dishevelled: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - dishevelled

Synonyms -

Cytological map position - 10B3-8

Function - signal transduction

Keywords - segment polarity, wingless pathway, tissue polarity

Symbol - dsh

FlyBase ID:FBgn0000499

Genetic map position - chrX:11,249,914-11,252,759

Classification - DEP domain, DIX domain, PDZ domain

Cellular location - cytoplasmic



NCBI link: Entrez Gene
dsh orthologs: Biolitmine
Recent literature
Mannava, A. G. and Tolwinski, N. S. (2015). Membrane bound GSK-3 activates Wnt signaling through Disheveled and Arrow. PLoS One 10: e0121879. PubMed ID: 25848770
Summary:
Wnt ligands and their downstream pathway components coordinate many developmental and cellular processes. In adults, they regulate tissue homeostasis through regulation of stem cells. Mechanistically, signal transduction through this pathway is complicated by pathway components having both positive and negative roles in signal propagation. This study examined the positive role of GSK-3/Zw3 in promoting signal transduction at the plasma membrane. Targeting GSK-3 to the plasma membrane activates signaling in Drosophila embryos. This activation requires the presence of the co-receptor Arrow-LRP5/6 and the pathway activating protein Disheveled. These results provide genetic evidence for evolutionarily conserved, separable roles for GSK-3 at the membrane and in the cytosol, and are consistent with a model where the complex cycles from cytosol to membrane in order to promote signaling at the membrane and to prevent it in the cytosol.

Mund, T., Graeb, M., Mieszczanek, J., Gammons, M., Pelham, H. R. and Bienz, M. (2015). Disinhibition of the HECT E3 ubiquitin ligase WWP2 by polymerized Dishevelled Open Biol 5. PubMed ID: 26701932
Summary:

Dishevelled is a pivot in Wnt signal transduction, controlling both β-catenin-dependent transcription to specify proliferative cell fates, and cell polarity and other non-nuclear events in post-mitotic cells. In response to Wnt signals, or when present at high levels, Dishevelled forms signalosomes by dynamic polymerization. Its levels are controlled by ubiquitylation, mediated by various ubiquitin ligases, including NEDD4 family members that bind to a conserved PPxY motif in Dishevelled (mammalian Dvl1-3). This study shows that Dvl2 binds to the ubiquitin ligase WWP2 and unlocks its ligase activity from autoinhibition. This disinhibition of WWP2 depends on several features of Dvl2 including its PPxY motif and to a lesser extent its DEP domain, but crucially on the ability of Dvl2 to polymerize, indicating that WWP2 is activated in Wnt signalosomes. Notch intracellular domains were shown to be substrates for Dvl-activated WWP2 and their transcriptional activity is consequently reduced, providing a molecular mechanism for cross-talk between Wnt and Notch signalling. These regulatory interactions are conserved in Drosophila whose WWP2 orthologue, Suppressor-of-deltex, downregulates Notch signalling upon activation by Dishevelled in developing wing tissue. Attentuation of Notch signalling by Dishevelled signalosomes could be important during the transition of cells from the proliferative to the post-mitotic state (Mund, 2015).

Weber, U. and Mlodzik, M. (2017). APC/CFzr/Cdh1-dependent regulation of planar cell polarity establishment via Nek2 kinase acting on Dishevelled. Dev Cell 40(1): 53-66. PubMed ID: 28041906
Summary:
The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase, well known for its role in cell-cycle progression. However, it has been linked to additional functions, mainly in neuronal contexts, when using the co-activator Cdh1/Fzr. A post-mitotic requirement is indicated for the APC/CFzr/Cdh1 in epithelial cell patterning and planar cell polarity (PCP) in Drosophila. PCP signaling is critical for development by establishing cellular asymmetries and orientation within the plane of an epithelium, via differential localization of distinct complexes of core PCP factors. Loss of APC/C function leads to reduced levels of Dishevelled (Dsh), a core PCP factor. The effect of APC/C on Dsh is mediated by Nek2 kinase, which can phosphorylate Dsh and is a direct APC/CFzr/Cdh1 substrate. This study has thus uncovered a pathway of regulation whereby APC/CFzr/Cdh1 negatively regulates Nek2, which negatively regulates Dsh, to ensure its proper stoichiometric requirement and localization during PCP establishment.
Wang, Y., Naturale, V.F. and Adler, P.N. (2017). Planar cell polarity effector Fritz interacts with Dishevelled and has multiple functions in regulating PCP. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 28258110
Summary:
The Planar cell Polarity Effector (PPE) genes inturned, fuzzy and fritz are downstream components in the frizzled/starry night signaling pathway, and their function is instructed by upstream Planar Cell Polarity (PCP) core genes such as frizzled and disheveled PPE proteins accumulate asymmetrically in wing cells and function in a protein complex mediated by direct interactions between In and Frtz and In and Fy. How the PCP proteins instruct the accumulation of PPE protein is unknown. This study found a likely direct interaction between Dishevelled and Fritz and Dishevelled and Fuzzy that could play a role in this. It has been previously shown that mild over expression of frtz rescues a weak in allele. To determine if this is due to extra Frtz stabilizing mutant In or due to Frtz being able to bypass the need for In, a precise deletion of the inturned gene (inPD) was generated. Mild overexpression of Fritz partially rescues inPD, indicating that fritz has In independent activity in PCP. Previous studies of PPE proteins used fixed tissues, and did not provide any insights into the dynamic properties of PPE proteins. In this study, CRISPR/Cas9 genome editing technology was used to edit the fritz gene to add a green fluorescent protein tag. fritzmNeonGreen provides complete rescue activity and works well for in vivo imaging. Data show that Fritz is very dynamic in epidermal cells and preferentially distributed to discrete membrane subdomains ("puncta"). Surprisingly, it was also found in in stripes in developing bristles.

Weber, U. and Mlodzik, M. (2017). APC/CFzr/Cdh1-Dependent Regulation of Planar Cell Polarity Establishment via Nek2 Kinase Acting on Dishevelled. Dev Cell 40(1): 53-66. PubMed ID: 28041906
Summary:
The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase, well known for its role in cell-cycle progression. However, it has been linked to additional functions, mainly in neuronal contexts, when using the co-activator Cdh1/Fzr. This study indicates a post-mitotic requirement for the APC/CFzr/Cdh1 in epithelial cell patterning and planar cell polarity (PCP) in Drosophila. PCP signaling is critical for development by establishing cellular asymmetries and orientation within the plane of an epithelium, via differential localization of distinct complexes of core PCP factors. Loss of APC/C function leads to reduced levels of Dishevelled (Dsh), a core PCP factor. The effect of APC/C on Dsh is mediated by Nek2 kinase, which can phosphorylate Dsh and is a direct APC/CFzr/Cdh1 substrate. This study has thus uncovered a pathway of regulation whereby APC/CFzr/Cdh1 negatively regulates Nek2, which negatively regulates Dsh, to ensure its proper stoichiometric requirement and localization during PCP establishment.
Hwang, S. H., Bang, S., Kang, K. S., Kang, D. and Chung, J. (2018). ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled. Biochem Biophys Res Commun. PubMed ID: 30497781
Summary:
Wnt signaling pathway plays critical roles in body axes patterning, cell fate specification, cell proliferation, cell migration, stem cell maintenance, cancer development and etc. Among the core components of Wnt signaling pathway, this study discovered that Dishevelled (Dsh) interacts with ULK1 and is phosphorylated by ULK1. Unexpectedly, the knockdown of ULK1 elicited a marked increase in Wnt/beta-catenin signaling. Multiple ULK1 phosphorylation sites existed on Dsh and many of them were located on the PDZ-DEP region. By using evolutionarily well conserved Drosophila Dsh, it was found that S239, S247 and S254 in the PDZ-DEP region are involved in phosphorylation of Dsh by ULK1. Among these, S247 and S254 were conserved in human Dsh. When phospho-mimetic mutants (2D and 2E Dsh mutants) of these conserved residues were generated and expressed in the eyes of the fruit flies, the activity of Dsh was significantly decreased compared to wild type Dsh. Through additional alanine scanning, it was further identified that S239, S247, S254, S266, S376, S554 and S555 on full length Dsh were phosphorylated by ULK1. In regards to the S266A mutation located in the PDZ domain among these phosphorylated residues, the results suggested that Dsh forms an SDS-resistant high molecular weight complex with beta-catenin and TCF in the nucleus in an S266 phosphorylation-dependent manner. Based on these results, it is proposed that ULK1 plays a pivotal role in the regulation of Wnt/beta-catenin signaling pathway by phosphorylating Dsh.
BIOLOGICAL OVERVIEW

