The apical determinants aPKC and dPatj regulate Frizzled-dependent planar cell polarity in the Drosophila eye: Bazooka has an antagonizing role on the aPKC-mediated inhibition of Fz1 activity in photoreceptor precursors

Planar cell polarity (PCP) is a common feature of many vertebrate and invertebrate epithelia and is perpendicular to their apical/basal (A/B) polarity axis. While apical localization of PCP determinants such as Frizzled (Fz1) is critical for their function, the link between A/B polarity and PCP is poorly understood. A direct molecular link is described between A/B determinants and Fz1-mediated PCP establishment in the Drosophila eye. Patj binds the cytoplasmic tail of Fz1 and is proposed to recruit aPKC, which in turn phosphorylates and inhibits Fz1. Accordingly, components of the aPKC complex and dPatj produce PCP defects in the eye. During PCP signaling, aPKC and dPatj are downregulated, while Bazooka is upregulated, suggesting an antagonistic effect of Bazooka on dPatj/aPKC. A model is proposed whereby the dPatj/aPKC complex regulates PCP by inhibiting Fz1 in cells where it should not be active (Djiane, 2005).

The C tail of Fz receptors regulates their localization and signaling activity. A short Fz Cterm governs apical localization, which is critical for effective Fz-PCP signaling. In contrast, a long Cterm (like that of Fz2) governs baso-lateral localization, promoting β-catenin signaling and preventing PCP activity. Thus, a striking feature of all core PCP proteins, including Fz1, is their apical localization within imaginal disc cells. Fz1 colocalizes partially with several components that regulate A/B polarity such as the Crumbs/Sdt/dPatj and Baz/aPKC/Par-6 complexes within the marginal domain, even though it is also present more basally relative to these complexes (Djiane, 2005).

Detailed sequence analysis of the Fz1 Cterm has revealed the presence of two clustered conserved PKC phosphorylation sites (Ser554 and Ser560 in Fz1). Given that aPKC expression in the apical domain overlaps with Fz1, a test was performed to see if aPKC can phosphorylate the Fz1 Cterm on the two conserved PKC sites in an in vitro kinase assay. Purified human aPKC protein phosphorylates in vitro a GST::Fz1 Cterm fusion protein. Furthermore, mutations of the two PKC consensus sites (Ser to Ala) prevent aPKC-mediated Fz1 phosphorylation, confirming that these sites are targets of aPKC (Djiane, 2005).

To investigate the importance of these phosphorylation sites in vivo, flies were generated carrying UAS-inducible transgenes of Fz1 mutant derivatives with either both serines mutated to alanine (Fz1-AA), inactivating the two prospective PKC sites, or both Serines mutated to Glutamic acid (Fz1-EE), mimicking phosphorylation. These transgenes were analyzed under sevenless (sev)-Gal4 control, which is expressed specifically in R3/R4 precursor cells just posterior to the MF during PCP establishment. Overexpression of wild-type Fz1 provides too much activity and interferes with the balance of Fz1 regulation within the R3/R4 pair, resulting in ommatidia with random R3/R4 cell fate decision and chirality, as well as symmetrical R3/R3 type ommatidia. Similarly, overexpression of Fz1-AA (with both aPKC sites inactivated; sev>Fz1-AA) induces ommatidia with random chirality and symmetrical clusters. In contrast, the phosphomimetic Fz1-EE (sev>Fz1-EE) shows hardly any effect. These data suggest that aPKC-mediated Fz1 phosphorylation inhibits Fz-PCP signaling activity (Djiane, 2005).

Since apical Fz1 localization is critical for its proper PCP signaling activity (Wu, 2004), it was hypothesized that the Fz1-EE mutation could affect the localization of the receptor. To investigate this possibility, the expression of the different myc tagged Fz1 transgenes was examined in imaginal discs (under en-Gal4 or dpp-Gal4 control). No difference between the expression of either Fz1-AA or Fz1-EE with that of wild-type Fz1 was found. These mutant Fz1 isoforms were expressed at similar levels and colocalized apically with aPKC, indicating that phosphorylation of Fz1 by aPKC does not affect Fz1's localization (Djiane, 2005).

A second feature critical to Fz1 signaling activity is its ability to recruit Dsh to the membrane. Interestingly, the aPKC sites partly overlap with the region of the Fz Cterm known to bind Dsh, raising the possibility that phosphorylation by aPKC could interfere with Dsh recruitment. Thus tests were performed to see whether Dsh recruitment is affected by phosphorylation of the Fz1 aPKC sites. S2 cells, which have no endogenous Fz, were transfected with Dsh-GFP and the different Fz1 mutants. In this assay, wild-type and both mutant forms of Fz1 recruit Dsh-GFP efficiently to the membrane. Then, whether overexpression of Fz1-AA and Fz1-EE can recruit Dsh-GFP to apical membranes like wild-type Fz1 in vivo (expressed with en-Gal4 in the posterior compartment of wing discs, where there is a sharp boundary between expressing and nonexpressing cells) was examined. Both Fz1-AA and Fz1-EE behave like wild-type Fz1, sequestering Dsh to the apical cell membrane in imaginal disc cells (Djiane, 2005).

In summary, it was shown that Fz1 can be phosphorylated in vitro by aPKC. Together with the in vitro results, the in vivo analysis of a phosphomimetic Fz1 mutant suggests that aPKC phosphorylation regulates Fz1 activity negatively and that this effect is not mediated by affecting Fz1 localization or Fz1-mediated Dsh membrane recruitment (Djiane, 2005).

In light of the importance of the aPKC sites in Fz1, it was of interest to determine how aPKC is recruited to the receptor. This could be mediated by direct binding or through a bridging factor, the most likely candidates being the A/B determinants that bind aPKC. Thus a two-hybrid interaction screen was conducted using the Fz1 Cterm as bait and components of different A/B protein complexes as prey. The closely related Fz2 Cterm was included as well as the Stbm Cterm as control baits. All components of the aPKC/Par-6/Bazooka apical complex were tested. For Baz, three different fragments were used: an N-terminal fragment involved in Baz dimerization (BazA), a central fragment with three PDZ domains involved in Par-6 binding (BazB), and a C-terminal fragment that binds aPKC (BazC). Similarly, all components of the Crb/Sdt/dPatj apical complex were tested except Crb (since Crb is a transmembrane protein) and the components of the more baso-lateral Scrib/Dlg/Lgl complex were analyzed. No direct interaction was detected between the Fz1 Cterm and aPKC, but, interestingly, Patj was found to be a specific binding partner of the Fz1 Cterm. No other protein was found to interact with the Fz1 Cterm, and in turn Patj did not interact with the Fz2 or Stbm Cterms. The interaction between the Stbm Cterm and Dlg was confirmed as was the interaction of aPKC with Par-6 and BazC (Djiane, 2005)

To confirm the Patj-Fz1 interaction in vivo, coimmunoprecipitation (CoIP) experiments were performed from Drosophila S2 cell extracts transfected with GFP fusion proteins with the Fz1 or Fz2 Cterms (GFP::Fz1 and GFP::Fz2, respectively). Patj could be co-immunoprecipitated from cells transfected with GFP::Fz1 but importantly not with GFP::Fz2 or GFP alone , demonstrating that Fz1 and Patj interact in Drosophila cells. A weak interaction was found between Fz1 and endogenous Baz and aPKC, suggesting the existence of one or several multiprotein complexes among Fz1, Patj, Baz, and aPKC. In contrast, other components of A/B protein complexes, such as Par-6 or Dlg, did not CoIP with either GFP::Fz1 or GFP::Fz2 (Djiane, 2005).

To map the Patj interaction domain with the Fz1 Cterm, GST pull-down experiments were performed. Patj is a modular protein containing a N-terminal L27 domain (previously referred to as MRE), mediating its interaction with Sdt, and four PDZ domains. Consistent with the yeast two-hybrid and CoIP results, in vitro translated full-length Patj bind the GST-Fz1 Cterm protein. The fourth PDZ domain of Patj is sufficient for direct binding to the Fz1 Cterm (Djiane, 2005).

Using the CoIP approach, the residues in the Fz1 Cterm required for Patj interaction were also mapped. Whereas the full-length Fz1 Cterm interacts with Patj, removing the last three residues (Fz1ΔBS) abolishes this interaction. The removal of an internal Cterm motif, encompassing the tryptophan critical for Dsh binding, retains Fz1 ability to bind Patj albeit to a lesser extent (Keyes, 2005).

These results support a direct interaction between the apical determinant Patj and the Fz1 Cterm and suggest that Patj could provide a link between Fz1 and aPKC, since Patj was shown in vitro to bind to aPKC either directly or indirectly through Par-6. The Fz1/Patj interaction is mediated by the fourth PDZ domain of Patj, requires the last three residues of Fz1, and is largely independent of the Fz motif that mediates Dsh binding (Djiane, 2005).

Apical localization is critical for PCP protein activity and particularly for Fz1, but until now no direct link between A/B polarity and PCP establishment has been described. This study shows that the apical determinants aPKC and Patj negatively regulate Fz-PCP signaling while Bazooka antagonizes this regulation. Patj binds directly to the Fz1 cytoplasmic tail, possibly recruiting aPKC, whose phosphorylation of two serine residues within the Fz1 Cterm inhibits the activity of the receptor in cells where signaling should not occur. This reveals a direct link between A/B polarity determinants and PCP establishment (Djiane, 2005).

This work provides the first evidence for a direct molecular link between A/B polarity determinants and PCP by demonstrating that the apical determinants aPKC, Patj, and Baz regulate Fz1 activity. This regulation is independent of Fz1 recruitment to the apical membrane, however, since none of the tested A/B determinants is actively responsible for it. For instance, deleting the Patj binding site in Fz1 or replacing the Fz1 Cterm for a shortened Fz2 Cterm, which cannot bind Patj, has no effect on Fz1 apical localization (Wu, 2004), excluding Patj as a recruiting or targeting factor in Fz1 apical localization. Furthermore, Fmi apical localization is unaffected in Patj and Baz mutants. Thus, although an intact A/B polarity is a prerequisite for PCP signaling, there is no mutual dependency for localizing the Patj/aPKC and the Fz-PCP complexes to the apical side of imaginal disc cells, where they can functionally interact (Djiane, 2005).

Other studies also support the existence of a link between A/B polarity and PCP. In the mouse, Looptail (Lp), the homolog of the Drosophila PCP gene stbm/Vang, interacts genetically with mScribble, a baso-lateral determinant conserved in flies. In particular, transheterozygous Lp/mScribble mice show PCP defects in the inner ear. In Drosophila, it has also been shown that PCP factors interact with A/B determinants. Recent work in the sensory organ precursor (SOP) cells has shown that the orientation of the two opposing domains of Dlg (anterior) and Baz (posterior) is dependent on Stbm and Fz activity (Djiane, 2005).

The downregulation of aPKC and Patj in the R3/R4 cells when Fz1 signals to induce PCP is consistent with a model whereby inhibitory phosphorylation of Fz1 mediated by aPKC is occurring throughout all eye disc cells, except in those that are required for PCP establishment at the time Fz1 signaling occurs. Fz1 activity is therefore always kept low outside of the PCP signaling window, and a release of this inhibition is required for PCP signaling to take place. It is noteworthy that overexpression of Fz1 always gives a robust GOF effect without requiring additional “input,” arguing that either the receptor is constitutively active or that a ligand is always present in nonlimiting amounts. In either scenario, it would be important to control Fz1 activity to prevent signaling at the wrong time and to allow limiting signaling components, such as Dsh, to be available for canonical Wnt/Fz-β-cat signaling when PCP signaling is not needed (Wu, 2004). This is particularly true in the eye disc, where cell fate determination and PCP occur almost simultaneously within a short time window. It is thus proposed that the downregulation of aPKC/Patj in the R3/R4 precursors, at the time of PCP establishment, determines when and in which cells Fz1 is active. A detailed analysis of the expression of Fz1 and Fmi in the non- R3/R4 cells reveals that they extend more basally than aPKC and Patj. Since the precise localization of the active Fz1 is unknown, it is possible that either another mechanism inactivates Fz1 more basally or that inactivation is not needed there (Djiane, 2005).

Furthermore, these results argue that high Baz levels in R3/R4 cells promote Fz1 signaling, possibly by antagonizing the inhibitory regulation of Fz1 by aPKC. Indeed, several lines of evidence suggest an inhibitory role of Baz on the activity of an aPKC complex. (1) In Drosophila embryonic neuroblasts, aPKC phosphorylates Lgl on the apical side of the cell to inhibit its function, restricting the active Lgl to the basal domain of the cell. This is mediated through direct binding of a Par-6/aPKC complex to Lgl, which can only occur after Baz is released from the Par-6/aPKC complex, suggesting a negative role of Baz on aPKC function. (2) Direct measurements of aPKC kinase activity on an exogenous substrate reveal that addition of purified Par-3, the vertebrate Baz homolog, inhibits aPKC kinase activity, whereas Par-6 enhances it. However, whether the aPKC inhibition by Par-3 is direct or indirect remains unclear. This antagonizing role of Bazooka on the aPKC-mediated inhibition of Fz1 activity in R3/R4 cells is further evidence of the tight regulation required for PCP establishment in the eye (Djiane, 2005).

In this model, the A/B determinants are acting upstream of PCP. Consistent with this, there is no effect on either aPKC or Patj expression in cell clones mutant for PCP genes. Similarly, the initial Baz enrichment in R3/R4 precursors is Fz/PCP independent. The later enrichment of Baz in R4 is, however, dependent on PCP signaling. This could correspond to a similar situation as observed in the SOP, in which the posterior relocalization of Baz is dependent on Fz1 activity (Djiane, 2005).

How does aPKC regulate Fz-PCP activity? The aPKC-mediated phosphorylation of the Fz1 Cterm inhibits its activity without affecting its apical localization or ability to recruit Dsh. The negative regulation must therefore occur by a different mechanism. One possibility is that Fz1 phosphorylation by aPKC inhibits a PCP-specific signal transduction to Dsh. Consistent with this hypothesis, similar point mutations in the conserved PKC sites of the canonical Wnt/β-cat-dedicated Fz2 (Fz2-AA and Fz2-EE), do not affect Fz2 ability to trigger a Wnt/β-cat response when overexpressed in the wing. Another possibility is that aPKC regulates Fz1 activity by promoting its destabilization or by increasing its turnover through the recycling pathway at the apical membrane. Further investigation will be required to answer these questions (Djiane, 2005).

The selective downregulation of Patj and upregulation of Baz in R3/R4 precursors define when and where Fz1, and therefore Fz-PCP signaling, is active. This scenario represents a permissive rather than an instructive requirement of aPKC, Patj, and Baz during PCP. Fz-PCP signaling components are widely expressed but only required at specific time points and in specific subsets of cells. As no activating PCP specific ligand is known, it is possible that alternate mechanisms control their activity. This study provides evidence for a negative regulation of PCP signaling by A/B polarity determinants, unveiling new mechanisms for regulating PCP. In addition to their importance during A/B polarity, a function has been revealed for the apical determinants Patj, Baz, and aPKC in regulating PCP and evidence is provided for a molecular link between apical-basal and planar cell polarity (Djiane, 2005).

aPKC controls microtubule organization to balance adherens junction symmetry and planar polarity during development

Tissue morphogenesis requires assembling and disassembling individual cell-cell contacts without losing epithelial integrity. This requires dynamic control of adherens junction (AJ) positioning around the apical domain, but the mechanisms involved are unclear. Atypical Protein Kinase C (aPKC) is required for symmetric AJ positioning during Drosophila embryogenesis. aPKC is dispensable for initial apical AJ recruitment, but without aPKC, AJs form atypical planar-polarized puncta at gastrulation. Preceding this, microtubules fail to dissociate from centrosomes, and at gastrulation abnormally persistent centrosomal microtubule asters cluster AJs into the puncta. Dynein enrichment at the puncta suggests it may draw AJs and microtubules together and microtubule disruption disperses the puncta. Through cytoskeletal disruption in wild-type embryos, a balance of microtubule and actin interactions was found to control AJ symmetry versus planar polarity during normal gastrulation. aPKC apparently regulates this balance. Without aPKC, abnormally strong microtubule interactions break AJ symmetry and epithelial structure is lost (Harris, 2007).

This study reveals the roles of aPKC during polarity establishment and elaboration in Drosophila embryos. In contrast to C. elegans, aPKC is not critical during initial polarity establishment; Baz and AJs are initially localized correctly and the embryonic epithelium can undergo initial morphogenesis. However, aPKC plays an early and striking role in maintaining the symmetrical organization of AJs, via effects on MT organization, and also plays an important later role in the elaboration of polarity (Harris, 2007).

aPKC's later role in polarity elaboration may reflect effects on multiple targets. aPKC is critical for the cortical localization of its normal binding partner PAR-6 and the apical determinant Crb. This latter effect is consistent with the fact that aPKC can phosphorylate Crb, and disruption of aPKC phosphorylation sites in Crb destabilizes Crb in the apical domain. Since Crb stabilizes AJs after gastrulation, this likely contributes to the eventual AJ breakdown in apkcm/z mutants. Crb may act in concert with PAR-6 or in parallel. aPKC can also phosphorylate and exclude the basolateral cue Lgl from the apical domain, and consequenct failure to exclude Dlg from the apical domain in apkcm/z mutants. Thus, apical invasion of basolateral cues may also contribute to the eventual loss of epithelial polarity in apkcm/z mutants (Harris, 2007).

However, it is unlikely that these global changes in apical-basal cell polarity are responsible for the early clustering of AJs into planar-polarized puncta in apkcm/z mutants. Indeed, most of these other polarity players affect polarity after gastrulation. crb mutants have normal spot junctions during gastrulation and early germband extension. Lgl and Dlg are not normally excluded from the apical domain until after gastrulation. Similarly, while mammalian aPKC can restrict PAR-1 to the basolateral domain of epithelial cells, Drosophila PAR-1 is not normally excluded from the apical domain at gastrulation. Thus, effects on Crb, Lgl/Dlg, and PAR-1 cannot easily account for the focusing of AJs and Baz into discrete planar-polarized puncta as apkcm/z mutants gastrulate (Harris, 2007).

Instead, the data suggest that aPKC regulates AJ symmetry by regulating MTs. MT regulation may be a common aPKC function. For example, Drosophila aPKC promotes MT stability at synaptic boutons of neuromuscular junctions, where aPKC forms a complex with Futsch (a MAP1B-like protein) and tubulin, recruiting Futsch to boutons to stabilize MTs. aPKC also regulates MT orientation as mammalian astrocytes and fibroblasts undergo directed migration during wound healing, while in MDCK cells, aPKC helps organize the MT cytoskeleton during ciliogenesis (Harris, 2007).

MT organization and reorganization play important roles in epithelial morphogenesis, and the data demonstrate that loss of aPKC disrupts these events. During Drosophila cellularization, strong MT nucleation from apical centrosomes is likely necessary for assembling lateral MTs that support the apical transport of lipids and proteins to form cell membranes. These MTs also help direct the initial apical positioning of AJs and Baz. During later development, the analysis of apkcm/z mutants indicates that centrosomal MTs can affect the symmetric positioning of AJs around the apical domain. Without aPKC activity, the centrosomes become abnormally dominant, bipolar cues, directing AJ clustering and thus disrupting AJ symmetry. Although this abnormal MT organization differs from changes to MT organization observed in AJ mutants, the possibility cannot be ruled out that there is feedback between MTs and AJs during epithelial morphogenesis and that aPKC may regulate these interactions. Indeed, such feedback is very likely and it will be critical to define MT-AJ cross talk mechanisms in future studies (Harris, 2007).

In apkcm/z mutants, MT-associated AJ/Baz puncta assemble at the dorsal and ventral sides of the cells. This suggests that MTs may normally function in AJ assembly at these newly formed cell-cell contacts. However, these polarized AJ assembly events must also be counterbalanced to maintain AJ symmetry and proper epithelial structure. Cytoskeletal inhibitor studies suggest that AJ symmetry may normally be regulated by a balance of MT-AJ and actin-AJ interactions at this stage -- actin appears to counteract MT-based AJ assembly at dorsal and ventral cell contacts. Actin as been shown to be enriched at anterior and posterior cell contacts, suggesting that it may be an early planar polarity cue at this stage. Perhaps this planar-polarized actin stabilizes a pool of AJs at anterior and posterior cell contacts, thereby counterbalancing MT-based AJ assembly at dorsal and ventral contacts. Alternatively, lower levels of actin at dorsal and ventral cell contacts could directly counteract MT-based AJ assembly at these sites. Distinguishing these possibilities requires further study. Nonetheless, the apkcm/z mutant phenotype appears to arise from a gain-of-function effect in which MTs become overactive and the proper balance between MT-AJ and actin-AJ interactions is lost. As a result, there is a break in AJ symmetry in apkcm/z mutants, MT-associated AJ puncta eventually become randomly positioned, and the epithelium dissociates (Harris, 2007).

The data suggest a speculative mechanistic model by which aPKC could normally regulate MT-AJ interactions. This study shows that MT association is responsible for the abnormal AJ asymmetry seen in apkcm/z mutants, and that Dynein accumulates at these abnormal AJ/Baz puncta. Since Dynein plays a role in apical transport of AJ/Baz proteins during cellularization, it is proposed that aPKC may normally regulate release of AJ/Baz complexes from Dynein, allowing a complete transport cycle. In the absence of this release, AJ/Baz complexes could maintain an abnormal association with MTs, and localized Dynein activity may pull the centrosomes and spot junctions together into the abnormal puncta seen in apkcm/z mutants. This abnormal cortical Dynein activity might also stabilize MTs emanating from the centrosomes, resulting in the persistent centrosomal MTs in the mutants. Alternatively, aPKC may function at the centrosomes to decrease MT nucleation or increase MT severing. Future experiments will illuminate these mechanisms and the generality of aPKC's role in controlling MT organization and AJ positioning (Harris, 2007).

lethal giant larvae is required with the par genes for the early polarization of the Drosophila oocyte

Most cell types in an organism show some degree of polarization, which relies on a surprisingly limited number of proteins. The underlying molecular mechanisms depend, however, on the cellular context. Mutual inhibitions between members of the Par genes are proposed to be sufficient to polarize the C. elegans one-cell zygote and the Drosophila oocyte during mid-oogenesis. By contrast, the Par genes interact with cellular junctions and associated complexes to polarize epithelial cells. The Par genes are also required at an early step of Drosophila oogenesis for the maintenance of the oocyte fate and its early polarization. This study shows that the Par genes are not sufficient to polarize the oocyte early and that the activity of the tumor-suppressor gene lethal giant larvae (lgl) is required for the posterior translocation of oocyte-specific proteins, including germline determinants. Lgl localizes asymmetrically within the oocyte and is excluded from the posterior pole. Phosphorylation of Par-1, Par-3 (Bazooka) and Lgl is crucial to regulate their activity and localization in vivo. Adherens junctions locate around the ring canals, which link the oocyte to the other cells of the germline cyst. However, null mutations in the DE-cadherin gene, which encodes the main component of the zonula adherens, do not affect the early polarization of the oocyte. It is concluded that, despite sharing many similarities with other model systems at the genetic and cellular levels, the polarization of the early oocyte relies on a specific subset of polarity proteins (Fichelson, 2010).

One general strategy to establish polarity within a cell is to create non-overlapping membrane domains along one specific axis. In most cell types, four complexes are involved in the formation of these domains: (1) Par-3-Par-6-aPKC, (2) Crb-Sdt-dPatj, (3) Scrib-Lgl-Dlg and (4) Par-1. However, the activities and interactions of these complexes depend on the cellular context. In single-cell systems that lack intercellular junctions, such as the C. elegans embryo and vertebrate hippocampal neurons, mutual inhibitions between the Par-3-Par-6-aPKC complex and Par-1 (PAR-2) seem sufficient to establish polarity. By contrast, in the follicular epithelium, the Crb-Sdt-dPatj complex acts redundantly with Par-3-Par-6-aPKC to define the apical side, whereas the Scrib-Lgl-Dlg complex cooperates with Par-1 on the lateral cortex. Consistent with this redundancy, expression of non-phosphorylatable forms of either Par-3 or Par-1 is not able to disrupt the apical-basal polarity of the follicle cells, although they both localize ectopically. This study shows that the early polarization of the oocyte is an intermediate case. The results suggest that the Crb-Sdt-dPatj complex does not act redundantly with the Par-3-Par-6-aPKC complex as it is not required, whereas Lgl could function with Par-1. Consistent with this hypothesis, this study showed that the expression of a non-phosphorylatable form of Par-1 is able to disrupt the early polarization of the oocyte, whereas a non-phosphorylatable form of Par-3 is not able to counter the activities of both Lgl and Par-1. In addition, it was found that Par-1 localizes on the fusome independently of Lgl further suggesting that both could act in parallel pathways (Fichelson, 2010).

The results show that phosphorylation plays a crucial role to regulate the activity and localization of Par-1, Par-3 and Lgl during early oogenesis, but to different extents in each case. Par-1 phosphorylation might not be crucial for its localization within the germarium, since it was found that the non-phosphorylatable and wild-type forms of Par-1 had a similar localization, although Par-1-AEM (apical-lateral exclusion motif (AEM), in which a conserved threonine is replaced by an alanine) appears a bit more cytoplasmic. Its overexpression, however, induces very strong and penetrant polarity defects in the germline. These results contrast with the follicular epithelium, where Par-1-AEM-GFP localizes ectopically to the apical membrane but does not affect the polarization of those cells. Thus, the main function of Par-1 phosphorylation in the germarium might be to downregulate its kinase activity. In addition, it was shown that the microtubule cytoskeleton is a probable target of Par-1 activity, since a strong reduction in microtubules was observed in oocytes expressing Par-1-AEM (Fichelson, 2010).

The localization of endogenous Par-3, wild-type Par-3-GFP and non-phosphorylatable Par-3 (Baz-S151A,S1085A-GFP) also appear identical. However, Baz-S151A,S1085A was unable to localize properly in the absence of the endogenous Par-3. This failure is probably due to the inability of this mutant form of Par-3 to homodimerize. Phosphorylation thus plays an important role for Par-3 localization. This non-phosphorylatable form of Par-3 is, however, still active, as it is able to rescue par-3-null homozygous clones in the follicular epithelium and is also sufficient to induce polarity defects in the oocyte when expressed at later stages of oogenesis. In this latter case, Baz-S151A,S1085A was expressed in the presence of the endogenous Par-3 and was able to reach the cortex of the oocyte and localize ectopically to the posterior pole. The results suggest that this ectopic Par-3 is probably made of heterodimers of endogenous and non-phosphorylatable forms of Par-3. The data further show clear differences with the follicular epithelium, where expression of the non-phosphorylatable form of Par-3 in par-3 mutant clones not only rescues the absence of endogenous protein, but also localizes properly at the apical side only. This could be a consequence of redundant mechanisms in the follicle cells (Fichelson, 2010).

