolysis hours later. In an evolutionarily conserved process, the ADP-ribosylated pool of Axin is preferentially increased immediately following Wnt exposure. ADP-ribosylation enhances Axin's association with phospho-LRP6, providing a mechanistic basis for the rapid switch in Axin function following Wnt stimulation. These results thus indicate that Tnks inhibition not only increases basal Axin levels, but also impedes the Wnt-dependent interaction between Axin and LRP6, suggesting a basis for the potency of Tnks inhibitors in Wnt-driven cancers. Thus, Tnks not only targets Axin for proteolysis independently of Wnt stimulation, but also promotes Axin's central role in Wnt pathway activation, which may be relevant to the context-dependent activation of Wnt signalling and the treatment of Wnt-driven cancers with Tnks inhibitors (Yang, 2016).

Wnt exposure induces biphasic regulation in the level of Axin, and a large increase in the level of ADP-ribosylated Axin immediately after stimulation. ADP-ribosylation enhances the interaction of Axin with phospho-LRP6, and promotes the activation of Wnt signalling. These findings lead to three major revisions of the current model for the role of Tnks in the activation of the Wnt pathway. First, Tnks serves bifunctional roles under basal conditions and after stimulation, revealing a remarkable economy and coordination of pathway components. Second, the results provide a mechanistic basis for the rapid reprogramming of Axin function in response to Wnt stimulation, and thereby reveal an unanticipated role for Tnks in this process. These findings suggest that Wnt exposure either rapidly increases the ADP-ribosylation of Axin or inhibits the targeting of ADP-ribosylated Axin for proteasomal degradation, through mechanisms yet to be elucidated. Finally, pharmacologic inactivation of Tnks was shown to diminish the interaction of Axin with LRP6, revealing a previously unknown mechanism through which small molecule Tnks inhibitors disrupt Wnt signalling, distinct from their known role in stabilizing the destruction complex inhibitors (Yang, 2016).

In the absence of Wnt stimulation, the concentration-limiting levels of Axin regulate its scaffold function in the destruction complex. As components of the destruction complex participate in other signalling pathways, the low levels of Axin were proposed to maintain modularity of the Wnt pathway. The new findings indicate that Axin levels are not only regulated in the absence of Wnt, but also regulated biphasically following Wnt stimulation. This sequential modulation of Axin divides activation of the pathway into an early, fast phase and a delayed long-term phase. During embryogenesis, the earliest expression of Wg triggers the rapid appearance of Axin in segmental stripes, which is a novel hallmark for the initial activation of the pathway. The findings reveal that Wnt exposure induces a rapid increase in the total level of Axin, and importantly, a preferential increase in the level of the ADP-ribosylated Axin. The early Axin stripes are absent in Tnks null mutant embryos and are also absent when the Tnks binding domain in Axin is deleted. Therefore, it is proposed that Axin ADP-ribosylation contributes to Axin stabilization and to the rapid response to Wg stimulation (Yang, 2016).

It is postulated that the initial increase in levels of ADP-ribosylated Axin jump-starts the response to Wnt stimulation by enhancing the Axin-LRP6 interaction, whereas the subsequent decrease in Axin levels prolongs the duration of signalling by reducing destruction complex assembly. Thus, Wnt stimulation induces rapid increases in the levels of not only cytoplasmic β-catenin, but also ADP-ribosylated Axin. Previous work that coupled mathematical modelling with experimental analysis revealed that several Wnt signalling systems were responsive to the relative change in β-catenin levels, rather than their absolute value. This dependence was proposed to impart robustness and resistance to noise and cellular variation. The current data raise the possibility that a similar principle applies to changes in Axin levels on the Axin-LRP6 interaction, as the marked increase in ADP-ribosylated Axin levels following Wnt stimulation is evolutionarily conserved. Thus, the relative change in levels of ADP-ribosylated Axin may promote signalling following Wnt exposure by facilitating the fold change in β-catenin levels (Yang, 2016).

The current findings have relevance for the context-specific in vivo roles of Tnks in Wnt signalling suggested in previous studies. Tnks inhibition disrupts Wnt signalling in a number of cultured cell lines, but in vivo studies in several model organisms suggested that the requirement for Tnks in promoting Wnt signalling is restricted to specific cell types or developmental stages. In mice, functional redundancy exists between the two Tnks homologues, such that Tnks single mutants are viable and fertile, whereas double mutants display embryonic lethality without overt Wnt-related phenotypes. However, a missense mutation in the TBD of Axin2 that is predicted to disrupt ADP-ribosylation resulted in either activating or inhibiting effects on Wnt signalling that were dependent on developmental stage. Tnks inhibitors resulted in the same paradoxical effects, suggesting complex roles in mouse embryonic development. Analogously, treatment of fish with Tnks inhibitors resulted in no observed defects in Wnt-mediated processes during development; however, the regeneration of injured fins in adults, a process that requires Wnt signalling, was disrupted (Yang, 2016).

Similarly, the finding that Drosophila Tnks null mutants are viable (Wang, 2016a; Wang, 2016b; Feng, 2014) was unexpected, as Tnks is highly evolutionarily conserved, and no other Tnks homologues exist in fly genomes. Nonetheless, the current studies reveal that a less than twofold increase in Axin levels uncovers the importance of Tnks in promoting Wg signalling during embryogenesis. Therefore, it is postulated that Tnks loss can be compensated during development unless Axin levels are increased, but that the inhibition of Wg signalling resulting from Tnks inactivation cannot be attributed solely to increased Axin levels. Furthermore, Drosophila Tnks is essential for Wg target gene activation in the adult intestine, and exclusively within regions of the gradient where Wg is present at relatively low concentration. Thus, the context-specific roles of Tnks observed in different model organisms may reflect the mechanisms described herein, which reveal that the Wnt-induced association of Axin with LRP6 occurs even in the absence of Axin ADP-ribosylation, but is markedly enhanced in its presence. It is postulated that by enhancing this interaction, Tnks-dependent ADP-ribosylation of Axin serves to amplify the initial response to Wnt stimulation, and thus is essential in a subset of in vivo contexts (Yang, 2016).

The recent discovery that Tnks enhances signalling in Wnt-driven cancers has raised the possibility that Tnks inhibitors will offer a promising new therapeutic option. Indeed, preclinical studies have supported this possibility. Tnks inhibitors were thought previously to disrupt Wnt signalling solely by increasing the basal levels of Axin, and thus by increasing destruction complex activity. However, the current findings indicate that the degree to which the basal level of Axin increases following Tnks inactivation is not sufficient to disrupt Wnt signalling in some in vivo contexts. Instead, the results reveal that Tnks inhibition simultaneously disrupts signalling at two critical and functionally distinct steps: by promoting activity of the destruction complex and by diminishing an important step in signalosome assembly: the Wnt-induced interaction between LRP6 and Axin. On the basis of these findings, it is proposed that the efficacy of Tnks inhibitors results from their combined action at both of these steps, providing a rationale for their use in the treatment of a broad range of Wnt-driven cancers. Therefore, these results suggest that in contrast with the current focus on tumours in which attenuation of the destruction complex aberrantly activates Wnt signalling (such as those lacking APC), the preclinical testing of Tnks inhibitors could be expanded to include cancers that are dependent on pathway activation by Wnt stimulation. These include the colorectal, gastric, ovarian and pancreatic cancers that harbour inactivating mutations in RNF43, a negative Wnt feedback regulator that promotes degradation of the Wnt co-receptors Frizzled and LRP6 (Yang, 2016).

Protein Interactions

The 20-amino acid repeat region in the central part of human APC down-regulates cytoplasmic beta-catenin levels in a colon carcinoma cell line (SW480), which lacks endogenous wild-type APC (Munemitsu, 1995). To test whether Drosophila Apc might have a similar function, an expression construct with a partial Apc cDNA containing the beta-catenin binding sites was generated and transiently transfected into the colon carcinoma cell line SW480. Forty-eight hours after transfection, the protein level and cellular localization of beta-catenin were measured by Western blot analysis and immunostaining, respectively. Apc significantly reduces the concentration of beta-catenin protein, with an efficiency ~60% that of human APC. Immunostaining shows that intracellular cytoplasmic beta-catenin decreases significantly, after introduction of the Apc fragment, but beta-catenin localizing at the plasma membrane is not significantly altered. These results demonstrate that Apc can down-regulate intracellular beta-catenin levels similar to that of human APC, suggesting that Apc is also a functional homolog of the human APC (Hayashi, 1997).

The existence of homologous beta-catenin binding sites in Drosophila Apc raises a question whether Apc interacts with the Drosophila homolog of beta-catenin, the Armadillo protein. To test this possibility an in vitro binding assay was carried out using a bacterially expressed Apc fusion protein containing beta-catenin binding sites and Arm protein translated in vitro. Arm binds to the Apc fragment containing the beta-catenin binding sites, but not to a control composed of a beta galactosidase fusion protein, suggesting that binding between Arm and the Apc fragment is specific. Altogether these results indicate that the beta-catenin binding sites in Apc can substitute for human APC in the down-regulation of beta-catenin, and that the same region interacts directly with Arm (Hayashi, 1997).

Drosophila Armadillo and its vertebrate homolog beta-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin, that then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered beta-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/beta-catenin is not essential for signaling. Tethered plakoglobin or beta-catenin might signal on their own or might act indirectly by elevating levels of endogenous beta-catenin. These hypotheses were tested in Drosophila by removing endogenous Armadillo. A series of mutant Armadillo proteins with altered intracellular localizations were generated, and these were expressed in wild-type and armadillo mutant backgrounds. Membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. Mutant forms of Armadillo were generated carrying either heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo’s nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners (Cox, 1999).