In Drosophila, Dishevelled is required in two distinct signaling pathways that share Frizzled receptors of similar structure, but have distinct intracellular signaling routes. Dishevelled phosphorylation is concurrent with Wingless pathway activation. Wingless serves to activate Dsh, which in turn inactivates Shaggy/Zeste white 3. In the absence of signaling, Armadillo is targeted for proteolytic degradation through phosphorylation by Shaggy. Arm accumulates in the cytoplasm and nucleus, where it activates Wg target genes by complexing with Pangolin/TCF transcription factors. Experiments suggest that upon Wnt signaling, part of the cytoplasmic pool of Dsh is phosphorylated and recruited to the membrane. Deletion analysis experiments have shown that the Dsh DEP domain is not required to induce Arm accumulation. The Dishevelled role in the tissue polarity pathway clearly indicates that, compared to its role in the Wingless pathway, different downstream components are involved. Rather, dominant genetic interactions and rescue experiments in the eye imaginal disc suggest that signaling is routed through a Rho/Rac and JNK/SAPK-like kinase cascade. Thus, Dsh is involved in two distinct Wnt signaling pathways, raising the question of how Dsh routes the information to the right effector cascade in each case (Boutros, 1999 and references).

dishevelled is an integral part of the wingless pathway. Wingless is secreted from anterior segmental compartments (for details, see wingless and engrailed), and is induced by Hedgehog, a protein made in posterior compartments. Wingless is required for the maintenance of engrailed synthesis, and for the accumulation of Armadillo protein (Klingensmith, 1994). Cells lacking dsh are unable to adopt the fates specified by Wingless.

Dishevelled is one element in a chain of interacting proteins that carry the wingless signal to sites within the cell, including the cell membrane and the nucleus. Phosphorylation is the most important biochemical mechanism for carrying signals between proteins. Dishevelled, a phosphoprotein, is synthesized uniformly throughout the embryo, but its level of phosphorylation varies with the level of wingless activity, (Yanagawa, 1995). Evidence that the serine threonine kinase shaggy/zeste white-3 is downstream of dishevelled suggests that it is not this kinase that is responsible for DSH phosphorylation, but that DSH phosphorylation is carried out by an as yet unidentified kinase (Yanagawa, 1995).

Phosphorylated DSH is correlated with high levels of cytoplasmic phosphorylated Armadillo. The function of this modification of ARM is not yet understood (Peifer, 1994), but certainly DSH and the wingless pathway are involved in cell surface integrity, acting through Armadillo on adherens junction stability (Klingensmith, 1994).

Most if not all biological organisms demonstrate an incredible (though imperfect) symmetry of structure and patterning. By symmetry is meant the positional repetition of a pattern on opposite sides of a dividing line or plane, distributed about a center or axis, resulting in geometric regularity. Symmetry is only one aspect of patterning. A second aspect, tissue polarity is exemplified by the pattern of epithelial hairs. For example, all the hairs of the wing surface point in the same direction. What is the biological basis of tissue symmetry and polarity? In Drosophila, planar cell polarity (PCP) signaling is mediated by the receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). PCP signaling controls the polarity of epithelial cells within a plane orthogonal to their apical-basal axis. One manifestation of this cellular polarity is the oriented organization of trichomes (cell hairs). In wild-type flies, the cell hair arising from each cell's distal vertex contributes to a parallel and specifically oriented array. Mutations in dsh, as well as additional genes including frizzled (fz), prickle (pk),inturned (in), fuzzy (fy),multiple wing hairs (mwh), and others all disrupt the polarity of the trichomes. The resulting mutant phenotypes include swirls and distortions of the hair polarity pattern, and in some instances, more than one trichome per cell. A putative signal transduction pathway has been proposed, which serves to polarize cells, allowing them to distinguish one side of the cell from the other, and to propagate this information from cell to cell. In this pathway, Frizzled, a seven-transmembrane protein (without apparent primary sequence homology to the G-protein-coupled receptors) acts as a receptor, functioning upstream of Dsh. Dsh then antagonizes the activities of Fuzzy, Inturned (both novel transmembrane proteins), and Mwh; in turn, it is proposed that these proteins regulate the cytoskeletal apparatus responsible for control of PCP. Mutations in RhoA affect this process, and experiments with dominant-negative mutants have implicated Cdc42 and Rac1 as additional effectors. Pk is proposed to function either in transmission of the signal to adjacent cells, or in interpretation of the directionality of the signal. Thus far, no ligand for the PCP pathway has been identified. Although a tentative signal transduction pathway has been proposed, the mechanism by which asymmetry is established in the responding cells is not understood (Axelrod and references, 1998).

In Drosophila, planar cell polarity signaling is mediated by the receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling also requires Dsh and may utilize DFz2 as a receptor. A heterologous system was used to examine the interaction of Dsh with Frizzled. mRNAs encoding Fz or Frizzled2 and a fusion of Dsh to green fluorescent protein (Dsh-GFP) were synthesized in vitro and injected into Xenopus embryos at the four-cell stage. Animal caps from stage 9 embryos, dissected to reveal the blastocoelar cells, were then examined by confocal microscopy. Dsh is recruited selectively to the membrane by Fz but not Frizzled2, and this recruitment depends on the DEP domain but not the PDZ domain in Dsh. When Fz is expressed simultaneously with Dsh-GFP, Dsh-GFP shows a qualitative redistribution to the membrane or cell cortex. Under these conditions, localization of Dsh-GFP to filopodia present on the blastocoelar (free) surfaces of the animal cap cells was also noticed. Staining with phalloidin (and Dsh-GFP) revealed that the filopodia contain filamentous actin. It is interesting to note that although the filopodia stain with Dsh-GFP, little or no Fz localizes there; at the cell cortex, the Fz and Dsh-GFP show imperfect colocalization. Fz staining is localized predominantly to the plasma membrane, and to a lesser extent to intracellular membranes (probably ER and/or Golgi) in these cells. This suggests that while Fz may induce localization of Dsh-GFP to the membrane and filopodia, it may do so by a mechanism other than direct binding. Frizzled2, the Wingless receptor, fails to induce membrane localization of Dsh, even in the presence of a functional Fz2 ligand (Axelrod, 1998).

Drosophila Dsh is a modular protein of unknown function that is well conserved in relation to its vertebrate homologs. Alignment of family members reveals three conserved domains. The first, a DIX domain, is similar to a domain in murine Axin (see Drosophila Axin), a recently described modulator of the Wnt1 pathway. The second contains a PDZ domain; PDZ domains recognize and bind short motifs at the carboxyl termini of proteins (but may bind other motifs as well). PDZ domains can also form dimers. The third domain, called DEP, is conserved among a set of proteins that have in common the ability to regulate various GTPases, including both heterotrimeric G proteins and Ras-like small GTPases. A mutation in the DEP domain impairs both membrane localization and the function of Dsh in PCP signaling, indicating that translocation is important for function. A single amino acid substitution in the DEP domain of Dsh is shown to confer a loss of function for PCP signaling, yet the mutant protein is functional for Wg signaling. This single amino acid substitution, coded for by the dsh1 allele, allows for translocation to the membrane, but is thought to impair the ability of Dsh1 to associate with its target at the membrane. This altered membrane interaction diminishes the ability of Dsh1 to function in PCP signaling (Axelrod, 1998).

Further genetic and molecular analyses suggest that conserved domains in Dsh function differently during PCP and Wg signaling, and that divergent intracellular pathways are activated. For example, the individual domains Dsh(DIX) and Dsh deleted for the PDZ domain, are each dominant negative for Wg signaling but have no effect on PCP signaling. Overexpression of Zeste white3, or an activated Arm protein, both involved in Wingless signaling, also fail to produce any effect on PCP. It is proposed that Dsh has distinct roles in PCP and Wg signaling. The PCP signal may selectively result in focal Fz activation and asymmetric relocalization of Dsh to the membrane, where Dsh effects cytoskeletal reorganization to orient prehair initiation. This analysis suggests that Dsh has different interactions in PCP and Wg signaling and predicts an additional genetic behavior (Axelrod, 1998).