It was found that Lgl localization is strikingly asymmetric in the early oocyte as it is completely absent from the posterior cortex from stage 1 to stage 5-6 of oogenesis. By contrast, it becomes specifically enriched at the posterior cortex from stage 7 onward. This is the first time that such an asymmetry is described within the germline so early during oogenesis for any protein. It was further demonstrated that this asymmetric localization depends on Lgl phosphorylation, as Lgl-3A localizes around the entire oocyte cortex. Phosphorylation thus plays an important role for Lgl localization. Surprisingly, the ectopic Lgl-3A localization is not sufficient to disrupt the early polarization of the oocyte. By contrast, the same Lgl-3A construct induces much stronger polarity defects than wild-type Lgl when overexpressed in embryonic neuroblasts or in the oocyte at later stages of oogenesis. One possible explanation is that at least one of the mutated Serine in Lgl-3A is also required for Lgl activity during the early stages of oogenesis. Another possibility is that an unknown redundant pathway is able to counteract Lgl activity in the germarium (Fichelson, 2010).

One question remaining from this work is the relationship between the localization of Par-1, Par-3 and Lgl, and their function. It is difficult to relate Par-1 localization on the fusome in region 1 of the germarium and the polarization defects induced by its absence in region 3. Furthermore, this study shows that Par-3 localizes around the ring canals with DE-Cadherin and Armadillo on genuine AJs, which are structures playing key roles in the polarization of many epithelia. However, the absence of Par-3 on these junctions in DE-Cadherin mutant clones does not perturb the polarization of the oocyte. The relevant localization of Par-3 for its polarizing activity in the germarium thus remains unknown. Finally, although Lgl localization is clearly asymmetric, excessive or ectopic localization only affect oogenesis after the oocyte becomes polarized (Fichelson, 2010).

Several arguments strongly point to the microtubule cytoskeleton and associated proteins as key targets of the polarity complexes in the germarium: (1) short treatments of microtubules depolymerizing drugs allow the restriction of cytoplasmic proteins into the oocyte in region 2 of the germarium but disrupt their localization to the posterior of the oocyte in region 3; (2) the Orb protein localizes into the oocyte in hypomorphic combinations of dhc64C, which encodes the heavy chain of the minus-end-directed molecular motor Dynein, but fails to translocate to the posterior pole of the oocyte; (3) In strong allelic combinations of Bicaudal D, a binding partner of the dynein-dynactin complex, Orb and centrosomes also fail to migrate to the posterior of the oocyte. Furthermore, in all three cases, the oocyte becomes polyploid later on and reverts to the nurse cell fate. This study shows that overexpression of Par-1-AEM strongly reduces the level of microtubules and induces identical phenotypes. This confirms that the earliest step of oocyte polarization depends on microtubules and is consistent with the well-established function of Par-1 mammalian homologues, the MARKs, in destabilizing microtubules. It is, however, more difficult to explain why the oocyte nucleus becomes polyploid and why the oocyte loses its identity. Although the Par proteins could have a separate function within the nucleus, depolymerizing the microtubules leads to an identical phenotype, which rather suggests that polyploidization of the oocyte nucleus is a direct consequence of the absence of microtubules. A failure to polarize is, however, not sufficient to induce polyploidization of the oocyte nucleus. Indeed, several mutants were found to retain Orb at the anterior of the oocyte but did not produce egg chambers with 16 nurse cells. The link between the cytoplasmic polarization of the oocyte and its nuclear identity thus remains unclear and is an exciting line for future investigations (Fichelson, 2010).

Atonal and EGFR signalling orchestrate rok- and Drak-dependent adherens junction remodelling during ommatidia morphogenesi

Morphogenesis of epithelial tissues relies on the interplay between cell division, differentiation and regulated changes in cell shape, intercalation and sorting. These processes are often studied individually in relatively simple epithelia that lack the complexity found during organogenesis when these processes might all coexist simultaneously. To address this issue, this study makes use of the developing fly retinal neuroepithelium. Retinal morphogenesis relies on a coordinated sequence of interdependent morphogenetic events that includes apical cell constriction, localized alignment of groups of cells and ommatidia morphogenesis coupled to neurogenesis. Live imaging was used to document the sequence of adherens junction (AJ) remodelling events required to generate the fly ommatidium. In this context, it was demonstrated that the kinases Rok and Drak function redundantly during Myosin II-dependent cell constriction, subsequent multicellular alignment and AJ remodelling. In addition, it was shown that early multicellular patterning characterized by cell alignment is promoted by the conserved transcription factor Atonal (Ato). Further ommatidium patterning requires the epidermal growth factor receptor (EGFR) signalling pathway, which transcriptionally governs Rho-kinase (rok) and Death-associated protein kinase related (Drak)-dependent AJ remodelling while also promoting neurogenesis. In conclusion, this work reveals an important role for Drak in regulating AJ remodelling during retinal morphogenesis. It also sheds new light on the interplay between Ato, EGFR-dependent transcription and AJ remodelling in a system in which neurogenesis is coupled with cell shape changes and regulated steps of cell intercalation (Robertson, 2013).

In Drosophila, Rok seems to be the main kinase responsible for phosphorylating the Myosin regulatory light chain (Sqh) during epithelial patterning and apical cell constriction. This is the case for the activation of MyoII during intercalation as germband extension proceeds, but also during various instances of compartment boundary formation and cell sorting situations in the embryo and in the wing imaginal disc. The current work reveals that in the constricting cells of the MF, Rok functions redundantly with Drak, a kinase recently shown to phosphorylate Sqh both in vitro and in vivo (Neubueser, 2010). It is noteworthy that previous work has shown that RhoGEF2 is not required for cell constriction in the MF, suggesting that perhaps another guanine exchange factor (GEF) might function redundantly with RhoGEF2 to promote cell constriction. These data on Drak reinforce the idea that redundancies exist in this context. Because the RhoA (Rho1 -- FlyBase) loss of function abolishes this cell response entirely, it would be expected that Drak function is regulated by RhoA. In addition, the current data indicate that Drak acts redundantly with Rok during MyoII-dependent multicellular alignment and AJ remodelling during ommatidia patterning. It will be interesting to test whether Drak functions in other instances of epithelial cell constriction or MyoII-dependent steps of AJ remodelling in other developmental contexts in Drosophila (Robertson, 2013).

This study demonstrates a two-tiered mechanism regulating the planar polarization of MyoII and Baz. In the constricting cells in the posterior compartment, MyoII and Baz are segregated from one another and this is exacerbated by the wave of cell constriction in the MF. Upon Ato-dependent transcription in the MF cells, this segregated pattern of expression is harnessed and these factors become planar polarized at the posterior margin of the MF. This is independent of the core planar polarity pathway including the Fz receptor and is accompanied by a striking step of multicellular alignment. Previous work has demonstrated that Ato upregulates E-Cad transcription at the posterior boundary of the MF. In addition, apical constriction leads to an increase in E-Cad density at the ZA. The current data are therefore consistent with both hh-dependent constriction and ato-dependent transcriptional upregulation of E-Cad promoting differential adhesion, thus leading to a situation in which the ato+ cells maximize AJ contacts between themselves and minimize contact with the flanking cells that express much less E-Cad at their ZA. This typically leads to a preferential accumulation of cortical MyoII at the corresponding interface. Such actomyosin cables are correlated with increased interfacial tension, and it is proposed that this is in turn responsible for promoting cell alignment. Unfortunately, the very small diameter of these constricted cells precludes direct measurements of the AJ-associated tension using laser ablation experiments (Robertson, 2013).

Supra-cellular cables of MyoII have been previously associated with cell alignment in various epithelia and have also been observed at the boundary of sorted clones, whereby cells align at a MyoII-enriched interface. Interestingly, this study found that the actomyosin cable defining the posterior boundary of the MF is also preferentially enriched for Rok, a component of the T1, MyoII-positive AJ in the ventral epidermis (Simoes Sde, 2010). This indicates an important commonality between actomyosin cable formation during cell sorting and the process of cell intercalation. However, unlike during intercalation, this study found that in the developing retina baz is largely dispensable for directing the pattern of E-Cad and actomyosin planar polarization. Further work will therefore be required to understand better the relationship between Baz and E-Cad at the ZA during ommatidia morphogenesis. It is speculated that the creation of a high E-Cad versus low E-Cad boundary in the wake of the MF might be sufficient to promote Rok and MyoII enrichment at the posterior AJs. This posterior Rok and MyoII enrichment might perhaps prevent E-Cad accumulation by promoting E-Cad endocytosis, as has been recently shown in the fly embryo (Robertson, 2013).

This study has used live imaging to define a conserved step of ommatidia patterning that consists of the coalescence of the ommatidial cells' AJs into a central vertex to form a 6-cell rosette. The corresponding steps of AJ remodelling require Rok, Drak, Baz and MyoII, a situation compatible with mechanisms previously identified during cell intercalation in the developing fly embryo. The steps of AJ remodelling required to transform lines of cells into 5-cell pre-clusters are transcriptionally regulated downstream of EGFR in a ligand-dependent manner. Interestingly, in the eye EGFR signalling is activated in the cells that form lines and type1-arcs in the wake of the MF and, thus, are undergoing AJ remodelling. Previous work examining tracheal morphogenesis in the fly has demonstrated that interfaces between cells with low levels versus high levels of EGFR signalling correlate with MyoII-dependent AJ remodelling in the tracheal placode. This situation resembles that which is described in this study in the wake of the MF. In the eye, however, it was found that EGFR signalling is not required to initiate cell alignment. Nevertheless, taken together with work in the tracheal placode and previous studies related to multicellular patterning in the developing eye, this work indicates a conserved function for the EGFR signalling pathway in promoting MyoII-dependent AJ remodelling. This leaves open several interesting questions; for example, it is not presently clear how EGFR signalling can promote discrete AJ suppression and elongation. It is, however, tempting to speculate that previously described links between EGFR signalling and the expression of E-Cad or Rho1 might play a role during this process (Robertson, 2013).

The Drosophila MAST kinase Drop out is required to initiate membrane compartmentalisation during cellularisation and regulates dynein-based transport

Cellularisation of the Drosophila syncytial blastoderm embryo into the polarised blastoderm epithelium provides an excellent model with which to determine how cortical plasma membrane asymmetry is generated during development. Many components of the molecular machinery driving cellularisation have been identified, but cell signalling events acting at the onset of membrane asymmetry are poorly understood. This study shows that mutations in drop out (dop; CG6498) disturb the segregation of membrane cortical compartments and the clustering of E-cadherin into basal adherens junctions in early cellularisation. dop is required for normal furrow formation and controls the tight localisation of furrow canal proteins and the formation of F-actin foci at the incipient furrows. This study shows that dop encodes the single Drosophila homologue of microtubule-associated Ser/Thr (MAST) kinases. dop interacts genetically with components of the dynein/dynactin complex and promotes dynein-dependent transport in the embryo. Loss of dop function reduces phosphorylation of Dynein intermediate chain, suggesting that dop is involved in regulating cytoplasmic dynein activity through direct or indirect mechanisms. These data suggest that Dop impinges upon the initiation of furrow formation through developmental regulation of cytoplasmic dynein (Hain, 2014).

This study is the first mutational analysis of a MAST kinase in any organism and demonstrates that the MAST kinase Dop plays an important role in plasma membrane cortex compartmentalisation during the generation of epithelial polarity in the fly. The results reported in this study demonstrate a requirement of Dop in the establishment of the furrow canal and the bAJ at the cycle 14 transition. The defect in bAJ formation is likely to be a consequence of a failure in the initial specification of the incipient furrows. It is proposed that Dop acts upstream in furrow canal formation by controlling the formation of F-actin-rich foci, which initiate the assembly of a specific furrow membrane cortex (Hain, 2014).

In mid-cellularisation stages, dop mutant phenotypes are reminiscent of embryos lacking the early zygotic gene bottleneck (bnk). In bnk mutants the initial formation of the cleavage furrows is normal, but then furrows close prematurely. Although it cannot be excluded that bnk might play a role in later defects associated with dop mutations, the primary defect in dop mutants concerned the lack of regular F-actin-rich furrows during the onset of cellularisation. Another early zygotic gene, nullo, is required for the proper recruitment of F-actin during furrow canal formation. Nullo and the actin regulator RhoGEF2 have been proposed to act in parallel pathways controlling processes that are distinct but both essential for F-actin network formation during the establishment of the furrow canal. Since early F-actin rearrangements are largely normal in nullo and RhoGEF2 single mutants, it is proposed that Dop is essential for the initial early focussing of F-actin, whereas Nullo and RhoGEF2 are required to elaborate and maintain F-actin levels to stabilise the furrows. The actin regulator enabled (ena) has been shown to act downstream of Abelson tyrosine kinase (Abl) in the redistribution of F-actin from the plasma membrane cortex into the furrows in both syncytial stages and cellularisation. Although ena would provide a good candidate for acting downstream of dop in the redistribution of F-actin, ena is already required for syncytial cleavages and the F-actin phenotypes in Abl mutants are much more severe than those that were found for dop mutants (Hain, 2014).

The similarity of syncytial cleavage furrows and the cleavage furrows at cellularisation raises the question of how they differ from each other. The molecular basis of the hexagonal pattern of the F-actin-rich cell cortex at the cleavage furrow relies upon the recycling endosome components Rab11 and Nuclear fallout (Nuf) and the actin polymerisation factors Dia and Scar/Arp2/3. In contrast to dop mutants, nuf, dia or Scar mutants indicate that these genes are required also for the dynamic redistribution of F-actin during syncytial development. Since Dop is a maternally supplied protein, its activity might be regulated by events triggered during the cycle 13-14 transition. The major difference between the furrows in syncytial stages and cellularisation is that metaphase furrows are formed during M phase, whereas cellularisation furrows are formed during G2 phase. Since Dop is a maternally supplied gene product, one would have to implicate regulation of Dop by zygotic factors to explain its phenotype at the cycle 13-14 transition. An alternative possibility is that Dop is regulated by phosphorylation or other post-translational modification through the cell cycle machinery and that, in the absence of Cdk1-dependent phosphorylation, its phosphorylation state is changed. This study provided evidence that Dop is indeed differentially post-translationally modified during syncytial versus cellular blastoderm stages. It is proposed that such cell cycle-dependent regulation of Dop may be crucial in transforming syncytial cleavages into persistent cellularisation furrows. Furthermore, the data suggest that this transition could require Dop-dependent regulation of dynein-associated microtubule transport (Hain, 2014).

The mechanisms for the initial localisation of Baz and E-cadherin are still unclear but, interestingly, dop is required for the localisation of both proteins. At the cycle 14 transition, E-cadherin and Arm puncta are associated with apical membrane projections and the homophilic association of these cadherin puncta is strengthened by membrane flow and is dependent on actin. Baz function allows these puncta to become tightened into sAJs. Thus, Dop might affect the stabilisation of the weakly interacting puncta either through cortical actin organisation or membrane flow. In addition to this early requirement for Baz localisation, Dop is also involved in clearing Baz from the basal cytoplasm during late cellularisation. The mechanism that eventually clears Baz from the basal cytoplasm depends on dynein-based transport. Therefore, Dop is required for dynein-based transport of different cargoes during cellularisation: lipid droplets, mRNA particles, Golgi and Baz. It is proposed that the main function of Dop in cellularisation is in regulating dynein-mediated transport of important cargos along microtubules (Hain, 2014).

This study presents the first evidence for regulation of dynein-mediated transport by a MAST family kinase. Dop is shown to controls phospho-Dic levels in a direct or indirect manner. The data are consistent with a model in which the initiation of furrow formation involves dynein-dependent transport that is controlled by Dop. In support of a role in membrane formation, this study found defects in the distribution of the recycling endosome and Golgi compartments in dop mutants. Interference with Rab11 function causes similar defects in Slam distribution as those shown by dop mutants. Therefore, Dop might control the transport of endomembrane compartments, which drive membrane growth. In addition, F-actin redistribution plays a major role in membrane cortical compartmentalisation in the initial stages of cellularisation. The focussing of F-actin to incipient furrows might involve a dynein-dependent shift of actin regulators or existing actin filaments to the furrow. An attractive hypothesis is that the translocation of F-actin and/or its regulators is coupled to an endomembrane compartment that is transported via microtubules towards the incipient furrow canals. Future studies should aim to determine which dynein cargos contribute to furrow formation and how Dop regulates Dic phosphorylation at the molecular level (Hain, 2014).

Protein Interactions

Several proteins, including Numb and Miranda, segregate into the basal daughter cell and are needed for the determination of its correct cell fate. Both the apical-basal orientation of the mitotic spindle and the localization of Numb and Miranda to the basal cell cortex are directed by Inscuteable, a protein that localizes to the apical cell cortex before and during neuroblast mitosis. The apical localizaton of Inscuteable requires Bazooka, a protein containing a PDZ domain that is essential for apical-basal polarity in epithelial cells. Bazooka localizes with Inscuteable in neuroblasts and binds to the Inscuteable localization domain in vitro and in vivo. In embryos lacking both maternal and zygotic bazooka function, Inscuteable no longer localizes asymmetrically in neuroblasts and is instead uniformly distributed in the cytoplasm. Mitotic spindles in neuroblasts are misoriented in these embryos, and the proteins Numb and Miranda fail to localize asymmetrically in metaphase. These results suggest that direct binding to Bazooka mediates the asymmetric localization of Inscuteable and connects the asymmetric division of neuroblasts to the axis of epithelial apical-basal polarity (Schober, 1999).

In Drosophila neuroblasts, inscuteable controls both spindle orientation and the asymmetric localization of the cell-fate determinants Prospero and Numb. Inscuteable itself is localized in an apical cortical crescent and thus reflects the intrinsic asymmetry of the neuroblast. Localization of Inscuteable depends on Bazooka, a protein containing three PDZ domains with overall sequence similarity to Par-3 of Caenorhabditis elegans. Bazooka and Inscuteable form a complex that also contains Staufen, a protein responsible for the asymmetric localization of Prospero messenger RNA. It is proposed that, after delamination of the neuroblast from the neuroepithelium, Bazooka provides an asymmetric cue in the apical cytocortex that is required to anchor Inscuteable. Since Bazooka is also responsible for the maintenance of apical-basal polarity in epithelial tissues, it may be the missing link between epithelial polarity and neuroblast polarity (Wodarz, 1999).

A direct interaction between Partner of Inscuteable and Baz in yeast two hybrid and GST pull-down experiments could not be demonstrated. However, Baz complexes with Insc in vivo, and directly interacts with Insc in vitro (Schober, 1999; Wodarz, 1999). Since Pins interacts with Insc, these observations suggest that Insc may be acting to link Baz to Pins. Several observations support this view. (1) For NBs and cells of mitotic domain 9, Pins does not localize apically in the absence of insc function. (2) Also supportive is the apparent temporal order in which these genes are recruited to the apical cortex of NB: Baz (while part of the epithelia), followed by Insc (during delamination), followed by Pins (after delamination). (3) In epithelial cells that do not express Insc, Pins and Baz do not colocalize; Baz is found on the apical cortex and Pins shows lateral cortical distribution, yet the ectopic expression of Insc (which localizes apically) is sufficient to recruit Pins to the apical cortex of these cells. All of the available data are consistent with the model that the formation and maintenance of an apical protein complex that imparts apical/basal polarity in NBs comprises the following events: cells in the neuroepithelium destined to become NBs have apical/basal polarity as evidenced by the apical localization of Baz; as these interphase cells delaminate, Insc is recruited to the apical complex in a Baz-dependent manner; Pins is in turn recruited to this complex and this involves interaction with Insc. Some as yet undefined events must occur between delamination (interphase) and mitosis that change the nature of this complex such that its maintenance becomes codependent on these three molecules (Yu, 2000).

Both insc and pins are required for the execution of the more downstream processes associated with asymmetric cell divisions and the relative roles of the two genes are at present unclear. However, some interesting distinctions can be made between the two genes. insc was originally isolated on the basis of its expression in neural precursors. Insc expression is restricted, conforms to the prepattern-proneural-neurogenic-panneural cascade, and links general neuronal differentiation programs to lineage information; Pins shows a wider expression pattern and becomes involved in asymmetric cell divisions only when a signal (i.e., insc) is active. Pins and Insc also appear to follow different routes to reach the apical cortex -- Pins apparently transiting via the membrane but not Insc, which suggests that other interactors may be involved in linking Pins to the cortex. Finally, the only known direct links to downstream events associated with asymmetric cell divisions appear to be mediated through Insc (Yu, 2000).

Polarized cells contain numerous membrane domains, but it is unclear how the formation of these domains is coordinated to create a single integrated cell architecture. Genetic screens of Drosophila embryos have identified three complexes, each containing one of the PDZ domain proteins -- Stardust (Sdt), Bazooka (Baz) and Scribble (Scrib) -- that control epithelial polarity and formation of zonula adherens. These complexes can be ordered into a single regulatory hierarchy that is initiated by cell adhesion-dependent recruitment of the Baz complex to the zonula adherens. The Scrib complex represses apical identity along basolateral surfaces by antagonizing Baz-initiated apical polarity. The Sdt-containing Crb complex is recruited apically by the Baz complex to counter antagonistic Scrib activity. Thus, a finely tuned balance between Scrib and Crb complex activity sets the limits of the apical and basolateral membrane domains and positions cell junctions. These data suggest a model in which the maturation of epithelial cell polarity is driven by integration of the sequential activities of PDZ-based protein complexes (Bilder, 2003).

Drosophila atypical protein kinase c associates with Bazooka and controls polarity of epithelia and neuroblasts

Atypical protein kinase C (aPKC) from Drosophila shows very high sequence similarity to PKClambda and PKCzeta from vertebrates and PKC-3 from C. elegans. Drosophila aPKC and Baz coimmunoprecipitate and directly bind to each other in a yeast two-hybrid assay. In embryos, both proteins colocalize in the apical cortex of almost all epithelial tissues and in neuroblasts. Apical localization of DaPKC in epithelia and neuroblasts is abolished in baz mutants, and vice versa: Baz is delocalized in DaPKC mutants. The phenotype of aPKC loss-of-function mutants resembles that of baz mutants, consistent with a close functional interdependence of both proteins. Together, these data provide in vivo evidence for an essential role of an atypical protein kinase C isoform in establishment and maintenance of epithelial and neuronal polarity (Wodarz, 2000).

To test whether aPKC and Baz colocalize, double-label immunofluorescence stainings of embryos was performed. aPKC and Baz are clearly colocalized in the epidermis and in neuroblasts. To determine the precise subcellular localization of aPKC and Baz with respect to the ZA, double-label immunofluorescence stainings were performed with antibodies against Arm, a component of the ZA and Baz. The merged image shows that Baz is localized apically to Arm. The same is true for aPKC. At the resolution of the confocal microscope, the possibility that the localization of Baz and DaPKC partially overlaps with Arm in the ZA cannot be ruled out (Wodarz, 2000).

Binding studies showing a physical association of aPKC and Baz, and colocalization of these two proteins suggests that they may functionally interact with each other. In stainings of baz mutant embryos derived from germ line clones (baz null embryos) with anti-aPKC antibody, apical localization of aPKC could not be detected in epithelia and neither could apical localization be detected in neuroblasts. Instead, aPKC was distributed in a diffuse fashion in the cytoplasm. baz null embryos also show a loss of membrane polarity that is evident by mislocalization of the basolateral transmembrane protein Nrt. In contrast to wild type, Nrt is not excluded from the apical plasma membrane. Moreover, the monolayered structure of the epidermis is lost and cells pile up on top of each other, as has been described before for sdt;baz double mutants (Wodarz, 2000).

To test whether mislocalization of Baz is sufficient to induce mislocalization of aPKC, Baz was overexpressed by means of the GAL4 system. Under these conditions, Baz is not confined to the apical cytocortex anymore and is found in more lateral and basal positions in epithelia and neuroblasts. Concomitantly, aPKC is also mislocalized and colocalized in ectopic positions with ectopic Baz, confirming that ectopic Baz can recruit aPKC to ectopic sites in the cytocortex (Wodarz, 2000).

It has been shown before that Baz is required for apical localization of Insc in neuroblasts and that Insc is required for stabilization of Baz in neuroblasts after delamination. A test was performed to see whether Baz and Insc are also required for localization of aPKC in neuroblasts. aPKC localization is indistinguishable from wild type in neuroblasts of inscP49/CyO heterozygous embryos, but is neither cortical nor apical in neuroblasts of inscP49 homozygous mutant embryos. In embryos lacking maternal Baz but carrying a paternal wild-type allele of baz (partial paternal rescue), asymmetric cortical localization of aPKC is detected in most neuroblasts at metaphase. However, aPKC crescents and metaphase plates are often misoriented with respect to the surface of the embryo, a phenotype that has also been observed at low penetrance in embryos lacking only zygotic expression of Baz. In embryos lacking both maternal and zygotic expression of Baz (baz null), aPKC is completely delocalized in neuroblasts and epithelial tissues. These results indicate that Baz is absolutely required for apical localization of aPKC in neuroblasts and epithelial tissues, while Insc is required for localization of aPKC only in neuroblasts. Baz levels are strongly reduced in neuroblasts of insc mutant embryos, most likely because Insc is required for stabilization of Baz. Thus, the effect of Insc on DaPKC localization is probably indirect and can be explained by the loss of Baz in insc mutant neuroblasts (Wodarz, 2000).

In the case of aPKC and Baz, the situation is more complicated. Consistent with a function as a scaffold, Baz is required for localization of the signaling protein aPKC. However, Baz itself is not properly localized in the absence of aPKC. It is easy to imagine how a structural multi-PDZ domain protein like InaD or Baz can localize a protein kinase, but how can aPKC be responsible for localization and stabilization of Baz? Baz possesses a PKC consensus phosphorylation site that is conserved between Baz, Par-3, and ASIP. Phosphorylation of this site by aPKC could be important to regulate binding of Baz to other proteins or to protect Baz from proteolytic degradation. It is also possible that aPKC binds simultaneously to Baz and another protein that may be required for localization of Baz. A detailed structure-function analysis of both Baz and aPKC will be necessary to clarify this issue (Wodarz, 2000).

Par-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila

To test whether there is a physical interaction between Bazooka and par-6, Drosophila embryo extracts were incubated with beads containing maltose-binding protein (MBP) or an MBP-Bazooka fusion protein. A significant amount of Par-6 protein can be detected by immunoblotting in the proteins bound to MBP-Bazooka, but not in the control. Whether this was due to direct binding of the two proteins was tested by incubating MBP and MBP-Bazooka beads with in vitro translated Par-6 protein. Par-6 binds to MBP-Bazooka, but not to MBP alone, and this interaction is not significantly altered in the presence of in vitro translated Inscuteable protein. These results suggest that Par-6 can directly bind to Bazooka. Despite this in vitro interaction, the two proteins could not be co-immunoprecipitated in vivo using anti-Bazooka or any of the different Par-6 antibodies generated. Thus, interaction between the two proteins maybe weak and not stable under the conditions needed to solubilize Bazooka. In vitro translated Par-6 protein does not bind to MBP-Inscuteable, which, together with the colocalization data and the binding of Bazooka to both Inscuteable and Par-6, suggests that binding of Par-6 to Inscuteable is indirect and occurs through Bazooka (Petronczki, 2001).

