BIOLOGICAL OVERVIEW

Planar cell polarity (PCP) is a common feature of many vertebrate and invertebrate epithelia and is perpendicular to the cellular apical/basal (A/B) polarity axis. While apical localization of PCP determinants such as Frizzled (Fz1) is critical for protein function, the relationship 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 (Pals1-associated tight junction protein) 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 Patj produce PCP defects in the eye. During PCP signaling, aPKC and Patj are downregulated, while Bazooka is upregulated, suggesting an antagonistic effect of Bazooka on Patj/aPKC. A model is proposed whereby the Patj/aPKC complex regulates PCP by inhibiting Fz1 in cells where it should not be active (Djiane, 2005).

Cellular polarity is a common feature in a broad variety of cell types in all metazoa. For instance, epithelial cells are polarized along their apical/basal (A/B) axis, with the apical side of the lateral membrane showing structural characteristics, such as close cell-cell contacts or adherens junctions (AJ), making it distinct from the basal side. A key feature in the establishment and maintenance of cell polarity is the segregation of different protein determinants to different regions of a cell. Epithelial cells in Drosophila express several polarizing protein complexes conserved in vertebrates. These are arrayed in an apical to basal order: (1) The Crumbs (Crb), Stardust (Sdt), PALS-1 Associated Tight Junction Protein (Patj) complex which is localized to the apical marginal membrane region. Crb is a transmembrane protein that binds through its intracellular domain the membrane-associated Guanylate kinase protein Sdt. Sdt in turn, binds the 4 PDZ domain protein Patj (Roh, 2002). While mutations in Crb or Sdt result in a failure to maintain A/B polarity in the embryo, mutations in Patj have no reported phenotype, arguing for a dispensable role of Patj in this complex (Pielage, 2003). (2) The Bazooka (Baz, a.k.a. Par-3), Par-6, atypical PKC (aPKC, a.k.a. PKCζ) complex also localized in the apical marginal region. Baz and Par-6 contain PDZ domains and bind aPKC in vertebrates and Drosophila. This complex is critical for epithelial A/B polarity and also for polarity of other cell types such as neuroblasts and oocytes in Drosophila and the C. elegans embryo. (3) Located basally to the AJ is the Scribble (Scrib), Discs large (Dlg), Lethal(2)giant larvae (Lgl) complex, responsible for baso-lateral identity. Mutations in these components result in epithelial disorganization, overgrowth, and an expansion of apical determinants. Although the expression domains of the different complexes are largely resolved, molecular interactions between proteins of distinct complexes exist. For instance, Patj can bind Par-6 in vitro (Nam, 2003), and a Par-6/aPKC complex can bind, phosphorylate, and inactivate Lgl in Drosophila neuroblast (Djiane, 2005 and references therein).

In addition to A/B polarity, many epithelia in vertebrates and invertebrates have a second axis of polarization, generally referred to as planar cell polarity (PCP), perpendicular to the A/B axis. In Drosophila, most external adult epithelial structures show PCP features. This is prominent in the distal orientation of wing hairs, the posterior orientation of bristles on the body wall, and the regular arrangement of ommatidia in the eye. Several conserved genes affecting PCP in most structures have been identified. These core PCP factors include the serpentine receptor Frizzled (Fz1), the cytoplasmic scaffold protein Dishevelled (Dsh), the 4-pass transmembrane protein Strabismus (Stbm; a.k.a. Van Gogh/Vang), the cytoplasmic protein Prickle (Pk), the cadherin Flamingo (Fmi; a.k.a. starry night/stan), and the Ankyrin repeat protein Diego (Dgo) (Djiane, 2005).

In the fly eye, PCP is established in the third instar eye imaginal disc posterior to the morphogenetic furrow (MF). Within developing ommatidial preclusters, the R3/R4 photoreceptor precursors are critical for PCP generation. The precursor closer to the midline (equator) of the eye field is thought to have higher Fz1 activity and will become R3; the neighboring polar cell gets induced as R4. Subsequently, the clusters rotate 90° (clockwise or counterclockwise) toward the midline. The R3/R4 cell fate decision is later translated into distinct chiral ommatidial forms in the dorsal and ventral halves of the adult eye (Djiane, 2005).

Similar to A/B polarity establishment, PCP protein complexes become asymmetrically localized during PCP generation. They are initially evenly distributed around the apical cortex of all eye disc cells and subsequently become enriched specifically in the critical R3/R4 pair posterior to the MF. As a result of interactions between PCP determinants, an asymmetry along the R3/R4 cell border is established, with Fz1, Dsh, and Dgo being found on the R3 side and Stbm and Pk only on the R4 side, reminiscent to similar events occurring during PCP generation in pupal wing cells (Djiane, 2005).

All core PCP proteins are located apically, suggesting that their function requires A/B polarity. The partial overlap between PCP proteins and several A/B determinants in the apical marginal region of epithelial cells also suggests potential molecular interactions. This study demonstrates a direct molecular link between A/B determinants and Fz1-mediated PCP establishment in the Drosophila eye. Patj binds Fz1, and is proposed to recruit aPKC, which in turn phosphorylates Fz1 to inhibit its function. Components of the aPKC complex and Patj are required for PCP establishment. Consistent with an inhibitory effect on Fz1, Patj, and to a lesser extent aPKC, expression is specifically downregulated in the R3/R4 pair where Fz1-PCP signaling is active. Finally, it is proposed that the upregulation of Bazooka in the R3/R4 cells prevents inhibition of Fz1 by the aPKC/Patj complex. These data support a model in which an aPKC/Patj complex inhibits Fz1-PCP activity in cells where it is not needed, allowing the use of shared components for other signaling events, such as Dsh for Wnt/β-catenin signaling (Djiane, 2005).

In summary, 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. However, this regulation is independent of Fz1 recruitment to the apical membrane, 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. 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 occur only 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 required only at specific time points and in specific subsets of cells. Since 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).

GENE STRUCTURE

cDNA clone length - 3258

(isoform A)

Bases in 5' UTR - 282

Exons - 4

Bases in 3' UTR - 360

PROTEIN STRUCTURE

Amino Acids - 871

Structural Domains

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 (Djiane, 2005).

date revised: 28 May 2008

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