Data in vivo suggest that among Arm’s known partners, cadherins have the highest affinity, with APC and dTCF (Pangolin) having lower and lowest affinities, respectively. Thus, in embryos with reduced levels of Arm, the remaining Arm is exclusively associated with cadherins, as assayed by immunolocalization and by function. About 70% of cellular Arm is cadherin-associated. When cadherin binding sites are saturated, excess Arm binds to APC/Axin, leading to its destruction and thus preventing accumulation of free Arm. While APC levels, at least in mammalian cells, are low, relative to the total pool of beta catenin, Arm bound to APC is rapidly targeted for destruction, thus opening the way for the binding of additional Arm. Normally the destruction machinery can not only dispose of all non-junctional Arm, but its resources will not even be fully employed, since Arm synthesis can be increased several-fold without biological consequences. However, when the destruction machinery is inactivated either by Wg signal or mutation, Arm is synthesized but not destroyed, and thus levels of Arm rise. APC can bind Arm but in all probability, the APC is rapidly saturated, allowing accumulation of sufficient Arm to allow dTCF to effectively compete for binding. DE-cadherin, dAPC, dTCF and any other possible unknown partners together account for virtually all the Arm in a normal embryo; little if any free Arm is present. This model helps explain the differences in localization of the Armadillo attached to a nuclear localization sequence (Arm-NLS) and Armadillo attached to a nuclear export signal (Arm-NES) in flies and frogs. In Xenopus, added NLS or NES signals dramatically altered Arm’s intracellular distribution as expected, while in Drosophila the distribution of wild type Armadillo, Arm-NLS and Arm-NES are indistinguishable. It is proposed that this reflects differences in the level of expression. In flies, mutant Arm accumulates at near wild-type levels, so its binding partners can accommodate the additional protein. Arm bound to cadherin at the plasma membrane is unavailable for nuclear import; likewise Arm in a complex with dTCF is not available for export. Thus Arm-NLS and Arm-NES localization is primarily determined by their binding partners, resulting in a near normal localization. In contrast, Arm-NLS and Arm-NES expression levels in Xenopus likely exceed those of either endogenous beta-catenin or its binding partners. Free Arm is thus accessible to the nuclear import and export machinery, allowing alteration of its localization. Given this, is nuclear localization of Arm a regulated step in Wg signaling in normal cells? The fact that a subset of cells accumulate cytoplasmic but not nuclear Arm suggests that nuclear import may be regulated. In the simplest situation, addition of an NLS ought to promote Arm nuclear accumulation and trigger signaling, while addition of an NES should antagonize signaling. However, heterologous targeting signals have only subtle effects on signaling. Arm-NES signals in the same fashion as does Arm-WT, while only a subset of the Arm-NLS lines are activated for signaling. In the case of Arm-NLS: in cells in which the destruction machinery is on, no free Arm is available for nuclear import or export. In cells with intermediate levels of Wg signaling, the destruction machinery may be slowed, allowing accumulation of cytoplasmic Arm in complex with APC, but not to sufficient levels to saturate APC and allow nuclear import. Only when signaling is fully activated would sufficient free Arm accumulate for nuclear import. Addition of an NLS would thus only alter the balance in cells near the signaling threshold. Further, if nuclear Arm is bound to dTCF, it may be inaccessible to the nuclear export machinery. The mechanisms by which Arm/beta-catenin enters nuclei remain unclear; dTCF-dependent and independent pathways may exist. The recent observation that beta Catenin may mediate its own nuclear transport, independent of importins, further complicates the issue. Additional levels of regulation may occur, beyond the simple regulation of Arm/beta Catenin stability (Cox, 1999 and references).

Wnt/Wingless directs many cell fates during development. Wnt/Wingless signaling increases the amount of beta-catenin/Armadillo, which in turn activates gene transcription. The Drosophila protein Axin is shown to interact with Armadillo and Drosophila APC. D-Axin was identified in a yeast two-hybrid screen for proteins that bind the Armadillo repeat domain of Arm. d-axin codes for a protein of 743 amino acids. A region near its N-terminus shows similarity to the regulator of G protein signaling (RGS domain), whereas its C-terminus contains a region homologous to a conserved sequence near the N-terminus of Dishevelled. Thus D-Axin has a domain structure very similar to that of proteins of the mammalian Axin family. Unlike mammalian Axin family members, which bind to GSK-3beta, D-Axin does not bind to the homologous protein Shaggy/Zeste white3. d-axin is expressed maternally and is ubiquitously expressed during development. Embryos devoid of maternal and zygotic d-axin have completely naked ventral cuticle, lacking all denticles (Hamada, 1999).

During wing disc development, Wg signaling is induced along the dorsoventral compartment boundary in the wing imaginal disc. Arm accumulates in the cytoplasm, associates with its partner Pangolin, and activates expression of target genes such as Distal-less. Mutation of d-axin results in the accumulation of cytoplasmic Armadillo and results in elevation of Distal-less. Ectopic expression of d-axin inhibits Wingless signaling. Hence, D-Axin negatively regulates Wingless signaling by down-regulating the level of Armadillo. It is speculated that the Axin family of proteins functions to establish a threshold to prevent premature signaling events caused by Wg/Wnt and to restrict areas that are capable of responding to Wg/Wnt. These results establish the importance of the Axin family of proteins in Wnt/Wingless signaling in Drosophila (Hamada, 1999).

Adherens junctions inhibit asymmetric division in the Drosophila epithelium: EB1 homologs are required for the symmetric epithelial division along the planar axis

Asymmetric division is a fundamental mechanism for generating cellular diversity. In the central nervous system of Drosophila, neural progenitor cells called neuroblasts undergo asymmetric division along the apical-basal cellular axis. Neuroblasts originate from neuroepithelial cells, which are polarized along the apical-basal axis and divide symmetrically along the planar axis. The asymmetry of neuroblasts might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. This study shows that by disrupting adherens junctions the symmetric epithelial division can be converted into asymmetric division. It was further confirmed that the adenomatous polyposis coli (APC) tumour suppressor protein is recruited to adherens junctions, and demonstrated that both APC and microtubule-associated EB1 homologs are required for the symmetric epithelial division along the planar axis. These results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions (Lu, 2001).

Drosophila neuroblasts delaminate from a polarized epithelial layer in the ventral neuroectoderm and divide asymmetrically along the apical-basal axis to produce larger apical neuroblasts and smaller basal ganglion mother cells. Previous studies identified Inscuteable (Insc) as a central protein in organizing neuroblast division. Insc provides positional information that couples mitotic spindle orientation with the basal localization of cell-fate determinants such as Numb and Prospero together with their respective adaptor proteins Partner of Numb (Pon) and Miranda (Lu, 2001).

The apical localization of Insc involves both a Baz-dependent initiation step and a maintenance step that requires Baz and Partner of Inscuteable (Pins). The expression of Baz and Pins in both neuroblasts and neuroepithelial cells suggests that these cells share certain apical-basal polarity information. Consistent with this notion is the observation that, when Pon is expressed ectopically in epithelial cells it is localized to the basal cortex, as in neuroblasts. Unlike neuroblasts, however, epithelial cells divide symmetrically along the planar axis and segregate ectopic Pon equally between the two daughter cells. These observations raise further questions: do epithelial cells have the ability to couple spindle orientation with protein localization, and segregate proteins asymmetrically between two unequally sized daughter cells? If so, what prevents them from executing this asymmetric division (Lu, 2001)?

To characterize epithelial division by monitoring it in live embryos, transgenic embryos were used expressing Pon and tau proteins fused with green fluorescent protein (GFP). During epithelial cell cycle, tau-GFP-labelled mitotic spindle is formed along the planar axis of the embryo, and Pon-GFP is initially uniformly associated with the cortex and then localized to a basal crescent. The mitotic spindle remains orientated along the planar axis throughout mitosis. After cytokinesis, the Pon-GFP crescent is bisected by the cleavage furrow and is equally distributed between two equally sized daughter cells. This in vivo analysis shows that the machinery for basal protein localization is intact in epithelial cells, but it is uncoupled from spindle orientation (Lu, 2001).

The uncoupling of spindle orientation with asymmetric protein localization in epithelial cells might be due to either a lack of such a coupling mechanism or the dominance of the coupling mechanism by yet another spindle-positioning mechanism. One of the hallmarks of epithelial cells is the adherens junction, which is composed of the cadherin-catenin complex and other associated proteins, is connected to the cytoskeleton, and is thought to be important in maintaining the planar organization of the epithelial monolayer. Therefore the possible role of adherens junction in orientating epithelial division was tested. The formation of adherens junction requires genes such as shotgun, crumbs (crb) and stardust. RNA interference (RNAi) was used to disrupt Crb function and analysed the effect on epithelial division (Lu, 2001).

Double-stranded (ds) crb RNA was injected into transgenic embryos expressing Pon-GFP and tau-GFP. In about 70% of crb(RNAi) embryos, the organization of the ectodermal epithelium was disrupted, with epithelial cells losing their columnar shape, adopting rounded morphology, and becoming separated from each other. Live imaging of epithelial divisions in these embryos revealed that nearly all the epithelial cells show a tight coupling between the positioning of Pon-GFP crescents and the orientation of the mitotic spindle. Pon-GFP crescents were found at basal and lateral positions and less frequently at apical positions on the cell cortex, and one of the spindle poles was positioned underneath the Pon-GFP crescent (Lu, 2001).