If Wg and planar polarity signaling utilize Dsh in a common fashion, then ectopic activation of one pathway should be able to cross-activate the other by promiscuously activating Dsh. In contrast, if each pathway utilizes Dsh in a distinct fashion, then ectopic activation might sequester Dsh in pathway-specific complexes, rendering it unavailable and therefore titrating the activity of the other pathway. These possibilities could best be tested under conditions in which Dsh is limiting. Overexpression of Fz causes a dominant gain-of-function PCP phenotype, and this phenotype is sensitive to the dose of dsh. Can Wg cross-activate Dsh activity for PCP signaling, or can it sequester Dsh? To investigate this question, it was first necessary to know if the Fz-overexpression phenotype would be either enhanced or suppressed by Wingless overexpression. Ectopic expression of Wg suppresses the Fz overexpression phenotype, suggesting that activation of Wg signaling may titrate the amount of Dsh available for PCP signaling. The reciprocal experiment was performed by asking if ectopic activation of the PCP pathway could interfere with Wg signaling. Because the ligand for PCP signaling is unknown, Fz was overexpressed during embryogenesis, and the cuticle phenotype analyzed. These embryos develop with lawns of denticles and are reminiscent of wg-mutant embryos, or those expressing dominant-negative Dsh constructs. The results suggest that titration can occur in this direction as well. The possibility cannot be ruled out that the titration observed in these experiments results from a promiscuous interaction between Wg and Fz, although this interaction may not occur in vivo. The observations are equally consistent with the possibility that under these conditions, activity of one pathway titrates the Dsh level available for the other (Axelrod, 1998).

Dishevelled appears to interact with the Notch pathway, and this interaction provides a direct link between Notch and wingless pathways. The dishevelled gene interacts antagonistically with Notch and its ligand Delta. A direct physical interaction between Dishevelled and the Notch carboxyl terminus, distal to the cdc10/ankyrin repeats, suggests a mechanism for this interaction. It is proposed that Dishevelled, in addition to transducing the Wingless signal, blocks Notch signaling directly, thus providing a molecular mechanism for the inhibitory cross talk observed between these pathways (Axelrod, 1996).

Frizzled family proteins have been described as receptors of Wnt signaling molecules. In Drosophila, the two known Frizzled proteins are associated with distinct developmental processes. Genesis of epithelial planar polarity requires Frizzled, whereas Dfz2 affects morphogenesis by wingless-mediated signaling. Dishevelled is required in both signaling pathways. Genetic and overexpression assays have been used to show that Dishevelled activates JNK cascades. In contrast to the action of wingless-pathway components, mutations in rhoA, hemipterous, basket, and jun as well as deficiencies removing the Rac1 and Rac2 genes show a strong dominant suppression of a Dishevelled overexpression phenotype in the compound eye. In an in vitro assay, expression of Dsh has been shown to induce phosphorylation of Jun, indicating that Dsh is a potent activator of the JNK pathway. Whereas the PDZ domain of Dsh, known to be required in the transduction of the wingless signal, is dispensable for signal-independent induction of Jun phosphorylation, the C-terminal DEP domain of Dsh is found to be essential. The planar polarity-specific dsh1 allele is found to be mutated in the DEP domain. These results indicate that different Wnt/Fz signals activate distinct intracellular pathways, and Dishevelled discriminates among them by distinct domain interactions (Boutros, 1998).

How can Fz/Dsh signaling be linked to small GTPase and JNK/MAPK pathways? Recent studies provided evidence that links G protein-coupled receptors, which share structural features with Fz proteins, to MAPK signaling through heterotrimeric G proteins and PI-3 kinases. It is intriguing to speculate that a subset of Fz proteins might signal through a similar pathway. It was also shown recently that XWnt5A and rFz2, in a heterologous assay, increase intracellular calcium via G proteins and phosphoinositol signaling. A mutation in the beta-subunit of a heterotrimeric G protein in C. elegans prevents correct spindle orientation, a process that is believed to be dependent on a Wnt and a Fz receptor, but not on Arm. Further studies regarding a possible involvement of PI-3K and G proteins in planar polarity signaling may provide additional insight to the diversity of Fz-related signaling pathways (Boutros, 1998 and references).

A signaling network for patterning of neuronal connectivity in the Drosophila brain

The precise number and pattern of axonal connections generated during brain development regulates animal behavior. Therefore, understanding how developmental signals interact to regulate axonal extension and retraction to achieve precise neuronal connectivity is a fundamental goal of neurobiology. This question was investigated in the developing adult brain of Drosophila. Extension and retraction is regulated by crosstalk between Wnt, fibroblast growth factor (FGF) receptor, and Jun N-terminal kinase (JNK) signaling, but independent of neuronal activity. The Rac1 GTPase integrates a Wnt-Frizzled-Disheveled axon-stabilizing signal and a Branchless (FGF)-Breathless (FGF receptor) axon-retracting signal to modulate JNK activity. JNK activity is necessary and sufficient for axon extension, whereas the antagonistic Wnt and FGF signals act to balance the extension and retraction required for the generation of the precise wiring pattern (Srahna, 2006).

Based on the observation that blocking Fz2 results in decreased numbers of dorsal cluster neuron (DCN) axons in the medulla, it was reasoned that Fz2 could be a receptor for a putative stabilization signal. Since Fz2 and Fz are partially redundant receptors for the canonical Wnt signaling pathway, expression of the canonical Wnt ligand Wingless (Wg) was investigated in the brain during pupation. However, no Wg expression was detected in the pupal optic lobes, suggesting that Wg is unlikely to be involved in regulating DCN axon extension. Therefore, the expression of Wnt5, which has been shown to be involved in axon repulsion and fasciculation in the embryonic CNS, was investigated. Anti-Wnt5 staining revealed widely distributed Wnt5 expression domains beginning at PF and lasting throughout pupal development and into adult life. Wnt5 is strongly expressed in the distal medulla and is also present on axonal bundles crossing the second optic chiasm.The number of DCN axons crossing to the medulla was examined in wnt5 mutant flies. The number of DCN axons crossing the optic chiasm is reduced from 11.7 to 7.9 in the absence of wnt5, suggesting that it may play a role in stabilizing DCN axons (Srahna, 2006).

Next, the requirement of the Wnt signaling adaptor protein Dsh was tested. In animals heterozygous for dsh6, a null allele of dsh, the average number of DCN axons crossing between the lobula and the medulla is reduced from 11.7 to 7.6 with 78.5% showing less than eight axons crossing. Signaling through Dsh is mediated by one of two domains. Signaling via the DIX (Disheveled and Axin) domain is thought to result in the activation of Armadillo/β-Catenin. DEP (Disheveled, Egl-10, Pleckstrin) domain-dependent signaling results in activation of the JNK signaling pathway by regulation of Rho family GTPase proteins during, for example, convergent extension movements in vertebrates. To uncover which of these two pathways is required for DCN axon extension the dsh1 mutant, deficient only in the activity of the DEP domain, was tested. Indeed, in brains from dsh1 heterozygous animals the number of extending axons was reduced from 11.7 to 7.4. In flies homozygous for the dsh1 allele the average number of axons crossing was further reduced to 4.7, with all the samples having less than six axons crossing. In contrast, the DCN-specific expression of Axin, a physiological inhibitor of the Wnt canonical pathway, did not affect the extension of DCN axons. Similarly, expression of a constitutively active form of the fly β-Catenin Armadillo also had no apparent effect on DCN extension. Finally, whether Wnt5 and Dsh interact synergistically was tested. To this end, wnt5, dsh1 trans-heterozygous animals were generated. These flies show the same phenotype as flies homozygous for dsh1, suggesting that Wnt5 signals through the Dsh DEP domain (Srahna, 2006).

To determine if dsh is expressed at times and places suggested by its genetic requirement in DCN axon outgrowth, the distribution of Dsh protein during brain development was examined. Dsh protein is ubiquitously expressed during brain development. High expression of Dsh is detected in the distal ends of DCN axons at about 15% PF shortly before they extend across the optic chiasm toward the medulla. In general, higher levels of Dsh were observed in the neuropil than in cell bodies (Srahna, 2006).

In summary, these data indicate that the stabilization of DCN axons is dependent on the Dsh protein acting non-canonically via its DEP domain (see Habas and Dawid Dishevelled and Wnt signaling: is the nucleus the final frontier? and Eisenmann's Wnt Signaling). Importantly, the axons that do cross in dsh mutant brains do so along the correct paths. This suggests that, like JNK signaling, Wnt signaling regulates extension, but not guidance, of the DCN axons (Srahna, 2006).