Par-6 is apically localized in asymmetrically dividing neuroblasts. To test whether the protein is required for asymmetric cell division, the distribution of Bazooka and Inscuteable were analyzed in neuroblasts of Par-6GLC embryos (embryos deficient for both maternal and zygotic par-6). Seventy-three per cent of the Par-6GLC mutant neuroblasts revealed homogeneous cytoplasmic distribution of Bazooka. In 27% of the mutant neuroblasts, Bazooka still shows some weak apical localization, but the strong apical crescents that are observed in 97% of the control neuroblasts were never seen. Whereas Inscuteable localizes asymmetrically at the apical cortex in 94% of the control neuroblasts, only 23% of the Par-6GLC mutant neuroblasts show clear Inscuteable crescents. In 44% of the mutant neuroblasts, the protein is partially delocalized, and in 32% Inscuteable is cytoplasmic. Thus, Par-6 is required for correct localization of both Inscuteable and Bazooka, even though the effect on Bazooka localization is stronger. Both Bazooka and Inscuteable are required for spindle orientation and asymmetric localization of Numb and Miranda (Petronczki, 2001).

Whether Par-6 is required in these processes was examined by staining Par-6GLC embryos for DNA and Miranda or Numb. Metaphase plates are frequently misoriented indicating a defect in spindle orientation. Statistical analysis showed that 25% of the neuroblast metaphase plates were misoriented by more than 60° relative to the horizontal plane, and 37% of the metaphase plates were misorientated between 30° and 60°. Although in control embryos Miranda localizes into a basal cortical crescent in 100% of all metaphase neuroblasts, no signs of asymmetric localization were detected in 80% of metaphase neuroblasts from Par-6GLC embryos. In 20% of Par-6 mutant metaphase neuroblasts, Miranda was excluded from the apical-most quarter of the neuroblast cortex, but a basal cortical crescent was never detected in these mutants. During anaphase and telophase, Miranda maintained its basal localization and segregated into the basal daughter cell in 100% of the control neuroblasts. In Par-6 mutant anaphase neuroblasts, Miranda concentrated at the cleavage furrow (77% or was actually indistinguishable from wild type (23%), indicating that there is a second, Par-6-independent mechanism involved in Miranda localization during late mitosis. Similar observations were made for Numb. Thus, Par-6 is required in neuroblasts for spindle orientation, for apical localization of Bazooka and Inscuteable, and for basal localization of Numb and Miranda during mitosis (Petronczki, 2001).

Partner of Inscuteable/Discs-large complex acts to localize Bazooka posteriorly during establishment planar polarity during asymmetric cell division

In the dorsal thorax (notum) of the Drosophila pupa, the pI cell divides unequally with its spindle axis aligned with the anterior-posterior (a-p) axis of the fly body. It produces two different daughter cells, pIIa and pIIb. During this division, Numb and its adaptor protein Partner of numb (Pon) form an anterior crescent and segregate unequally into the anterior pIIb cell. In fz mutant pupae, the division of the pI cell is oriented randomly relative to the a-p axis and the Numb crescent does form, but at a random position. Thus, Fz is not required to establish planar asymmetry per se, but is necessary to orient the axis of the asymmetric cell division. This indicates that additional genes may be required for establishing, rather than orienting, planar asymmetry in the pI cell (Bellaïche, 2001 and references therein).

Fz organizes the actin cytoskeleton at the site of hair formation. Planar polarity in the pI cell is established by a mechanism that involves a remodeling of the previously established apical-basal polarity. During the pI cell division, Baz and DaPKC relocalize from the apical cortex to the posterior lateral cortex, while Dlg and Pins accumulate asymmetrically at the anterior lateral cortex. This redistribution along the a-p axis leads to the formation of two complementary planar domains at the cell cortex. This mechanism of polarity establishment is distinct from the one described in Drosophila neuroblasts. In these cells, Pins is recruited via Insc by Baz to the apical cortex, and acts in a Dlg-independent manner to maintain the Baz/DmPAR-6/DaPKC/Insc complex at the apical cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell (Bellaïche, 2001).

In the dividing pI cell, Numb and Pon colocalize at the anterior pole of the lateral cortex, marked with Fasciclin3 (Fas3), below the adherins junction (AJ), marked with DE-Cadherin (Shotgun). In epithelial cells in interphase, Baz colocalizes with Shotgun at the AJ around the apical cortex. In the pI cell, Baz accumulates at the posterior cortex during mitosis. Prior to chromosome condensation, this accumulation is seen at the level of the AJ. Then, during prophase and metaphase, Baz forms a posterior crescent below AJ and opposite to Numb. At telophase, the pIIa cell inherits a higher level of Baz than its sister cell. DaPKC shows a similar distribution to Baz in the pI cell (Bellaïche, 2001).

In neuroblasts, a key function of the Baz/DaPKC/DmPAR-6 complex is to recruit the Insc and the Pins proteins. However, in the pI cell, Insc is not expressed and Pins does not colocalize with Baz at the posterior cortex. Rather, it localizes to the anterior pole in early prophase and colocalizes with Numb at the anterior lateral cortex at metaphase (Bellaïche, 2001).

Because DaPKC and Baz have a dual function in epithelial polarity and asymmetric neuroblast division, it was hypothesized that genes required for epithelial polarity might also regulate planar polarity in the pI cell. To test this hypothesis, the planar distribution of various proteins known to be distributed asymmetrically along the apical-basal axis of epithelial cells was examined. Of these, only Dlg was identified as a protein localizing asymmetrically along the planar axis in the pI cell. Dlg overlaps with Fas3 below the AJ in interphase cells. In dividing pI cells, Dlg redistributes in part along the planar a-p axis. From late prophase onward, Dlg becomes enriched at the anterior cortex, where it colocalizes with Numb and Pins. During this time, Dlg does remain detectable at the posterior lateral cortex. At telophase, a higher level of Dlg segregates into the pIIb cell. Thus, the accumulation of Dlg/Pins and Baz at opposite poles of the cell defines two complementary cortical domains oriented along the a-p planar axis of the pI cell. The position of the mitotic spindle at metaphase correlates with the localization of these two cortical domains. The posterior spindle pole is positioned near the accumulation of Baz, and the anterior spindle pole lies near the accumulation of Dlg. In both pI and epidermal cell, the mitotic spindle poles are found below the AJ, which appear to remain functional since they retain their ability to recruit Arm (Bellaïche, 2001).

To determine the possible function of Baz in the planar polarization of the pI cell, clones of baz mutant cells were studied in the notum. Loss of baz activity does not affect the localization of Shotgun and Dlg, indicating that apical-basal polarity in the notal epithelium is maintained in the absence of Baz. In the dividing pI cell, Numb either does not localize asymmetrically or forms a weak crescent at the anterior cortex at prometaphase. In contrast, Pins localizes asymmetrically at the cortex of the pI cell during division. Moreover, baz mutant pI cells divide within the plane of the epithelium with a normal a-p orientation with Pins localizing at the anterior cortex. This shows that baz is required for the asymmetric localization of Numb but is not essential to establish asymmetry nor to orient polarity along the a-p axis (Bellaïche, 2001).

The role of Pins and Dlg in localizing Baz asymmetrically was examined. In pins mutant pI cells, Baz accumulates at the posterior cortex at metaphase, but the asymmetry is less pronounced than in wild-type cells. This raises the possibility that Pins participates in the asymmetric localization of Baz. In dlg1P20 mutant pupae, Baz is correctly localized to the apical posterior cortex prior to chromosome condensation, but does not form a cortical crescent below the AJ during late prophase and prometaphase. Instead, Baz accumulates in the cytoplasm and remains cortical only at the level of the AJ. Thus, the initial posterior localization of Baz at the level of the AJ does not depend on the activity of the GUK domain of Dlg, but its cortical localization below the AJ does require dlg activity. It is concluded that planar polarization of the pI cell cannot be maintained without Dlg activity (Bellaïche, 2001).

To test whether the initial Dlg-independent localization of Baz at the posterior cortex depends on Fz signaling, the distribution of Baz was studied in fz mutant pupae. In wild-type pupae, a clear accumulation of Baz is seen at the level of the AJ in 61% of the interphase pI cells. By contrast, an asymmetric distribution of Baz at the apical cortex is detected in only 19% of the interphase pI cells in fz mutant pupae. In the remaining 81% of the cells, the asymmetric accumulation of Baz is either weak or similar to that seen in the surrounding epithelial cells. This indicates that Fz signaling regulates the initiation of the asymmetric localization of Baz at the posterior cortex. At metaphase, however, Baz and Pins form misoriented crescents relative to the a-p axis that localize at opposite poles in fz mutant pI cells. It is concluded that the formation of the two opposite Baz and Pins domains does not depend on fz activity, and that planar asymmetry can be established in the absence of Fz signaling. However, as previously seen for pins, the asymmetric distribution of Baz is less pronounced in fz mutant pI cells than in wild-type cells. Moreover, Dlg is distributed around the entire cell cortex, indicating that Fz signaling is required for the anterior accumulation of Dlg (Bellaïche, 2001).

Since Pins localizes asymmetrically in a Fz-independent manner, it was asked whether Pins is necessary to localize Baz at one pole of the pI cell in the absence of Fz. It was found that Baz localizes uniformly around the basal-lateral cortex in 82% of the fz;pins double mutant pI cells at metaphase. Moreover, although Numb forms a crescent at anaphase in pI cells mutant for pins or fz, no Numb crescent is seen at either metaphase or anaphase in fz;pins double mutant pI cells. Consistently, loss of fz activity enhances the pins bristle loss phenotype. These data show that Pins and Fz act in a redundant manner to exclude Baz from the anterior cortex and to establish planar asymmetry in the pI cell (Bellaïche, 2001).

These results show that Pins localizes to the anterior cortex in a Baz-independent manner, in an orientation opposite that of Baz, as does Numb. Pins cooperates with Fz to exclude Baz from the anterior cortex of the pI cell. In contrast, in neuroblasts, Pins localizes in a Baz-dependent manner to the apical pole, opposite Numb, and stabilizes the Insc/Baz/DmPAR-6/DaPKC complex. Nevertheless, Pins promotes the localization of Numb in both cell types (Bellaïche, 2001).

One important difference between pI cells and neuroblasts is the lack of insc expression in pI cells. To test the functional significance of this lack of Insc, Insc was expressed in the pI cell. Under these circumstances, Insc and Pins localize at the anterior cortex. Insc triggers the anterior relocalization of Baz, while Numb forms a posterior crescent at anaphase. The pI cell division remains planar. This contrasts with the effect of Insc in epithelial cells. In these cells, Insc localizes apically and orients the spindle along the apical-basal axis. This further indicates that the apical-basal polarity is remodeled in the pI cell. It is concluded that the ectopic expression of Insc is sufficient to reverse the planar polarity axis of the pI cell and to modify the activity of Pins relative to Baz. In the absence of Insc, the Dlg/Pins complex excludes Baz, while expression of Insc leads to the formation of a Pins/Insc/Baz complex. In both cases, Numb localization is opposite that of Baz (Bellaïche, 2001).

A conserved oligomerization domain in Drosophila Bazooka/PAR-3

The PAR-3/PAR-6/aPKC complex is required to establish polarity in many different cell types, including the C. elegans zygote and epithelial and neuronal cells in Drosophila and mammals. In each context, the components of this complex display a mutually dependent asymmetric cortical localization. PAR-6 is a direct effector of Rho family GTPases and binds to and regulates aPKC. Mammalian PAR-3 (mPar3) can associate with transmembrane proteins and may link the complex to the membrane, but this can account for only part of the requirement for this protein in the complex. The function of a novel conserved domain, CR1, of PAR-3 has been investigated using computational, biochemical, and genetic approaches. Sequence-structure comparison by FUGUE predicts that CR1 has the same structural fold as a bacterial oligomerization domain. CR1 of the Drosophila homolog, Bazooka (BAZ), mediates oligomerization in vitro and in vivo. Furthermore, deletion of CR1 disrupts BAZ localization in both epithelial cells and the germline and strongly impairs BAZ function in epithelial polarity. These results indicate that this domain is important for the localization and activity of the PAR-3/PAR6/aPKC complex and define a new role for PAR-3 in assembling higher order protein complexes (Benton, 2003).

CR1 is not homologous to any known protein domain, but Position-Specific Iterated BLAST searches reveal significant similarity to an N-terminal region of unknown function in mammalian histidine ammonia lyases (HAL). Since this primary structural analysis was uninformative, the identification of higher order structural homologs of CR1 was undertaken using the FUGUE program. FUGUE aligns a given query sequence with a database of proteins whose structures have been determined to calculate a statistical score (Z score) of the likelihood of the query adopting a particular known fold, using environment-specific amino acid substitution tables and structure-dependent gap penalties. The former are derived from the analysis of the occurrence of each amino acid in each type of structural environment, and the latter take into account the possible variability in the lengths of surface turns and loops in otherwise structurally homologous proteins. The sensitivity and accuracy of the search is enhanced by generating a consensus query sequence from a multiple alignment of all primary sequence homologs (in this case, all PAR-3 and HAL proteins). With BAZ CR1 (amino acids 1-83) as a query, FUGUE identifies one 'likely' match: E. coli DinI. DinI is similar in length to CR1 and forms a ßalphaßßalpha structure, but does not show significant primary sequence homology to CR1. The similarity in the predicted structure of CR1 to DinI is therefore unlikely to reflect a common evolutionary origin of these proteins, but may reflect a shared biochemical property. Notably, both DinI and a homologous protein in coliphage 186, Tum, have been reported to form dimers or tetramers in vitro. Furthermore, two enzymes (cytokine D-dopachrome tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase) that have, like CR1, a similar fold but no obvious sequence homology to DinI can also form multimers. These observations suggest that the protein fold adopted by DinI may represent a common oligomerization domain (Benton, 2003).

CR1 mediates oligomerization. Using the yeast two-hybrid system, it was found that both BAZ and mPar3 CR1 can indeed self-associate. This interaction is direct, since a purified MBP-fusion protein of the BAZ N terminus precipitates full-length BAZ translated in vitro. To determine whether BAZ self-associates in vivo, a full-length BAZ:GFP fusion protein was immunoprecipitated from embryonic extracts using an anti-GFP antibody and a Western blot of the immunoprecipitate was probed with an anti-BAZ antibody. Endogenous BAZ was found to efficiently copurify with this fusion protein. The amount of endogenous protein in the immunoprecipitate is in fact about twice that of the tagged protein, suggesting that each molecule of BAZ binds to several other molecules to form higher order complexes. In contrast, a GFP-tagged truncated version of BAZ, lacking the oligomerization domain (BAZ-deltaN:GFP), fails to coimmunoprecipitate endogenous BAZ, indicating that this domain is essential for BAZ oligomerization in vivo (Benton, 2003).

To assess the importance of this property, the subcellular localization of these GFP-fusion proteins was first analyzed, as well as a tagged version of the BAZ N terminus containing CR1 (BAZ-N:GFP). BAZ:GFP displays an identical distribution to endogenous BAZ in follicular epithelial cells: it colocalizes with aPKC along the apical cortex and concentrates at the zonula adherens marked by Armadillo (ARM; Drosophila ß-catenin). Almost no GFP fluorescence is detected elsewhere in these cells, indicating that BAZ is very efficiently recruited to these sites. In contrast, BAZ-deltaN:GFP is largely diffuse in the cytoplasm of epithelial cells, although a small proportion of the protein is detected at the apical cortex. These observations indicate that the ability of BAZ to oligomerize is important to concentrate it apically. The distribution of BAZ-N:GFP is similar to that of BAZ-deltaN:GFP: most of the protein is cytoplasmic, but a very small fraction localizes to the zonula adherens. Since BAZ-N:GFP contains the oligomerization domain, this localization is likely to be mediated by its association with the endogenous full-length protein (Benton, 2003).

When expressed in the germline, BAZ:GFP is efficiently recruited to the cortex of germ cells. The protein does not localize uniformly along the cortex, however, but forms discrete foci, which are reminiscent of the 'clusters' of PAR-3, PAR-6, and aPKC around the anterior cortex of the C. elegans embryo. BAZ-deltaN:GFP does not concentrate in such foci and, as in follicle cells, is largely diffuse within the cytoplasm. BAZ-N:GFP displays a striking distribution in the germline, in huge spherical cytoplasmic aggregates of up to 10 microm diameter. Such aggregates, albeit smaller, are also observed in follicle cells, and these may reflect the ability of CR1 to self-associate to form oligomeric complexes (Benton, 2003).

To determine the functional importance of BAZ oligomerization, the ability of these truncated proteins to rescue baz mutant phenotypes was assessed. When full-length BAZ:GFP is expressed in baz mutant follicle cell clones, it efficiently rescues epithelial polarity, as assessed by the localization of aPKC, the formation of the zonula adherens (detected by ARM staining), and cell morphology. BAZ-deltaN:GFP displays partial rescuing activity and can be detected, albeit weakly, at the zonula adherens in morphologically normal cells, indicating that its recruitment to this site must depend upon interactions with proteins other than endogenous BAZ. In approximately half of the egg chambers, however, mutant cells display either abnormal polarity and morphology or, more frequently, appear to have been lost from the epithelium (no gaps are ever seen in baz clonal egg chambers that express BAZ:GFP). Thus, the N terminus of BAZ is important, but not absolutely essential, for its function in the follicular epithelium. As expected, BAZ-N:GFP does not significantly rescue baz mutant phenotypes, and almost all egg chambers contain gaps in the epithelium (Benton, 2003).

The functional properties of these proteins was analyzed by scoring their ability to rescue the embryonic lethality of baz zygotic mutants. The full-length protein efficiently rescues embryonic lethality, and the cuticles of the few unhatched embryos are almost wild-type, containing only a very small dorsal hole. In contrast, the truncated proteins are either severely (BAZ-deltaN:GFP) or completely (BAZ-N:GFP) compromised in their ability to rescue lethality. In both cases, the cuticles of the dead embryos are fragmented or contain large dorsal holes, similar to control baz mutant embryos. These results indicate that the oligomerization domain of BAZ is important in vivo (Benton, 2003).

These data demonstrate that oligomerization is critical for the efficient apical localization of BAZ in epithelial cells; this is essential for its function in establishing apical-basal polarity. This property is likely to be conserved, since CR1 of mPar3 also self-associates. Indeed, these results provide an explanation for the inhibitory effect of overexpression of an N-terminal portion of mPar3 on the polarization of cultured hippocampal neurons. This region lacks the PDZ domains and the aPKC binding site, but still contains the CR1 domain, and may therefore exert its dominant-negative effect by associating with and inhibiting the oligomerization of endogenous mPar3. No dominant-negative effects of BAZ-N:GFP were detected, but this could reflect the fact that most of the protein is sequestered in apparently harmless cytoplasmic aggregates. CR1 may therefore have a higher affinity for itself than for the full-length protein, and the ectopic expression levels of this domain achieved in Drosophila cells may be insufficient for it to exert the inhibitory effects observed in the mammalian system (Benton, 2003).

It is unclear how the BAZ/PAR-6/aPKC complex is targeted to the apical membrane in epithelia, but evidence from other organisms indicates that this is likely to be mediated by the association of BAZ with the cytoplasmic tail of an apical transmembrane protein. The observation that the removal of CR1 disrupts its localization suggests that this domain is essential to stabilize the association of the complex with the apical membrane. Since CR1 is not sufficient for significant localization, it seems unlikely that it binds directly to an apical anchor. A more probable explanation for its role is that it increases the avidity of the interaction with the apical membrane by generating oligomeric complexes that contain multiple binding sites for such anchors. The self-association of CR1 may also contribute to the function of the complex by allowing a single membrane-tethered molecule of BAZ to nucleate the formation of higher order BAZ/PAR-6/aPKC complexes along the cortex. This may serve as an amplification mechanism by generating, for example, a high local concentration of aPKC activity in response to an upstream spatial cue. Moreover, the ability of BAZ to form an interconnected protein network could underlie its function in establishing a physical barrier to prevent the lateral movement of membrane or cortical proteins (Benton, 2003).

Two other proteins involved in epithelial polarity, Discs Lost (DLT: now redefined as Drosophila Patj) and Lethal(2) Giant Larvae (LGL), have also been observed to self-associate in vitro and/or in vivo. These proteins are thought to be components of distinct protein complexes that act in conjunction with the BAZ/PAR-6/aPKC complex to establish apical-basal asymmetries in Drosophila epithelia. While the biological significance of oligomerization has not yet been demonstrated for either DLT or LGL, these observations, together with this functional analysis of BAZ, suggest that the ability to form interconnected protein networks or complexes is fundamental to the establishment of cell polarity (Benton, 2003).

Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division: Bazooka-atypical PKC-Par6-Inscuteable, are responsible for asymmetric Miranda localization

Drosophila neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical-basal axis, and unequal-sized daughter cells appear. This study identified a Drosophila mutant in the Ggamma1 subunit of heterotrimeric G protein, which produces Ggamma1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gß13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gß13F to the membrane, indicating an essential role of cortical G1-Gß13F signaling in asymmetric divisions. In Ggamma1 and Gß13F mutant NBs, Pins-Gαi, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka-atypical PKC-Par6-Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gßgamma and other mutants indicates a predominant role of Partner of Inscuteable-Gi in spindle orientation. It is thus suggested that the two apical signaling pathways have overlapping but different roles in asymmetric NB division (Izumi, 2004).

Because the Gß13F-Ggamma1 complex, which distributes uniformly in the cortex, functions in asymmetric organization of the spindle, differential activation or inactivation of Gßgamma signaling must occur in the apical-basal direction. Two apical signaling pathways are implicated in the apical-basal difference in spindle development in a redundant fashion. What is the relationship between the apical signals and the Gßgamma signal? Spindle size is reduced by an increase in the amount of Gßgamma, but a lack of Gßgamma results in formation of a large, symmetric spindle. These findings raise the possibility that spindle development is suppressed by the Gßgamma signal, which is repressed by the presence of an apical complex on the apical side in the wild-type cells, resulting in a large apical and small basal spindle. This model suggests that the apical complex acts upstream of the Gßgamma signal. In contrast, elimination of Gß13F affects the localization of the apical components: Pins becomes uniformly distributed and Galphai becomes undetectable. In addition, Ggamma1N159 and G圩F mutations appear to destabilize the localization of the components in the Baz-DaPKC pathway, as judged by the reduced staining by their antibodies (although this may be an indirect consequence of the mislocalization of Pins-Galphai). The Gßgamma signal is thus required for normal distribution of the components of both apical pathways, consistent with the idea that the apical pathways acts downstream of the Gßgamma signal in regulating spindle asymmetry. Tests for epistasis between the apical pathways and the Gßgamma signal are needed to clarify their relationship in the regulation of spindle organization (Izumi, 2004).

The effects of Ggamma1N159 and G圩F mutations on cell-size asymmetry are remarkable but different from those in double mutants in which both apical pathways are disrupted simultaneously, where daughter cell sizes are completely equal. The cell-size ratio of GMCs to their sibling NBs shows a broad distribution: from 0.6 to 1 in the Ggamma1 (and G圩F) mutants. This residual asymmetry in daughter cell size is due to Baz-DaPKC activity. The components of this pathway indeed distribute asymmetrically in Ggamma1 (and G圩F) mutant NBs in which Pins-Galphai activity is no longer asymmetric (Pins is uniformly distributed and Galphai is absent) (Izumi, 2004).

Why does this polarized Baz-DaPKC activity cause less asymmetry in daughter cell size in spite of the redundant function of the Baz-DaPKC pathway and Pins-Galphai? Antibody staining for Baz, DaPKC, and DmPar-6 suggests that their levels and their polarized distribution are weakened in Ggamma1 (and G圩F) mutants. A possible explanation is that low levels of polarized Baz-DaPKC activity confer only low levels of asymmetry to the daughter cell size in the absence of polarized Pins-Galphai. Thus, the degree of cell-size asymmetry resulting from NB divisions may depend on the dosage of the components of one apical pathway when the other is absent or uniformly distributed. In contrast, Miranda localization does not appear to be severely impaired in Ggamma1N159 and G圩F mutants until late embryonic stages, indicating that the polarized Baz-DaPKC activity in these mutants is sufficient to localize Miranda. Therefore, full asymmetry in daughter cell size may require relatively higher levels of Baz-DaPKC activity than does polarized distribution of cell-fate determinants does (Izumi, 2004).

In Ggamma1N159 and G圩F mutants, Insc has a distribution different from the other components of the Baz-DaPKC pathway. In most of these mutant NBs, Insc distributes broadly to both the cytoplasm and the cortex in a slightly asymmetric way, but Baz, DaPKC, and DmPar-6 localize asymmetrically in the cortex. The cytoplasmic distribution of Insc is also slightly asymmetric in pins mutant NBs. It is not known whether cytoplasmic Insc is functional. Interestingly, Insc distribution often appears to correlate better with the asymmetry in daughter cell size than do the other components of the Baz-DaPKC pathway in Ggamma1N159 and G圩F mutants: in most telophase NBs that are cleaving into two equal daughters, DaPKC and DmPar-6 are excluded from the daughter GMC, but Insc tends to distribute evenly to both daughter cells. This occurs also in pins mutants, in which ~15% of NBs divide equally but most NBs divide unequally. In pins NBs cleaving equally, Insc is found equally in the cytoplasm of both daughter cells, but DaPKC and DmPar-6 remain in the newly forming NB; in unequally dividing NBs, all three components are found preferably on the NB side. These observations raise the intriguing possibility that Insc has more important roles in the generation of spindle asymmetry than do the other components of the Baz-DaPKC pathway. Because the absence of Baz results in mislocalization of Insc and vice versa, it is technically difficult to discriminate Insc-specific from Baz-specific functions. It may be Insc or some unknown Insc-associating effectors, rather than Baz, that functions in parallel with Pins-Galphai in the establishment of cell-size asymmetry (Izumi, 2004).

The question of whether the two apical pathways have redundant functions in aspects of NB division other than cell-size asymmetry has been elusive. In this paper, examination of Ggamma1N159 and G圩F mutant NBs, as well as those overexpressing baz, suggests that the asymmetric localization of Miranda depends solely on polarized Baz activity and not on Pins-Galphai function. Miranda always distributes on the cortical side, opposite the distribution of Baz in these mutants and in the wild-type. This also occurs for sensory precursor cells in the peripheral nervous system: in sensory precursor cell division Insc is not expressed, and Pins and Baz distribute on cortical sides opposite to each other, unlike in NBs; however, both Miranda and Numb localize to the cortex opposite Baz, as seen in NBs (Izumi, 2004).