After cytokinesis, Pon-GFP was segregated to one of the two similarly sized daughter cells. Asymmetric segregation of Pon-GFP to one of two similarly sized daughter cells was also observed in crb zygotic mutant embryos. Immunostaining of crb(RNAi) embryos with antibodies against Asense, Prospero and Insc indicated that epithelial cells do not express these neuronal markers, suggesting that the ability of these cells to undergo asymmetric division is not a result of cell-fate change (Lu, 2001).

Overexpression of the membrane-bound cytoplasmic tail of Crb (Crb-intra) causes similar disorganization of the epithelium as seen in crb mutants. Therefore the effect of overexpressing Crb-intra on epithelial division was examined. As observed in crb(RNAi) embryos, epithelial cells overexpressing Crb-intra showed coupling of the mitotic spindle with the Pon-GFP crescent and asymmetric segregation of Pon-GFP to one of the daughter cells. Thus, when the formation of the adherens junction is disrupted, epithelial cells switch from a symmetric to an asymmetric division pattern (Lu, 2001).

In addition to its function in localizing Insc and regulating division axis in the neuroblasts, Baz is also required for the formation of adherens junction and the maintenance of epithelial polarity. Nextthe function of Baz in epithelial division was investigated. The baz(RNAi) embryos showed overall disruption of epithelium organization similar to that observed in crb(RNAi) embryos. Unlike in crb(RNAi) embryos, however, epithelial cells in baz(RNAi) embryos divide in a symmetric fashion, with Pon-GFP distributed uniformly around the cell cortex throughout mitosis and the mitotic spindle orientated in random directions. After cytokinesis, two equally sized daughter cells were produced and Pon-GFP was equally distributed between them (Lu, 2001).

Daughter cell size asymmetry in neuroblast division is largely unaffected in baz(RNAi) embryos. It was also observed that in crb(RNAi) epithelial cells Baz can still be localized into a crescent but the crescent is mispositioned and that Pon-GFP is always localized to the opposite side of the Baz crescent. This suggests that, although mispositioned, Baz is still functional in directing Pon-GFP localization in crb(RNAi) embryos. To test whether the coupling of Pon-GFP localization with spindle orientation observed in crb(RNAi) embryos is Baz dependent, double RNAi was performed by co-injecting a mixture of baz and crb dsRNAs. Epithelial divisions in the co-injected embryos looked similar to baz single-injected embryos, with Pon-GFP segregated equally between two equally sized daughter cells. It is therefore concluded that epithelial cells depend on Baz to couple spindle orientation with protein localization when the adherens junction is disrupted (Lu, 2001).

To investigate the molecular mechanism underlying the planar positioning of spindles by the adherens junction, the function of proteins associated with the adherens junction was examined. A ubiquitously expressed, epithelial-cell-enriched APC (E-APC) is localized to the adherens junction, and, in shotgun and crb mutants, this adherens junction localization of E-APC is disrupted. The human APC protein interacts with a microtubule-associated EB1 protein, and the yeast homologue of EB1 (Bim1), together with the cortical marker Kar9, has been implicated in a search-and-capture mechanism of spindle positioning. Therefore the function of E-APC in epithelial cell division was tested (Lu, 2001).

In about 60 of the E-APC(RNAi) embryos, the positioning of Pon-GFP crescent and orientation of mitotic spindle became tightly coupled during epithelial division. At cytokinesis, epithelial cells divided asymmetrically to produce two unequally sized daughter cells, and Pon-GFP was always segregated to the smaller daughter cell. The asymmetric segregation of Pon-GFP and the ability to undergo unequal cytokinesis all depend on Baz, because in baz and E-APC double RNAi embryos, Pon-GFP is equally segregated to two similarly sized daughter cells . Therefore, in the absence of E-APC, epithelial cells divide asymmetrically in a Baz-dependent fashion. This suggests that adherens-junction-associated E-APC promotes spindle positioning along the planar axis and prevents the coupling of spindle positioning with asymmetric basal protein localization (Lu, 2001).

To test whether E-APC functions with EB1 to orientate the mitotic spindle, RNAi was performed on a closely related fly homologue of EB1 (dEB1 ). In dEB1(RNAi) embryos, the epithelial divisions were also asymmetric, producing two unequally sized daughter cells, with Pon-GFP segregated to the smaller cell. It was observed that the penetrance of dEB1(RNAi) phenotype (~20%) is lower than that of E-APC(RNAi). Since there is strong maternal contribution of dEB1, the low penetrance might be due to a perdurance of maternal dEB1 protein. Alternatively, it might be due to functional compensation by two other distantly related EB1 homologues in the fly genome. It has been noted that E-APC lacks the carboxy-terminal domain that is required for interaction with EB1, and no direct interaction was observed between E-APC and EB1 in in vitro binding assays. It therefore remains to be determined whether the two are functionally linked together in vivo through some cofactor(s), or whether E-APC functions mainly to maintain adherens junction integrity and EB1 interacts with other unidentified molecules to orientate spindles (Lu, 2001).

These results indicate that two sets of polarity cues exist for spindle positioning in epithelial cells: a planar polarity cue mediated by the adherens junction and an apical-basal polarity cue regulated by Baz. The division pattern of wild-type epithelial cells suggests that the planar polarity cue is normally dominant over the apical-basal polarity cue. Epithelial cells within the procephalic neurogenic region (PNR) that express endogenous Insc or epithelial cells outside of the PNR that express ectopic Insc are known to orientate their mitotic spindle along the apical-basal axis during division. This suggests that the dominance of planar polarity over apical-basal polarity can be overcome by the expression of Insc. The normal appearance of the adherens junction in epithelial cells in the PNR, together with the observation that these cells divide along the planar axis and maintain their normal monolayer organization in insc mutant, suggests that Insc functions by strengthening the apical-basal polarity instead of weakening the planar polarity through changing the behaviour of the adherens junction (Lu, 2001).

When neuroblasts delaminate from the epithelium layer, they undergo morphological changes from columnar to round shape, lose their contacts with the surrounding cells and thus the adherens junction structures. This situation may be reminiscent of epithelial cells in adherens-junction mutants in which the planar polarity cue is lost. In both cases, the Baz-mediated polarity pathway takes over. That one polarity cue can dominate over another cue in orientating division axis may have its precedents in other organisms. Budding yeast can divide in either an axial or a bipolar pattern. Mutations in genes such as AXL1, BUD3, BUD4 and BUD10/AXL2 result in loss of polarity cue for axial bud formation and the cells divide in a bipolar fashion. This suggests that axial and bipolar cues coexist and that the axial cue is normally dominant over the bipolar cue. During mammalian cortical neurogenesis, neural progenitors switch from early symmetric divisions to later asymmetric divisions. It will be interesting to determine whether similar mechanisms and molecules are used to control this division symmetry switch in mammals. These results on E-APC highlights the importance of tumour suppressors in regulating not only cell growth but also polarity and asymmetric division (Lu, 2001).

Shortstop recruits EB1/APC1 and promotes microtubule assembly at the muscle-tendon junction

Shortstop (Shot) is a Drosophila Plakin family member containing both Actin binding and microtubule binding domains. In Drosophila, it is required for a wide range of processes, including axon extension, dendrite formation, axonal terminal arborization at the neuromuscular junction, tendon cell development, and adhesion of wing epithelium. To address how Shot exerts its activity at the molecular level, the molecular interactions of Shot with candidate proteins was investigated in mature larval tendon cells. Shot colocalizes with the complex between EB1 and APC1 and with a compact microtubule array extending between the muscle-tendon junction and the cuticle. It is suggested that EB1 and APC1 become associated with the muscle-tendon basal hemiadherens junction in postmitotic tendon cells following their association with Shot. Shot forms a protein complex with EB1 via its C-terminal EF-hands and GAS2-containing domains. In tendon cells with reduced Shot activity, EB1/APC1 dissociate from the muscle-tendon junction, and the microtubule array elongates. The resulting tendon cell, although associated with the muscle and the cuticle ends, loses its stress resistance and elongates. These results suggest that Shot mediates tendon stress resistance by the organization of a compact microtubule network at the muscle-tendon junction. This is achieved by Shot association with the cytoplasmic faces of the basal hemiadherens junction and with the EB1/APC1 complex (Subramanian, 2003).

Tendon cells undergo maturation during larval stages. In third instar larvae, different tendon cells acquire distinct shapes according to their orientation and the type of muscles to which they are connected. Initially, the localization of Shot was characterized relative to MT and F-actin organization in mature tendon cells of flat, opened third instar larvae. Shot and Tubulin staining overlapped within the entire cell. A unique domain at the focal plane of the muscle-tendon junction exhibits a compact MT array, which overlapped Shot staining. This domain was not detected when the optical section was taken 0.5 microm more internal to the junction focal plane. An optical cross-section perpendicular to the muscle-tendon junction site shows that the MT-Shot array extends from the muscle-tendon junction to the cuticle. Moreover, the MT array is oriented in the same direction as the microfilaments of the muscle cells, as shown by EM analysis. Thus, Shot and MTs are colocalized within a unique subcellular domain in the tendon cell that connects the muscle-tendon junction and the cuticle (Subramanian, 2003).