Wnt signaling to Dsh requires the Fz receptors. To examine if the effect of Wnt5 on DCN axon extension is also mediated by Fz receptors, the number of DCN axons crossing the optic chiasm in was counted fz, fz2, and fz3 mutants. There was no significant change in the number of axons crossing in the brain of fz3 homozygous animals. In contrast, in brains heterozygous for fz and fz2, the number of the axons crossing was reduced from 11.7 to 6.6 (fz) and 6.9 (fz2), with 71% and 85.7%, respectively, showing less than eight axons crossing. These data suggest that DCN axons respond to Wnt5 using the Fz and Fz2 receptors, but not Fz3. To determine whether the Fz receptors act cell-autonomously in individual DCNs, single-cell clones doubly mutant for fz and fz2 were generated and the number of DCN axons crossing the optic chiasm was counted. In contrast to wild-type cells, where 37% of all DCN axons cross, none of the fz, fz2 mutant axons reach the medulla. To test whether wnt5, fz, and fz2 genetically interact in DCNs, flies trans-heterozygous for wnt5 and both receptors were examined. Flies heterozygous for both wnt5 and fz mutations show a strong synergistic loss of DCN axons (11.7 to 3.7) and in fact have a phenotype very similar to that of flies homozygous for dsh1. Flies doubly heterozygous for wnt5 and fz2 also show a significant decrease in DCN axons (5.7), compared with either wnt5 (~8) or fz2 (8.5) mutants. These data indicate that the genetic interaction between wnt5 and fz is stronger than the interaction between wnt5 and fz2 (Srahna, 2006).

Examination of the expression domains of Fz and Fz2 in the developing brain supports the possibility that they play roles in stabilizing DCN axons. Both Fz and Fz2 are widely expressed in the developing adult brain neuropil. In addition, Fz is expressed at higher levels in DCN cell bodies (Srahna, 2006).

The observation that the wnt5 null phenotype can be enhanced by reduction of Fz, Fz2, or Dsh suggests that another Wnt may be partially compensating for the loss of Wnt5. To test this possibility, flies heterozygous for either wnt2 or wnt4 were examined. wnt2 heterozygotes display a reduction of DCN axon crossing from 11.7 to 7.3, whereas no phenotype was observed for wnt4. Thus, wnt2 and wnt5 may act together to stabilize the subset of DCN axons that do not retract during development. In summary, these results support the model that Wnt signaling via the Fz receptors transmits a non-canonical signal through Dsh resulting in the stabilization of a subset of DCN axons (Srahna, 2006).

Data is provided that supports the hypothesis that the regulation of JNK by Rac1 modulates DCN axon extension. As such attempts were made to determine how Wnt signaling might interact with Rac1 and JNK. The opposite phenotypes of dsh and Rac1 loss-of-function suggest that they might act antagonistically. To determine if Rac1 is acting upstream of, downstream of, or in parallel to Dsh in DCN axon extension, dominant-negative Rac1 was expressed in dsh1 mutant flies. If Rac1 acts upstream of Dsh, the dsh1 phenotype (i.e., decreased numbers of axons crossing the optic chiasm) is expected. If Rac1 acts downstream of Dsh, the Rac1 mutant phenotype (i.e., increased number of axons crossing) would be expected If they act in parallel, an intermediate, relatively normal phenotype is expected. Increased numbers of axon crossing were observed, suggesting that Rac1 acts downstream of Dsh during DCN axon extension and that Dsh may repress Rac1 (Srahna, 2006).

Next, whether Dsh control of DCN axon extension is mediated by the JNK signaling pathway acting downstream of Wnt signaling was tested, as the similarity of their phenotypes suggests. If this were the case, activating JNK signaling should suppress the reduction in Dsh levels. Conversely, reducing both should show a synergistic effect. Therefore the JNKK hep was expressed in dsh1 heterozygous flies and it was found that the hep gain-of-function is epistatic to dsh loss-of-function. Furthermore, reducing JNK activity by one copy of BSK-DN in dsh1 mutant animals results in a synergistic reduction of extension to an average of 0.8 axons with 60% showing no axons crossing and no samples with more than three axons. In summary, the results of genetic analyses suggest that Wnt signaling via Dsh enhances JNK activity through the suppression of Rac1 (Srahna, 2006).

Dsh appears to promote JNK signaling and to be expressed in DCN axons prior to their extension toward the medulla early in pupal development. Since JNK signaling is required for this initial extension, it may be that Dsh also plays a role in the early extension of DCN axons. To test this possibility, DCN axon extension was examined at 30% pupal development in dsh1 mutant brains. In wild-type pupae, essentially all (~40) DCN axons extend toward the medulla. In contrast, in dsh1 mutant pupae, a strong reduction in the number of DCN axons crossing the optic chiasm between the lobula and the medulla was observed (Srahna, 2006).

Although the genetic data indicate that Dsh- and Rac-mediated signaling have sensitive and antagonistic effects on the JNK pathway, they do not establish whether the Dsh-Rac interaction modulates JNK's intrinsic activity. To test this, the amount of phosphorylated JNK relative to total JNK levels in fly brains was evaluated by Western blot analysis using phospho-JNK (P-JNK) and pan-JNK specific antibodies. Then it was determined if Dsh is indeed required for increased levels of JNK phosphorylation. Dsh1 mutant brains showed a 25% reduction in P-JNK consistent with a stimulatory role for Dsh on JNK signaling. The reduction caused by loss of Dsh function is reversed, when the amount of Rac is reduced by half, consistent with a negative effect of Rac on JNK signaling downstream of Dsh. These data support the conclusion that Dsh and Rac interact to regulate JNK signaling by modulating the phosphorylated active pool of JNK (Srahna, 2006).

Taken together, these data suggest that during brain development DCN axons extend under the influence of JNK signaling. A non-canonical Wnt signal acting via Fz and Dsh ensures that JNK signaling remains active by attenuating Rac activity. In contrast, activation of the FGFR activates Rac1 and suppresses JNK signaling. These data support a model whereby the balance of the Wnt and FGF signals is responsible for determining the number of DCN axons that stably cross the optic chiasm. To test this model, FGFR levels were reduced, using the dominant-negative btl transgene, in dsh1 heterozygous flies. It was found that simultaneous reduction of FGF and Wnt signaling restored the number of axons crossing the optic chiasm to almost wild-type levels (10.2, with 33% of the samples indistinguishable from wild-type, suggesting that the two signals in parallel, act to control the patterning of DCN axon connectivity (Srahna, 2006).

These data suggest the following model of DCN axon extension and retraction. DCN axons extend due to active JNK signal. These axons encounter Wnt5 and probably Wnt2 as well, resulting in activation of Disheveled. Disheveled, via its DEP domain, has a negative effect on the activity of the Rac GTPase, thus keeping JNK signaling active. After DCN axons cross the second optic chiasm they encounter a spatially regulated FGF/Branchless signal that activates the FGFR/Breathless pathway. Breathless in turn activates Rac, which inhibits JNK signaling in a subset of axons. These axons then retract back toward the lobula. The wide expression of the different components of these pathways and the modulation of JNK phosphorylation by Dsh and Rac in whole-head extracts strongly suggests that this model may apply to many neuronal types (Srahna, 2006).

Rapid disruption of Dishevelled activity uncovers an intercellular role in maintenance of Prickle in core planar polarity protein complexes

Planar polarity, the coordinated polarization of cells in the plane of a tissue, is important for normal tissue development and function. Proteins of the core planar polarity pathway become asymmetrically localized at the junctions between cells to form intercellular complexes that coordinate planar polarity between cell neighbors. This study combined tools to rapidly disrupt the activity of the core planar polarity protein Dishevelled, with quantitative measurements of protein dynamics and levels, and mosaic analysis, to investigate Dishevelled function in maintenance of planar polarity. Mechanistic insight is provided into the hierarchical relationship of Dishevelled with other members of the core planar polarity complex. Notably, it was shown that removal of Dishevelled in one cell causes rapid release of Prickle into the cytoplasm in the neighboring cell. This release of Prickle generates a self-propagating wave of planar polarity complex destabilization across the tissue. Thus, Dishevelled actively maintains complex integrity across intercellular junctions (Ressurreicao, 2018).

Defining the roles of individual components in signaling networks can be a significant challenge. This is particularly so when the network is not a simple linear pathway, if components play more than one role in the cell (pleiotropy), and if there is 'adaptation' such that over time, the pathway adjusts to the effects of experimental manipulations. However, in many cases, these difficulties can be bypassed through methods that rapidly alter protein activities (Ressurreicao, 2018).

Consistent with this, it has been recently shown that spatiotemporal activation of gene expression is an effective tool for dissecting feedback interactions during planar polarity patterning in the Drosophila wing (Warrington, 2017). The current work used methods for rapidly disrupting protein function to probe the role of the Dsh protein in planar polarity. The main finding is that Dsh regulates Pk membrane association in core planar polarity complexes, acting cell-non-autonomously to prevent its relocalization to the cytoplasm. Notably, this role for Dsh is specifically revealed when Dsh is rapidly depleted from core protein complexes but not in the simple dsh loss-of-function situation, when instead a largely mobile fraction of Pk is seen associated with cell junctions. It is speculated that a Dsh-dependent signal normally passes between cells via the core protein complexes to maintain Pk recruitment. When this signal is disrupted, Pk rapidly leaves the junctions. However, in the long-term absence of Dsh, Pk can return to cell junctions, where it is speculated to weakly associates with cell membranes by virtue of being prenylated (Ressurreicao, 2018).