Phosphorylation of the Lethal (2) giant larvae protein by DaPKC directs the localization of cell-fate determinants to the basal cell cortex. When baz is overexpressed in NBs, ectopically distributed Baz excludes Miranda from the Baz region and DaPKC colocalizes with the ectopic Baz. In contrast, a decrease in Baz activity in the wild-type results in cytoplasmic localization of DaPKC and uniform cortical distribution of Miranda. All these findings suggest that the Baz-directed localization of DaPKC excludes Miranda from the apical cortex via Lethal (2) giant larvae phosphorylation. In the absence of Baz, Miranda is eventually concentrated to the budding GMC during telophase by unknown mechanisms, a phenomenon called 'telophase rescue'. This phenomenon did not occur by depleting both baz activity and Gßgamma signaling, suggesting that telophase rescue involves Gßgamma signaling or asymmetric Pins-Galphai localization (Izumi, 2004).

The absence of any single component of the apical complex has the same effect on spindle orientation during NB division, which is normally perpendicular to the apical-basal axis. Thus, proper orientation of the spindle has been thought to require all the apical components. However, observations on epithelial cells and mitotic domain 9 cells indicated that the spindle always points to the location of Pins when Pins is localized in the cell. This alignment of the spindle toward Pins occurs irrespective of the localization of the Baz-pathway components. For instance, wild-type epithelial cells divide parallel (Pins direction) but not perpendicular to the apical-basal axis (Baz direction); so do most epithelial cells and mitotic domain 9 cells in G圩F and Ggamma1 mutants. Therefore, the Pins-Galphai pathway, rather than the Baz-DaPKC complex, is likely to play a dominant role in controlling spindle orientation (Izumi, 2004).

In most NBs in pins, G圩F, and Ggamma1 mutants, the spindle is oriented in the direction of Baz localization and therefore follows the localization of the cell-fate determinants. This coincidence results in the determinants' virtually normal segregation to one daughter cell despite the random orientation of division. Thus, only when Pins-Galphai are absent or uniformly distributed in NBs, polar Baz activity appears to be capable of directing spindle orientation. Alternatively, the mitotic spindle may position the Baz-DaPKC complex over one spindle pole (Izumi, 2004).

In the NB in which the Baz-DaPKC pathway is depleted, Pins-Galphai can still localize asymmetrically and orient the spindle. Interestingly, the Pins crescent forms in random orientations in this situation, leading to random spindle orientation. This fact suggests that the Baz-DaPKC complex or its combination with Pins-Galphai is necessary to orient the Pins-Galphai crescent in the apical direction of the NB, raising an intriguing possibility that there are unknown mechanisms by which formation of the apical complex occurs on the apical side. This postulated mechanism may involve interactions with neighboring epithelial cells (Izumi, 2004).

What is the molecular mechanism by which Pins-Galphai orient the spindle? It is interesting to assume that Pins has the ability to attract the spindle pole. This idea is consistent with previous evidence; although epithelial cells do not normally express Insc, its ectopic expression in these cells recruits Pins-Galphai to the apical cortex and reorients the mitotic spindle in the apical-basal direction. The C. elegans homologues of Pins, GPR-1/GPR-2, interact with Galphai/Galphao and a coiled-coil protein, LIN-5, which is required for GPR-1/GPR-2 localization. All these molecules are indeed involved in the regulation of forces attracting spindles during early cleavages. Although Lin-5 has no obvious homologue in other species, functional homologues may regulate Pins localization and/or the connection between the spindle pole and Pins in Drosophila. Furthermore, the C. elegans gene ric-8 (see Drosophila Ric-8), which interacts genetically with a Galphao gene, is also required for embryonic spindle positioning. Its homologue in mammals acts as a guanine nucleotide exchange factor for Galphao, Galphaq, and Galphai. An analysis of the Drosophila RIC-8 homologue may give insight into the mechanisms by which Pins-Galphai regulate spindle orientation (Izumi, 2004).

Echinoid is a component of adherens junctions that cooperates with DE-Cadherin to mediate cell adhesion

Echinoid is an immunoglobulin domain-containing transmembrane protein that modulates cell-cell signaling by Notch and the EGF receptors. In the Drosophila wing disc epithelium, Echinoid is a component of adherens junctions that cooperates with DE-Cadherin in cell adhesion. Echinoid and β-catenin (a DE-Cadherin interacting protein) each possess a C-terminal PDZ domain binding motif that binds to Bazooka/PAR-3; these motifs redundantly position Bazooka to adherens junctions. Echinoid also links to actin filaments by binding to Canoe/AF-6/afadin. Moreover, interfaces between Echinoid- and Echinoid+ cells, like those between DE-Cadherin- and DE-Cadherin+ cells, are deficient in adherens junctions and form actin cables. These characteristics probably facilitate the strong sorting behavior of cells that lack either of these cell-adhesion molecules. Finally, cells lacking either Echinoid or DE-Cadherin accumulate a high density of the reciprocal protein, further suggesting that Echinoid and DE-Cadherin play similar and complementary roles in cell adhesion (Wei, 2005).

Several observations prompted the study of Ed as a canonical CAM in the monolayered wing imaginal disc. Thus, mitotic recombination clones of cells mutant for the null allele ed1x5 exhibit rounded and smooth contours, in contrast to clones of wild-type cells that show wiggly shapes. This indicated that ed- /- cells have distinct adhesive properties and assort with themselves rather than with the surrounding ed+/- M+/- cells. (ed1x5 clones were M+, since without a growth advantage they hardly survive). It was also observed that Ed was absent from the membrane of the heterozygous cells that contacted the mutant cells, a finding consistent with the observation that Ed forms homophilic interactions and that these are required to incorporate/stabilize Ed at the cell membrane. Finally, Ed was found to localize basally to the apical marker Crb and apically to the basolateral marker Dlg. In fact, Ed colocalizes with both DE-Cad and Arm, and, therefore, it might be part of AJs. AJs are structures important for cell-cell contact and recognition. So, these results suggested that Ed plays a role in cell-cell adhesion (Wei, 2005).

Whether Ed affects components of AJs was examined by analyzing the localization of Arm within ed mutant clones. Arm strongly accumulates at the apical membranes of ed- /- cells, and these cells have a reduced apical surface. Both effects are clear in small clones, but cells within larger clones (over hundreds of cells) had both the density of Arm and the apical surface more similar to those of the wild-type cells. Similar observations were made with DE-Cad and Actin. It is suggested that the increased concentration of these molecules in small clones most probably results from the apical constriction as supported by the accumulation of nonmuscle myosin II, without a net per cell increment of these proteins. Alternatively, it could result from increased stability of these proteins. The apical constriction continued through the SJs and ended at the planes just below the GJs as revealed by an Innexin antibody. Hence, these ed- /- cells adopt a bottle shape. In contrast, the apposed ed- /- and ed+/- cells that form the border of the clone enlarge and adopte a rectangular shape. At this interface, the ed- /- cells often contacted the heterozygous cells by their long sides, as if in an attempt to minimize the number of cells that formed the interface (Wei, 2005).

Interestingly, Arm and DE-Cad, but not Actin, are depleted at the interface membrane of both small and large clones. This suggests that ed- /- and ed heterozygous cells discriminate one another and that AJs do not form properly in between them (Wei, 2005).

ed clones are surrounded by an Actin 'cable'. High-magnification images suggest that the cable is contained within the ed heterozygous cells surrounding the clone and that it is therefore generated by these cells. Several observations suggest that this Actin cable exerts a force. The cells surrounding an ed clone elongate toward the clone and accumulate nonmuscle myosin II at the interface membrane, as if attempting to cover the space exposed by the apically constricted ed- /- cells. This effect is reminiscent of the stretching of the leading-edge cells that will cover the underlying amnioserosa during dorsal closure of the embryos. In the wing disc, the boundary that separates the dorsal (D) and ventral (V) regions of the wing pouch has the shape of a smooth arc and contains an actin 'fence'. After the second instar, this boundary corresponds to a compartment border that imposes absolute restrictions to cell lineages. Large ed- /- clones close to or touching this boundary displace it toward the clones. In contrast, ed clones that straddle the boundary do not overtly distorted it, although the boundary could be less smooth within the clone. (Straddling clones might be originated before the compartment border was established or might be formed of D and V clones that fuse together). Moreover, the Actin cable surrounding the clones fuse with the Actin fence at the D/V boundary, suggesting that the distortion of this boundary is effected through this Actin linkage. Control ed+ M+ clones do not induce such distortions. These observations suggest that the Actin cable may contribute to the roundish shape of the ed clones and help confine their cells (Wei, 2005).

DE-Cad is a classical homophilic cell adhesion molecule of AJs. It interacts with β-catenin/Arm, which in turn binds α-catenin. Through the association between α-catenin and F-Actin, DE-Cad establishes links between cells that connect to the Actin cytoskeleton. This study shows that Ed is another CAM that, at the resolution of confocal microscopy, is also located at the AJs of imaginal disc cells. While cells in clones mutant for ed still seem to form normal AJs, the cells at the border of the clone seem impaired in forming them. It is hypothesized that this may help them segregate from surrounding ed+/- cells. Ed was identified as a binding partner for PDZ proteins that, similarly to Arm, helps localize Baz to AJs. Moreover, it was found that through the binding of Cno, Ed, like DE-Cad/β-catenin, may link to F-Actin. Hence, Ed has functions in cell-cell adhesion similar to those of DE-Cad (Wei, 2005).

The differential adhesion hypothesis proposes that cell sorting may be driven by differences in the quantity and/or quality of adhesive molecules displayed on the surface of cells. In keeping with this hypothesis, it was found that ed- /- cells sort out from ed+/- cells, as shown by the remarkably round shapes and smooth contours of the ed clones. Moreover, their differential adhesiveness is also manifest by the fusion of different ed clones to yield composite but still roundish clones. It is suggested that contraction of the apically enriched Actin network and of the actin cable surrounding the clone, possibly by interaction with nonmuscle myosin II also present there, may contribute to the the apical constriction of the ed- /- cells. It was also observed that the interface between ed+/- and ed- /- cells is depleted of DE-Cad, Arm and Baz, besides completely lacking Ed. This strongly suggests that this interface is deficient in AJs and probably helps to insulate ed- /- cells from the surrounding ed heterozygous cells. It is hypothesized that this deficiency of AJs, which may reduce adhesion between ed+/- and ed- /- cells, and the inward-pulling force generated by apical constriction and the actin cable may help create the smooth and rounded contour of the clones at the level of AJs. At the plane of SJs, the clonal boundary is not as smooth. This may be due to the presence of normal levels of SJs, since seemingly wild-type amounts of Dlg were detected at the interface membrane. Normal levels of SJs may allow the clones to remain integrated in the epithelium. It is stressed that when ed clones grow large, the apical constriction disappears, suggesting that the forces responsible for this constriction become insufficient or no longer operate. If the force is exerted, at least in part, by the Actin cable surrounding the clone, as in a purse-string mechanism, it would make sense that this force becomes ineffectual as the number of cells within the clone increases. Remarkably, these differences of apical cell constriction observed in small and large ed clones have a correlate on the adult wing blade: small clones display an increased density of trichomes, implying that their cells are small or more tightly packed, whereas large clones have cells of normal size. This indicates that the apical constriction is retained through imaginal disc eversion, when the disc epithelium changes from columnar to planar (Wei, 2005).

In the embryonic epithelium, Baz, localized to both AJs and the marginal zone, is the initial apical regulator. How is Baz recruited to the apical domain? In the follicular epithelium, Baz is localized to this domain through lateral exclusion mediated by PAR-1/14-3-3 and apical anchoring by Crb/Sdt/Patj. The data support an additional mechanism to localize Baz to the apical domain. Both Ed and Arm can bind Baz through their C-terminal PDZ binding motif and therefore they may redundantly localize Baz to AJs. Indeed, the localization of Baz to AJs is relatively normal in the absence of either one. Most Baz is lost only when both Arm and Ed are depleted, as occurs at the interface membrane of ed clones or in large shg clones where Ed gradually breaks down. In the latter case, there is good colocalization between Baz and the sites maintaining residual Ed. It is suggested that in the epithelium of the wing disc, Baz localizes to AJs by the combined effects of its binding to Ed/Arm and the lateral exclusion of PAR-1/14-3-3. Additionally, apical anchoring of Baz may be mediated by direct association between the Baz and Crb apical complexes. During early embyogenesis, Ed is also present at pseudocleavage furrows. This observation, together with the ability of Ed to localize Baz to AJs, may explain the finding that during cellularization, Baz can accumulate apically in the absence of Arm. Ed also binds to the PDZ domain of Cno and mediates its localization to AJs, where Cno interacts with F-Actin either directly or indirectly through the association with Polychaetoid/ZO-1. Interestingly, the evolutionally conserved EIIV domain of Ed binds Baz and Cno in a mutually exclusive manner. Thus, the concentrations of and differential affinities between Ed, Baz, and Cno should determine their dynamic equilibrium at AJs (Wei, 2005).

Although Baz is critical to form AJs in the blastoderm and in the follicular epithelium, removal of Baz (or Par-6) from cells of the wing disc does not affect the localization of DE-Cad or Ed to AJs. This is consistent with the report that in imaginal discs, Baz does not affect the localization of DE-Cad and Dlg but is required for the asymmetric localization of cell fate determinants. Together, these results suggest that in wing discs, the Baz complex is not critical for the formation of AJs, and that the effect of the loss of Ed on AJs formation/maintenance is not due to Baz depletion (Wei, 2005).

Several similarities between the roles of DE-Cad and Ed in the wing disc epithelium are worth noting. Both Ed and DE-Cad are CAMs that establish homophilic interactions and localize to AJs. The absence of either Ed or of DE-Cad in cells of small clones causes their apical constriction and strong segregation from wild-type cells, giving rise to smooth round borders. In both cases, the mutant cells are impaired in forming AJs with neighboring wild-type or heterozygous cells and are surrounded by an Actin cable. Ed interacts with Cno, and DE-Cad with Arm, and both Cno and Arm directly or indirectly associate with F-Actin. Thus, Ed and DE-Cad represent two distinct classes of CAMs, with widely different chemical compositions, that connect to F-Actin, contribute to cell adhesion in the wing disc, and seem to have partially overlapping functions (Wei, 2005).

In contrast, DE-Cad and Ed differ in their ability to regulate the apical/basal cell polarity. Ed affects components of AJs, but not those of the apical Crb and the basolateral Dlg complexes. In contrast, DE-Cadherin is necessary for Crb localization, but similarly to Ed, it is not required for Dlg localization. Furthermore, the maintenance of Ed at AJs requires DE-Cad. In contrast, localization of DE-Cad to AJs is independent of Ed. Interestingly, the DE-Cad/Arm complex is not essential for the formation of the follicular epithelium, but upon removal of this complex, the integrity of the epithelium is lost slowly over the period of several days. This suggests that other molecules may be maintaining the epithelial structure. During stages 1 to 10 of oogenesis Ed is mainly expressed in the follicle cells, and these cells, if mutant for ed, show at low frequency a multilayered structure with disrupted expression of some polarity markers. Thus, it will be of interest to elucidate whether, in this epithelium, Ed and DE-Cad/Arm also play partially redundant roles in cell adhesion and apical/basal polarity. While both Ed and DE-Cad contribute to cell adhesion and recognition, it is unclear whether each molecule imparts specific recognition properties to cells, so that the final cell-cell affinity results from the sum of distinct affinities mediated by these different CAMs. More specifically, can an increased level (density) of DE-Cad replace the absence of Ed? The results showing that ed- /- cells, with either normal levels (in large clones) or high density (in small clones) of DE-Cad, do not intermix with wild-type cells suggests that the binding specificity provided by a given CAM is not overruled by a higher level (density) of a different CAM. Moreover, the cell sorting properties conferred by Ed cannot account for the separation of cells at both sides of the A/P compartment boundary of the wing disc because A and P cells do not intermingle within composite ed, smo double mutant clones. (Similarly, DE-Cad is not responsible for the sorting out of A and P cells. Hence, cell-cell adhesion in the wing disc appears to depend on multiple CAMs (Ed, DE-Cad, etc.), each imparting specific cell recognition properties. Although Ed and its C-terminal EIIV motif are conserved in invertebrates, no clear vertebrate homolog with 7 Ig domains and a PDZ domain binding motif has been found. Nectin1-4 comprises a family of 3 Ig domain-containing CAM that have several differentially spliced forms and localize to AJs. Most spliced forms share a conserved C-terminal E/A-X-Y-V that binds the PDZ domain of Afadin. Moreover, this motif also interacts with Par-3, the vertebrate homolog of Baz. In spite of these similarities, overexpression of either nectin 1-α or 3-α does not rescue the remarkable clonal phenotype of ed (Wei, 2005).

Direct association of Bazooka/PAR-3 with the lipid phosphatase PTEN reveals a link between the PAR/aPKC complex and phosphoinositide signaling

Cell polarity in Drosophila epithelia, oocytes and neuroblasts is controlled by the evolutionarily conserved PAR/aPKC complex, which consists of the serine-threonine protein kinase aPKC and the PDZ-domain proteins Bazooka (Baz) and PAR-6. The PAR/aPKC complex is required for the separation of apical and basolateral plasma membrane domains, for the asymmetric localization of cell fate determinants and for the proper orientation of the mitotic spindle. How the complex exerts these different functions is not known. The lipid phosphatase PTEN directly binds to Baz in vitro and in vivo, and colocalizes with Baz in the apical cortex of epithelia and neuroblasts. PTEN is an important regulator of phosphoinositide turnover that antagonizes the activity of PI3-kinase. Pten mutant ovaries and embryos lacking maternal and zygotic Pten function display phenotypes consistent with a function for PTEN in the organization of the actin cytoskeleton. In freshly laid eggs, the germ plasm determinants, oskar mRNA and Vasa, are not localized properly to the posterior cytocortex and pole cells do not form. In addition, the actin-dependent posterior movement of nuclei during early cleavage divisions does not occur and the synchrony of nuclear divisions at syncytial blastoderm stages is lost. Pten mutant embryos also show severe defects during cellularization. The data provide evidence for a link between the PAR/aPKC complex, the actin cytoskeleton and PI3-kinase signaling mediated by PTEN (von Stein, 2005).

In order to find molecules that bind to Baz/PAR-3 and that may provide a link between the PAR/aPKC complex and the cortical cytoskeleton, a yeast two-hybrid screen was performed using the three PDZ domains of Baz as bait. Three independent clones of the lipid phosphatase PTEN were isolated that specifically bind to Baz. PTEN catalyzes the dephosphorylation of phosphoinositide lipids at the D3 position of the inositol ring, One substrate of particular importance is the lipid phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P3], which is converted by the activity of PTEN to phosphatidylinositol (4,5) bisphosphate [PtdIns(4,5)P2]. PtdIns(3,4,5)P3 is produced by activation of phosphatidylinositol 3-kinase (PI3-kinase) in response to stimulation by a multitude of growth factors and cytokines. Interestingly, PtdIns(3,4,5)P3 locally activates Cdc42 by recruitment of guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP specifically on Cdc42. Moreover, in mammalian cells PtdIns(3,4,5)P3 recruits phosphoinositide dependent kinase 1 (PDK1; Pk61C – FlyBase) which activates aPKC by direct phosphorylation of a conserved threonine residue in the activation loop of the kinase. Thus, PtdIns(3,4,5)P3 is likely to activate two components of the PAR/aPKC complex, Cdc42 and aPKC. Because PTEN is predicted to antagonize the activation of both Cdc42 and aPKC by lowering the level of PtdIns(3,4,5)P3 in the plasma membrane, the association of PTEN with Baz may have a significant impact on the activity of these two key components of the PAR/aPKC complex (von Stein, 2005 and references therein).

PI3-kinase signaling and PTEN have been implicated in the polarization of Dictyostelium amoebae in response to a source of chemoattractant. PI3-kinase and PTEN are localized to the leading edge and uropod, respectively, in a very dynamic fashion. PI3-kinase signaling also appears to be required for directed migration of leukocytes. In both cases, PI3-kinase and PTEN are thought to participate in a self-sustaining loop that intracellularly amplifies the shallow concentration gradient of the chemoattractant. PI3-kinase and PTEN also affect the polarization of hippocampal neurons in culture and, more specifically, the localization of PAR-3 and aPKC to the tip of the neurite that is going to become the axon. Thus, there is increasing evidence that PTEN and the PAR/aPKC complex may cooperate in the control of cell polarity (von Stein, 2005 and references therein).

In Drosophila, the function of PTEN has mainly been studied with respect to its role in the regulation of growth and proliferation in larval and adult tissues. Pten mutant cells have elevated PtdIns(3,4,5)P3 levels and are larger than wild-type cells owing to increased growth. Clones of Pten mutant cells in imaginal discs also show subtle defects in the organization the actin cytoskeleton. Pten interacts genetically with components of the insulin signaling pathway including the insulin receptor, the insulin receptor substrate IRS-1/Chico, PI3-kinase and protein kinase B (PKB). These findings provided solid evidence for an antagonistic relationship between PTEN and PI3-kinase and show that the regulation of phosphoinositide levels is the main vital function of PTEN (von Stein, 2005).

This study shows that PTEN directly binds to Baz/PAR-3 and colocalizes with Baz in the apical cortex of epithelia and neuroblasts. Pten mutant embryos lacking maternal and zygotic Pten function show defects during early embryonic development that point to a function for Pten in the organization of the actin cytoskeleton. Removal of Pten function from the germline in ovaries causes abnormal actin organization in nurse cells and in the oocyte. It is proposed that recruitment of PTEN by Baz may contribute to the polarization of the actin cytoskeleton, most likely by creating local differences in the balance between PtdIns(3,4,5)P3 and PtdIns(4,5)P2 in the plasma membrane. Moreover, PTEN may affect the activity of two key components of the PAR/aPKC complex, aPKC and Cdc42. The binding of PTEN to Baz provides the first molecular link between the PAR/aPKC complex, the actin cytoskeleton and phosphoinositide signaling (von Stein, 2005).

Therefore, Baz/PAR-3 and PTEN directly bind to each other and colocalize in the apical cortex of neuroblasts and epithelia. What could be the physiological meaning of this interaction? Evidence for a functional link between the PAR/aPKC complex and PI3-kinase signaling comes from a recent study that showed that both pathways are required for polarization of cultured hippocampal neurons. In this system, the PAR/aPKC complex localizes to the tip of the outgrowing axon and its localization is abolished upon overexpression of PTEN. However, no information is available on the mechanism of how PTEN interacts with the PAR/aPKC complex in this system (von Stein, 2005).

Mammalian PTEN contains a canonical PDZ-binding motif at its C terminus, and this motif has been reported to interact with the multi-PDZ proteins MAGI-2 and MAGI-3. Both PDZ proteins localize to tight junctions in mammalian epithelia and cooperate with PTEN to control the activity of the downstream kinase PKB/Akt, indicating that subcellular targeting of PTEN may be important for its biological activity. This hypothesis is supported by studies of a deletion mutant of PTEN lacking the PDZ-binding motif. Although this mutant retains lipid phosphatase activity, its activity differed from the full-length wild-type form of PTEN in several biological assays. Together, these observations demonstrate that targeting of PTEN to a specific subcellular location may be essential for its proper function in the control of cell polarity. The data show that PTEN is specifically recruited to the apical plasma membrane of epithelia and neuroblasts by direct association with Baz/PAR-3, a key regulator of cell polarity (von Stein, 2005).

In order to address the issue of whether Pten activity is required for the control of cell polarity in Drosophila, the phenotype of mutant ovaries and embryos lacking maternal and zygotic Pten activity was analyzed. The organization of the actin cytoskeleton in nurse cells and in the oocyte of Pten germ-line clones becomes abnormal from stage 9 onwards, resulting in the production of smaller, misshapen eggs. Ptenmat,zyg embryos show defects in the axial expansion of nuclei during nuclear division cycles 4-7 and fail to synchronize the cell cycle in syncytial blastoderm nuclei. In addition, pole cells are strongly reduced in number or are missing altogether, which is accompanied by the failure to maintain oskar mRNA and Vasa protein localization at the posterior pole. Very similar phenotypes have been reported for embryos treated with the actin depolymerizing drug cytochalasin D and for mutants in genes that are required for the organization of the actin cytoskeleton. Mutations in the gene shackleton also show defects in axial expansion and lack pole cells, but the posterior localization of oskar mRNA is normal, indicating that defects in axial expansion alone are sufficient to cause the lack of pole cells. Interestingly, although germ plasm determinants were mislocalized or absent in early Ptenmat,zyg embryos, they were still localized normally during oogenesis, pointing to a function for Pten in maintenance, rather than establishment, of germ plasm determinant localization. Studies on ovaries and embryos mutant for the actin-binding protein tropomyosin II give essentially the same results. Thus, all of the phenotypes of Ptenmat,zyg mutant ovaries and embryos described here can be related to a function for PTEN in actin-dependent processes (von Stein, 2005).

The links between PTEN and actin are obviously the substrate and the product of the enzymatic activity of PTEN, PtdIns(3,4,5)P3 and PtdIns(4,5)P2. Both phosphoinositide lipids are important regulators of the actin cytoskeleton. PtdIns(4,5)P2 acts mostly by direct binding to actin-associated proteins that link the actin cytoskeleton to the plasma membrane or by binding to proteins that are involved in the initiation of de novo actin polymerization, e.g., profilin and WASP. PtdIns(3,4,5)P3 in turn acts on the actin cytoskeleton via recruitment of guanine nucleotide exchange factors (GEFs) for the small GTPases Rac1, Rho and Cdc42, which can activate WASP proteins and the Arp2/3 complex. Because the subcellular localization of endogenous PTEN is not known, it is not possible to predict at present how exactly PTEN may affect the organization of the actin cytoskeleton during early embryonic development. However, the fact that overexpressed PTEN2 colocalizes with PtdIns(4,5)P2 to the junctional region of epithelial cells indicates that PTEN may locally alter the balance between PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in the plasma membrane, leading to a modification of the actin cytoskeleton in defined regions of the cytocortex. Studies of PTEN knockout cells and Pten mutants in Drosophila have indeed shown that loss of Pten leads to a significant increase in the amount of PtdIns(3,4,5)P3 in the plasma membrane (von Stein, 2005).

Surprisingly, PTEN does not appear to be required for the control of apicobasal polarity of neuroblasts and epithelia, despite its apical colocalization with Baz in these two cell types. The asymmetric localization of cell fate determinants to the basal cortex of mitotic neuroblasts requires both an intact actin cytoskeleton and the activity of the PAR/aPKC complex. Thus, the PAR/aPKC complex must be communicating with the actin cytoskeleton, but how this occurs is unknown. The finding that mutations in Pten lead to severe defects in several actin dependent processes during oogenesis and early embryonic development support the hypothesis that PTEN may provide a link between the PAR/aPKC complex and the actin cytoskeleton in neuroblasts and epithelia. However, this link may not be essential in these cell types because of functional redundancy in the system that controls the levels of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 at the plasma membrane. Functional redundancy has recently been uncovered for the pathways that control the different cell size of neuroblasts and ganglion mother cells during asymmetric neuroblast division. During this division, the activity of either the PAR/aPKC complex or the Pins/Galphai complex alone is sufficient to generate two daughter cells of different size. Only the simultaneous inactivation of both complexes leads to loss of cell size asymmetry. Alternatively, even if the balance between PtdIns(4,5)P2 and PtdIns(3,4,5)P3 at the plasma membrane were altered in neuroblasts and epithelia of Ptenmat,zyg embryos, alterations in the biological activity of downstream components of the system may compensate for this imbalance. In support of this interpretation, the loss of Pten function that are described in this study affects only a subset of actin-dependent processes in oogenesis and early embryogenesis, while a participation of PTEN in other actin-dependent processes may be masked by the redundant activities of additional actin effectors (von Stein, 2005).

