bazooka
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).
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).
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).
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).
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).
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).
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).
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, 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 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).
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).
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).
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