What might be the nature of the intercellular signal from Dsh to Pk? It is suggested that it passes via the Fmi homodimers that form between cells, as numerous lines of evidence indicate these are essential for cell-cell signaling in planar polarity. A simple possibility is that Dsh binding to Fz induces a conformational change in the complex that passes via the Fmi homodimers to alter the conformation of bound Stbm, thus creating a Pk binding site. Such molecular signaling events mediated by allostery are common features of ligand-receptor interactions. In support of the model that the Dsh signal is transduced via a change in Fz behavior, it is noted that following Dsh disruption, Fz distribution and stability is altered faster than those of Fmi and Stbm (Ressurreicao, 2018).

A related mechanism is suggested by recent observations that the core proteins incorporate into intercellular complexes non-stoichiometrically and that all components contribute to complex stability. These findings are interpreted as suggesting that core complex stability is dependent on a phase transition mediated by multivalent interactions between the core proteins, with Dsh playing a critical role. The rapid destabilization of Fz after Dsh depletion may be a result of loss of multivalent binding interactions mediated between the different domains of Dsh, as also occurs in Wnt signalosome assembly, over time leading to a reduction in multivalent binding interactions between Fmi and Stbm. This would thus produce a gradual 'loosening' of the complex that would result in release of Pk from its binding interactions with Fmi and Stbm. Some support for this model comes from the observation that super-resolution microscopy immediately after Dsh disruption shows subtle changes in the size and distribution of Fmi in junctional puncta (Ressurreicao, 2018).

A striking observation is that if Dsh fails to maintain Pk recruitment in intercellular complexes, free Pk can destabilize Dsh in the same cell, leading to release of Pk in the neighboring cell and a wave of core planar polarity complex destabilization. This observation both supports previous work showing that physiological levels of Pk can effectively destabilize Fz-Dsh complexes at cell junctions (Warrington, 2017) and highlights the importance of sequestering Pk into 'proximal' complexes, to prevent unregulated activity of Pk within the cell. It is proposed that Fmi mediates essential intercellular signals from Fz-Dsh in 'distal' core complexes that actively maintain Pk in 'proximal' core protein complexes. In turn, this promotes the effective segregation of distal and proximal complexes to opposite cell ends, driven in part by destabilizing feedback interactions between Pk and Dsh in the same cell (Ressurreicao, 2018).

Wnt regulation: is exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex

Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector beta-catenin. The multiprotein destruction complex binds and phosphorylates beta-catenin, and transfers it to the SCF-TrCP E3-ubiquitin ligase for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing beta-catenin. Recent work clarified underlying mechanisms, but important questions remain. This study explored beta-catenin transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. This study found that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axin's RGS domain plays an important role. Elevating Dishevelled levels in Drosophila embryos has paradoxical effects, promoting the ability of limiting levels of Axin to turn off Wnt signaling. When Dishevelled levels were elevated, it forms its own cytoplasmic puncta, but these do not recruit Axin. Superresolution imaging in mammalian cells raises the possibility that this may result by promoting Dishevelled:Dishevelled interactions at the expense of Dishevelled:Axin interactions when Dishevelled levels are high (Schaefer, 2020).

During embryonic development, cells must choose fate based on their position within the unfolding body plan. One key is cell-cell signaling, by which cells communicate positional information to neighbors and ultimately direct downstream transcriptional programs. A small number of conserved signaling pathways play an inordinately important role in these events in all animals. These include the Hedgehog, Notch, Receptor Tyrosine kinase, BMP/TGFβ, and Wnt pathways, which influence development of most tissues and organs. These same signaling pathways regulate tissue stem cells during tissue homeostasis and play critical roles in most solid tumors. Due to their powerful effects on cell fate and behavior, evolution has shaped dedicated machinery that keeps each signaling pathway definitively off in the absence of ligand (Schaefer, 2020).

In the Wnt pathway, signaling is turned on and off by regulating stability of the key effector β-catenin (βcat). In the absence of Wnt ligands, newly synthesized βcat is rapidly captured by the multiprotein destruction complex . Within this complex, the protein Axin acts as a scaffold, recruiting multiple partners. Axin and adenomatous polyposis coli (APC) bind βcat and present it to the kinases casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3) for sequential phosphorylation of a series of N-terminal serine and threonine residues on βcat (Schaefer, 2020).

It has become increasingly clear that the destruction complex is not a simple four-protein entity. Instead, Axin directs assembly of destruction complex proteins into what the field originally described as 'puncta.' These are now recognized as examples of supermolecular, nonmembrane bound cellular compartments, referred to as biomolecular condensates. Condensate formation is driven by Axin polymerization via its DIX domain, by APC function, and by other multivalent interactions (Schaefer, 2020).

Ubiquitination by E3 ubiquitin ligases is a key mechanism for regulating protein stability. Once the destruction complex templates βcat phosphorylation, the most N-terminal phosphorylated serine forms part of the core of a recognition motif for a Skp-Cullin-F-box (SCF)-class E3 ubiquitin ligase. This E3 ligase ubiquitinates βcat for proteasomal destruction. SCF-class E3 ligases include Cullin1 (Cul1), Skp1, F-box proteins, and Ring box (RBX) subunits, which work together to bind substrates and attach multiple ubiquitin moieties. Cul1 is the scaffold of the complex, at one end binding Rbx1 and its associated E2-Ubiquitin proteins and at the other end binding Skp1. Skp1(SkpA in Drosophila) links Cul1 and the F-box protein-in this case, βTrCP. βTrCP (Slimb in Drosophila) contains the substrate recognition domain of the E3 ligase. The βcat recognition site spans the WD40 repeats on the C-terminal end of βTrCP. This domain forms a propeller structure with a pocket that binds only to phosphorylated proteins. βTrCP can bind multiple phospho-proteins and thus regulate diverse cell signaling pathways (e.g., NFκB and Hedgehog signaling). After βTrCP-βcat binding, βcat is poly-ubiquitinated and can now be recognized by the proteasome. While down-regulation of βcat levels via protein degradation is a key function of the destruction complex, understanding of how βcat is transferred from the complex to the SCF E3 ligase is a key unanswered question (Schaefer, 2020).

Two classes of models seem plausible. In the first class of models, the E3 ligase is a physical entity separate from the destruction complex-this would fit with the many roles for the SCFSlimb E3 ligase, which binds and ubiquitinates diverse phospho-proteins, ranging from the Hedgehog effector Ci/Gli to the centrosome assembly regulator PLK4 . However, given the abundance of cellular phosphatases, this model has a potential major problem. Phosphorylated βcat released free from the destruction complex into the cytoplasm would likely be rapidly dephosphorylated, preventing its recognition by the E3 ligase. Consistent with this, earlier work revealed that APC helps prevent βcat dephosphorylation within the destruction complex. In a second class of models, the SCFSlimb E3 ligase might directly dock on or even become part of the destruction complex, either by direct interaction with destruction complex proteins or by using phosphorylated βcat as a bridge. In this model, once βcat is phosphorylated it could be directly transferred to the E3 ligase, thus preventing dephosphorylation of βcat by cellular phosphatases during transit. Immunoprecipitation (IP) experiments in animals and cell culture revealed that βTrCP can co-IP with Axin, APC, βcat, and GSK3, and that Wnt signals reduce Axin:βTrCP co-IP. However, these studies did not examine whether βTrCP or other components of the E3 are recruited to the destruction complex, leaving both models an option, especially if βTrCP acts as a shuttling protein between complexes (Schaefer, 2020).