Besides its function in the regulation of actin, PTEN may regulate the catalytic activity of aPKC, a core component of the PAR/aPKC complex that directly binds to Baz. The mammalian homologs of aPKC, the atypical PKC isoforms l and zeta, require phosphorylation by the upstream kinase PDK1 in order to become fully active. PDK1 is recruited to the plasma membrane by direct binding of its pleckstrin homology (PH) domain to PtdIns(3,4,5)P3. PDK1, PTEN and several downstream effectors of the PI3-kinase signaling pathway in Drosophila show strong genetic interactions and are crucial for the regulation of cell growth and proliferation. Biochemical evidence has been obtained that aPKC is a substrate for PDK1 and it is proposed that aPKC is phosphorylated in response to elevated PtdIns(3,4,5)P3 levels. According to this hypothesis, PTEN would be a negative regulator of the kinase activity of aPKC (von Stein, 2005).

In addition to PDK1, PtdIns(3,4,5)P3 recruits GEFs that activate the small GTPases Cdc42 and Rac1. Intriguingly, active GTP-bound Cdc42 is also a component of the PAR/aPKC complex in mammalian cells and in Drosophila. GTP-bound Cdc42 binds directly to the CRIB domain of PAR-6 and this interaction might elevate the kinase activity of aPKC, as has been shown in mammalian cells. Thus, PtdIns(3,4,5)P3 might activate aPKC both by recruitment of PDK1, which directly phosphorylates aPKC, and by recruitment of GEFs, which activate aPKC via Cdc42 and PAR6. Studies on PTEN knockout cells have indeed shown that PTEN inhibits Rac1 and Cdc42. The presence of PTEN in one complex together with aPKC, Cdc42 and PAR-6 should therefore lead to inhibition of both pathways that activate aPKCs, revealing a novel way to control the activity of a key component of the PAR/aPKC complex (von Stein, 2005).

Regulated and polarized PtdIns(3,4,5)P3 accumulation, involving Bazooka and PTEN, is essential for apical membrane morphogenesis in photoreceptor epithelial cells

In a specialized epithelial cell such as the Drosophila photoreceptor, a conserved set of proteins is essential for the establishment of polarity, its maintenance, or both -- in Drosophila, these proteins include the apical factors Bazooka, Atypical protein kinase C, and Par6 together with E-cadherin. However, little is known about the mechanisms by which such apical factors might regulate the differentiation of the apical membrane into functional domains such as an apical-most stack of microvilli or more lateral sub-apical membrane. In photoreceptors Bazooka (D-Par3) recruits the tumor suppressor lipid phosphatase PTEN to developing cell-cell junctions (Zonula Adherens or ZA). ZA-localized PTEN controls the spatially restricted accumulation of optimum levels of the lipid PtdIns(3,4,5)P3 within the apical membrane domain. This in turn finely tunes activation of Akt1, a process essential for proper morphogenesis of the light-gathering organelle, consisting of a stack of F-actin rich microvilli within the apical membrane. Spatially localized PtdIns(3,4,5)P3 has been shown to mediate directional sensing during neutrophil and Dictyostelium chemotaxis. It is concluded that a conserved mechanism also operates during photoreceptor epithelial cell morphogenesis in order to achieve normal differentiation of the apical membrane (Pinal, 2006).

Although localized accumulation of PtdIns(3,4,5)P3 is thought to be an essential player in generating the polarity required for directed cell migration, Par proteins have been shown to play a key role in the establishment of polarity in many other biological contexts. This study has found a direct connection between these two pathways in the retinal epithelium and shows that, in photoreceptors, Baz recruits PTEN to the developing ZA and thus promotes PtdIns(3,4,5)P3 degradation and PtdIns(4,5)P2 biosynthesis in that membrane domain. Importantly, ZA-localized PTEN is required for precise regulation of the accumulation of PtdIns(3,4,5)P3 in the entire apical membrane. Although localized rhabdomeric PtdIns(3,4,5)P3 likely involves localized activation of PI3-Kinase, the juxtaposition of PTEN2 (a splice variant of PTEN that contains a C-terminal PDZ binding domain as does mammalian PTEN) to the source of PtdIns(3,4,5)P3 biosynthesis appears essential for achieving the optimal fine tuning of the PtdIns(3,4,5)P3-dependent Akt1 activation. Activated Akt1 is in turn important for controlling the precise localization of Crumbs (Crb) and D-PATJ within the photoreceptor apical membrane and for achieving proper microvilli morphogenesis. This could be due to a direct regulation of the Crb complex by Akt1 or to a more general role for Akt1 in apical membrane differentiation. Interestingly, Akt1 activation occurs precociously within the photoreceptor apical membrane in the absence of PTEN. This is consistent with a previous report that photoreceptor differentiation is accelerated in the absence of PTEN function, but it may also merely reflect the fact that normal levels of Akt activation at these early stages are too low to be detected in this system. Correlating with the early onset and over-activation of Akt 1 detected in the absence of PTEN function, disruption of the microvilli is readily observed early during photoreceptor differentiation (Pinal, 2006).

Later, during photoreceptor morphogenesis, both Baz and PTEN2 progressively localize to the nascent rhabdomere, at a time when microvilli are extending to reach their mature length. Although the arrangement of microvilli is already defective at this stage, part of the PTEN mutant phenotype (i.e., deformed rhabdomeres with short microvilli) might be due to a role for PTEN during this late phase of morphogenesis. Although PtdIns(4,5)P2 can be detected in the developing ZA before the onset of microvilli induction within the apical membrane, correlating with the location of PTEN2:GFP, this phosphatidylinositol species is also found in the developing rhabdomere later in development. Some PtdIns(4,5)P2 may diffuse into the apical membrane from the ZA, but it is also likely to be produced by PTEN2 in the apical domain itself from 60% pupal development onward, perhaps maintaining a precise balance between PtdIns(3,4,5)P3 and PtdIns(4,5)P2 (Pinal, 2006).

These data indicate that precise regulation of phophoinositide levels in a range of polarized cells is critical for regulating appropriate cytoskeleton-dependent responses such as microvilli morphogenesis or protruding activity in migrating cells. This study has demonstrated an important role for Akt in the process of apical membrane differentiation and, in particular, rhabdomere morphogenesis. It will be interesting to test whether this molecule is also important for the directed migration of neutrophils and macrophages or for defining neuronal polarity, for which localized PtdIns(3,4,5)P3 and Par-3 are also crucial. PTEN and Akt are both major effectors of the insulin signaling pathway regulating cell growth, and it is likely that this pathway is coupled to effectors of the cell cytoskeleton to accommodate modulation of cell size. Interestingly, loss of function of TSC1 or TSC2 (tuberous sclerosis complex), important downstream targets of Akt, leads to rhabdomeric phenotypes that are very similar to that of PTEN and, in particular, to split rhabdomeres interrupted by segments of Crb/D-PATJ stalk-like membrane. This observation raises the possibility that the cytoskeletal or membrane effector(s), or both, of the pathway described in this study lie downstream of the TSC1/2 complex (Pinal, 2006).

PTEN is one of the most frequently mutated genes in human cancer. Based on the results presented in this study, it is proposed that in addition to modulating growth and promoting cell survival, the increase in Akt activity could lead to instabilities within the apical membrane of mutant epithelial cells and that these instabilities might contribute to metastatic and invasive phenotypes by facilitating PtdIns(3,4,5)P3-dependent migratory activity (Pinal, 2006).

Antagonistic functions of Par-1 kinase and protein phosphatase 2A are required for localization of Bazooka and photoreceptor morphogenesis in Drosophila

Establishment and maintenance of apical basal cell polarity are essential for epithelial morphogenesis and have been studied extensively using the Drosophila eye as a model system. Bazooka (Baz), a component of the Par-6 complex, plays important roles in cell polarity in diverse cell types including the photoreceptor cells. In ovarian follicle cells, localization of Baz at the apical region is regulated by Par-1 protein kinase. In contrast, Baz in photoreceptor cells is targeted to adherens junctions (AJs). To examine the regulatory pathways responsible for Baz localization in photoreceptor cells, the effects of Par-1 on Baz localization were studied in the pupal retina. Loss of Par-1 impairs the maintenance of AJ markers including Baz and apical polarity proteins of photoreceptor cells but not the establishment of cell polarity. In contrast, overexpression of Par-1 or Baz causes severe mislocalization of junctional and apical markers, resulting in abnormal cell polarity. However, flies with similar overexpression of kinase-inactive mutant Par-1 or unphosphorylatable mutant Baz protein show relatively normal photoreceptor development. These results suggest that dephosphorylation of Baz at the Par-1 phosphorylation sites is essential for proper Baz localization. This study also shows that the inhibition of protein phosphatase 2A (PP2A) mimics the polarity defects caused by Par-1 overexpression. Furthermore, Par-1 gain-of-function phenotypes are strongly enhanced by reduced PP2A function. Thus, it is proposed that antagonism between PP2A and Par-1 plays a key role in Baz localization at AJ in photoreceptor morphogenesis (Nam, 2007).

Clonal analysis suggests that Baz is crucial for targeting or maintenance of Par-6 and aPKC. In contrast, Baz protein expression was not significantly reduced in par-6 or apkc null clones, although Baz protein distribution was mislocalized to the basolateral region. This implies that Baz plays a nodal role among the Par-6 complex proteins. A caveat in this analysis is that par-6 and apkc mutant clones are very small (1-2 ommatidia) compared to the relatively large baz mutant clones, raising the possibility that par-6 and apkc mutant clones analyzed may represent rare escaper cells that survive with weaker phenotypes. However, this may not be the case because nearly identical phenotypes were seen in more than 50 par-6 or apkc mutant clones. The data suggesting the central role for Baz in Par-6 and PKC localization are also consistent with studies on embryonic epithelia, in which Baz localization to the membrane precedes localization of Par-6, implying Par-6-independent membrane localization of Baz. The requirement of Baz for the localization of Par-6/aPKC but not vice versa suggests that initial Baz localization may be independent of Par-6 and aPKC, as reported in embryonic epithelia (Nam, 2007).

Similar relationships among Par-6 complex proteins have also been observed during asymmetric cell division in C. elegans. PAR-3, the homolog of Baz, is properly localized in the absence of either PKC-3 (aPKC) or PAR-6, whereas it is indispensable for localization of both PKC-3 and PAR-6. These studies suggest that Baz/Par-3 is a major component in the control of cell polarity in diverse systems, including the photoreceptors in Drosophila. However, the Par-6 complex may exist in different compositions with unique functions, depending on various developmental contexts. For instance, Baz/Par-3 and Par-6 colocalize to the apical cortex of dividing neuroblasts in the Drosophila CNS and in dividing cells in C. elegans embryos, whereas Baz is distinctly localized to the AJ basal to the Par-6 domain in the apical membrane of photoreceptors. It is also striking that whereas Baz is not critically required for photoreceptor differentiation in the larval eye imaginal disc, it becomes crucial during pupal eye development. These data suggest that Baz is required for specific developmental events such as junctional reorganization and rhabdomere formation, although it is expressed in photoreceptors from the time of neuronal fate specification as well as in undifferentiated cells prior to retinal development (Nam, 2007).

Par-1 is a key regulator of Baz localization in ovarian follicle cells. In this system, Par-1 is localized to the basolateral membrane and is essential for exclusion of Baz expression from the basolateral membrane. During early embryogenesis, Par-1 is transiently restricted to the lateral membrane, but at mid-gastrulation it is localized near the apical domain immediately below the region of spot adherens junction (SAJ). Par-1 is required for the restriction of SAJ, preventing the expansion of the E-Cad SAJ marker into the lateral membrane, but not affecting Crb-Dpatj apical markers (Nam, 2007).

In contrast to follicle and embryonic epithelia, in eye imaginal discs, par-1 LOF clones show no significant apical basal polarity defects, suggesting that Par-1 is not required for cell polarity in eye imaginal disc epithelia. This raises a question of whether Par-1 function is dispensable for photoreceptor morphogenesis. In this study, analysis was focused on pupal eye development, since some cell polarity genes such as crb are not required in larval imaginal discs, although they become essential later during the pupal stage when the retina undergoes dramatic reorganization of cell junctions and the apical basal pattern in the photoreceptor cells. Analysis of pupal eyes suggests that Par-1 is required for the distal-proximal growth or maintenance of apical and AJ domains of photoreceptor cells, as Baz and apical markers often fail to form continuous AJ and rhabdomeres along the distal-proximal axis of the retina in par-1 null mutant clones. These phenotypes are similar to the defects shown previously in the eyes of Crb complex mutants. Like par-1 mutations, loss of these gene functions also affects the extension/maintenance of AJ and rhabdomeres but not apical basal cell polarity. Since Baz is essential for proper targeting of Par-6 and Crb complex proteins, loss of Par-1 function may result in mislocalization of Crb complex through affecting Baz localization, although it is possible that Par-1 may also be directly involved in localization of Crb complex proteins independent of Baz. In the eye imaginal disc, it has been reported that Par-1 is localized to the apical-marginal zone and AJ. In the pupal eye, it was also found that Par-1 is enriched in the apical region of photoreceptor clusters, although a low level of Par-1 is also detected broadly along the basolateral membrane. Thus, Par-1 localization is not restricted to the basolateral membrane but appears to be regulated in a complex pattern in different cell types (Nam, 2007).

In ovarian follicle cells, phosphorylation of Baz by Par-1 is required for proper localization of Baz to the apical region of the cells, and BazSA mutated proteins are abnormally localized to the basolateral membrane. In contrast, the current data show that under conditions of overexpression, Baz protein mutated at Par-1 phosphorylation sites is targeted to the AJ whereas wild-type Baz is ectopically localized. Overexpressed wild-type Baz may be abnormally targeted to non-AJ sites, but it is also possible that ectopic Baz may recruit AJ proteins to form ectopic AJs. Nonetheless, the data suggest that, in photoreceptor cells, Par-1-dependent phosphorylation is not essential for initial localization of Baz to AJ. Instead, dephosphorylation of Baz may be a key for the localization of Baz to AJ. This explanation is consistent with the data that the pattern of AJ and apical markers is severely disrupted by overexpression of Par-1 but not by loss of Par-1, suggesting that Baz phosphorylation by Par-1 must be suppressed to maintain photoreceptor cell polarity. The data provide genetic evidence to support that Mts is a major enzyme responsible for antagonizing the effects of Par-1-dependent Baz phosphorylation (Nam, 2007).

PP2A has been implicated in the regulation of tight junction formation in MDCK epithelial cells by interacting with aPKC. However, it is unlikely that the Mts role in Baz localization is mediated through aPKC. First, wild-type and mutant BazSA are localized to completely different sites even though both have an intact phosphorylation site for aPKC. Second, the phenotype of Par-1 overexpression is mimicked by inhibition of Mts but not by loss of aPKC. The idea of specific antagonism between Par-1 and Mts is also supported by the enhancement of the Par-1 overexpression phenotype by reduction of mts gene dosage but not apkc. Thus, a model is proposed in which the localization of Baz to AJ in photoreceptors cells during early pupal eye development depends on the removal of Par-1-mediated phosphorylation by Mts PP2A activity. In this model, Mts plays a pivotal role in regulation of Baz localization and consequent maintenance of photoreceptor cell polarity. On the contrary, Par-1 does not play an essential role for the initial targeting of Baz to AJ, but it is required for growth or stability of AJs and rhabdomeres during photoreceptor morphogenesis. It will be interesting to see whether the antagonistic interaction of Par-1 and PP2A plays an important role in regulation of Baz localization and function in various developmental contexts in Drosophila and other animal species (Nam, 2007).

PP2A antagonizes phosphorylation of Bazooka by PAR-1 to control apical-basal polarity in dividing embryonic neuroblasts

Bazooka/Par-3 (Baz) is a key regulator of cell polarity in epithelial cells and neuroblasts (NBs). Phosphorylation of Baz by PAR-1 and aPKC is required for its function in epithelia, but little is known about the dephosphorylation mechanisms that antagonize the activities of these kinases or about the relevance of Baz phosphorylation for NB polarity. This study found that protein phosphatase 2A (PP2A) binds to Baz via its structural A subunit. By using phospho-specific antibodies, it was shown that PP2A dephosphorylates Baz at the conserved serine residue 1085 and thereby antagonizes the kinase activity of PAR-1. Loss of PP2A function leads to complete reversal of polarity in NBs, giving rise to an 'upside-down' polarity phenotype. Overexpression of PAR-1 or Baz, or mutation of 14-3-3 proteins that bind phosphorylated Baz, causes essentially the same phenotype, indicating that the balance of PAR-1 and PP2A effects on Baz phosphorylation determines NB polarity (Krahn, 2009).

Apical-basal polarity of NBs is controlled by a relatively small number of proteins which assemble into protein complexes localized to the NB cortex in an asymmetric fashion. These cortical proteins interact with each other in a functional hierarchy. At the top of the hierarchy is Baz, because it can localize to the apical NB cortex in loss-of-function mutants for any of the other factors, including PAR-6, aPKC, Insc, Pins, and others (Krahn, 2009).

This study shows that Baz gets frequently mislocalized to the basal NB cortex when it is moderately overexpressed or when it is excessively phosphorylated at S1085, either by overexpression of PAR-1 or by loss-of-function of PP2A. It is expected that similar antagonistic activities of kinases and phosphatases regulate the phosphorylation state of additional sites of Baz/PAR-3 that are relevant in different cellular contexts. Loss of function of 14-3-3ζ and to a lesser extent of 14-3-3epsilon causes mislocalization of endogenous Baz in NBs, whereas overexpression of 14-3-3ζ and 14-3-3epsilon suppresses the mislocalization of overexpressed Baz. It is therefore suggested that the ratio of Baz phosphorylated at S1085 to the amount of available 14-3-3 determines whether Baz gets mislocalized to the basal cortex. In this model, the 14-3-3 proteins function as a buffer to inactivate mislocalized, phosphorylated Baz. This inactivation could be explained by the inhibition of aPKC binding to Baz upon association of 14-3-3 with Baz. If the amount of overexpressed Baz exceeds the buffering capacity of 14-3-3, this would lead to the formation of active Baz/aPKC complexes at the basal cortex. These basally localized, active Baz/aPKC complexes may in turn affect the localization of PAR-1. The mammalian aPKC homolog PKCζ can phosphorylate PAR-1 at a conserved serine residue, and this phosphorylation causes a strong reduction of PAR-1 kinase activity and the release of PAR-1 from the plasma membrane. If the same was true in Drosophila, it would explain the total reversal of NB polarity, because the now basally localized aPKC would phosphorylate PAR-1, which would cause its release from the membrane and the establishment of a new apical cortical domain at the previously basal cortex (Krahn, 2009).

PAR-1, 14-3-3 proteins, and PP2A are strongly expressed during oogenesis, and maternal contributions may account for difficulties identifying requirements during early embryogenesis. In contrast, eliminating maternal expression of these genes results in phenotypes too severe to allow the study of neurogenesis. However, overexpression of a dominant-negative form of Mts from early neurogenesis onward also caused polarity reversal only in late-stage NBs. While this experiment does not exclude the possibility that the late onset of polarity reversal in NBs is due to the perdurance of the maternal gene products, it points to a fundamental difference in the mechanism of how NB polarity is controlled immediately after delamination as opposed to subsequent asymmetric divisions. The majority of late-stage NBs showing polarity reversal were not in direct contact with the overlying epithelium and thus may rely exclusively on intrinsic polarity cues, in contrast to NBs that have just delaminated and maintain contact to the overlying epithelium. Late-stage NBs lacking contact to the overlying epithelium show a higher variability of spindle orientation as compared to early-stage NBs in close contact to the epithelium. Thus, late-stage NBs may be particularly sensitive to changes in the phosphorylation state and general activity level of Baz, because they rely on Baz as the main cue for orienting their polarity axis (Krahn, 2009).

It is interesting to note that mutations uncoupling spindle orientation from the localization of cell fate determinants commonly show fully random spindle orientation, including a variety of oblique orientations. In contrast, hyperphosphorylation of Baz at S1085 resulted very rarely in oblique orientations, and spindles were always aligned with the asymmetric crescents of cell fate determinants. Although a good explanation for why there is a strong bias for either total reversal of polarity or misorientation of the spindle by 90° is not available, the findings point to the existence of a spatial cue functioning upstream of Baz that defines a polarity axis perpendicular to the plane of the epithelium (Krahn, 2009).

Polarized subcellular localization of Jak/STAT components is required for efficient signaling: Par-3-dependent STAT apical localization

Three protein complexes control polarization of epithelial cells: the apicolateral Crumbs and Par-3 complexes and the basolateral Lethal giant larvae complex. Polarization results in the specific localization of proteins and lipids to different membrane domains. The receptors of the Notch, Hedgehog, and WNT pathways are among the proteins that are polarized, with subcellular receptor localization representing an important aspect of signaling regulation. For example, in the WNT pathway, differential DFz2 receptor localization results in activation of either the canonical or the planar polarity pathway. Despite the large body of research on the vertebrate JAK/STAT pathway, there are no reports indicating polarized signaling. By using the conserved Drosophila JAK/STAT pathway as a system, it was found that the receptor and its associated kinase are located in the apical membrane of epithelial cells. Unexpectedly, the transcription factor STAT is enriched in the apicolateral membrane domain of ectoderm epithelial cells in a Par-3-dependent manner. These results indicate that preassembly of STAT and the receptor/JAK complex to specific membrane domains is a key aspect for signaling efficiency. These results also suggest that receptor polarization in the ectoderm cell membrane restricts the cell's response to ligands provided by neighboring cells (Sotillos, 2008).

Besides setting up epithelial polarity, apicobasal complexes also modulate the subcellular compartmentalization or localized activation of various signaling molecules. The JAK/STAT signaling pathway is involved in processes ranging from immune response to organogenesis. In the vertebrate-signaling model, inactive STAT is shuttling from the cytoplasm to the nucleus. Ligand binding to the dimerized receptor results in the activation of JAK bound to the receptor. JAK phosphorylates itself and the receptor, creating docking sites for STAT. Inactive cytoplasmic STAT now binds to the phosphoreceptor/JAK complex, where it is phosphorylated by the kinase. Phosphorylated STAT is imported to the nucleus, where it activates the transcription of target genes. In contrast to vertebrates, in which the JAK/STAT core-signaling elements are highly redundant, the Drosophila pathway is composed of only three ligands, Unpaired (Upd), Unpaired2, and Unpaired3; one receptor, Domeless (Dome); one JAK, Hopscotch (Hop); and one transcription factor, STAT92E. Therefore, Drosophila was used as a model to investigate the polarization of the pathway (Sotillos, 2008).

dome, hop, and stat92E mRNAs are maternally provided and ubiquitously transcribed in the embryo. To analyze their protein subcellular localization, specific antibodies were used or functional tagged proteins were expressed by using UAS-dome, UAS-hop-Myc, and UAS-STAT92E-GFP. These constructs were expressed by using either mesodermal or ectodermal Gal4 drivers, and the subcellular localization of the proteins was analyzed, paying special attention to three organs where the endogenous ligand is expressed and the pathway is active: the posterior spiracles (ectodermal origin), the pharyngeal musculature (mesodermal), and the hindgut (an ectodermal tube surrounded by mesoderm) (Sotillos, 2008).

In the pharynx, as expected for a receptor, Dome localizes to the membrane, and does so in a dotted pattern that could correspond to endocytic vesicles. Hop-myc localizes to the cytoplasm, obscuring any membrane localization. This is due to the high levels of Hop-myc expressed, saturating the receptor binding sites and accumulating in the cytoplasm, as simultaneous coexpression of Hop-myc with the receptor relocates Hop to the membrane. This depends on the cytoplasmic domain of Dome, as it also occurs with a construct missing the extracellular domain but not with constructs missing the intracellular domain. STAT is detected in the cytoplasm and is more concentrated in the nuclei, as expected from the activation of the pathway in the pharynx. All of these observations agree with current knowledge of JAK/STAT activation based on vertebrate studies (Sotillos, 2008).

In contrast to the mesoderm, analysis of ectoderm cells shows a different picture. Both in the hindgut and the posterior spiracles, the Dome receptor localizes on the apical membrane. Hop is again cytoplasmic, but after coexpression with Dome both proteins localize to the apical membrane. Surprisingly, by using a specific antibody it was observed that STAT concentrates on the apical membrane of all embryonic ectodermal cells irrespective of the level of activation of the pathway. And, in cells in which the pathway is active, STAT also localizes to the nucleus. The signal detected by the antibody is specific; the same result by using a STAT-GFP fusion protein. STAT membrane localization is more prominent in cells in which the pathway is inactive; for instance, in the trunk epidermis or the spiracle after stage 15. This suggests that STAT translocates from the subapical membrane to the nucleus after pathway activation, returning to the membrane after inactivation (Sotillos, 2008).

To determine if STAT-GFP membrane localization is due to any other of the pathway's components, STAT-GFP localization was analyzed in upd, dome, or hop null mutants. STAT does not disappear from the membrane in a deficiency that removes all three Upd ligands. STAT membrane localization is not affected in null mutants for either dome or hop, demonstrating that apical STAT localization is independent of the pathway (Sotillos, 2008).

STAT localizes to the membrane domain in which the apical complexes are located. This, and the fact that STAT does not localize to the membrane in the mesoderm where Crb and Par-3 complexes are not formed, suggests the apical complexes could be recruiting STAT. To test this, different apical complex proteins were expressed in the mesoderm, and their capacity to modify STAT subcellular localization was studied. Neither the expression of Crb nor aPKC (another member of Par-3 complex) is able to translocate STAT to the membrane. In contrast, expression of Par-3 results in efficient membrane translocation of STAT and STAT-GFP. Moreover, STAT-GFP and Par-3 coimmunoprecipate from embryo extracts overexpressing STAT-GFP and par-3, pointing to Par-3 as the molecule responsible of STAT apical localization. In accordance, STAT-GFP is lost from the membrane in par-3 zygotic mutants, whereas in crb null mutants, where the polarity is highly compromised and Par-3 localization is severely affected, STAT remains in the membrane of cells only where Par-3 is still present. Similarly, in null aPKC embryos, STAT-GFP exclusively remains apical in cells in which Par-3 still localizes at the membrane. Thus, STAT recruitment is independent of Crb or aPKC and may directly depend on Par-3 (Sotillos, 2008).