These immunofluorescent localization studies suggest a distinct abundance of MTs and MFs at both sides of the muscle-tendon junction. While MFs are highly enriched at the muscle side, MTs are detected mainly at the tendon side. To address whether this organization reflects differences in the distribution of additional junction-associated proteins, the larval flat preps were stained for PSß-integrin, Paxillin, and P-tyrosine, and their relative distribution at the focal plane of the muscle-tendon junction was analyzed. In all preparations, Shot marks the outlines of the tendon cell. About half of the PSß-integrin staining overlaps Shot staining, whereas the other half is located at the muscle membrane. Paxillin is more abundant on the muscle side. Interestingly, staining for P-tyrosine, which marks the extent of tyrosine-phosphorylated proteins (including Paxillin, Src, and others) is restricted to the tendon side of the junction and is tightly associated with the plasma membrane. Thus, although PSß-integrin distribution appears to be equal at both sides of the muscle-tendon hemiadherens junction, the molecular composition on both sides of these junctions appears to be distinct, as demonstrated for EB1, APC1, Paxillin, and the extent of P-tyrosine reactivity. It is tempting to speculate that these unequal protein distributions might relate to the enrichment of MTs and Shot on the tendon cell side (Subramanian, 2003).

EB1 is an evolutionarily conserved protein that binds the plus ends of growing MTs. It was first identified as a binding partner for the adenomatous polyposis coli tumor suppressor, APC. The EB1/APC complex is involved in regulation of MT polymerization and MT association with distinct subcellular domains. For example, in yeast, the EB1 homolog (BIM1) has been shown to modulate MT dynamics and link MTs to the cortex. Drosophila EB1 (Rogers, 2002) is important for the proper assembly, dynamics, and positioning of the mitotic spindle. Its association with APC2 in apical cell-cell adherens junctions is suggested to be essential for parallel spindle orientation (Lu, 2002) and for neuroblast asymmetric cell division (Subramanian, 2003).

Biochemical data suggest that the association of the C-terminal EF-GAS2 domain with EB1 is MT independent. A direct physical interaction between the C-terminal EF-GAS2 domain and alpha-tubulin had been suggested by a yeast two-hybrid screen and by the ability to precipitate purified Tubulin by the GAR and GSR domains (both included within the C terminus of the mammalian ACF7). These domains lack the EF-hands motif. EB1 is detected in association with the entire Shot C-terminal domain containing EF-hands. At this stage, no additional information is available regarding the site responsible for EB1 association (Subramanian, 2003).

The data suggest that Shot association with the basal muscle-tendon junction is EB1 independent (since it is detected even in the absence of EB1/APC1); hence, it is suggested that EB1 and APC1 become associated with the muscle-tendon basal hemiadherens junction in postmitotic tendon cells following their association with Shot. The assembly of the MT-rich domain may be induced by either their direct association with Shot or with EB1/APC1, or with both. Interestingly, EB1, APC1, and Shot are not observed at the cell-cell adherens junctions formed between the tendon cell and its neighboring ectodermal cells (Subramanian, 2003).

There is no direct evidence for the polarity of MTs within the compact Shot/MT-rich domain, since no differential EB1 localization was detected in this domain. Other studies suggest that MTs in the entire epidermis are arranged at a polar orientation in which their plus ends face the basal pole and their minus ends face the cuticle. Similarly, in the pupal wing, MTs have been shown to be arranged with their plus ends facing the basal hemiadherens junctions. Thus, it is likely that in the tendon cells (which are part of the epidermal layer), MTs are similarly arranged, i.e., with their plus ends facing the basal hemiadherens junction (Subramanian, 2003).

The experiments show that reduced Shot activity leads to a significant tendon cell elongation, occurring presumably following muscle contractions. What is the mechanism allowing the MTs to elongate in the mutant tendon cell? An interesting possibility is that the MTs are connected to the muscle-tendon junction through their plus ends via their association with EB1 and Shot, and that this arrangement arrests further MT polymerization and maintains the MTs in a polarized arrangement. Following dissociation of the Shot/EB1/APC1 complex and the reduction of Shot activity, MTs undergo further polymerization and extension, leading to the significant elongation of the tendon cell. The newly formed MTs are not well connected to the cell cortex, thus leading to cell breakdown upon further muscle contraction. Support for the involvement of Shot in mediating MT-polarized organization emerges from recent analysis of Shot function in mushroom body neurons of the Drosophila adult brain. Distinct Nod-ßgal reactivity suggests that MT polarity within the axons is distinct from that of dendrites in wild-type mushroom body neurons. In neurons mutant for shot, the polarity of MTs in the axons is reversed and resembles that of dendrites (Subramanian, 2003).

Shot may perform a similar function in the organization of a compact and polarized array of MTs in the adult wing epithelium, as well as within the ligament cells of embryonic sensory chordotonal organs (Subramanian, 2003).

Studies with the different Shot domains show that the Actin binding domain, but not the Plakin domain, is capable of driving specific localization of both domains to the F-actin layer at the muscle-tendon junction. The thin Actin layer at the muscle-tendon junction may therefore be essential for the recruitment of Shot into the cytoplasmic faces of the hemiadherens domains. The C terminus containing the EF-hands and GAS2 domains is also capable of localizing at the muscle-tendon junction domain. This localization may be attributed to its association with endogenous EB1, as well as with MTs that are already arranged in the larval tendon cells, or with endogenous Shot. The Plakin domain on its own did not show specific subcellular localization, suggesting that it does not bind to proteins that are highly localized in the tendon cell. Alternatively, proteins that may form a complex with this domain may be engaged in existing protein complexes and therefore are not accessible to the exogenous Plakin protein. None of the Shot structural domains show a dominant-negative effect when overexpressed in tendon cells or in wing imaginal discs; this finding suggests that the proteins to which they bind are not present in limited amounts (Subramanian, 2003).

Interestingly, in larvae tendon cells, both the Actin-Plakin domain of Shot and the Shot C-terminal EF-GAS2 domain exhibit similar distribution. When transfected into Schneider cells, each domain shows a distinct subcellular localization. The Actin-Plakin-GFP was detected at the leading edge and in most cases did not overlap EB1, while the EF-GAS2 domain overlapped EB1 and decorated MTs. Similar studies with Drosophila Shot and ACF-7, the mammalian Shot ortholog, show that the Actin binding domain and the EF-GAS2 domain are associated with Actin MFs and with MTs, respectively, in transfected cells. However, the M1 domain in ACF-7 (similar to the Plakin domain) has been shown to be associated with MTs in the transfected cells, while the Shot-Plakin domain does not exhibit significant association with MTs. These differences may reflect differential distribution of yet uncharacterized ACF-7 binding proteins within the mammalian cells (Subramanian, 2003).

What could be the connection between Shot activity and reduced F-actin content? Recent studies suggest that MT disassembly activates Rho by the release of GEFs that are specifically associated with and inhibited by MTs. In tendon cells, no unique association was detected of GEF (Pebble) or Rho with the MT-rich domain. Therefore, the relevance of these factors is not clear. Recently, it was shown that in Drosophila embryonic tracheal cells, activated RhoA mimicks the Shot loss-of-function phenotype; this finding suggests a similar inverse correlation between Actin polymerization by RhoA and the loss of shot function. Thus, the activity of Shot in organizing MTs to special subcellular sites via its association with EB1/APC1, and the inhibition of F-actin in these sites, may be relevant to other tissues in which Shot plays an essential role (Subramanian, 2003 and references therein).

The MT network is essential for a wide array of cellular functions. Shot, a multidomain Plakin family member, is essential for arranging a compact network of MTs in tendon cells. This is achieved by the association of Shot with the cytoplasmic faces of the muscle-tendon junction and presumably by the subsequent recruitment of the EB1/APC1 complex to these sites. In tendon cells, this unique MT organization is essential to resist muscle contraction (Subramanian, 2003).

A roles of Dishevelled and APC 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).

The subcellular distribution of Axin-GFP was studied at late embryonic stages, i.e., in epidermal cells that are no longer stimulated by Wingless ('-Wg cells'). In these -Wg cells, conspicuous green dots are seen throughout the cytoplasm. Similar dots have been observed in vertebrate cells expressing tagged Axin; these dots are associated with vesicles. Interestingly, most of the Axin-GFP dots coincide with dots of E-APC staining. E-APC is the main APC protein expressed in the Drosophila embryonic epidermis; many of the E-APC dots accumulate in apicolateral regions along the plasma membrane. This can be seen in young embryos that have just begun to express Axin-GFP, but, in older embryos in which Axin-GFP has accumulated to high levels, E-APC is largely delocalized from the plasma membrane and is recruited into the cytoplasmic Axin-GFP dots, presumably by direct binding. It is likely that these dots represent the Axin destruction complex. Thus, in -Wg cells, this complex appears to be located predominantly in the cytoplasm, where it actively promotes the degradation of Armadillo (Cliffe, 2003).

Next, Axin-GFP was expressed in embryos without APC function, i.e., in embryos that express a mutant E-APC protein (N175K) and lack the second APC protein (dAPC) that acts redundantly with E-APC. These APC double mutants show very few Axin-GFP dots, and the green fluorescence appears mostly diffuse or grainy. Indeed, the staining of the mutant E-APC N175K protein itself appears grainy and is much less dotty than the staining of wild-type E-APC. The few remaining dots colocalize with Axin-GFP dots. Thus, E-APC is required for the formation of the Axin-GFP dots, indicating that the N175K mutant cannot promote Axin complex formation (Cliffe, 2003).

