Protein Interactions

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).

Apicobasal cell polarity is crucial for morphogenesis of photoreceptor rhabdomeres and adherens junctions (AJs) in the Drosophila eye. Crumbs (Crb) is specifically localized to the apical membrane of photoreceptors, providing a positional cue for the organization of rhabdomeres and AJs. The Crb complex consisting of Crb, Stardust (Sdt) and Discs-lost (Dlt) colocalizes with another protein complex containing Par-6 and atypical protein kinase C (aPKC) in the rhabdomere stalk of photoreceptors. Loss of each component of the Crb complex causes age-dependent mislocalization of Par-6 complex proteins, and ectopic expression of Crb intracellular domain is sufficient to recruit the Par-6 complex. The absence of Par-6 complex proteins results in severe mislocalization and loss of Crb complex. Dlt directly binds to Par-6, providing a molecular basis for the mutual dependence of the two complexes. These results suggest that the interaction of Crb and Par-6 complexes is required for the organization and maintenance of apical membranes and AJs of photoreceptors (Nam, 2003).

The strong dependence of Crb localization on Sdt and Dlt suggests that Crb may be destabilized or may not be targeted to the membrane in the absence of Sdt or Dlt. It is intriguing that Sdt and Dlt are lost only partially in the absence of Crb. The findings of a direct interaction between Dlt and Par-6 suggest that Sdt-Dlt can still be targeted to the membrane in the absence of Crb through the binding of Dlt to the Par-6 complex. However, it is important to note that Dlt is essentially lost in sdt mutant clones and vice versa. This raises an intriguing possibility that Dlt or Sdt are dependent on each other in vivo to be targeted to the apical membrane via binding to either Crb or Par-6. This mutual dependency between Dlt and Sdt may explain why Dlt and Sdt are lost in the absence of the other, rather than being associated with the Par-6 complex (Nam, 2003).

The interaction between the Crb and Par-6 complexes is mediated by the PDZ3 region of Dlt and the N-terminal domain of Par-6. The N-terminal domain of Par-6 is also used for binding aPKC. Therefore, a potential function of Dlt is to bind Par-6 in competition with aPKC or to facilitate the interaction of Par-6 with aPKC or other Par-6 binding proteins. Mutant analysis indicates that loss of Dlt and Sdt in sdt- clones causes mislocalization of both Crb and Par-6 complex proteins. This suggests that Sdt-Dlt interaction provides a scaffold to recruit Crb complex to the Par-6 complex and enhance the stability of these two complexes rather than functioning as a competitor for aPKC (Nam, 2003).

Proteins in Crb and Par-6 complexes consist of multiple functional domains which may be involved in diverse protein-protein interactions. A recent study has shown that in mammalian cell culture systems the PDZ domain of Par-6 binds not only Par-3 but also the N terminus of Pals1. These results suggest that the crosstalk between the Crb and Par-6 complexes is mediated by multiple domain-specific interactions. Evidence from genetic analysis using mutants suggests that the crosstalk between the two complexes is mutually required for normal organization of apical membranes and AJs in vivo, and also provides a basis for partial redundancy of these complexes in the organization of photoreceptor cell polarity. Interestingly, when either Crb or Sdt is lost, mislocalization or elimination of other associated components including Par-6 complex proteins becomes more severe in the age-dependent manner. This suggests that the Crb complex may be required for the maintenance rather than the formation of the Par-6 complex. The age-dependent degenerative phenotype may be related to the requirement of extensive apical membrane growth to make rhabdomeres and AJs along the growing axis of photoreceptors during pupal stage. Loss of any one component of the Crb complex is likely to be increasingly more detrimental as the process of membrane reorganization proceeds. In crb- or sdt- mutants, significant fractions of Par-6 complex proteins remain in the membrane despite the age-dependent and progressive mislocalization of apical markers. By contrast, loss of Par-6 or aPKC results in mislocalization of Dlt from the apical membrane. This suggests that the Par-6 complex plays essential functions for membrane localization of Crb complex proteins. Furthermore, both Par-6 and aPKC seem to be important for survival and/or proliferation of retinal cells because mutant clones were very small compared with adjacent twin spots and often completely disrupted, probably due to cell death. This is consistent with the findings of frequent apoptosis in aPKC- or par-6- embryos (Nam, 2003).