To analyze if JAK/STAT polarization is functionally relevant, genetic interactions with polarity mutants were tested. Heterozygous polarity mutants or stat92E embryos are viable and normal. In contrast, embryos simultaneously heterozygous mutant for stat92E and either par-3, aPKC, or crb present phenotypes associated to JAK/STAT loss of function, including malformation of the posterior spiracles and abnormal segmentation. A specific readout of the pathway's activity was studied, analyzing the expression of a crb-spiracle enhancer that is directly activated by JAK/STAT (Lovegrove, 2006). The expression of this enhancer is severely reduced in zygotic par-3 mutants simultaneously heterozygous for stat92E, compared to its expression in heterozygous stat92E embryos or zygotic par-3 mutants. In contrast, the expression of the JAK/STAT independent ems-spiracle enhancer is not affected in the same genetic backgrounds. The capability of Par-3 to induce STAT membrane localization and the strong genetic interaction between stat92E and cell-polarity mutations indicate that the apical polarization of JAK/STAT components is required for full-signaling efficiency in the ectoderm (Sotillos, 2008).

Next, whether the apical localization of all JAK/STAT transducer components in the ectoderm results in signaling occurring exclusively through this membrane domain was tested. For this purpose the posterior hindgut, where JAK/STAT is required in the ectoderm and in the mesoderm surrounding it, was analyzed. Upd expressed from the most anterior ectodermal cells of the hindgut activates in the ectoderm ventral veinless (vvl) and upregulates in the mesoderm dome through the dome-MESO enhancer. Thus, vvl and the dome-MESO autoregulatory enhancer can be used as readouts for JAK/STAT activation in the different hindgut tissues (Sotillos, 2008).

If signaling in the ectoderm were transduced exclusively through the apical membrane, it would be expected that vvl activation on the hindgut would not be possible if Upd is presented from the basal side. To test this Upd was expressed either in the ectoderm or in the mesoderm, and its effect on vvl activation in the ectoderm was analyzed. As a positive control the expression of dome-MESO was analyzed. When expressed throughout the ectoderm, Upd induces ectopic expression of dome-MESO in the mesoderm and of vvl in the ectoderm, behaving as the endogenous Upd. In contrast, when Upd is expressed throughout the mesoderm, dome-MESO is ectopically activated, whereas vvl is not. The unresponsiveness of the ectoderm cells to Upd from the mesoderm is consistent with the endogenous receptor being apically localized in the hindgut ectoderm and, thus, unable to receive any mesoderm signal (Sotillos, 2008).

Many proteins involved in the establishment and maintenance of cell polarity also modulate signaling pathways by modifying or restricting the localization of their signaling components. Precise subcellular distribution may help the activation of the pathway or restrict its activity by sequestering key elements. This study has shown that in the epithelial cells the localization of JAK/STAT components is highly polarized. The apical restriction of the receptor can influence transduction, since only ligand presented to the apical side of the epithelium would be detected. This may be of relevance after septic injury, when circulating haemocytes secrete the Upd3 cytokine into the haemolymph. In this case, the secreted ligand would activate its targets in the fat body without stimulating the ectoderm epithelial cells, since the cell junctions efficiently block Upd diffusion to the apical side (Sotillos, 2008).

Par-3-dependent STAT apical localization is intriguing. The localization of STAT to the subapical membrane seems important for signal transduction, since mutations reducing the amount of cell polarity proteins enhance stat loss of function phenotypes and reduce the activation of direct pathway targets. It is proposed that in ectodermal cells, where the receptor and the kinase locate apically, the existence of a subapical pool of STAT facilitates its rapid translocation to the activated receptor, increasing signaling efficiency. Future research should resolve whether this is achieved simply by the increased local concentration of apical STAT facilitating receptor binding or if there exists some dedicated machinery to translocate STAT from the subapical region to the active receptor similar to the one involved in nuclear import. It is interesting to note that crb expression is upregulated by JAK/STAT signaling in the follicle cells and in the posterior spiracles. Since Crb helps maintaining Par-3 in the apical membrane, upregulation of crb by STAT might increase apical Par-3, reinforcing signal transduction by increasing the apical concentration of STAT (Sotillos, 2008).

Linking cell cycle to asymmetric division: Aurora-A phosphorylates the Par complex to regulate Numb localization

Drosophila neural precursor cells divide asymmetrically by segregating the Numb protein into one of the two daughter cells. Numb is uniformly cortical in interphase but assumes a polarized localization in mitosis. This study shows that a phosphorylation cascade triggered by the activation of Aurora-A is responsible for the asymmetric localization of Numb in mitosis. Aurora-A phosphorylates Par-6, a regulatory subunit of atypical protein kinase C (aPKC). This activates aPKC, which initially phosphorylates Lethal (2) giant larvae (Lgl), a cytoskeletal protein that binds and inhibits aPKC during interphase. Phosphorylated Lgl is released from aPKC and thereby allows the PDZ domain protein Bazooka to enter the complex. This changes substrate specificity and allows aPKC to phosphorylate Numb and release the protein from one side of the cell cortex. These data reveal a molecular mechanism for the asymmetric localization of Numb and show how cell polarity can be coupled to cell-cycle progression (Wirtz-Peitz, 2008).

Since the discovery of Numb asymmetry, several proteins required for Numb localization have been identified, but how they cooperate remained unclear. This paper describes a cascade of interactions among these proteins that culminates in the asymmetric localization of Numb in mitosis. In interphase, Lgl localizes to the cell cortex, where it forms a complex with Par-6 and aPKC. At the onset of mitosis, AurA phosphorylates Par-6 in this complex, thereby releasing aPKC from inhibition by Par-6. Activated aPKC phosphorylates Lgl, causing its release from the cell cortex. Since Baz competes with Lgl for entry into the Par complex, the disassembly of the Lgl/Par-6/aPKC complex allows for the assembly of the Baz/Par-6/aPKC complex. Baz is a specificity factor that allows aPKC to phosphorylate Numb on one side of the cell cortex. Since p-Numb is released from the cortex (Nishimura, 2007; Smith, 2007), these events restrict Numb into a cortical crescent on the opposite side (Wirtz-Peitz, 2008).

The data show that Lgl acts as an inhibitory subunit of the Par complex. Given that Par-6 inhibits aPKC activity until the onset of mitosis, why would an additional layer of regulation be required? Like all phosphoproteins Numb is in a dynamic equilibrium between the phosphorylated and unphosphorylated states. Too high a rate of phosphorylation shifts this equilibrium toward the phosphorylated state, mislocalizing Numb into the cytoplasm. Too low a rate shifts it toward the unphosphorylated state, mislocalizing Numb around the cell cortex. Importantly, these data show that only the Baz complex can phosphorylate Numb. Assuming an abundance of Lgl over cortical Par-6, an increase in aPKC activity would translate into a comparatively small increase in the levels of Baz complex. This is because assembly of the Baz complex requires free subunits of Par-6 and aPKC, which become available only once the pool of cortical Lgl has been completely phosphorylated. Therefore, it is proposed that Lgl acts as a molecular buffer for the activity of the Par complex toward Numb. This maintains Numb phosphorylation within a range that is sufficiently high to exclude Numb from one side of the cell cortex but sufficiently low to permit the cortical localization of Numb to the other side (Wirtz-Peitz, 2008).

What is the evidence for this model? Lgl3A, a nonphosphorylatable mutant of Lgl in which the three aPKC phosphorylation sites are mutated to Ala, infinite buffering capacity, induces the mislocalization of Numb around the cell cortex. Conversely, in lgl mutants, having no buffering capacity, Numb is mislocalized into the cytoplasm. Moreover, the model predicts the loss of buffering capacity in the lgl mutant to be offset by an increase in the amount of substrate, since this would render the excess activity of the Par complex limiting. Indeed, overexpression of Numb in lgl mutants restores the cortical localization of Numb as well as its cortical asymmetry (Wirtz-Peitz, 2008).

The results indicate that Lgl gain- and loss-of-function phenotypes are entirely accounted for by the role of Lgl in inhibiting the assembly of the Baz complex. Previously, however, it was thought that the asymmetric phosphorylation of Lgl by aPKC restricts an activity of Lgl to the opposite side of the cell cortex. Based on this model, it was subsequently proposed that Lgl mediates the asymmetric localization of cell fate determinants by inhibiting the cortical localization of myosin-II. In addition, the role of the yeast orthologs of Lgl in exocytosis led to speculation that Lgl establishes an asymmetric binding site for cell fate determinants by promoting targeted vesicle fusion. However, the data show that Lgl asymmetry is extremely transient, and that the protein is completely cytoplasmic from NEBD onward. Lgl cannot therefore interact with any cortical proteins in prometaphase or metaphase, when myosin-II was reported to localize asymmetrically, or establish a stable landmark for vesicle fusion. Interestingly, a recent study demonstrated that yeast Lgl inhibits the assembly of SNARE complexes by sequestering a plasma membrane SNARE (Hattendorf, 2007). This mechanism is reminiscent of fly Lgl sequestering Par-6 and aPKC from interaction with Baz, suggesting that the defining property of Lgl-family members is not a specific role in exocytosis, but a more generic role in regulating the assembly of protein complexes (Wirtz-Peitz, 2008).

The data identify Numb as a key target of aPKC in tumor formation and suggest that Lgl acts as a tumor suppressor in the larval brain by inhibiting the aPKC-dependent phosphorylation of Numb. Although it is tempting to conclude that tumor formation in lgl mutants results from the missegregation of Numb, missegregation of Numb in numbS52F or upon expression of Lgl3A does not cause neuroblast tumors. How might this be explained? During mitosis, unphosphorylated cortical Numb is inherited by the differentiating daughter. At the same time, Baz and aPKC are excluded from this daughter, which limits Numb phosphorylation after exit from mitosis. In the subsequent interphase, some differentiating daughters reexpress members of the Baz complex (Bowman, 2008), but Numb continues to be protected from phosphorylation since cortical Lgl prevents the reassembly of the Baz complex. Thus, Lgl acts both in mitosis and interphase to maximize the amount of unphosphorylated Numb in the differentiating daughter cell (Wirtz-Peitz, 2008).

In lgl mutants, Numb phosphorylation is increased in mitosis, and less unphosphorylated Numb is segregated into the basal daughter cell. Moreover, the assembly of the Baz complex is unrestrained in the subsequent interphase, which is exacerbated by the missegregation of aPKC into both daughter cells. Together, these defects minimize the amount of unphosphorylated Numb in the differentiating daughter cell (Wirtz-Peitz, 2008).

Why is the amount of unphosphorylated Numb critical for differentiation? Recently, it was shown that aPKC-dependent phosphorylation of Numb inhibits not only its cortical localization, but also its activity, owing to the reduced affinity of p-Numb for its endocytic targets (Nishimura, 2007). Therefore, ectopic phosphorylation of Numb leads to its inactivation, transforming the basal daughter cell into a neuroblast in a manner similar to mutation of numb. Consistent with this model, studies in SOP cells have documented ectopic Notch signaling in lgl mutants. Although the numbS52F mutant and Lgl3A overexpression also lead to missegregation of Numb, the levels of active unphosphorylated Numb are increased rather than decreased in these cases and are sufficient to support differentiation (Wirtz-Peitz, 2008).

The data also provide additional insight into the mechanism of tumor formation in aurA mutants. In aurA mutants, the differentiating daughter cell inherits less Numb because Numb is mislocalized around the cell cortex. At the same time, aPKC is missegregated into the differentiating daughter cell, where it promotes Numb phosphorylation in the subsequent interphase. Together, these events result in subthreshold amounts of unphosphorylated Numb in some basal daughter cells, transforming these into neuroblasts. This model explains why aurA mutants are characterized by reduced aPKC activity in mitosis, but are nonetheless suppressed by aPKC mutations, since a lack of aPKC in the differentiating daughter cell restores threshold amounts of unphosphorylated Numb (Wirtz-Peitz, 2008).

The data reveal that Lgl inhibits Numb phosphorylation to maintain Numb activity, whereas AurA promotes Numb phosphorylation in mitosis to ensure its asymmetric segregation. It is concluded that Lgl and AurA act on opposite ends of a regulatory network that maintains appropriate levels of Numb phosphorylation at the appropriate time in the cell cycle (Wirtz-Peitz, 2008).

There are few reports of polarized vertebrate JAK/STAT signaling. However, analysis of the subcellular localization of two IL-6 receptors in MDCK epithelial cells has shown that gp130 localizes basolaterally and CNTF-R apically. Also, in the mammary glands, the IL-4Ra receptor is localized apically in luminal cells during gestation and lactation. Recently, activated STAT3 has been transiently detected at the membrane in the nascent cell-cell contacts of squamous cell carcinoma of the head and neck. In vertebrates the Par-3 complex functions as a regulator of junction biogenesis. It will be interesting to investigate whether Par-3 also mediates the localization of STAT3 in the membrane. The results suggest that JAK/STAT polarization in epithelia may be a general feature (Sotillos, 2008).

Assembly of Bazooka polarity landmarks through a multifaceted membrane-association mechanism

Epithelial cell polarity is essential for animal development. The scaffold protein Bazooka (Baz/PAR-3) forms apical polarity landmarks to organize epithelial cells. However, it is unclear how Baz is recruited to the plasma membrane and how this is coupled with downstream effects. Baz contains an oligomerization domain, three PDZ domains, and binding regions for the protein kinase aPKC and phosphoinositide lipids. With a structure-function approach, this study dissected the roles of these domains in the localization and function of Baz in the Drosophila embryonic ectoderm. A multifaceted membrane association mechanism localizes Baz to the apical circumference. Although none of the Baz protein domains are essential for cortical localization, it was determined that each contributes to cortical anchorage in a specific manner. It is proposed that the redundancies involved might provide plasticity and robustness to Baz polarity landmarks. Specific downstream effects were identified, including the promotion of epithelial structure, a positive-feedback loop that recruits aPKC, PAR-6 and Crumbs, and a negative-feedback loop that regulates Baz (McKinley, 2012).

The PDZ domains of Baz are dispensable for its localization. The current results show that this is due to redundant mechanisms in other parts of the protein. In fact, each PDZ domain plays a unique role in Baz positioning and activity in the Drosophila embryonic ectoderm. The following main roles were identified for the PDZ domains: PDZ1 and PDZ3 recruit Baz to the apical domain, PDZ2 mediates downstream effects on epithelial structure and PDZ1 promotes the turnover of Baz. Each domain also has minor effects that might result from distinct activities or secondary effects of their main activities: PDZ1 and PDZ3 have non-essential but detectable effects on epithelial structure and PDZ2 promotes weak membrane binding (McKinley, 2012).

PDZ1 and PDZ3 activities involve at least two sub-regions of the domains. PDZ domains typically use their peptide-binding pocket to bind the C-termini of their protein partners, but regions outside of these pockets can also mediate interactions. PDZ1 promotes apical surface and circumferential localization independently of its peptide-binding pocket, and its peptide-binding pocket plays a distinct role in promoting Baz turnover. These opposing activities might form a negative-feedback loop that regulates localization of Baz. By contrast, PDZ3 appears to solely promote Baz localization. It can promote apical surface and circumferential localization independently of its peptide-binding pocket, whereas its peptide-binding pocket specifically promotes circumferential anchorage. The binding partners that engage these sites are unknown. However, in vitro studies have shown that the C-termini of Arm and Ed can bind Baz PDZ1-3 in tandem, and that the C-terminus of PTEN can bind Baz PDZ2-3. Binding partners for regions outside the peptide-binding pockets have not been identified for Baz PDZ domains, but the binding of rat PAR-3 PDZ2 to PIPs involves outside regions, as does the binding of C. elegans PAR-3 PDZ1 to PAR-6 (McKinley, 2012).

A major function of Baz PDZ domains is to maintain the protein around the apical circumference. PDZ1 and PDZ3 use peptide-binding-pocket-independent mechanisms to generally localize Baz to the apical domain, but Baz is focused around the apical circumference through mechanisms involving the peptide-binding pockets of these domains. Without these pockets, Baz can saturate its remaining apical anchors and mislocalizes in puncta over the apical surface. PDZ1 appears to prevent this mislocalization by reducing protein levels below saturation, but PDZ2 and PDZ3 might directly bind circumferential proteins or promote an active redistribution of Baz. This activity is weaker for PDZ2 versus PDZ3 (with the former requiring oligomerization and the latter not) and it is possible that the localization activity of PDZ2 is a by-product of its binding to downstream effectors localized to the apical circumference. Thus, it is proposed that PDZ3 has the most direct role in anchoring Baz around the apical circumference (McKinley, 2012).

Because Baz can localize to the apical membrane without its PDZ domains, other localization mechanisms are also involved. The results clarify the importance of two additional mechanisms. The first involves dynamic interactions with apical polarity proteins. Baz has been shown to recruit aPKC to the apical domain as epithelial polarity is first established and to maintain aPKC during later stages. However, aPKC normally localizes at the apical surface with PAR-6 and the Crb complex above Baz and adherens junctions. When BazδPDZ1-3 forms puncta over the apical surface domain it recruits aPKC, PAR-6 and Crb, but the proteins then segregate locally, mimicking their associations around the apical circumference. Indeed, BazδPDZ1-3 might separate from the apical polarity proteins by two known mechanisms: the release of aPKC after it phosphorylates its binding site on Baz, and the loss of PAR-6 binding as a result of competition with Crb. The segregation of aPKC, PAR-6 and Crb from BazδPDZ1-3 suggests that they would not form a stable anchorage site for Baz. However, removal of the aPKC binding region from BazδPDZ1-3 (but not full-length Baz) severely weakens its cortical localization. This suggests that a positive feedback loop exists between Baz and aPKC to maintain localization of each protein. It is proposed that the proteins undergo continuous cycles of attraction and local repulsion to maintain their close but non-overlapping positioning around the apical domain (McKinley, 2012).

An additional Baz localization mechanism involves PIPs. A conserved region of the C-terminal tail of Baz has been shown to bind PIPs (Krahn, 2010), and it was found that the apical surface puncta of BazδPDZ1-3 colocalize with plasma membrane domains enriched with PIPs. Although deletion of the PIP binding region had no effect on full-length Baz (Krahn, 2010), deleting it and the PDZ domains together strongly disrupts plasma membrane binding. Thus, the aPKC binding region and the PIP binding region might both mediate apical localization of Baz in the absence of the PDZ domains. These anchorage mechanisms are also dependent on the oligomerization of Baz, because BazδOD+δPDZ1-3 shows minimal cortical localization. Moreover, the mechanisms appear to support each other because the aPKC binding region cannot compensate for the loss of the PIP binding region from BazδPDZ1-3 and vice versa. Also, membrane binding is abrogated with deletion of the Baz C-terminus, including the aPKC and PIP binding regions (Krahn, 2010). Perhaps the continuous cycles of attraction and local repulsion between Baz, aPKC, PAR-6 and Crb are partly staged on a platform of PIPs (McKinley, 2012).

Interactions with these proteins and lipids might also explain the ability of Baz to partially maintain epithelial structure without its PDZ domains. Indeed, deletion of the aPKC binding region from full-length Baz abrogates its rescue activity (Krahn, 2010), as does deletion of the OD and the PIP binding region, when expressed with a weaker driver, but not a stronger driver (McKinley, 2012).

The results indicate that there are at least five sites in Baz, in addition to its OD, that are involved in membrane localization. No single site is essential, and different combinations of interactions are sufficient for anchorage. This suggests that the individual anchorage mechanisms are relatively weak, as has been shown for the PIP binding region (Krahn, 2010). Cortical localization through multiple weak interactions might provide plasticity and robustness for the role of Baz/PAR-3 as a multifunctional polarity landmark (McKinley, 2012).

The membrane-association mechanism of Baz would allow fine regulation of protein positioning. For example, Baz becomes planar polarized around the apical domain to regulate germband extension in the Drosophila embryo. Rho kinase has been shown to reduce Baz at anterior and posterior cell edges by phosphorylating the Baz C-terminus and inhibiting PIP binding. However, Baz is not fully lost from these edges. Thus, planar polarity might arise from a partial set of membrane-association mechanisms acting along anterior-posterior edges and a more complete set acting at dorsal-ventral edges. Apical localization of Baz is also altered in amnioserosa cells to regulate apical constriction during dorsal closure. Here, Baz forms apical surface puncta in addition to its circumferential localization. Although the mechanisms for this redistribution are unclear, the work suggests that it might involve weakening of PDZ domain activities. Intriguingly, Ed, an in vitro binding partner of the PDZ domains, is specifically absent in the amnioserosa. However, this might not fully explain the redistribution because Baz appears to be localized normally in most ectodermal cells of ed mutants. A more dramatic cellular reorganization occurs as neuroblasts delaminate from the epithelium. As this occurs, adherens junctions and Crb are lost from the cells, but Baz is retained apically and engages with new partners to direct asymmetric cell division after delamination. The mechanisms regulating Baz during this transition are unknown, but its multifaceted membrane-association mechanism might ensure robust apical localization as Baz exchanges molecular interaction networks (McKinley, 2012).

Redundancies in Baz/PAR-3 scaffold activity might also have permitted co-evolution with polarity networks to organize eggs, single-cell embryos, epithelial cells, neurons and stem cells. Indeed, roles for Baz/PAR-3 PDZ domains appear to have diverged. In C. elegans, PDZ2, but not PDZ1 or PDZ3, is essential for embryogenesis, as in Drosophila, but PDZ2 of C. elegans PAR-3 was also shown to be required for proper localization, in contrast to Baz PDZ2. Also, mammalian PDZ2 has also been shown to mediate membrane binding through PIPs, but key residues involved in the interaction are not conserved in Drosophila or C. elegans (McKinley, 2012).

The Baz localization mechanism appears to be unique among characterized polarity scaffold proteins. Other scaffolds also involve multiple mechanisms, but typically there is a primary mechanism that localizes the scaffold to the membrane and secondary mechanisms that focus localization to a particular site. For example, the leucine-rich repeats of Scribble are crucial for its cortical localization in Drosophila epithelia, whereas its second PDZ domain promotes septate junction localization. In Drosophila, the Hook domain of Discs Large is crucial for plasma membrane targeting, whereas particular PDZ domains promote septate junction localization in epithelia and synapse localization in neurons. Similar 'twostep' localization mechanisms have been described for C. elegans and mammalian Discs large, mammalian PSD-95, Drosophila Inscuteable and Pins and Drosophila Stardust. Contrasting these mechanisms, no single site in Baz is essential for membrane recruitment in ectodermal cells. However, any of these mechanisms could be context dependent. Scaffolds shown to localize through a two-step mechanism in one context might use a multifaceted membrane-association mechanism in another, and Baz could localize by one-step or two-step mechanisms in other cell types or developmental stages (McKinley, 2012).

This study has identified a multifaceted membrane-association mechanism that localizes Baz to the apical circumference in epithelial cells. This mechanism integrates with downstream pathways, involving both negative- and positive-feedback loops, which regulate Baz and epithelial polarity. It is important to define the partners for the interaction sites involved, and to dissect how these interactions are controlled (McKinley, 2012).

Interactions between the PDZ domains of Bazooka (Par-3) and phosphatidic acid: in vitro characterization and role in epithelial development

Bazooka (Par-3) is a conserved polarity regulator that organizes molecular networks in a wide range of cell types. In epithelia, it functions as a plasma membrane landmark to organize the apical domain. Bazooka is a scaffold protein that interacts with proteins through its three PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains and other regions. In addition, Bazooka has been shown to interact with phosphoinositides. This study shows that the Bazooka PDZ domains interact with the negatively charged phospholipid phosphatidic acid immobilized on solid substrates or in liposomes. The interaction requires multiple PDZ domains, and conserved patches of positively charged amino acid residues appear to mediate the interaction. Increasing or decreasing levels of diacylglycerol kinase or phospholipase D - enzymes that produce phosphatidic acid - reveal a role for phosphatidic acid in Bazooka embryonic epithelial activity but not its localization. Mutating residues implicated in phosphatidic acid binding revealed a possible role in Bazooka localization and function. These data implicate a closer connection between Bazooka and membrane lipids than previously recognized. Bazooka polarity landmarks may be conglomerates of proteins and plasma membrane lipids that modify each other's activities for an integrated effect on cell polarity (Yu, 2012).

These results identified an in vitro interaction between Baz PDZ domains and PA. Each PDZ domain appears to have a positive surface patch capable of binding PA, but multiple sites must be present in Baz for PA binding. By manipulating the enzymes responsible for generating PA and mutating the PA-binding sites in Baz, Baz-PA interactions appear to have minimal, or context-specific, effects on Baz recruitment to the plasma membrane and may influence downstream effects of Baz important for epithelial structure (Yu, 2012).

The abilities of GST-tagged Baz PDZ1-3, PDZ1-2, and PDZ2-3, but not single PDZ domains, to bind PA indicate that each PDZ domain can contribute to PA binding but that GST dimers of single PDZ domains are insufficient for binding. These data suggest that the binding interaction involves multiple low-affinity sites that require tandem organization and/or oligomerization for an effective interaction with PA. Consistent with this requirement, the full-length Baz protein contains an oligomerization domain that is expected to form extended multimers through front-to-back interactions between positively and negatively charged faces on the domain, based on studies of the homologous mammalian Par-3 domain. Such extended oligomerization may allow Baz to form platforms for engaging and stabilizing local pools of PA. It may also explain the ability of PDZ3 to confer PA binding site-dependent localization and activity in the BazPDZ1-2 construct. Indeed, with the oligomerization domain deleted, mutation of the PA binding site of PDZ3 had no detectable effect on the localization of a construct missing PDZ1 and PDZ2 (Yu, 2012).

The oligomerization of Baz is expected to draw multiple binding partners together to form Baz polarity landmarks. In addition, multiple redundant binding sites have been shown to recruit Baz to the apical circumference of Drosophila embryonic ectodermal cells (McKinley, 2012 ). However, by stripping these recruitment sites from the protein or by analyzing sensitive pools of the protein, it was difficult to find evidence for PA recruiting Baz to the plasma membrane. Alternatively, PA may influence effects downstream of Baz. Some of these effects could involve parallel pathways, but the ability of Baz to bind PA in vitro and the effects of mutating the PA binding site in vivo suggest that PA functions at Baz polarity landmarks. Unfortunately, the lack of probes for PA prevents the visualization of PA in developing epithelial cells (Yu, 2012).

Of the known binding partners of Baz, two have been shown to be influenced by PA in other systems -- phosphoinositides and aPKC. Baz can directly bind the lipid phosphatase PTEN and its substrate PIP3, suggesting that it may facilitate the production of phosphatidylinositol 4,5-biphosphate, which has been shown to direct apical membrane identity in other systems. The Baz phosphoinositide-binding site also contributes to its cortical localization through a mechanism involving multiple low-affinity interactions. PA can influence phosphoinositide production and thus could potentially affect the cortical localization of Baz via phosphoinositides, but the apparent role of PA in mediating effects downstream of Baz suggests that any role in phosphoinositide signaling might be downstream. As discussed, the plasma membrane recruitment of DOCK2 is initiated by PIP3 and stabilized by PA. Such cooperation may be prevalent, as a number of other proteins appear to have binding sites for both PA and phosphoinositides. Thus an accumulation of PA at Baz polarity landmarks could influence the effects of phosphoinositides in the local membrane (Yu, 2012).