The N175K mutant bears a missense mutation in a surface residue of its Armadillo repeat domain, and its loss of function is due to its inability to associate with the plasma membrane. This results in naked cuticles, the hallmark of ubiquitous Wingless activation. Intriguingly, the N175K mutant is a fully stable protein that retains its Axin binding site. It binds to Axin as efficiently as wild-type E-APC in vitro. Thus, the inability of the N175K mutant protein to associate with the plasma membrane appears to be the sole reason for its failure to promote Axin complex assembly (Cliffe, 2003).

Expression of Axin-GFP in the APC double mutant embryos restores their mutant phenotype partially toward normal. Thus, Axin-GFP is less active in these mutants; this finding confirms that Axin function depends on APC. This dependence is strong but not absolute, and it is likely to reflect the role of APC in promoting Axin complex assembly. Moreover, overexpression of Axin-GFP compensates to some extent for the loss of APC. This parallels the results in APC mutant cancer cells in which overexpressed Axin proteins can bypass the function of APC; this finding suggested that APC has a regulatory role with regard to Axin. This regulatory role could be to target Axin to a specific subcellular location: one would expect APC-mediated targeting to be less critical at elevated levels of Axin expression (Cliffe, 2003).

Axin-GFP expression was examined next in the epidermis of 3- to 6-hr-old embryos; at this stage, stripes of +Wg cells alternate with stripes of -Wg cells. As in older embryos, conspicuous dots of Axin-GFP are scattered throughout the cytoplasm of -Wg cells. Strikingly, in +Wg cells, these dots are associated almost exclusively with apicolateral regions of the plasma membrane. This is observed neither in the epidermis of older embryos that lack Wingless expression nor in wingless mutants. Conversely, coexpression of Wingless with Axin-GFP causes a relocation of virtually all Axin-GFP dots to the plasma membrane and also restores the membrane-associated staining of E-APC in older embryos. Thus, Wingless signaling is both necessary and sufficient for relocation of the Axin-GFP dots to the plasma membrane. Notably, a FRET signal between Axin and LRP-5 has been observed in Wnt-stimulated mammalian cells; this result suggested a Wnt-induced recruitment of Axin to the plasma membrane. This result is the first direct demonstration that Wnt signaling triggers a relocation of Axin to the plasma membrane (Cliffe, 2003).

Axin-GFP levels were examined by Western blot analysis to confirm that Axin-GFP is expressed at moderate levels as an intact full-length fusion protein. Coexpression with Wingless does not change these levels of Axin-GFP, although this analysis can only detect a maximal reduction to 50%. The exposure of these embryos to ubiquitous Wingless was 0-8 hr, so the inability to detect a decrease in Axin-GFP levels in response to Wingless is not inconsistent with the previously determined half-life of tagged mammalian Axin of 4 hr under Wnt signaling conditions. Under these experimental conditions, the main effect of Wingless signaling is clearly a relocation of Axin to the plasma membrane rather than a destabilization of Axin (Cliffe, 2003).

It was asked whether relocation of Axin-GFP to the plasma membrane might be sufficient for its inactivation. If so, overexpressed Wingless should block the excessive activity of Axin-GFP. This is only partly true: some restoration of naked cuticle (predominantly along the midline) is seen in embryos coexpressing Wingless and Axin-GFP compared to embryos expressing Axin-GFP alone. Thus, a component upstream of Axin but downstream of Wingless may be limiting in the inactivation of Axin. The relocation of Axin to the plasma membrane may be a necessary first step toward its inactivation (Cliffe, 2003).

To identify further components of the Wingless pathway that are required for this relocation, 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).

Membrane bound forms of activated Armadillo ('Arm*', i.e., forms lacking their N termini) show significantly more signaling activity than Arm* without a membrane-targeting domain; this finding led to the suggestion that Armadillo exerts its signaling function in the cytoplasm rather than in the nucleus. However, overexpression of membrane-targeted Arm* causes a dramatic relocation of Axin-GFP, and of E-APC, to the plasma membrane throughout the embryonic epidermis, presumably by direct binding. This mimics the Wingless-induced membrane relocation of Axin-GFP, except that the membrane-targeted Arm* relocates Axin-GFP and E-APC to the entire lateral membrane where it itself is localized. No such relocation is seen under conditions of ubiquitous high levels of untargeted Arm*. The striking relocation of Axin-GFP to the plasma membrane by the membrane-targeted Arm* may cause its inactivation even in cells that are only weakly stimulated by Wingless; thus, this finding provides an alternative explanation for the increased activity of membrane bound Armadillo (Cliffe, 2003).

This work provides evidence that the assembly of Axin complex in the cytoplasm depends on a membrane-targeting function of E-APC. This function may also affect targeting to internal membranes, or vesicles, suggesting that the Axin complex may be associated with vesicles. In support of this, overexpressed Axin is associated with vesicles in Xenopus embryos. Furthermore, Dsh (which is required for the Wingless-induced membrane relocation of Axin) is also associated with vesicles, and to some extent with the plasma membrane, in vertebrate and Drosophila cells. Indeed, Axin and Dsh colocalize after overexpression in vertebrate cells. Notably, the DIX domain of the mammalian Dsh protein Dvl-2 contains a phospholipid binding motif that is conserved in the DIX domain of Axin, and targeting of Dvl-2 to vesicles by this motif is essential for its function in controlling the degradation of β-catenin (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).

Characterization of Drosophila Eb1, a potential partner of APC

EB1, a parter of APC, is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homolog (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, dynamics of single microtubules in real time were visualized. Depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. These results reveal crucial roles for EB1 in mitosis, which is postulated to involve its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex (Rogers, 2002).

Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome

Stem cell self-renewal can be specified by local signals from the surrounding microenvironment, or niche. However, the relation between the niche and the mechanisms that ensure the correct balance between stem cell self-renewal and differentiation is poorly understood. This study shows that dividing Drosophila male germline stem cells use intracellular mechanisms involving centrosome function and cortically localized Adenomatous Polyposis Coli tumor suppressor protein to orient mitotic spindles perpendicular to the niche, ensuring a reliably asymmetric outcome in which one daughter cell remains in the niche and self-renews stem cell identity, whereas the other, displaced away, initiates differentiation (Yamashita, 2003).

Adult stem cells maintain populations of highly differentiated but short-lived cells such as skin, intestinal epithelium, or sperm through a critical balance between alternate fates: Daughter cells either maintain stem cell identity or initiate differentiation. In Drosophila testes, germline stem cells (GSCs) normally divide asymmetrically, giving rise to one stem cell and one gonialblast, which initiates differentiation starting with the spermatogonial transient amplifying divisions. The hub, a cluster of somatic cells at the testis apical tip, functions as a stem cell niche: Apical hub cells express the signaling ligand Unpaired (Upd), which activates the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway within GSCs to maintain stem cell identity (Yamashita, 2003).

Analysis of dividing male GSCs by expression of green fluorescent protein (GFP)-α-tubulin in early germ cells revealed that in 100% of the dividing stem cells observed, the mitotic spindle was oriented perpendicular to the hub-GSC interface throughout mitosis, with one spindle pole positioned within the crescent where the GSC contacted the hub. Stem cell division was rare, averaging one dividing stem cell observed per 5 to 10 testes (~2% of total stem cells) in 0- to 2-day-old adults. Spindles were not oriented toward the hub in gonialblasts (Yamashita, 2003).

Drosophila male GSCs maintained a fixed orientation toward the hub throughout the cell cycle, unlike Drosophila embryonic neuroblasts or the Caenorhabditis elegans P1 cell, in which spindle orientation is established during mitosis by a programmed rotation of the spindle. The single centrosome in early interphase GSCs was consistently located adjacent to the hub. After centrosome duplication, one centrosome remained adjacent to the hub, whereas the other migrated to the opposite side of the nucleus. The mechanisms responsible for Drosophila GSC spindle orientation may differ between sexes. In female GSCs, the spectrosome, a spherical intracellular membranous structure, remains localized next to the apical cap cells, where it may help anchor the spindle pole during mitosis. In interphase male GSCs, in contrast, the spectrosome was often located to the side, whereas at least one centrosome held the stereotyped position adjacent to the hub (Yamashita, 2003).

To investigate centrosome function in orientation of male GSCs, males were analyzed that were null mutant for the integral centrosome component centrosomin (cnn), which is required for normal astral microtubule function. In cnn mutant males, mitotic spindles were not oriented toward the hub in ~30% of the dividing GSCs examined. In an additional 10% to 20%, spindles were properly oriented, but the proximal spindle pole was no longer closely associated with the cell cortex at the hub-GSC interface and the entire spindle was displaced away from the hub. The frequency of spindle orientation defects was highest in metaphase. Loss of function of cnn also partially randomized the interphase centrosome positioning in male GSCs. In more than 35% of the cnn mutant GSCs with duplicated centrosomes that were scored, neither centrosome was positioned next to the hub (Yamashita, 2003).

The number of germ cells associated with the hub was increased 20% to 30% in cnn mutant males, from an average of 8.94 GSCs per hub in the wild type to 11.89 GSCs per hub in cnnHK21/cnnHK21 and 10.69 GSCs per hub in cnnHK21/cnnmfs3. Hub size was not significantly different in cnn compared with wild-type males. In cnn testes with many stem cells, GSCs appeared crowded around the hub and often seemed attached to the hub by only a small region of cell cortex. Finite available physical space around the hub may limit the increase in stem cell number in cnn mutant males (Yamashita, 2003).