A second set of outstanding questions concerns the mechanisms by which Wnt signaling down-regulates βcat destruction. Wnt signaling is initiated when Wnt ligands interact with complex multiprotein receptors, comprised of Frizzled family members plus LRP5/6. This receptor complex recruits the destruction complex to the plasma membrane via interaction of Axin with the phosphorylated LRP5/6 tail and with the Wnt effector Dishevelled (Dvl in mammals/Dsh in Drosophila). This leads to down-regulation of the destruction complex, reducing the rate of βcat destruction. Current data suggest destruction complex down-regulation occurs via multiple mechanisms, some rapid and others initiated more slowly. These include direct inhibition of GSK3 by the phosphorylated LRP5/6 tail, inhibition of Axin homo-polymerization by competition with hetero-polymerization with Dsh, competition between Dsh and APC2 for access to Axin, targeting Axin for proteolytic destruction, and blockade of βcat transfer to the E3 ligase. Recent work has explored the role of Dsh. Overall protein levels of Axin, APC2 and Dsh in Drosophila embryos experiencing active Wnt signaling are within a fewfold of one another, suggesting that competition is a plausible mechanism for destruction complex down-regulation (Schaefer, 2018). The competition model is also consistent with the effects of elevating Axin levels, which makes the destruction complex more resistant to turn-down. However, somewhat surprisingly, elevating Dsh levels had only modest consequences on cell fate choices, and Dsh only assembled into Axin puncta in cells receiving Wingless signals, suggesting that Dsh may need to be 'activated' by Wnt signals in order to effectively compete with APC for Axin and thus mediate destruction complex down-regulation. Candidate phosphorylation sites and kinases potentially involved in this activation have been identified. Intriguingly, when Axin, APC, and Dvl were expressed in mammalian cells, potential competition between APC and Dvl for interaction with Axin was revealed. This study examined in vivo the effects of simultaneously altering levels of Dsh and Axin, testing aspects of the competition model, and combined this with analysis of how Dsh and Axin affect one another's assembly into puncta in a simple cell culture model, using structured illumination superresolution microscopy (Schaefer, 2020).

Wnt signaling plays key roles in development and disease by regulating the stability of its effector βcat. In the absence of Wnt signals, βcat is phosphorylated by the Wnt-regulatory destruction complex, ubiquitinated by an SCF-class E3 ubiquitin ligase, and destroyed by the proteasome. Binding of Wnt ligands to their Frizzled/LRP receptors stabilizes βcat via the cytoplasmic effector Dsh. This study explored two important questions in the field: Is there a direct transfer of βcat from the destruction complex to the E3 ligase, and how does Dsh interaction with the destruction complex protein Axin regulate destruction complex function (Schaefer, 2020)?

Regulating the stability of βcat is the key step in Wnt signaling. The SCFSlimb E3 ligase was first identified as the relevant E3 regulating βcat levels in 1998. It specifically recognizes βcat after its sequential phosphorylation by CK1 and GSK3, and the most N-terminal phosphoserine is a key part of the binding site for the F-box protein Slimb/βTrCP. Phosphatase activity in the cytoplasm can rapidly dephosphorylate this residue, raising the question of how βcat is transferred to the E3 ligase without being dephosphorylated. Earlier work offered two clues. First, βTrCP can co-IP with Axin and APC, suggesting it may associate, at least transiently, with the destruction complex, providing a potential transfer mechanism. Consistent with this, stabilizing Axin using Tankyrase inhibitors led to colocalization of βTrCP and Tankyrase with the destruction complexes that assemble in response. However, it was not clear if this occurred by a direct interaction of βTrCP with destruction complex components via bridging by phosphorylated βcat or occurred because other components of the SCFSlimb E3 ligase were recruited more directly, with βTrCP recruited as a secondary consequence. A second clue emerged from analyses revealing that one role for APC is to prevent dephosphorylation of βcat while it is in the destruction complex, protecting the βTrCP binding site (Schaefer, 2020).

Two plausible models were suggested by these data. In the first, the entire SCFSlimb E3 ligase might be recruited to the destruction complex, allowing direct transfer of phosphorylated βcat between the two complexes. In a second model, βTrCP could serve as a shuttle, binding to phosphorylated βcat at the destruction complex and shuttling it to a place where the E3 assembled and ubiquitinated βcat (Schaefer, 2020).

This study explored interactions of the E3 ligase with the destruction complex using cell biological assays in SW480 cells. Ready recruitment was observed of the βTrCP homologue Slimb to destruction complex puncta by Axin, butrecruitment by APC2 was not observed, consistent with earlier assays by co-IP. Slimb recruitment did not require the βcat-binding site of Axin, making it less likely that recruitment occurs solely via bridging by βcat. However, it was enhanced by the RGS domain of Axin-future work to assess whether this involves a direct interaction or whether an indirect one is warranted. There are conserved residues in the RGS domain that are not necessary for the APC-Axin interaction, some of which form a pi helix, and it will be interesting to further explore the function of these residues. Both the region containing the N-terminus plus the F-box of Slimb and that including its WD40 repeats could be separately recruited into Axin puncta, suggesting it may be recruited by multiple interactions-in the case of the WD40 repeats, this could include bridging by phosphorylated βcat. Once again, direct binding assays in vitro would provide further insights, building on earlier assays suggesting a multipartite binding interaction. Superresolution imaging suggests the interaction between Slimb and Axin is intimate, consistent with direct binding. FRAP data, on the other hand, reveal that Slimb can come in and out of the complex, similar to the behavior of Axin and APC (Schaefer, 2020).

In contrast to the strong recruitment of Slimb to destruction complex puncta, two other core components of the SCFSlimb E3 ligase, Skp1 and Cul1, were not avidly recruited. The occasional recruitment seen could reflect interactions with endogenous βTrCP in the puncta. Coexpression of SkpA or Cul1 with Slimb slightly enhanced recruitment, but this was still not as robust as the recruitment of Slimb itself. IP/mass spectroscopy data and earlier work are consistent with the presence of all three core SCFSlimb E3 ligase proteins in the destruction complex, but suggest they may be present at lower levels than core destruction complex proteins. One possibility is that Slimb/βTrCP usually acts as a shuttle, but its presence occasionally recruits the other E3 proteins. Another possibility is that the entire SCFSlimb E3 ligase docks on the destruction complex transiently to accept phosphorylated βcat, ubiquitinate it, and then transfer it to the proteasome. Consistent with this possibility, inhibiting Tankyrase not only stimulates association of βTrCP with Axin but also leads to recruitment of the proteasome itself to the destruction complex-intriguingly, proteasome inhibition reduces destruction complex assembly, though this effect appears to be indirect due to effects on Axin2 levels. Further analyses will be needed to discriminate between these possibilities (Schaefer, 2020).

Additional work is also needed to explore how βcat transfer to the E3 ligase is regulated. Direct targeting of βcat to the E3, by fusing the F-box of Slimb with the βcat-binding sites of Tcf4 and E-cadherin, is sufficient to stimulate βcat destruction, independent of the destruction complex, but in vivo the destruction complex plays a critical role. Several pieces of data are consistent with the idea that transfer of βcat to the E3 ligase is the step regulated by Wnt signaling, rather than phosphorylation of βcat, with APC having an important role. Further exploration of this process will be welcome (Schaefer, 2020).

It has been clear for more than two decades that Dsh is a key effector of Wnt signaling. However, its precise mechanisms of action are complex and not fully understood. Current data suggest that Dsh is recruited to activated Frizzled receptors via its DEP domain. Dsh then helps ensure the Wnt-dependent phosphorylation of LRP5/6, leading to receptor clustering, facilitating Axin recruitment, and thus inhibiting GSK3. Dsh homo-polymerization, via its DIX domain, and hetero-polymerization with Axin, along with DEP-domain dependent Dsh cross-linking, are then thought to lead to down-regulation of the destruction complex and thus stabilization of βcat (Schaefer, 2020).

Intriguingly, in Drosophila embryos Dsh, Axin, and APC are present at levels within a few-fold of one another. Many current models suggest that relative ratios of these three proteins are critical to the signaling outcome, with APC and Dsh competing to activate or inhibit Axin, respectively. Consistent with this, substantially elevating Axin levels in vivo, using Drosophila embryos as a model, renders the destruction complex immune to down-regulation by Wnt signaling. Subsequent work revealed that the precise levels of Axin are critical-elevating Axin levels by two- to fourfold has little effect, while elevation by ninefold is sufficient to constitutively inactivate Wnt signaling. One might then predict that elevating Dsh levels would have the opposite effect, sequestering Axin and thus stabilizing βcat and activating Wnt signaling. While very high levels of Dsh overexpression can have this effect, it was previously surprising to learn that sevenfold elevation of Dsh levels only had a subtle effect on Wnt signaling and thus had little effect on embryonic viability (Schaefer, 2018). The data further suggested that Dsh is only recruited into Axin puncta in cells that received Wg signal, in which puncta are recruited to the plasma membrane, even though seemingly similar levels of Dsh were present in Wnt-OFF cells (Schaefer, 2018). This opened the possibility that a Wnt-stimulated activation event, such as Dsh phosphorylation, might be required to facilitate Dsh interaction with Axin and thus Axin inactivation. In this scenario, elevating Dsh levels in cells without this activation event, for example, in Wnt-OFF cells, would not alter signaling output (Schaefer, 2020).