Baz also binds aPKC to dynamically form the PAR complex. It is intriguing that PA has been shown to bind mammalian aPKC (PKCzeta) in vitro and addition of PA to cell extracts activates the kinase. Similarly, exogenous PLD can increase PKCzeta activity in cell culture. More recently, DGK activity has been linked to increased PKCzeta/iota activity as part of a signaling pathway linking hepatocyte growth factor to Rac activation and membrane ruffling in mammalian cell culture. Thus local recruitment of PA to Baz polarity landmarks has the potential to affect local aPKC activity (Yu, 2012).

PDZ domains of many proteins have been shown to bind phosphoinositides. The current results expand the lipid repertoire that PDZ domains can bind. For Baz specifically, these data implicate a closer and more complex connection to membrane lipids than previously recognized. Thus Baz polarity landmarks can be viewed as conglomerates of proteins and plasma membrane lipids that likely modify each other's activities for an integrated effect on cellular organization (Yu, 2012).

Src kinases mediate the interaction of the apical determinant Bazooka/PAR3 with STAT92E and increase signalling efficiency in Drosophila ectodermal cells

Intercellular communication depends on the correct organization of the signal transduction complexes. In many signalling pathways, the mechanisms controlling the overall cell polarity also localize components of these pathways to different domains of the plasma membrane. In the Drosophila ectoderm, the JAK/STAT pathway components are highly polarized with apical localization of the receptor, the associated kinase and the STAT92E protein itself. The apical localization of STAT92E is independent of the receptor complex and is due to its direct association with the apical determining protein Bazooka (Baz). This study found that Baz-STAT92E interaction depends on the presence of the Drosophila Src kinases. In the absence of Src, STAT92E cannot bind to Baz in cells or in whole embryos, and this correlates with an impairment of JAK/STAT signalling function. It is believed that the requirement of Src proteins for STAT92E apical localization is mediated through Baz, since can Src can be co-precipitated with Baz but not with STAT92E. This is the first time that a functional link between cell polarity, the JAK/STAT signalling pathway and the Src kinases has been established in a whole organism (Sotillos, 2013).

In vertebrates, there is strong evidence showing that, besides JAK, the Src kinases can activate STAT signalling. First, STAT3 and STAT5 are crucial downstream factors in Src-induced transformation. Second, Src kinase activation has been shown to result in STAT tyrosine phosphorylation. By contrast, in Drosophila the available data suggest that the involvement of Src in STAT92E activation is probably marginal. This is shown by the fact that in Drosophila the phenotypes of null stat92E alleles are very similar to those of mutants in which the function of the JAK kinase, the receptor or of all ligands, is abolished, indicating that STAT92E activation via alternative kinases occurs in a minority of tissues. One of the few cases reported of JAK-independent STAT92E activation occurs during the proliferation and migration of the embryo pole cells where STAT92E is activated by the Ras/Raf pathway downstream of the Torso tyrosine kinase receptor. The study of mutations in the C-terminal Src kinase (Csk), a negative regulator of Src signalling, offers indirect evidence to suggest that Src may activate STAT92E in the Drosophila eye. Eyes that lack Csk are larger than normal, a phenotype also observed when the canonical JAK/STAT pathway is ectopically activated during eye development. Moreover, Csk clones exhibit higher levels of STAT92E expression, which has been interpreted as being due to the induction of a STAT92E positive-feedback loop. However, these results do not clarify whether Src-induced STAT92E activation is due to direct STAT92E phosphorylation or caused by indirect regulation (Sotillos, 2013).

In the ectoderm, where the JAK/STAT pathway is highly polarized, efficient signalling requires STAT92E apical localization achieved through interaction with the membrane-associated apical polarity protein Baz (Sotillos, 2008). This study obtained several pieces of evidence showing that Src is also required for the correct STAT92E membrane localization and signalling. First, although heterozygous stat92E or Src mutant embryos are normal, double heterozygous stat92E and Src mutant embryos display phenotypes that resemble a partial JAK/STAT signalling failure. Second, the expression of a direct target of STAT92E is downregulated in a Src mutant background. Third, decrease of Src gene activity affects STAT92E localization to the membrane of epithelial cells or to the membrane of mesodermal cells expressing Baz. Fourth, in S2R+ cultured cells STAT92E co-localizes with Baz only when co-expressed with Src42A or Src64B. The data also show that tyrosine 711 is not required for membrane localization, demonstrating that this function of Src is independent of STAT92E activation (Sotillos, 2013).

Although it was possible to co-precipitate Baz with STAT92E and Baz with Src42A, co-precipitation of Src42A with STAT92E was not achieved. This suggests that the interaction between Src42A and STAT92E is either too labile to be detected or that Src modifies Baz and that this allows the recruitment of STAT92E to Baz. The data also suggest that the kinase activity of Src42A is dispensable for STAT92E-Baz interactions, as a kinase-dead isoform of Src42A is able to rescue membrane localization in Src mutant backgrounds to the same level that a constitutively activated form can. Although a direct activation of STAT92E in response to Src and growth factors was possible in the minority of cell types, the genetic interactions do not reveal any phenotype apart from those of the canonical JAK/STAT pathway (Sotillos, 2013).

In addition to the parallel dimers formed by SH2 interaction with phosphor-Tyrosine in activated STATs (Mohr, 2012), vertebrate STAT proteins have been shown to form 'inactive' antiparallel dimers mediated by the region that includes the N-terminal and the coiled-coil domains (Mao, 2005; Neculai, 2005; Ota, 2004). In Drosophila, active phosphorylated STAT92E also forms parallel homodimers through an SH2-phosphotyrosine interaction. This study suggests that STAT92E may also form inactive homodimers mediated through the N-terminal coiled-coil domain. Thus, formation of STAT92E dimers prior to pathway activation could be an ancestral STAT characteristic, reinforcing Drosophila as a model for studying vertebrate STAT signalling (Sotillos, 2013).

Previous papers have described the requirement of a STAT92E-Baz interaction for efficient JAK/STAT signalling (Sotillos, 2008). This study has uncovered the domains in both molecules involved in this physical interaction. In the case of STAT92E the region was narrowed down to the transactivation (TA) domain of the protein: only the most C-terminal part of the molecule is able to localize to the apical membrane cortex on its own and to co-precipitate with Baz. Moreover, when the TA domain is removed, the C-terminal domain is unable to localize to the cell membrane where Baz is located and reduces its ability to co-precipitate with Baz, probably owing to the loss of binding to the N-terminal part of Baz. However, this construct is still able to bind to the C-terminal part of Baz, indicating that another domain in this fragment is also involved in this interaction. In vertebrates, the TA domain has been shown to be crucial for the regulation of the activity of STAT through the interaction with several proteins. This study has added a new function to this domain as a mediator of the interaction of STAT92E with Ba (Sotillos, 2013).

Baz is a scaffolding protein that is able to interact with various proteins and lipids through different regions. Interaction with STAT92E requires both the Baz N-terminal region (1-317) that includes the oligomerization domain and the C-terminal region (1048-1464) that includes the phosphatidylinositol-binding site. Given that both Baz N- and C-terminal domains are conserved, and that STAT92E TA domain is conserved in vertebrate STATs, it is possible that the PAR3-STAT interaction is a conserved feature of JAK/STAT signalling. S2 cell and embryo experiments show a requirement of Src in the STAT92E-Baz interaction. Paradoxically, it is possible to precipitate STAT92E in the absence of Src using the N- and the C-terminal fragments of Baz in vitro. Since in vitro Baz fragments are being used that most probably have a different conformation from the full-length protein, this paradox can be explained if, in vivo, Src function was required to change the conformation of Baz or to displace another protein that interferes with the binding, events that would not take place in the in vitro binding (Sotillos, 2013).

In summary, these results show that Src42A and Src64B are required redundantly in the ectoderm to allow STAT92E to bind to Baz. This interaction leads to the priming of JAK/STAT signalling by concentrating inactive STAT92E dimers apically near the polarized receptor kinase complex, contributing in this way to the efficient canonical JAK/STAT signalling. Although it cannot be completely discarded that future studies in Drosophila may find some specific cell type in which direct STAT92E activation by Src kinases exists, the data indicate that, in general, there is no direct STAT92E activation by Src in Drosophila. It is speculated that the existence of complexes where STAT, PAR3 and Src interact might have allowed the evolution of STAT-activation shortcuts that in vertebrates would have led to Src directly phosphorylating STAT in tyrosine 700 without the intervention of JAK or the canonical receptors. Considering the relevance of these proteins in development and disease, future studies should address whether apical STAT localization through PAR3-Src activity is also functioning in the vertebrate lineage (Sotillos, 2013).

Peptide binding properties of the three PDZ domains of bazooka (Drosophila par-3)

The Par complex is a conserved cell polarity regulator. Bazooka/Par-3 is scaffold for the complex and contains three PDZ domains in tandem. PDZ domains can act singly or synergistically to bind the C-termini of interacting proteins. Sequence comparisons among Drosophila Baz and its human and C. elegans Par-3 counterparts indicate a divergence of the peptide binding pocket of PDZ1 and greater conservation for the pockets of PDZ2 and PDZ3. However, it is unclear whether the domains from different species share peptide binding preferences, or if their tandem organization affects their peptide binding properties. To investigate these questions, phage display screens were used to identify unique peptide binding profiles for each single PDZ domain of Baz. Comparisons with published phage display screens indicate that Baz and C. elegans PDZ2 bind to similar peptides, and that the peptide binding preferences of Baz PDZ3 are more similar to C. elegans versus human PDZ3. Next the peptide binding preferences of each Baz PDZ domain was quantified using single identified peptides in surface plasmon resonance assays. In these direct binding studies, each peptide had a binding preference for a single PDZ domain (although the peptide binding of PDZ2 was weakest and the least specific). PDZ1 and PDZ3 bound their peptides with dissociation constants in the nM range, whereas PDZ2-peptide binding was in the microM range. To test whether tandem PDZ domain organization affects peptide binding, a fusion protein containing all three PDZ domains and their normal linker regions was examined. The binding strengths of the PDZ-specific peptides to single PDZ domains and to the PDZ domain tandem were indistinguishable. Thus, the peptide binding pockets of each PDZ domain in Baz are not obviously affected by the presence of neighbouring PDZ domains, but act as isolated modules with specific in vitro peptide binding preferences (Yu, 2014).

PI(4,5)P2 produced by the PI4P5K SKTL controls apical size by tethering PAR-3 in Drosophila epithelial cells

The control of apical-basal polarity in epithelial layers is a fundamental event in many processes, ranging from embryonic development to tumor formation. A key feature of polarized epithelial cells is their ability to maintain an asymmetric distribution of specific molecular complexes, including the phosphoinositides PI(4,5)P2 and PI(3,4,5)P3. The spatiotemporal regulation of these phosphoinositides is controlled by the concerted action of phosphoinositide kinases and phosphatases. Using the Drosophila follicular epithelium as a model system in vivo, this study shows that PI(4,5)P2 is crucial to maintain apical-basal polarity. PI(4,5)P2 is essentially regulated by the PI4P5 kinase Skittles (SKTL), whereas neither the phosphatase PTEN nor the PI(4,5)P3 kinase DP110 lead to loss of apical-basal polarity. By inactivating SKTL and thereby strongly reducing PI(4,5)P2 levels in a single cell of the epithelium, the disassembly was observed of adherens junctions, actin cytoskeleton reorganization, and apical constriction leading to delamination, a process similar to that observed during epithelial-mesenchymal transition. Evidence is provided that PI(4,5)P2 controls the apical targeting of PAR-3/Bazooka to the plasma membrane and that the loss of this polarized distribution is sufficient to induce a similar cell shape change. Finally, it was shown that PI(4,5)P2 is excluded from the cell apex and that PAR-3 diffuses laterally just prior to the apical constriction in a context of endogenous invagination. All together, these results indicate that the PIP5 kinase SKTL, by controlling PI(4,5)P2 polarity, regulates PAR-3 localization and thus the size of the apical domain (Claret, 2014).

The results indicate that PI(4,5)P2 distribution is critical for BAZ tethering and AJ apical localization control in order to regulate apical constriction in the follicular epithelium. As PI(4,5)P2 is the major source of PI(3,4,5)P3, a joint implication of PI(3,4,5)P3 in this process cannot be ruled out. However, the results indicate that PI(3,4,5)P3 alone is not implicated during the constriction in anterodorsal cells (ADCs). The apical levels of PI(3,4,5)P3 increase contrary to what is expected if it was jointly implicated together with PI(4,5)P2. Furthermore, the overexpression of SKTL modulates PI(4,5)P2 levels in ADCs without changing the level of PI(3,4,5)P3. Hence, altogether and in accordance with previous observations in other Drosophila tissues, the results suggest that PI(4,5)P2 is the important phosphoinositide during the regulation of apical constriction. Interestingly, the results further reveal an efficient regulatory process that tightly limits the apical amount of SKTL, especially during tubulogenesis (Claret, 2014).

Drosophila or mammalian PAR-3/BAZ has been shown to bind to a broad spectrum of phosphoinositides in vitro. This paper reports that in vivo, PI(4,5)P2 is crucial for BAZ membranous localization, but that PI(3,4,5)P3 is dispensable. AJs are also affected by PI(4,5)P2 depletion, though potentially indirectly. In fact, it has been shown that BAZ and the actin cytoskeleton are two major contributors to AJ stability (Claret, 2014).

Previously work has shown that in the Drosophila oocyte, SKTL is required for BAZ and PAR-1 localization to the cortex. However, the mutual exclusion between PAR-1 and BAZ prevented assessing which one is delocalized first. In FCs at stage 9/10, the apical-basal polarity is very stable, perhaps because of AJs. In these cells, evidence is provided that PI(4,5)P2 specifically affects BAZ, but not PAR-1, and that BAZ localization relies mostly on the PI4P5K SKTL, rather than the PI3 phosphatase PTEN. The results, together with previous biochemical studies, suggest that PI(4,5)P2 is crucial for BAZ apical localization (Claret, 2014).

In Drosophila, the function of MOE with regards to AJs seems to vary depending on the tissue. In the wing disc, moe mutant cells can be extruded basally from the pseudoepithelium, suggesting than in this tissue MOE is important for epithelium integrity. However, in photoreceptor cells, MOE is dispensable for AJ maintenance and is rather important for apical morphogenesis. In FCs, MOE inactivation upon PI(4,5)P2 removal or MOE depletion has no effect on apical domain size and epithelium integrity. Therefore, the delamination of PI(4,5)P2 depleted cells seems to be independent of MOE (Claret, 2014).

It is proposed that the apical downregulation of PI(4,5)P2 during dorsal appendage morphogenesis may be crucial for the onset of tubulogenesis, when cells should constrict their apices in order to allow epithelial invagination. Indeed, a more drastic downregulation of PI(4,5)P2 by inactivation of SKTL triggers columnar epithelial cells to adopt a bottle shape before delamination, resembling an EMT. It should be noted that an active participation of wild-type neighboring cells in the delamination of sktl mutant cells cannot be excluded. Surprisingly, during the first steps of tubulogenesis, phospholipid polarity is totally reversed, with PI(3,4,5)P3 apical and PI(4,5)P2 lateral. This inversion does not appear to be the simple outcome of DP110 activation and production of PI(3,4,5)P3 from PI(4,5)P2 at the apex. Therefore, another process must contribute to the decrease in PI(4,5)P2 at the apical membrane. Interestingly, the PI(4,5)P2 phosphatase OCRL interacts with clathrin-coated pits and the GTPase RAB5, which is particularly enriched in the subapical region in ADCs. Thus, it is possible that endocytosis of PI(4,5)P2 leads to its apical exclusion. Likewise, this may involve a concomitant downregulation of the activity of the PI4P5K SKTL (Claret, 2014).

Finally, evidence is provided that the level of PI(4,5)P2 required for polarity maintenance in Drosophila epithelia is controlled by the PI4P5K SKTL, rather than by the tumor-suppressor gene PTEN. These results suggest that the control of PI4P5K activity is important during normal developmental but could also be implicated in pathological processes such as metastasis (Claret, 2014).

A Par-1-Par-3-centrosome cell polarity pathway and its tuning for isotropic cell adhesion

To form regulated barriers between body compartments, epithelial cells polarize into apical and basolateral domains and assemble adherens junctions (AJs). Despite close links with polarity networks that generate single polarized domains, AJs distribute isotropically around the cell circumference for adhesion with all neighboring cells. How AJs avoid the influence of polarity networks to maintain their isotropy has been unclear. In established epithelia, trans cadherin interactions could maintain AJ isotropy, but AJs are dynamic during epithelial development and remodeling, and thus specific mechanisms may control their isotropy. In Drosophila, aPKC prevents hyper-polarization of junctions as epithelia develop from cellularization to gastrulation. This study shows that aPKC does so by inhibiting a positive feedback loop between Bazooka (Baz)/Par-3, a junctional organizer, and centrosomes. Without aPKC, Baz and centrosomes lose their isotropic distributions and recruit each other to single plasma membrane (PM) domains. Surprisingly, loss- and gain-of-function analyses show that the Baz-centrosome positive feedback loop is driven by Par-1, a kinase known to phosphorylate Baz and inhibit its basolateral localization. Par-1 was found to promote the positive feedback loop through both centrosome microtubule effects and Baz phosphorylation. Normally, aPKC attenuates the circuit by expelling Par-1 from the apical domain at gastrulation. The combination of local activation and global inhibition is a common polarization strategy. Par-1 seems to couple both effects for a potent Baz polarization mechanism that is regulated for the isotropy of Baz and AJs around the cell circumference (Jiang, 2015).

The identification of Par-1 as an inducer of Baz-centrosome co-recruitment is surprising given its well-established role in inhibiting Baz complex formation in Drosophila, C. elegans, and mammalian systems. It is proposed that Par-1 contributes to both global inhibition and local promotion of Baz complex assembly, providing a simple and potent Baz polarization mechanism (Jiang, 2015).

The Baz-centrosome positive feedback loop is evident from the specific accumulation of Baz next to cortical centrosomes, the MT requirement for Baz accumulation, the Baz requirement for centrosome recruitment, and the dynein role for drawing Baz and centrosomes together. Significantly, Par-1 is also necessary and sufficient for the loop and seems to have two direct roles. One is promotion of astral microtubules around the centrosome, an effect consistent with known effects of Par-1 on MT regulators, but requiring further elucidation in the Drosophila embryo. The other is the phosphorylation of Baz at Ser-151 and Ser-1085. These modifications have well-characterized inhibitory effects on Baz cortical association, but strikingly, they are also enriched where the Baz-centrosome positive feedback loop occurs and appear necessary for Baz entry into the loop. It is speculated that phospho-regulated Baz-14-3-3 protein interactions mediate further protein interactions, or induce conformational changes, important for Baz-MT association. Indeed, 14-3-3 proteins can bridge MT motors, a Par-3 conformational change induces direct MT binding, Par-3 directly binds a dynein subunit, and other links to MTs are known (Jiang, 2015).

Although the Par-1-Par-3-centrosome pathway can be a potent Baz polarization mechanism, it is normally attenuated within a homeostatic system. During early cellularization, Par-1 localizes over the entire PM and presumably phosphorylates Baz and MT regulators. In response, it is proposed that Baz is continually displaced and diffuses over the PM but is additionally primed for MT interactions. Simultaneously, the two centrosomes found atop each nucleus would provide the positional information for localizing Baz around the apical circumference through dynein-mediated MT associations. As Baz accumulates, it recruits aPKC to the apical domain, from where aPKC then displaces Par-1. Normally, this Baz-aPKC-Par-1 negative feedback loop seems to keep the Par-1-Baz-centrosome pathway in check. In the absence of aPKC, the Par-1- Baz-centrosome pathway continues unabated, leading to excessive Baz and centrosome polarization, loss of AJ isotropy, and later epithelial dissociation (Jiang, 2015).

Intriguingly, focused accumulations of Par-3 and AJs colocalize with cortical centrosomes during C. elegans intestinal development and during zebrafish collective cell migration. Moreover, Par-1 induces centrosomal MT interactions with AJs during human liver lumen formation in vitro and is needed for Baz-centrosome associations during the asymmetric division of Drosophila germline stem cells. Thus, induction of the Par- 1-Par-3-centrosome pathway, with regulated shifts to aPKC or Par-1 activities, may be generally relevant to developmental transitions of animal tissues (Jiang, 2015).

Drosophila MAGI interacts with RASSF8 to regulate E-Cadherin-based adherens junctions in the developing eye

Morphogenesis is crucial during development to generate organs and tissues of the correct size and shape. During Drosophila late eye development, interommatidial cells (IOCs) rearrange to generate the highly organized pupal lattice, in which hexagonal ommatidial units pack tightly. This process involves the fine regulation of adherens junctions (AJs) and of adhesive E-Cadherin (E-Cad) complexes. Localized accumulation of Bazooka (Baz), the Drosophila PAR3 homolog, has emerged as a critical step to specify where new E-Cad complexes should be deposited during junction remodeling. However, the mechanisms controlling the correct localization of Baz are still only partly understood. This study shows that Drosophila Magi, the sole fly homolog of the mammalian MAGI scaffolds, is an upstream regulator of E-Cad-based AJs during cell rearrangements, and that Magi mutant IOCs fail to reach their correct position. They uncovered a direct physical interaction between Magi and the Ras association domain protein RASSF8 through a WW domain-PPxY motif binding, and showed that apical Magi recruited the RASSF8-ASPP complex during AJ remodeling in IOCs. Further, this Magi complex was required for the cortical recruitment of Baz and of the E-Cad-associated proteins α- and β-catenin. They propose that, by controlling the proper localization of Baz to remodeling junctions, Magi and the RASSF8-ASPP complex promote the recruitment or stabilization of E-Cad complexes at junction sites (Zaessinger, 2015).

As Magi is the sole Drosophila homolog of the three vertebrate MAGI scaffolds, it offers a powerful system with which to investigate the functions of these important proteins. Using newly generated null alleles, this study has shown that Magi coordinates the number and packing of IOCs in the developing Drosophila pupal eye by regulating AJ dynamics. Magi is necessary in the IOCs to localize the RASSF8-ASPP complex correctly during their junctional remodeling. This ensures the integrity of E-Cad-based junctions and the correct localization of Baz, α- and β-catenin. Based on these observations and on the growing evidence of a role for Baz in AJ remodeling, a model is proposed whereby, during AJ remodeling in IOCs, Magi recruits the RASSF8-ASPP complex, which helps to localize Baz at the membrane and regulates the sites of E-Cad accumulation (Zaessinger, 2015).

Junction remodeling is a key step during morphogenesis, in which cells in a tissue change position and neighbors. For instance, in the developing pupal eye, IOCs found between ommatidia organize as a single row of cells. During this process existing contacts are eliminated and new ones are established by remodeling E-Cad-based junctions. In Magi mutants, rearrangement defects and some incorrect localization of IOCs were observed. At the same time, E-Cad-based AJs were interrupted in Magi mutant cells. It is proposed that this defect in AJ remodeling leads to IOCs remaining at the wrong place in the lattice. The most parsimonious model is that the defects in AJ remodeling trigger the defects in cell numbers seen in Magi mutants by preventing apoptosis, although it was not possible to fully substantiate this as the effect of Magi on apoptosis was not statistically significant. If the model is correct, it still remains unclear how disrupted junctions would lead to a failure in apoptosis. One possibility is that IOCs only receive the correct 'death signal' when they have rearranged to contact the correct cells. Thus, in Magi mutants, the defective AJs would lead to apoptosis failure because the IOCs did not attain their position in the 'death zone' to receive the killing signal (Zaessinger, 2015).

These junctional defects are reminiscent of those seen for magi-1 mutants in the nematode C. elegans, in which magi-1 loss of function enhanced the defects caused by cadherin and catenin mutations and disrupted cell migration during enclosure (Lynch, 2012). MAGI scaffolds are thus implicated in the fine regulation of AJs in both flies and nematodes. A similar role has been suggested for MAGI proteins in mammalian epithelial cells. In overexpression studies, human MAGI1 reduced the Src-induced invasiveness of MDCK cells and stabilized E-Cad-mediated intercellular aggregation (Kotelevets, 2005). By analogy, the overexpression phenotype of Drosophila Magi could thus be due to stronger AJs, although this remains to be experimentally tested. The overexpression effects of MAGI-1b were sensitive to PTEN and AKT activities (Kotelevets, 2005) and mammalian MAGI scaffolds have also been implicated in PTEN activation through their direct binding to PTEN. However, this study did not detect any physical interaction between Drosophila Magi and Pten, and the overexpression phenotype of Magi, at least in the Drosophila eye, appeared insensitive to Pten. Although these are negative observations, they suggest that in Drosophila Magi and Pten do not form a complex to regulate AJs (Zaessinger, 2015).

Despite its effects on eye development, Magi mutants exhibit slightly enlarged wings. Whether this is dependent on E-Cad belt integrity and AJ dynamics remains to be established. The fact that ASPP shows a very similar wing phenotype supports this model (Zaessinger, 2015).

Rather than binding and modulating the activity of Pten, this analysis supports a model whereby Magi, by binding to the RASSF8-ASPP complex, recruits and stabilizes Baz at the membrane. Accumulation of Baz has been shown to specify and initiate the formation of new AJs both in cellularizing embryos and in photoreceptors. It is proposed that Baz recruited at the membrane of IOCs will in turn promote the stabilization or the proper distribution around the cell cortex of AJ material. Since biochemical and genetic experiments suggest that RASSF8 and Magi act together in a complex, it is proposed that the effects of Magi on AJs and on Baz membrane recruitment are mediated by RASSF8, and are thus likely to involve ASPP. Indeed, mammalian ASPP2 binds PAR3 and is required for PAR3 localization at junctions both in cell culture and in the mouse neuroepithelium. This suggests that Magi might control Baz localization through ASPP. However, Baz membrane recruitment is unlikely to be the only step to form correct AJs downstream of Magi/RASSF8/ASPP. Previous studies have implicated C-terminal Src kinase (Csk) and its action on Src kinase, and the relationships between Magi, Baz and Csk should be investigated in the future (Zaessinger, 2015).

During IOC remodeling, Magi therefore appears to be a crucial upstream regulator of AJs. However, the mechanisms governing Magi membrane localization are still unknown. One hypothesis is that the membrane recruitments of different AJ components and regulators are dependent on each other in stabilization loops. However, this is unlikely to be the case for Magi as it is still perfectly localized at the membrane in ASPP, RASSF8 and baz mutants, and in ASPP; RASSF8 double mutants (Zaessinger, 2015).