As suggested by the increased stem cell number, there were several cases in both live and fixed samples from cnn males in which a stem cell that had recently divided with a mitotic spindle parallel to the hub-GSC interface produced two daughter cells that retained contact with the hub, a finding that was not observed in the wild type. GSCs were also observed dividing with a misoriented/detached spindle that lost attachment to the hub, probably explaining the mild increase in stem cell number relative to the frequency of misoriented spindles (Yamashita, 2003).

The normal close attachment of one spindle pole to a region of the GSC next to the hub and the effects of cnn mutants on centrosome and spindle orientation suggest that a specialized region of the GSC cell cortex touching the hub might provide a polarity cue toward which astral microtubules from the centrosome and spindle pole orient. High levels of DE-cadherin (fly epithelial cadherin) and Armadillo (Arm; fly ß-catenin) colocalized at the hub-GSC interface, as well as at the interface between adjacent hub cells, marked by high levels of Fas III. High levels of DE-cadherin and Arm were not detected around the rest of the GSC surface. Forced expression of DE-cadherin-GFP specifically in early germ cells confirmed that DE-cadherin in GSCs colocalized to the hub-GSC interface (Yamashita, 2003).

DE-cadherin and Armadillo at the hub-GSC interface may provide an anchoring platform for localized concentration of Apc2, one of two Drosophila homologs of the mammalian tumor suppressor gene Adenomatous Polyposis Coli (APC), which in turn may anchor astral microtubules to orient centrosomes and the spindle. Immunofluorescence analysis revealed Apc2 protein localized to the hub-GSC interface. In apc2 mutant males, GSCs were observed with mispositioned centrosomes, misoriented spindles, or detached spindles. Both the average number of stem cells and hub diameter increased in apc2 mutant males compared with that of the wild type. Unlike in cnn mutants, GSCs did not appear crowded around the hub in apc2 males, perhaps as a result of the enlarged hub (Yamashita, 2003).

The second Drosophila APC homolog, apc1, may also contribute to normal orientation of the interphase centrosome and mitotic spindle. Apc1 protein localized to centrosomes in GSCs and spermatogonia during late G2/prophase, after centrosomes were fully separated but before nuclear envelope breakdown. Apc1 was not detected at centrosomes from prometaphase to telophase. Spindle orientation and centrosome position were perturbed in GSCs from apc1 males, and the number of stem cells per testis and the diameter of the hub both slightly increased in apc1 mutant testes compared with those of the wild type (Yamashita, 2003).

It is proposed that the reliably asymmetric outcome of male GSC divisions is controlled by the concerted action of (1) extrinsic factor(s) from the niche that specify stem cell identity, and (2) intrinsic cellular machinery acting at the centrosome and a specialized region of the GSC cortex located at the hub-GSC interface to orient the cell division plane with respect to the signaling microenvironment. Astral microtubules emanating from the centrosome may be captured by a localized protein complex including Apc2 at the GSC cortex where it interfaces with the hub, similar to the way in which cortical Apc2 may orient mitotic spindles in the syncytial embryo or epithelial cells (Yamashita, 2003).

Mechanisms that orient the mitotic spindle by attachment of astral microtubules to specific cortical sites may be evolutionally conserved. In budding yeast, spindle orientation is controlled by capture and tracking of cytoplasmic microtubules to the bud tip, dependent on Kar9, which has weak sequence similarity to APC proteins. Kar9 has been localized to the spindle pole body and the cell cortex of the bud tip, reminiscent of the localization of Drosophila Apc1 at centrosomes and Apc2 at the cell cortex (Yamashita, 2003).

Polarization of Drosophila male GSCs toward the hub could result simply from the geometry of cell-cell adhesion. GSCs appear to be anchored to the hub in part through localized adherens junctions. Homotypic interactions between DE-cadherin on the surface of hub cells and male GSCs could concentrate and stabilize a patch of DE-cadherin. The resulting localized DE-cadherin cytoplasmic domains could then provide localized binding sites for ß-catenin and Apc2 at the GSC cortex. Although binding of E-cadherin and APC to ß-catenin is thought to be mutually exclusive, APC could be anchored at the cortical patch through the actin cytoskeleton, which in turn could interact with ß-catenin/α-catenin (Yamashita, 2003).

Orientation of stem cells toward the niche appears to play a critical role in the mechanism that ensures a reliably asymmetric out-come of Drosophila male GSC divisions, consistently placing one daughter within the reach of short-range signals from the hub and positioning the other away from the niche. Oriented stem cell division may be a general feature of other stem cell systems, helping maintain the correct balance between stem cell self-renewal and initiation of differentiation throughout adult life (Yamashita, 2003).

The APC tumor suppressor binds to C-terminal binding protein to divert nuclear ß-Catenin from TCF

Adenomatous polyposis coli (APC) is an important tumor suppressor in the colon. APC antagonizes the transcriptional activity of the Wnt effector ß-catenin by promoting its nuclear export and its proteasomal destruction in the cytoplasm. This study reports a third function of APC in antagonizing ß-catenin involving C-terminal binding protein (CtBP). APC is associated with CtBP in vivo and binds to CtBP in vitro through its conserved 15 amino acid repeats. Failure of this association results in elevated levels of ß-catenin/TCF complexes and of TCF-mediated transcription. Notably, CtBP is neither associated with TCF in vivo nor does mutation of the CtBP binding motifs in TCF-4 alter its transcriptional activity. This questions the idea that CtBP is a direct corepressor of TCF. The evidence indicates that APC is an adaptor between ß-catenin and CtBP and that CtBP lowers the availability of free nuclear ß-catenin for binding to TCF by sequestering APC/ß-catenin complexes (Hamada, 2004).

To identify proteins that bind to APC in Drosophila embryos, crude embryonic extracts were incubated with bacterially expressed Drosophila E-APC fused to glutathione-S-transferase (GST). Analysis of associated proteins by MALDI mass spectrometry reveals dCtBP as an unexpected binding partner of E-APC. CtBP was initially discovered as a cellular protein binding to the C terminus of the adenovirus E1A protein, which suppresses its transformation potential. CtBP is a transcriptional corepressor in mammals and binds to various DNA binding proteins via a short conserved motif P-h-D-L-S-x-R/K. Mammals have a second CtBP relative, CtBP2, which also recognizes this motif and whose function overlaps that of CtBP (Hamada, 2004).

Intriguingly, a motif similar to P-h-D-L-S-x-R/K is found in each of the 15 amino acid repeats (15R) of APC and of Drosophila E-APC. These repeats can bind to ß-catenin but cannot promote its proteasomal destruction; the latter requires the Axin binding motifs of APC. Therefore, there is no known function of the 15Rs in the downregulation of ß-catenin. The interaction between an individual 15R and ß-catenin has been characterized at the structural level. The presumed CtBP binding motif shares some but not all of the residues in the C-terminal half of the 15R that are engaged in the interaction with ß-catenin (Hamada, 2004).

Binding between E-APC and dCtBP was confirmed in vitro by pull-down assays between bacterially expressed GST-dCtBP and in vitro translated E-APC. This binding is comparable to that between E-APC and Armadillo (Drosophila ß-catenin); however, Armadillo does not bind directly to GST-dCtBP. A small region spanning the two 15Rs of E-APC fused to GST is sufficient for binding to in vitro translated dCtBP, while a triple alanine substitution ('AxAxA') in the P-h-D-L-S motif of each 15R (in the context of the C-terminal half of E-APC) almost completely abolishes binding to dCtBP. The same is true for the binding between human CtBP and a central fragment of APC (residues 918-1698) that binds efficiently to GST-CtBP, while its mutant version AxAxA binds poorly. APC(918-1698) contains two further putative CtBP binding motifs that were substituted in addition ('AxAxAplus'). This further reduced the binding to GST-CtBP (by >16%); no binding whatsoever was detectable with a GST-LEF-1 control. Importantly, both APC mutants bind to ß-catenin equally well as the wild-type. Likewise, both mutants retain the ability to reduce the overall levels of coexpressed HA-tagged ß-catenin in transfected APC mutant cancer cells, though a low level of endogenous ß-catenin can still be detected by immunofluoresence in these transfected cells. Thus, the binding between APC and CtBP is specific and conserved and neither appears to affect APC's binding to ß-catenin nor its ability to promote the destruction of cytoplasmic ß-catenin (Hamada, 2004).

APC is also associated with CtBP in mammalian cells: endogenous CtBP can be coimmunoprecipitated with endogenous APC, and vice versa, in 293T cells and in HCT116 colorectal cancer cells that express wild-type APC. Furthermore, in APC mutant cancer cells, the resident APC truncations can be coimmunoprecipitated in SW480 cells, but not in COLO320 cells. Notably, the 15Rs are retained only in the APC truncation of the former, but not of the latter. Thus, the association of APC with CtBP in mammalian cells depends on its 15Rs (Hamada, 2004).

Few colorectal carcinomas express APC truncations that lack the 15Rs. COLO320 is one of the rare colorectal cancer cell line of this type. Interestingly, this line exhibits exceptionally high TCF-mediated transcription. This suggests that the 15Rs may harbor an activity that is critical for the downregulation of the transcriptional activity of TCF (Hamada, 2004).