The simplest versions of the antagonism model, involving competition between formation of Axin/APC versus Axin/Dsh complexes, would also suggest that elevating Dsh levels should alleviate effects of elevating Axin. This was tested directly, expressing high levels of Dsh maternally and lower levels of Axin zygotically. It was anticipated that elevating Dsh levels would blunt the effects of elevating levels of Axin. Instead, a substantial surprise was in store: elevating levels of Dsh enhanced the ability of Axin to resist turndown by Wnt signaling, thus leading to global activation of the destruction complex and inactivation of Wnt signaling. This was true whether effects were assessed on cell fate choice, Arm levels, or expression of a Wnt-target gene. Intriguingly, the data were also consistent with the possibility that elevating Dsh levels may alter Axin:APC interactions in Wnt-ON cells-this might provide a clue to an underlying mechanism (Schaefer, 2020).

What could explain this paradoxical result? The current data do not provide a definitive answer but do open some intriguing possibilities and new questions. In the view of the authors, part of the explanation will be that Wg-dependent 'activation' of Dsh is required for it to interact with and thus down-regulate Axin. Consistent with this, Dsh phosphorylation can regulate its ability to homopolymerize. By elevating Dsh levels, the capacity of this activation system might have been exceeded. High levels of 'nonactivated' Dsh, while unable to interact with Axin, might still interact with other key proteins involved in destruction complex down-regulation, sequestering them in nonproductive complexes. For example, Dsh can bind CK1, which has complex roles in Wnt regulation With key proteins sequestered, the system might become less able to inactivate the slightly elevated levels of Axin present, thus leading to constitutive activity of the destruction complex. In this speculative scenario, it is not the relative levels of Axin and Dsh that are key but the relative levels of Axin and 'active Dsh' (Schaefer, 2020).

The results of our SIM experiments may also provide insights. The ability of Axin and Dsh to both homo- and hetero-polymerize means free monomers must make a choice. It is likely this is a regulated choice, though the mechanism of regulation remains unclear. The experiments with SW480 cells, while overly simple, may provide an illustration of how the homo-/hetero-polymerization balance can shift. In cells in which both Axin and Dsh were expressed at relatively low levels, puncta contained both proteins, and internal structure was consistent with some level of hetero-polymerization. In contrast, when levels of Dsh were significantly higher, Axin and Dsh tended to segregate into separate, adjoining puncta, suggesting the balance was shifted to homo-polymerization, though the polymers retained the ability to dock on one another. If similar events occur on elevating Dsh expression in Drosophila embryos, segregation could allow Axin to remain in functional destruction complexes, even in Wnt-ON cells, while Dsh localized to separate puncta sequestered other Wnt-regulating proteins, potentially explaining how elevating Dsh expression could paradoxically down-regulate Wnt signaling. Elevating Dsh levels may also lead it to preferentially associate with itself, as was suggested by the SIM data in SW480 cells-this could recruit endogenous Dsh away from its normal localization with the destruction complex, thus preventing it from participating in inactivating Axin. Defining the mechanisms that determine the relevant affinities of each protein for itself versus for its partner will be informative. Intriguingly, a similar docking was observed rather than coassembly behavior when the puncta formed by Axin and those formed by the Arm repeat domain of APC2 were imaged-this may be another example where relative affinities of proteins for themselves versus their binding partners differ (Schaefer, 2020).

Very recent work provides important new insights in this regard. Yamanishi (2019) determined the structure of the heterodimer of the DIX domains of Dsh and Axin and also measured their relative affinities for one another. Another study used cryo-electron microscopy to solve the structure of Dsh filaments and also measured affinities of DIX domains of Dsh and Axin. The results of the second group contrast, with the first group suggesting Dsh homodimerization is an order of magnitude more favorable than Axin homodimerization, while heterodimerization is intermediate in affinity, and the other suggesting Axin homodimerization is most favorable. Resolution of this will be important, as how Dsh acts to turn down destruction complex activity by heterodimerization is assessed. It also is interesting given in vivo observations that APC may help stabilize Axin homo-polymerization . These data also may help explain the results in SIM, where segregation of Dsh and Axin is favored in some circumstances. Defining the in vivo regulatory mechanisms that modulate homo- and heteropolymerization will be an important goal. Together the results leave more questions than answers but suggest that there are important features of Wnt signaling in vivo yet to be uncovered. Further cell biological and biochemical experiments in vivo, combined with new mathematical models of the suspected competition, will be extremely useful (Schaefer, 2020).

Endosomal Wnt signaling proteins control microtubule nucleation in dendrites

Dendrite microtubules are polarized with minus-end-out orientation in Drosophila neurons. Nucleation sites concentrate at dendrite branch points, but how they localize is not known. Using Drosophila, this study found that canonical Wnt signaling proteins regulate localization of the core nucleation protein gammaTubulin (gammaTub). Reduction of frizzleds (fz), arrow (low-density lipoprotein receptor-related protein [LRP] 5/6), dishevelled (dsh), casein kinase Igamma, G proteins, and Axin reduced gammaTub-green fluorescent protein (GFP) at branch points, and two functional readouts of dendritic nucleation confirmed a role for Wnt signaling proteins. Both dsh and Axin localized to branch points, with dsh upstream of Axin. Moreover, tethering Axin to mitochondria was sufficient to recruit ectopic gammaTub-GFP and increase microtubule dynamics in dendrites. At dendrite branch points, Axin and Dsh colocalized with early endosomal marker Rab5, and new microtubule growth initiated at puncta marked with Fz, Dsh, Axin, and Rab5. It is proposed that in dendrites, canonical Wnt signaling proteins are housed on early endosomes and recruit nucleation sites to branch points (Weiner, 2020).

Neurons extend long branched processes from a central cell body. This shape is incompatible with a centrosomal microtubule organizing center (MTOC). Mature neurons are therefore among the ranks of differentiated cells that have noncentrosomal microtubule arrays. It is particularly important to understand how neuronal microtubules are organized because the distance from the primary site of synthesis in the cell body to functional sites in axons and dendrites can be large and, therefore, place heavy demands on microtubule-based transport. In humans, slight disruptions in microtubule regulators or motors can manifest as neurodegenerative disease, underscoring neuronal reliance on perfectly orchestrated microtubule-based transport (Weiner, 2020).

If neuronal microtubules are not anchored to the centrosome, how are they organized? In all neurons so far examined, axonal microtubules have their dynamic plus ends oriented away from the cell body (plus-end-out). In dendrites of vertebrate neurons, microtubules are mixed polarity. In invertebrate neurons (Drosophila and Caenorhabditis elegans), axons have the same plus-end-out microtubule organization as vertebrates, but mature dendrites have almost all minus-end-out microtubules. In immature Drosophila dendrites, microtubules are mixed polarity and only gradually resolve to the minus-end-out mature arrangement. Thus, although the final arrangement of microtubules in vertebrate and invertebrate dendrites is somewhat different, they are the same during dendrite development (Weiner, 2020).

Although the arrangement of neuronal microtubules is clearly noncentrosomal, the source of axonal and dendritic microtubules has been controversial. Two major models for generating axonal and dendritic microtubules have been proposed. The first is that neuronal microtubules are nucleated at the centrosome, or perhaps elsewhere in the cell body, and then released for transport/sliding into axons and dendrites. This model has substantial support, including recent analyses with newer techniques. For example, live imaging of microtubules with plus-tip (+TIP) tracking proteins and photoconvertible αTubulin has provided evidence for directional transport of microtubules into and out of developing axons in mammalian and Drosophila neurons (Weiner, 2020).

The second model is that nucleation sites are found outside the cell body and that microtubules are generated locally in axons and dendrites. Evidence for this model came from the observation that centrosomal γTubulin (γTub), the core microtubule nucleation protein, decreases gradually over time and that centrosome ablation does not disrupt axon formation. Similarly, the centriole is not surrounded by γTub in Drosophila neurons in vivo, and it is dispensable for neuronal microtubule organization. One way to reconcile these two models is to assume that both are important and that, very early in neuronal development, microtubule sliding can dominate, whereas later in development and in mature neurons, microtubules are primarily locally nucleated (Weiner, 2020).

In some cell types, the Golgi complex recruits nucleation sites, and small Golgi outposts can be found in both mammalian and Drosophila dendrites. Thus, it was proposed that the Golgi might act as a noncentrosomal MTOC in dendrites. However, subsequent analysis of γTub and Golgi outposts, including a strategy to deplete Golgi from dendrites, called this proposal into question (Weiner, 2020).