Another possibility is that Magi would require mature AJs with E-Cad to be at the membrane. No direct correlation was found between E-Cad accumulation around the apical membrane and Magi membrane localization. For instance, in ASPP; RASSF8 double-mutant cells, E-Cad belt interruptions were detected either without or with Magi, indicating that Magi localization does not require E-Cad directly. Furthermore, an extensive domain mapping of Magi failed to identify a single domain (WW or PDZ) that would be required for Magi recruitment, suggesting that it might be independent of these domains or that several redundant mechanisms may be at play. The nature of the signal required for Magi membrane localization thus remains to be uncovered (Zaessinger, 2015).

Even though Magi binds to RASSF8 directly and both proteins function together during Drosophila eye morphogenesis, their mutant phenotypes are not identical. First, RASSF8 mutants have a wing rounding phenotype, which is absent in Magi mutants. Second, whereas RASSF8 has a significant role in the global developmental apoptosis rate in the pupal eye, no significant effect could be detected for Magi and ASPP. Taken together, this suggests that the assembly of a Magi-RASSF8-ASPP complex might be context dependent or that RASSF8 has Magi-independent functions (Zaessinger, 2015).

Although the human N-terminal RASSF (RASSF7-10) proteins lack any PPxY motifs, one is present in ASPP2 and has been shown to bind to MAGI1. It is therefore possible that MAGI-ASPP complexes are formed in all organisms but the precise mode of interaction differs: mediated by RASSF8 in the fly, but direct in humans (Zaessinger, 2015).

MAGI scaffolds have been suggested to play a role in tumorigenesis. First, they are bound and inactivated by several viral oncoproteins. Second, MAGI1 has been shown to exhibit tumor suppressor activity in colorectal cancer cell lines in xenograft model. Finally, mutations in MAGI2 and MAGI3 are reported in colon, prostate and breast cancers. Documented alterations include deletion of the second WW motif of MAGI2 and a MAGI3:AKT3 fusion leading to a disruption of MAGI3. Based on the current work, it is proposed that these are loss-of-function mutations. It would be interesting to investigate whether changes in AJ dynamics are associated with these MAGI mutations in human cancers and whether they contribute to tumorigenesis (Zaessinger, 2015).

Magi is associated with the Par complex and functions antagonistically with Bazooka to regulate the apical polarity complex

The mammalian MAGI proteins play important roles in the maintenance of adherens and tight junctions. The MAGI family of proteins contains modular domains such as WW and PDZ domains necessary for scaffolding of membrane receptors and intracellular signaling components. Loss of MAGI leads to reduced junction stability while overexpression of MAGI can lead to increased adhesion and stabilization of epithelial morphology. However, how Magi regulates junction assembly in epithelia is largely unknown. This study investigated the single Drosophila homologue of Magi to study the in vivo role of Magi in epithelial development. Magi is localized at the adherens junction and forms a complex with the polarity proteins, Par3/Bazooka and aPKC. A Magi null mutant was generated and found to be viable with no detectable morphological defects even though the Magi protein is highly conserved with vertebrate Magi homologues. However, overexpression of Magi results in the displacement of Baz/Par3 and aPKC and leads to an increase in the level of PIP3. Interestingly, it was found that Magi and Baz function in an antagonistic manner to regulate the localization of the apical polarity complex. Maintaining the balance between the level of Magi and Baz is an important determinant of the levels and localization of apical polarity complex (Padash Barmchi, 2016).

A common component of junctional and polarity complexes is modular scaffolding proteins that are capable of binding to each other as well as recruiting other proteins to the complex. Magi proteins are evolutionarily conserved scaffolding proteins and contain multiple domains including a N-terminal catalytically inactive GUK domain, two WW domains and five to six PDZ (PSD95/Dlg/ZO-1) domains (Dobrosotskaya, 1997). There are three MAGI proteins in vertebrates (MAGI-1,2,3) all with multiple splice isoforms. MAGI-1 and MAGI-3 are relatively ubiquitously expressed and localize to a range of junctions including epithelial tight junctions. MAGI-2 (also known as AIP1/S-SCAM/ARIP1) is expressed in the nervous system as a synaptic protein and within glomerular podocytes in the kidney and plays important role in scaffolding synaptic proteins such as NMDA receptors and Neuroligin, the tip-link protocadherin Cadherin23, the Kir4.1 K(+) channel, as well as kidney proteins such as nephrin and JAM4 (Padash Barmchi, 2016).

Within epithelia and endothelia, MAGI-1 and -3 are localized at tight junctions and form a structural scaffold for the assembly of junctional complexes. MAGI-1 also localizes and plays a role in modulating adherens junction adhesion through scaffolding beta-catenin and PTEN. MAGI-1 overexpression stabilizes adherens junctions and epithelial cell morphology through increased E-cadherin and β-catenin recruitment. Silencing of MAGI-1 has the opposite effect with decreased adherens junction adhesion and reduced focal adhesion formation leading to anchorage-independent growth and migration in vitro. MAGI-1 overexpression suppresses the invasiveness of MDCK cells, as well as suppresses tumor growth and spontaneous lung metastasis through the increased recruitment of PTEN or β-catenin and E-cadherin (Padash Barmchi, 2016).

Overall, MAGI proteins play important roles in the stabilization of cell-cell interactions and as such Magi is a key target in polarized epithelia during cell death and viral infection. For instance, MAGI-1 is cleaved by activated caspases during apoptosis, a process thought to mediate the disassembly of cell-cell contacts (Gregorc, 2007). MAGI proteins are also targeted by a number of oncogenic viruses: it is aberrantly sequestered in the cytoplasm by Adenovirus E4orf1, and is targeted for degradation by the E6 oncoprotein of high-risk human papillomavirus. E6-mediated degradation of MAGI-1 in cultured epithelial cells leads to loss of tight-junction integrity (Padash Barmchi, 2016 and references therein).

There is a high degree of conservation of protein structure and function in the invertebrate homologues of Magi in particular with regards to epithelial junction formation and maintenance. In C. elegans, Magi-1 plays a role in the segregation of different cell adhesion complexes into distinct membrane domains along the lateral plasma membrane. In Drosophila, Magi binds Ras association domain protein 8 (RASSF8) and modulates adherens junctions remodeling in late eye development during interommatidial cell (IOC) rearrangements. In this context Magi function is necessary to recruit the polarity protein Par-3 (Drosophila Bazooka, Baz) to the remodeling adherens junction. However, the association of Drosophila Magi or any Magi homologue with any components of the Par polarity complex in stable epithelia has not been determined (Padash Barmchi, 2016).

The Par complex consisting of Par-3/Par-6/aPKC localizes to tight junctions where MAGI is present in vertebrate epithelial cells and is necessary for assembly of this junctional complex as well as for separation of the apical region of the plasma membrane from the basolateral domain. In Drosophila epithelial cells, the Par complex localizes to the apicolateral membrane and demarcates the boundary between the apical and basolateral membrane regions. Mutant embryos for any member of this complex show loss of apicobasal polarity and disruption in the integrity of epithelia. Although the members of the Par complex are important for the establishment of cell polarity, some of the core components of this complex such as Baz are dispensable for the maintenance of cell polarity during later stages of development. Baz localizes to adherens junction and mutant clones of baz in wing imaginal discs are fully viable with no polarity or adherens junction defects. Similarly, Magi function in AJ stability has been determined in many systems, but surprisingly loss of Drosophila Magi has no effect on established, stable AJs (Zaessinger, 2015). Little is known about the convergence of Magi and Par complex function at the adherens junctions and it is possible that Baz and Magi function in established epithelia are redundant. Therefore this study investigated the role of Magi in the established and stable epithelia of the Drosophila wing imaginal disc to test the potential interactions between Magi and members of the Par complex (Padash Barmchi, 2016).

Drosophila Magi was found associated with the PAR polarity complex and is localized at the adherens junction with Baz, Par-6, and aPKC. Overexpression of Magi resulted in the reduction of apical polarity proteins from the membrane and these interactions required the second half of the Magi protein containing the four PDZ domains. Overexpression of Baz resulted in a reduction of Magi from the membrane but an increase in aPKC and Par-6. While Magi mutants were viable with no polarity defects, Magi levels were found to be antagonistic with Baz, and a balance between the two was found to be necessary to regulate the level and localization of Par complex (Padash Barmchi, 2016).

PDZ domain-containing proteins form scaffolding protein complexes with a wide range of roles including cell polarity and signaling. As a MAGUK protein, Magi is part of a scaffold that interacts with members of the polarity complex at the adherens junctions in the epithelia of the imaginal disc. The scaffolding function of Magi has been well established in other systems. In vertebrates epithelial cells MAGI-1 has been shown to act as structural scaffold at tight junctions and adherens junctions. In C. elegans, Magi-1 localizes apical to adherens junction and functions as an organizer to ensure that different cell adhesion complexes are segregated into distinct membrane domains along the lateral plasma membrane. In neuronal cells MAGI-2/S-SCAM was also shown to cluster the cell adhesion molecule Sidekick, and the AMPA and NMDA glutamate receptors at the synapse (Padash Barmchi, 2016).

Given the strong conservation of the Magi protein it is surprising that null mutants of Drosophila Magi exhibit no lasting cellular defects (other than transient defects in the interommatidial cells of the pupal eye and null animals are fully viable. Similarly in C. elegans, magi-1 null worms are healthy with only a few embryos (1.3%) with defects during the ventral enclosure stage. As Magi is highly conserved, it is plausible that Magi may only act in response to cell stress, DNA damage or some other trigger. For example, loss of p53 does not disrupt cellular function under normal conditions and p53 null flies or mice are viable with no cellular defects. However, the role of p53 in response to DNA damage is well established and when these animals are exposed to irradiation apoptosis is not induced. Alternatively, Magi function might be redundant with other components of the apical polarity complex or another protein and that loss of both is necessary for the disruption of cellular function. Core scaffolding components of the apicobasal polarity complex are dispensable for maintaining polarity in the wing imaginal disc epithelia supporting the idea of redundancy in this system. For instance, somatic clones of loss of function mutations in crb, sdt and baz have no effect on the polarity in the wing disc epithelia of the 3rd instar larvae. Baz is a strong candidate for redundancy with Magi given the localization to the adherens junction and function as a PDZ scaffolding protein. As loss of baz in the wing imaginal disc does not disrupt the polarity of wing disc epithelia this leads to the hypothesis that Baz and Magi are redundant. However, somatic clones of a baz null mutant in a Magi mutant background did not lead to a loss of cell polarity or apoptosis. While the two scaffolding proteins do not appear to functionally interact, it was observed that Magi and Baz are in a protein complex and their close proximity within the wing columnar epithelia also suggests a common complex. Overexpression of Magi displaces Baz and aPKC from the apical membrane and, likewise overexpression of Baz displaces Magi from the membrane. The simultaneous over-expression of Magi and Baz suppresses the changes caused by their individual expression, suggesting a balance or competition between the two proteins. The maintenance of a balance between Magi and Baz might be due to a direct physical competition between these two proteins or opposite effects on a common mediator or interactor (Padash Barmchi, 2016).

Baz and vertebrate MAGI proteins bind the lipid phosphatase PTEN and thus the Magi-Baz interaction and balance could be influenced by changes in the level of phosphoinositides such as PtdIns(4,5)P2 (PIP2) or PtdIns(3,4,5)P3 (PIP3). In polarized epithelia, PIP2 is found within the apical domain and PIP3 restricted to the basal-lateral domain. Baz localization in polarized epithelia depends on PIP2 and on the PI4P5 kinase Skittles. Baz in turn can be a positive regulator of PIP2 levels at the plasma membrane by local recruitment of the lipid phosphatase PTEN. This study observed an increase in PIP3 levels with increased expression of Magi, which may reflect the loss of Baz and a loss of PTEN recruitment to the membrane. This study was not able to assess changes in PTEN levels at the membrane with available antibodies. However it was observe that the recruitment of Magi or Baz was not affected in Pten mutant cells. Similarly the changes in PIP3 levels are unlikely to be the cause of Baz loss in the presence of increased Magi as co-expression of PTEN and Magi still resulted in the loss of Baz from the membrane. Prior studies on Magi in Drosophila in the pupal eye did not detect any physical interaction between Drosophila Magi and Pten, and the phenotypes generated by overexpression of Magi in the Drosophila eye were not affected by Pten mutants. Therefore it is likely that loss of Baz in the presence of increased Magi in the wing imaginal disc and vice versa is through competition for a protein component (Padash Barmchi, 2016).

In the developing eye Magi forms a protein complex with RASSF8 (the N-terminal Ras association domain-containing protein) and ASPP (Ankyrin-repeat, SH3-domain, and proline-rich-region containing protein), and this complex plays a role during remodeling of the adherens junctions in the interommatidial cells (IOCs) (Zaessinger, 2015). When IOCs rearrange to create the pupal lattice, this process requires regulation of the E-Cadherin complex where RASSF8 and ASPP regulate adherens junction remodeling and integrity through regulation of Src kinase activity. Magi recruits the RASSF8-ASPP complex in the process of adherens junction remodeling and there are defects in IOC rearrangement in Magi mutants where AJs are frequently interrupted. In the eye the Magi-RASSF8-ASPP complex is necessary for the cortical recruitment of Baz and of the adherens junction proteins α- and β-catenin. A model has been proposed where Magi-RASSF8-ASPP complex functions to localize Baz to remodeling junctions to promote the recruitment or stabilization of E-Cad complexes (Zaessinger, 2015). However, it is not thought that the RASSF8-ASPP complex is the point of competition between Magi and Baz within the wing imaginal disc. In the wing imaginal disc Magi and the RASSF8-ASPP complex are localized to the adherens junction domain independently (Zaessinger, 2015) and while RASSF8 mutants have a wing rounding phenotype, Magi mutants do not. Furthermore no differences were observed in Baz, Ecad or Arm distribution in Magi somatic loss of function clones in the wing imaginal disc. Finally the Magi WW domains are required for the interaction with RASSF8 (Zaessinger, 2015), while the overexpression of the Magi transgene that contains the PDZ domains led to a reduction in Baz suggesting that second half of the Magi protein containing the PDZ domains contains the important sites for this competition (Padash Barmchi, 2016).

Therefore, a strong possibility to explain the reciprocal effects of overexpression is that Baz and Magi compete for a common binding site. Magi was found to interacte with both Baz and aPKC; the latter two are known to interact directly. However, it is unlikely that the shared site is through physical scaffolding of aPKC, as high levels of wild type aPKC had no effect on either Magi or Baz and was not able rescue the changes in Baz levels and localization caused by Magi overexpression. In addition the overexpression of Magi also led to a reduction in aPKC. It is unlikely that the loss of Baz is responsible for this displacement as aPKC is not mislocalized in Baz clones and Baz is not mislocalized in Par-6, aPKC or Cdc42 null clones. Further investigation is required to explore the mechanisms that underlie Magi interactions with components of the apical polarity complex and the adherens junction complex (Padash Barmchi, 2016).

Meru couples planar cell polarity with apical-basal polarity during asymmetric cell division

Polarity is a shared feature of most cells. In epithelia, apical-basal polarity often coexists, and sometimes intersects with planar cell polarity (PCP), which orients cells in the epithelial plane. From a limited set of core building blocks (e.g. the Par complexes for apical-basal polarity and the Frizzled/Dishevelled complex for PCP), a diverse array of polarized cells and tissues are generated. This suggests the existence of little-studied tissue-specific factors that rewire the core polarity modules to the appropriate conformation. In Drosophila sensory organ precursors (SOPs), the core PCP components initiate the planar polarization of apical-basal determinants, ensuring asymmetric division into daughter cells of different fates. This study shows that Meru, a RASSF9/RASSF10 homologue, is expressed specifically in SOPs, recruited to the posterior cortex by Frizzled/Dishevelled, and in turn polarizes the apical-basal polarity factor Bazooka (Par3). Thus, Meru belongs to a class of proteins that act cell/tissue-specifically to remodel the core polarity machinery (Banerjee, 2017).

Polarity is a fundamental feature of most cells and tissues. It is evident both at the level of individual cells and groups of cells (e.g. planar cell polarity (PCP) in epithelia. However, despite the fact that different cell types use a common set of molecules to establish and maintain polarity (Par complexes, Fz-PCP pathway), the organization of polarized cells and cell assemblies varies dramatically across different species and tissues. This implies the existence of factors that act in a cell or tissue-specific manner to modulate/rewire the core polarity machinery into the appropriate organization. Despite many advances in understanding of polarity in unicellular and multicellular contexts, little is known about the identity or function of such factors (Banerjee, 2017).

An example of polarity remodeling is the process of asymmetric cell division (ACD), where cells need to rearrange their polarity determinants into a machinery capable of asymmetrically segregating cell fate determinants, vesicles and organelles, as well as controlling the orientation of the mitotic spindle. ACDs result in two daughter cells of different fates and occur in numerous cell types and across species. Well-studied examples include budding in Saccharomyces cerevisiae, ACD in the early embryo of Caenorhabditis elegans, or ACD of progenitor cells in the mammalian stratified epidermis and neural stem cells in the mammalian neocortex. In Drosophila melanogaster, the study of germline stem cells, neuroblasts (neural stem cells) and sensory organ precursors (SOPs) has greatly contributed to understanding of the cell biology and molecular mechanisms of ACD (Banerjee, 2017).

SOPs (or pI cells) divide asymmetrically within the plane of the epithelium into pIIa and pIIb daughter cells. pIIa and pIIb themselves divide asymmetrically to give rise to the different cell types of the external sensory organs (bristles), which are part of the peripheral nervous system and allow the adult fly to sense mechanical or chemical stimuli. Individual SOPs are selected by Notch-dependent lateral inhibition from multicellular clusters of epithelial cells expressing proneural genes (proneural clusters) (Banerjee, 2017).

The unequal segregation of cell fate determinants (the Notch pathway modulators Numb and Neuralized), which specifies the different fates of the daughter cells, requires their asymmetric localization on one side of the cell cortex prior to mitosis. This is achieved by remodeling the PCP and apical-basal polarity systems in the SOP, and by orienting the spindle relative to the tissue axis. The epithelial sheet that forms the pupal notum (dorsal thorax), where the best-studied SOPs are located, is planar polarized along the anterior-posterior tissue axis, with the transmembrane receptor Frizzled (Fz) and its effector Dishevelled (Dsh) localizing to the posterior side of the cell cortex, while the transmembrane protein Van Gogh (Vang, also known as Strabismus) and its interactor Prickle (Pk) are found anteriorly. The apical-basal polarity determinants central to SOP polarity are the PDZ domain-containing scaffold protein Bazooka (Baz, or Par3), atypical Protein Kinase C (aPKC) and Partitioning defective 6 (Par6), which localize apically in epithelial cells and the basolaterally localized membrane-associated guanylate kinase homologues (MAGUK) protein Discs-large (Dlg). In most epithelial cells, these proteins localize uniformly around the cell cortex, whereas in SOPs they show a striking asymmetric localization during mitosis: the Baz-aPKC-Par6 complex is found at the posterior cell cortex, opposite an anterior complex consisting of Dlg, Partner of Inscuteable (Pins) and the G-protein subunit Gαi. The Fz-Dsh complex provides the spatial information for the Baz-aPKC-Par6 complex, while Vang-Pk positions the Dlg-Pins-Gαi complex (likely through direct interaction between Vang and Dlg). The asymmetric distribution of the polarity determinants then directs the positioning of cell fate determinants at the anterior cell cortex. Additionally, Fz-Dsh and Pins orient the spindle along the anterior-posterior axis by anchoring it on both sides of the cell via Mushroom body defective (Mud, mammalian NuMA) and Dynein (Banerjee, 2017).

The planar symmetry of the Baz-aPKC-Par6 complex in SOPs is initially broken in interphase via Fz-Dsh, and is independent of the Dlg-Pins-Gαi complex. Once this initial asymmetry is established, the core PCP components become dispensable for Par complex polarization at metaphase due to the mutual antagonism between the opposing polarity complexes, which then maintains asymmetry during cell division. Indeed, Baz is still polarized in fz mutants during mitosis, but losing both pins and fz results in Baz spreading uniformly around the cortex. Crucially, it is unclear how Fz-Dsh can transmit planar information to the Baz-aPKC-Par6 complex in SOPs but not in neighboring epithelial cells. The cell-type dependent coupling between PCP and apical-basal polarity suggests the involvement of unknown SOP-specific factors in this process (Banerjee, 2017).

The four N-terminal RASSFs (Ras association domain family) in humans (RASSF7-10) have been associated with various forms of cancer, but the exact processes in which these scaffolding proteins act remain mostly elusive. Drosophila RASSF8, the homologue of human RASSF7 and RASSF8, is required for junctional integrity via Baz. Interestingly, human RASSF9 and RASSF10 were found in an interaction network with Par3 (the mammalian Baz homologue) and with several PCP proteins. The Drosophila genes CG13875 and CG32150 are believed to be homologues of human RASSF9 and RASSF10, respectively and remarkably, CG32150 mRNA is highly enriched in SOPs (Banerjee, 2017).

This study shows that Meru, encoded by CG32150, is an SOP-specific factor, capable of linking PCP and apical-basal polarity. Meru localizes asymmetrically in SOPs based on the polarity information provided by Fz/Dsh, and is able to recruit Baz to the posterior cortex (Banerjee, 2017).

PCP provides the spatial information for the initial polarization of SOPs at interphase, resulting in the planar polarization of Baz, which is uniformly localized prior to SOP differentiation. How Fz/Dsh communicate with Baz and enable its asymmetric enrichment was unknown. Based on the current results and previous findings, the following model is proposed for the role of Meru in SOP polarization. Upon selection and specification of SOPs, Meru expression is transcriptionally activated by the AS-C transcription factors (Reeves and Posakony, 2005). At interphase, planar-polarized Fz/Dsh recruit Meru to the membrane and hence direct its polarization. Meru in turn positions and asymmetrically enriches Baz, promoting the asymmetry of aPKC-Par6. Upon entry into mitosis, Meru is also required to retain laterally localized Baz, thus supporting the antagonism between the opposing Dlg-Pins-Gαi and Baz-aPKC-Par6 complexes, ultimately enabling the correct positioning of cell fate determinants (Banerjee, 2017).

The meru mutant cell fate phenotype (bristle duplication or loss) is weaker than the baz loss-of-function phenotype, which results in loss of entire SOPs. This is likely due to two factors: (1) unlike meru mutants, the full baz mutant phenotype is the result of a complete loss of Baz in all cells of the SOP lineage, which is known to cause multiple defects including apoptosis of many sensory organ cells as well as cell fate transformations; (2) since a small amount of Baz is retained at the cortex of some meru mutant cells, it is likely that this residual Baz can still be polarized through the antagonistic activity of Pins at metaphase and thus partially rescues SOP polarization. Indeed, it was observed that reduction of pins or baz levels by RNAi strongly enhanced the meru cell specification phenotype. Conversely, supplying excess levels of Baz in a meru mutant background presumably restores sufficient Baz at the cortex to rescue the meru specification defect, as long as Pins is present to drive asymmetry at mitosis. (Banerjee, 2017).

While a decrease in cortical Baz can account for the cell specification defects in meru mutants, it does not explain the spindle orientation phenotypee. This abnormal spindle alignment could either be due to a decrease in Fz/Dsh levels/activity, or a decrease in the ability of Dsh to recruit the spindle-tethering factor Mud. No gross abnormalities were detected in Fz levels in meru mutants, though the presence of Fz in all neighboring cells would make it difficult to detect subtle decreases in SOPs. Further work will be required to understand Meru's role in spindle orientation (Banerjee, 2017).

Analysis of Meru in Drosophila is in agreement with the association of human RASSF9 and RASSF10 with both Par3 and PCP proteins previously reported. However, while the interaction with Dsh is conserved between the fly and human proteins, the transmembrane protein Vangl1 (the mammalian homologue of Vang), rather than its antagonist Fz was recovered in the mammalian proteomic analysis. This could reflect species-specific differences or altered polarity in the transformed human embryonic kidney 293 cells used for the mammalian work. Although Meru (CG32150) was classified as a potential homologue of RASSF10, alignment of the protein sequences showed similar sequence identities for both human RASSF9 (31%) and RASSF10 (26%). Thus, further functional work on Meru, its Drosophila paralogue CG13875, as well as mammalian RASSF9 and RASSF10 is required to understand the evolutionary and functional relationships between these proteins (Banerjee, 2017).

Little is known about the in vivo functions of either RASSF9 or RASSF10 in other species. Xenopus RASSF10 is prominently expressed in the brain and other neural tissues of tadpoles, potentially indicating a function in neurogenesis, a process where ACDs are known to take place. Interestingly, mouse RASSF9 shows a cell-specific expression in keratinocytes of the skin and loss of RASSF9 results in differentiation defects of the stratified epidermis. Considering that Par3 is required for ACD of basal layer progenitors of the stratified epidermis this raises the exciting prospect that RASSF9 might regulate ACD in the mammalian skin (Banerjee, 2017).

The polarization of cells and tissues is essential for their architecture and ultimately allows them to fulfill their function. The polarity machinery can be considered as a series of modules that are combined in a cell or tissue-specific manner, and hence requires specific factors that can create a polarity network appropriate to each tissue and cell type. This study has identified Meru as an SOP-specific factor, which is able to link PCP (Fz-Dsh) with apical-basal polarity (Baz). The PCP proteins Vang and Pk promote the positioning of the opposing Dlg-Pins-Gαi complex. Although Vang can directly bind to Dlg, the SOP and neuroblast-specific factor, Banderuola (aka Wide Awake) was recently shown to be required for Dlg localization and could thus constitute a link between the two polarity systems on the opposite side of the cortex (Banerjee, 2017).

There is increasing evidence that cell-type specific rewiring of the polarity modules may be a widespread phenomenon. For instance, in different parts of the embryonic epidermis, Baz is planar polarized by Rho-kinase or by the Fat-PCP pathway, while in the retina, Vang is responsible for Baz polarization. Apical-basal polarity can also operate upstream of PCP in some systems, as in Drosophila photoreceptor specification, where aPKC restricts Fz activity by inhibitory phosphorylation in a subset of photoreceptor precursors. Thus, tissue-specific factors are likely to operate in a number of different contexts (Banerjee, 2017).

The interplay between PCP and apical-basal polarity is also evident in other species, as Dishevelled has been reported to promote axon differentiation in rat hippocampal neurons by stabilizing aPKC, while Xenopus Dishevelled is required for Lethal giant larvae (Lgl) basal localization in the ectoderm. Interestingly, both mammalian Par3 and the Vang homologue Vangl2 are required for progenitor cell ACD in the developing mouse neocortex, raising the question as to whether PCP and apical-basal polarity are also connected in mammalian ACDs. It is therefore proposed that tissue-specific factors such as Meru might enable the diversity and plasticity observed across different polarized cells and tissues by rewiring the core polarity systems (Banerjee, 2017).

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

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