To test whether the binding of CtBP to the 15Rs is functionally relevant, a complementation assay was used of APC mutant cancer cells based on a luciferase reporter linked to TCF binding sites (pTOPFLASH). This quantitative assay is highly specific for TCF-mediated transcription and serves as a fairly direct readout of exogenous APC function in restoring low levels of TCF transcription. COLO320 cells show very high TOPFLASH values, >2× higher than those of SW480 cells and up to 5× higher than those of other APC mutant colorectal cancer cells. These values are reduced substantially after cotransfection with APC(918-1698), which spans the 15Rs and the 5'-most nuclear export signal (NES1506) and Axin binding site. Similar APC fragments have previously been found to efficiently reduce the ß-catenin levels in SW480 cells. In contrast, the AxAxA mutant is less active in reducing TOPFLASH values, and AxAxAplus is even less active. The control values of pFOPFLASH (containing mutant TCF sites) are low and unchanged by the mutants. It is concluded that the binding between APC and CtBP is critical for the APC-mediated downregulation of the transcriptional activity of ß-catenin. The residual activities of AxAxA and AxAxAplus in this assay are likely to reflect their ability to promote Axin-mediated destruction and nuclear export of ß-catenin; note that APC(918-1698) and its mutant versions shuttle in and out of the nucleus, as judged by their nuclear accumulation after exposure to leptomycin B (Hamada, 2004).

Evidence has indicated that APC can sequester nuclear ß-catenin and keep it from binding to TCF and activating transcription. This sequestration can be demonstrated experimentally if an APC fragment is targeted to the nucleus by linkage to a nuclear localization signal (NLS): this causes a dramatic nuclear accumulation of endogenous ß-catenin, but these high levels of nuclear ß-catenin are ineffective in stimulating TCF-mediated transcription. This therefore provides an assay for measuring the sequestration of nuclear ß-catenin by APC (Hamada, 2004).

NLS-fusions of the AxAxA and AxAxAplus mutants were tested in this sequestration assay. Interestingly, the mutant NLS-fusions are less active in reducing TOPFLASH values than their wild-type controls. These differences are significant since the expression levels of wild-type and mutant NLS-fusions are essentially the same. Notably, the loss of function of the AxAxA and AxAxAplus mutants in reducing ß-catenin activity is exacerbated in this sequestration assay where the levels of nuclear ß-catenin are high. This suggests a role of the APC-CtBP interaction in sequestering nuclear ß-catenin (Hamada, 2004).

A possible model is that APC binds to free nuclear ß-catenin in competition with TCF and targets ß-catenin to CtBP (by being an adaptor between these two proteins), thus diverting ß-catenin away from TCF. CtBP, being anchored at specific sites within the nucleus, could act as a "sink" for APC/ß-catenin complexes, thus shifting the binding equilibrium of ß-catenin yet further away from TCF (Hamada, 2004).

Three lines of evidence support this model: (1) ß-catenin can be detected in a complex with CtBP in SW480, but not in COLO320 cells, whose APC truncation can bind neither CtBP nor ß-catenin; (2) in COLO320 cells transfected with NLS-fusions of APC, it is estimated that the levels of endogenous TCF-4/ß-catenin complexes are 1.5×-2× higher in the case of AxAxAplus compared to the wild-type control. These increased levels of TCF-4/ß-catenin complexes are likely to be the basis for the high TCF-mediated transcription in the complementation assays. (3) In CtBP mutant mouse cells expressing tagged LEF-1, 2×-3× more endogenous ß-catenin can be coimmunoprecipitated with LEF-1 than in the corresponding parental control cells (heterozygous for both alleles). The total levels of ß-catenin are the same in the two cell lines, as are the amounts of APC bound ß-catenin. The latter two lines of evidence indicate that CtBP reduces the availability of ß-catenin for binding to TCF (Hamada, 2004).

If so, absence of CtBP should result in elevated levels of TCF-mediated transcription. Indeed, the basal TOPFLASH activity (due to endogenous TCF/ß-catenin) in CtBP mutant cells is increased ~3.7× compared to their control cells. Furthermore, cotransfection of activates ß-catenin (S33A mutant) and Lef-1 stimulate TOPFLASH activity to higher levels in CtBP mutant cells compared to the control. By comparison, <2× differences are detected in transcriptional activity between mutant and wild-type cells if FOPFLASH or an SV40-based control reporter (pRL-SV) are tested. Indeed, the activity levels of the internal control renilla reporter (pRL-CMV) are the same in both cell lines. Therefore, Lef-1-mediated transcription is more sensitive to CtBP loss than the transcription mediated by other transcription factors. Thus, CtBP appears to antagonize TCF-mediated transcription in a relatively specific way (Hamada, 2004).

It has been reported that Xenopus CtBP can bind to XTcf-3 and antagonize the transcription of TCF target genes in the early Xenopus embryo. It was noted that TCF-3 and TCF-4 factors possess CtBP binding motifs and suggested that CtBP may be a corepressor of these TCFs. Potentially, this could explain the increased basal levels of TCF-mediated transcription in CtBP mutant cells compared to their parental controls. However, it is unlikely to explain the increased levels of Lef-1-stimulated transcription, given that Lef-1 is a TCF factor that lacks CtBP binding motifs (Hamada, 2004).

In vivo association between CtBP and TCF had never been demonstrated, so this was examined in comparison to the in vivo association between CtBP and APC. First, it was asked whether endogenous CtBP and TCF-4 coimmunoprecipitate in colorectal cancer cells, given that TCF-4 is expressed in these cells. ß-catenin coimmunoprecipitates with TCF-4, as expected; however, CtBP is not detectable in the same TCF-4 immunoprecipitate. Conversely, while APC coimmunoprecipitates with CtBP, TCF-4 does not. Thus, endogenous CtBP is associated with APC, but not with TCF, in colorectal cancer cells. Notably, the same is true in 293T cells in which TCF is transcriptionally inactive: endogenous CtBP is associated with APC and ß-catenin, but not with endogenous TCF-4. It is concluded that TCF is not detectable in a complex with CtBP, regardless of cell type and transcriptional activity (Hamada, 2004).

It has been reported that exogenous TCF-4 can repress TOPFLASH transcription in transfected simian COS cells (that lack E1A expression) in a CtBP-dependent manner, while a C-terminal truncation of TCF-4 without the CtBP binding motifs (such as those arising from frameshift mutations in TCF-4 in some microsatellite-unstable colorectal carcinomas) does not respond to overexpressed CtBP in this assay. These experiments were repeated by comparing the activities of mutant TCF-4, whose two CtBP binding motifs were mutated in the same way as those of APC (TCF-4 AxAxA with triple alanine substitutions in residues 1, 3, and 5 of the P-h-D-L-S-x-R/K motif) and its wild-type control in TOPFLASH assays, and in their response to overexpressed CtBP. Overexpressed TCF-4 can repress TOPFLASH transcription in a dose-dependent manner in transfected SW480 and COS cells. However, the AxAxA TCF-4 mutant was similarly inhibitory, despite being expressed at slightly higher levels than wild-type TCF (especially at low doses of transfected plasmid). Furthermore, the mutant was equally responsive to coexpressed CtBP as the wild-type TCF-4. Therefore, although the AxAxA mutation affects the activity of APC(918-1698) in TCF-specific transcription assays, the same mutation in TCF-4 does not affect its activity in these assays. In agreement with this, a comparable double mutation of the CtBP binding motifs in XTcf-3 does not reduce its repressive potential in Xenopus embryos. Note that this double mutation does reduce the in vitro binding of XTcf-3 to CtBP, and so does the AxAxA double mutant of TCF-4. However, the in vitro binding between CtBP and TCF-4 is ~10× less strong than that between TCF-4 and ß-catenin. Thus, the in vitro binding between CtBP and TCF, although apparently specific, is very weak indeed. It may be spurious, given the lack of a detectable association between these proteins in vivo (Hamada, 2004).

In summary, no evidence was obtained for a significant physical or functional interaction between CtBP and TCF. These results thus question the idea that CtBP functions generally as a corepressor of TCF factors. It is agreed that the TCF-4 frameshift mutations observed in microsatellite-unstable colorectal carcinomas are passenger mutations without any functional relevance for TCF-mediated transcription or tumorigenesis (Hamada, 2004).

It was asked whether dCtBP might antagonize Armadillo-mediated transcription during Drosophila development. However, this is not straightforward to test, since dCtBP mutants show highly pleiotropic mutant phenotypes: null mutant embryos are grossly abnormal and do not develop beyond early stages, due to failing interactions between dCtBP and segmentation gene products. This precludes a meaningful analysis of dTCF target gene expression in these mutants. And although dCtBP has been implicated in antagonizing dTCF transcription in the developing midgut, this is an indirect effect mediated by the DNA binding protein Brinker to which CtBP can bind. Likewise, CtBP loss in the mouse causes pleiotropic mutant phenotypes, one of which, unexpectedly, mimics loss of Wnt signaling, but this could also be an indirect effect of CtBP binding to another target protein outside the Wnt pathway (Hamada, 2004).

Thus, to explore the regulatory relationship between dCtBP and Armadillo during development, it was asked whether dCtBP loss would affect the phenotypic consequences of overactive or depleted Armadillo. This is indeed the case: lowering the dose of dCtBP enhances the rough eye phenotype caused by activated Armadillo, but the same condition suppresses the wing nick phenotype due to Armadillo depletion in cells whose stimulation by Wingless is required for normal wing margin formation. These genetic interactions are similar to those of negative components of the Wnt pathway that downregulate Armadillo, such as Drosophila Axin and APC, consistent with dCtBP antagonizing Armadillo. Again, it is emphasized that this antagonism is unlikely to be due to dCtBP being a direct corepressor of dTCF, given that the latter does not contain any CtBP binding motifs. The results suggest that the antagonism between CtBP and Armadillo/ß-catenin is conserved and operates in multiple tissues and cell types (Hamada, 2004).