Within Drosophila dendrites, γTub is concentrated at branch points. A previous study identified proteins that localize a different microtubule regulator, adenomatous polyposis coli (Apc) 2, to branch points. It was reasoned that some or all of this machinery might be used to position γTub to the same region. Whether any of the Apc2 localization proteins act upstream of γTub-green fluorescent protein (GFP) in dendrites was tested. Surprisingly, a subset of Wnt signaling proteins was required to localize γTub-GFP to dendrite branch points, regulate dendritic microtubule polarity, and nucleate microtubules in dendrites in response to axon injury. The required proteins include the seven transmembrane domain Frizzled (Fz) proteins (Wnt receptors), Arrow (Arr, a Wnt coreceptor), heterotrimeric G proteins, Dishevelled (Dsh), Casein kinase I (CK1)γ, and Axin. Axin seems to be the key output protein of this pathway because it was sufficient to recruit γTub to ectopic sites in dendrites. Within branch points, Fz, Axin, and Dsh were found on puncta that colocalized with Rab5. In addition, new end-binding protein 1 (EB1) comets at polymerizing microtubule plus ends initiated from puncta marked with Fz, Arr, Dsh, Axin, and Rab5. It is proposed that Wnt signaling proteins localize to early endosomes at dendrite branch points and function there to control local microtubule nucleation. Although it has previously been shown that Wnt signaling proteins can function from endosomes, identification of microtubule nucleation as an output of endosomal Wnt proteins is quite unexpected (Weiner, 2020).

It was particularly intriguing to find integral membrane signaling proteins required for noncentrosomal microtubule nucleation. Although Wnt signaling has been linked to microtubule plus-end regulation in axon growth cones and regulation of microtubule stability and spindle orientation, the only connection to the minus end is localization of some cytoplasmic Wnt signaling proteins like Axin to the centrosome in dividing cells. This study demonstrated that a Wnt signaling pathway acts upstream of microtubule nucleation in a postmitotic cell. Not only were many canonical Wnt signaling proteins required for γTub-GFP to accumulate at branch points, but Axin and Dsh themselves concentrated at branch points. In addition, the scaffolding protein Axin was able to recruit γTub-GFP and the nucleation activator Cnn to mitochondria when tethered to them. Moreover, reduction of Wnt signaling proteins phenocopied loss of γTub in two functional nucleation assays, indicating that most or all dendritic nucleation occurs downstream of this pathway. Although this pathway seems to be the major regulator of dendritic nucleation, neurons are quite resilient to its loss under baseline conditions, and the simple ddaE neurons have normal arbor shape. This is likely because parallel pathways can be used to generate new minus ends. For example, microtubule severing can be used to generate new plus and minus ends and amplify microtubule number. In many cell types, minus ends generated when a microtubule is severed are recognized by minus-end binding proteins in the calmodulin-regulated spectrin-associated protein (CAMSAP)/Patronin family. In C. elegans, γTub-mediated microtubule nucleation has been shown to act in parallel and quite redundantly with Patronin to regulate microtubule organization. Recent work has shown that Patronin-mediated minus-end growth is an important regulator of dendritic microtubules in Drosophila, so it is possible that microtubule severing in conjunction with Patronin recruitment to minus ends can compensate for nucleation under most normal circumstances. Consistent with this hypothesis, phenotypes from reduction of nucleation or Patronin become more evident after severe stress, including axon [29] or dendrite [77, 98] injury (Weiner, 2020).

Although it was consistently found that partial loss of function (RNAi or heterozygous mutants) for fz, fz2, arr, dsh, Gao, Gas, and Axin reduced γTub localization and/or function, no evidence was found that β-catenin/Arm, the key transcription factor that is the output of canonical Wnt signaling, was involved. In addition, an Arm protein trap showed clear expression in epidermal cells but was not seen in dendritic arborization neurons (da neurons). Because Axin itself was sufficient to recruit γTub, there was no strong rationale for a transcriptional regulator to mediate signaling between fz/arr and microtubule nucleation. It is proposed that canonical Wnt signaling proteins are co-opted in dendrites to directly recruit nucleation complexes to endosomes. Because this is a variant of canonical Wnt signaling that unexpectedly seems not to involve β-catenin, this pathway is termed apocryphal Wnt signaling in reference to the Apocrypha, ancient writings found in only some versions of the Bible (Weiner, 2020).

The involvement of arr as well as dsh and Axin suggests that a signalosome might be involved in dendritic Wnt signaling. Signalosomes form when wnt ligands bind to Fz and LRP5/6 at the plasma membrane, triggering recruitment and multimerization of Dsh and Axin. The normal output of signalosome formation is release of β-catenin from the destruction complex and its subsequent stabilization and transit to the nucleus to activate transcription. Signalosomes assemble at the plasma membrane. Endocytosis generally seems to promote Wnt signaling, although in many contexts the signalosome itself is disassembled upon endocytosis. It is not clear whether signalosomes persist after endocytosis, though in some Drosophila cells, Dsh and Arr are localized to endosomes. In dendrites, puncta of Fz, Dsh, and Axin colocalized with Rab5, suggesting that a stable signaling complex is present on endosomes in mature neurons. The initiation of comets from these puncta indicates that endosomes are likely the key site where Wnt signaling proteins promote nucleation. Colocalization of tagged Golgi proteins with Rab5 suggests that the previous association between Golgi markers and nucleation could have been due to leakage into endosomes. In addition, the identification of plasma membrane proteins acting upstream of γTub in dendrites suggests a more general role for the Golgi in the cell body by controlling secretion of Arr and Fz (Weiner, 2020).

Wnt signaling receptors have been classically studied at the plasma membrane, where they bind extracellular ligands that can be autocrine or paracrine in nature. A requirement for arr and fz upstream of γTub in dendrites suggests that a Wnt ligand is likely involved. Failure of neuronal wntless knockdown to reduce γTub-GFP at branch points favors the hypothesis that the ligand may be secreted from a neighboring cell. In the embryo, wingless (wg)/Wnt-1 is made in a patch of epithelial cells adjacent to developing dendritic arborization neurons and helps pattern dendrite orientation in ddaE . It would be very interesting if surrounding cells influenced the microtubule cytoskeleton in mature neurons through Fz and Arr at the plasma membrane. This signaling pathway is particularly intriguing in the context of regeneration or during neurodegenerative disease. During axon regeneration, the initial injury response involves a nucleation-dependent increase in microtubule dynamics, which serves a neuroprotective role. Modulating Wnt signaling could therefore influence neuroprotection in dendrites. In addition, this study found that this pathway is required during dendrite regeneration to position nucleation sites in regrowing dendrites. Interestingly, G protein coupled receptors (GPCRs) represent 33% of all Food and Drug Administration-approved drug targets, and as part of this family, Fz presents a possible target (Weiner, 2020).

Local microtubule nucleation also occurs in axons. As Rab5 endosomes are present throughout axons, it will be interesting to determine whether Wnt signaling proteins can be recruited to axonal early endosomes and whether they recruit nucleation proteins in this part of the cell. It is also possible that a link between Wnt signaling, endosomes, and nucleation could exist more broadly in other cell types. Indeed, the localization of Axin to centrosomes suggests that even in mitotic cells, parts of this relationship are conserved. Intriguingly, endosomal membranes are concentrated around the centrosome, and Rab5 reduction disrupts mitosis, so it is possible that Wnt signaling proteins, endosomes, and nucleation function together at centrosomes (Weiner, 2020).


GENE STRUCTURE

cDNA clone length - 2.6 kb

Bases in 5' UTR - 213

Bases in 3' UTR - 519


PROTEIN STRUCTURE

Amino Acids - 623

Structural Domains

A deletional analysis of dsh has identified several conserved domains essential for activity including a so-called GLGF/DHR motif found in Drosophila discs-large(a tumor suppressor gene) and its relatives (Yanagawa, 1995), an opa repeat, and several PEST sequences (Klingensmith, 1994). The GLGF/DHR motif is also found in a vertebrate postsynaptic density protein (PSD-950) and an erythrocyte membrane protein (Klingensmith, 1994).

All Dishevelled proteins have three highly conserved domains. An N-terminal DIX (Dishevelled-Axin) domain extends for 80 amino acids and is also found in the Axin protein, which seems to be a scaffolding factor for Wnt signaling. Sequence analysis reveals that Dsh has a PDZ domain. Many other signaling proteins contain PDZ domains that can bind to a conserved stretch of amino acids at the C-termini of receptors to form homotypic complexes with other PDZ domains. Multi-domain PDZ domain proteins have been proposed to integrate signaling molecules into larger complexes. A third domain at the C-terminal part of Dsh, called the DEP (Dishevelled-EGL-10-Pleckstrin), can be found in signaling factors such as the RGS protein family, that are involved in G protein signaling. It is not yet known whether DEP domains play an active role in regulating G proteins. Although Dsh and its domains are highly conserved from Drosophila and C. elegans to mouse and human, Dsh does not have an apparent catalytic biochemical activity, nor have any clear interacting partners for Dsh been identified (Boutros, 1999).


date revised: 10 April 2008 
Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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