This study has presented evidence that CtBP binds to APC directly and specifically via the conserved 15Rs of APC and that the association of the two proteins in vivo is functionally relevant since it is required for the full activity of APC in reducing TCF-mediated transcription in colorectal cancer cells. In contrast, no evidence was found for a direct physical or functional interaction between CtBP and TCF in mammalian cells, calling into question whether CtBP acts generally as a transcriptional corepressor of TCF factors (Hamada, 2004).

Instead, the evidence suggests that CtBP antagonizes TCF-mediated transcription by cooperating with APC to sequester nuclear ß-catenin. This sequestration could be a safeguard function of APC, operating in parallel to (and to some extent redundantly with) its other functions in promoting nuclear export and degradation of ß-catenin. It is proposed that APC sequesters ß-catenin by targeting it to CtBP, thus lowering the pool of free nuclear ß-catenin that is available for binding to TCF. The sequestration of the APC/ß-catenin complex by CtBP may be based on spatial segregation within the nucleus (e.g., anchoring of the complex at specific subnuclear bodies). Whatever the precise mechanism, the observed functional cooperation between CtBP and APC in colorectal cancer cells suggests a role of CtBP as a tumor suppressor in the colon (Hamada, 2004).

Modulation of the ligand-independent traffic of Notch by Axin and Apc contributes to the activation of Armadillo in Drosophila

There is increasing evidence for close functional interactions between Wnt and Notch signalling. In many instances, these are mediated by convergence of the signalling events on common transcriptional targets, but there are other instances that cannot be accounted for in this manner. Studies in Drosophila have revealed that an activated form of Armadillo, the effector of Wnt signalling, interacts with, and is modulated by, the Notch receptor. Specifically, the ligand-independent traffic of Notch serves to set up a threshold for the amount of this form of Armadillo and therefore for Wnt signalling. In the current model of Wnt signalling, a complex assembled around Axin and Apc allows GSK3 (Shaggy) to phosphorylate Armadillo and target it for degradation. However, genetic experiments suggest that the loss of function of any of these three elements does not have the same effect as elevating the activity of β-catenin. This study shows that Axin and Apc, but not GSK3, modulate the ligand-independent traffic of Notch. This finding helps to explain unexpected differences in the phenotypes obtained by different ways of activating Armadillo function and provides further support for the notion that Wnt and Notch signalling form a single functional module (Muñ-Descalzo, 2011).

Cells expressing ArmS10, a form of Arm that is insensitive to phosphorylation by GSK3, do not overgrow and remain integrated in the epithelium. Clones of cells mutant for Axin, a central element of the Arm destruction complex, exhibit very high levels of Arm, some of which can be found in the nucleus, and exhibit overgrowths and round edges suggestive of defects in cellular recognition. These phenotypes are related to, but distinct from, those caused by expression of ArmS10 and support the contention that Axin exerts controls on the activity of Arm that are additional to those mediated through its role as a scaffold for GSK3. The effects of Axin loss of function are reminiscent of those caused by expression of ArmS10 in cells with compromised Notch function. Since these effects are caused by the loss of the ligand-independent traffic of Notch, this study tested whether Axin exerts some effect on the traffic of Notch (Muñ-Descalzo, 2011).

Clones of cells mutant for Axin did not show alterations in ligand-dependent Notch signalling, although they exhibited a mild but reproducible increase in Notch protein on the apical side, and overexpression of Axin reduced the amount of Notch present at the cell surface. These observations suggest that Axin regulates the amount of Notch at the cell surface. To test whether this control is exerted by targeting the endocytosis and traffic of Notch, label and chase experiments were performed with Notch. Under the experimental conditions and focusing the analysis in the pouch of the wing imaginal disc, labelled Notch disappeared from the cell surface within 10 minutes of the chase and could be found in punctate intracellular structures, presumably vesicles associated with endocytic traffic. Performing the same assay in the absence of Axin revealed that the endocytosis and traffic of Notch is impaired in Axin mutant cells, and after 30 minutes a substantial amount of Notch could still be detected on the cell surface. This suggests that Axin is involved in, or can influence, the traffic of Notch. Performing the same experiment in discs overexpressing Axin, a decrease was observed in the amount of Notch over time. Altogether, these results suggest that Axin contributes to the removal of Notch from the cell surface and to targeting it for degradation (Muñ-Descalzo, 2011).

Regulation of the activity of Arm by Notch is mediated by its ligand-independent traffic as shown by the activity of chimeric receptors in which the extracellular domain of Notch has been substituted by the extracellular domain of CD8 (CeN) or Torso (TN; Tor - FlyBase). Since Wingless signalling promotes the traffic and degradation of these receptors and cells lacking Axin have elevated levels of Wnt signalling, this study examined what would happen to the stability of CeN in this situation. Surprisingly, the levels of CeN remained largely unchanged in clones of cells mutant for Axin, suggesting that in the absence of Axin, despite high levels of Wnt signalling, CeN cannot be degraded . This could be because Axin is required for the degradation of CeN or because this degradation is dependent on Wnt and Dsh but not on Axin. A contribution of Axin is favoured by the observations that overexpression of Axin reduces, and Axin loss of function increases, Notch levels (Muñ-Descalzo, 2011).

A functional relationship between Axin and Notch is also highlighted by the observation that, in tissue culture, simultaneous reductions of Notch and Axin induce very high levels of Arm activity. However, in vivo, simultaneous loss of both Notch and Axin leads to a suppression of the growth induced by the loss of Axin alone, a phenotype that is associated with extensive cell death and perhaps reflects a synergy of the roles of each protein in apoptosis. For this reason, to test the synergy between the two proteins in determining Arm activity in vivo, a NotchRNAi construct was expressed that reduces, but does not abolish, Notch function in clones of cells mutant for Axin. Under these conditions, there is no apoptosis and larger outgrowths than those promoted by the loss of Axin alone were observed. These phenotypes indicate a synergistic effect of the mutations and suggest that Axin is involved in the modulation of Notch while it traffics through the cell (Muñ-Descalzo, 2011).

Apc, a second element of the Arm destruction complex, is encoded in Drosophila by Apc1 (Apc - FlyBase) and Apc2, which play redundant roles in the regulation of Wnt signalling. In order to test whether Apc is also involved in the traffic of Notch, clones of cells mutant for Apc1 and Apc2 were generated in wing imaginal discs and the traffic of Notch was assessed. In these clones, cells exhibited very similar phenotypes to those of Axin mutants in terms of growth, overall shape and levels of Arm. In addition, they exhibited altered traffic of Notch. However, instead of being clearly localised in vesicles or in the cell membranes, as in the case of Axin mutant cells, Notch protein appeared as a 'fuzzy' stain throughout the cytoplasm of the Apc1/2 mutant cells that was not associated with any subcellular structure. Axin and Apc have been shown to play functionally related, but distinct, roles in the regulation of Arm/δ-catenin and these differences might extend to their effects on Notch (Muñ-Descalzo, 2011).

The function of Axin and Apc is to provide a scaffold for the phosphorylation of Arm/β-catenin by Sgg/GSK3. Since, in mammalian systems, GSK3 has been shown to phosphorylate Notch and there are reports of interactions between Notch and Sgg in Drosophila, tests were performed to see whether Sgg has an effect on the traffic of Notch. Clones of cells mutant for sgg displayed elevated levels of Arm but no discernible effects on the endocytosis and traffic of Notch. This is consistent with the observation that Sgg is not required for the effects of Notch on Wnt signalling (Muñ-Descalzo, 2011).

In addition to their interactions with Wnt signalling, Axin and Apc display interactions with other signalling pathways and, in the case of Apc, with the cytoskeleton. These additional interactions might contribute to the differences between the effects of activated Arm and the loss of function of Axin and Apc. Notwithstanding this, the results reveal a function of Axin and Apc in the traffic of Notch. Previous studies have shown that compromising the traffic of Notch elevates the activity of an activated form of Arm. In Axin or Apc1,Apc2 mutant clones, in addition to the elevation of active Arm, the traffic of Notch is compromised and probably contributes to the increase in Arm activity. In this situation, the levels and activity of Arm would be higher than those resulting from the expression of an activated form of Arm alone. There is evidence that Axin functions in the regulation of Arm activity in a manner that is independent of its role as a scaffold for GSK3. Some of these effects could be mediated through its role in the endocytosis and traffic of Notch, which also could traffic with a GSK3-independent form of Arm (Muñ-Descalzo, 2011).

These results underscore the inadequacy of the notion that Wnt signalling flows through a linear pathway to target the destruction complex and promote β-catenin transcriptional activity. Although this framework helps to explain some of the effects associated with Wnt signalling, it is inconsistent with the observation that, in many instances, changes in the concentration of Arm/β-catenin are insufficient to promote transcriptional activity. While the axis Wnt-Dsh-Axin/Apc-β-catenin is the backbone of Wnt signalling, it is clear that there are additional elements that are not simply modulatory add-ons. In this regard, the interactions between Wnt and Notch signalling are a recurrent theme in developmental biology and disease and might not reflect a simple functional convergence in specific processes at the transcriptional level. The results presented in this study reinforce the notion that Wnt and Notch configure a molecular device (Wntch), in which the mutual control of their activities serves to regulate the assignation of cell fates with the effect of Notch providing a buffer to fluctuations in the resting levels of Arm (Muñ-Descalzo, 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).

Apc-like: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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