An important distinction of Par-6 complex in the photoreceptors from other epithelia is the localization of Baz. Baz localizes with Crb complex in the subapical membrane or both the subapical region and AJ in the Drosophila embryonic epithelia. Vertebrate Par-3 also localizes to the apical tight junction in vertebrate epithelial cells. By contrast, Baz in the photoreceptors is specifically positioned in the AJs basal to the all other proteins in the Crb/Par-6 complexes. Baz and Arm are recruited together to ectopic membrane sites by misexpression of CrbJM, suggesting that Baz is an integral component of AJ. However, Baz is not recruited by CrbPBM, whereas Par-6 and aPKC can be ectopically recruited by CrbPBM rather than CrbJM. Therefore, Baz appears to be recruited to AJ independently of Par-6/aPKC (Nam, 2003).

Intriguingly, despite its specific localization to AJs, loss of Baz results in most severe disruption of AJ as well as the more apical Dlt domain. It has been proposed that the Par-6/aPKC cassette is recruited to the site of cell-cell contact and then moves along the most apical zone of the developing cell-cell contact. In this process, an important step for cell polarity formation is to tether the cytoplasmic Par-6/aPKC complex to the site of cell-cell contact at the membrane, which is mediated by the interaction of Par-3 and a membrane protein JAM. Therefore, the results that baz mutation causes loss of Dlt and AJs support the crucial role of Baz in the initial step of cell polarization. However, the distinct localization of Baz from Par-6 and aPKC in the photoreceptors suggests that the mode of Baz localization varies in different systems. In photoreceptors, Baz may be targeted to the membrane with Par-6 but be sorted out from Par-6 in subsequent steps of polarization to remain in the AJs, whereas Par-6-aPKC-Baz cassette remains together in the complex in other epithelia. In contrast to Baz, aPKC localizes to both rhabdomere stalk and AJ, suggesting that Baz and Par-6 are completely separated during polarization while aPKC is not sorted from both Par-6 and Baz. The critical function of Baz in the localization of Crb complex in the rhabdomere stalk is consistent with the requirement of Baz for Crb localization in embryonic epithelia. However, the requirement of Baz in the embryo appears to be dependent on the stage of development since Crb distribution in the absence of Baz becomes normal in late embryos. On the contrary, such stage-dependent recovery of Crb complex localization has not been observed in baz- photoreceptor cells (Nam, 2003).

Recent studies have shown that mutations in human CRB1 cause RP12 and LCA, severe recessive retinal diseases, emphasizing the importance of Crb family proteins in the eyes of mammals including humans. The Drosophila Crb and human CRB1 are localized in analogous subcellular membrane domains of photoreceptors, the rhabdomere stalk and the inner segment in Drosophila and human photoreceptors, respectively. Besides similar subcellular localization, Crb and human CRB1 are functionally conserved. Age-dependent photoreceptor defects in the crb mutant also provide analogy to age-dependent retinal degeneration in RP12/LCA patients. These studies here imply that hCRB1 may function as a protein complex with homologs of Sdt and Dlt and such a complex may interact with a homologous Par-6 complex. Whether such homologous human genes are the targets of inherited retinal diseases such as RP remains to be studied (Nam, 2003).

Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and epithelia: Mutant neuroblasts lack apical localization of Par6 and Lgl

Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype; reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes (Rolls, 2003).

One of the more unexpected findings is that aPKC mutant neuroblasts show normal Baz/Par3 apical localization. The Baz/Par3-Par6-aPKC complex has been suggested to form a functional unit that is interdependent for localization in C. elegans, mammals, and Drosophila. Baz/Par3 shows normal apical localization in aPKC mutant neuroblasts, showing that normal Baz/Par3 localization can occur without being part of the Par3-Par6-aPKC complex. In addition, neuroblasts lacking apical aPKC and Par6 still form a molecularly defined apical cortical domain containing Baz/Par3, Insc, Pins, and Dlg. These results lead to the proposal of a hierarchy for apical protein localization in neuroblasts: Baz/Par3-Insc-Pins-Dlg --> aPKC --> Par6-Lgl. This hierarchy is consistent with recent biochemical analyses in which a protein complex was isolated containing Par6-aPKC-Lgl, but not Baz/Par3. It is suggested that aPKC may be required to anchor the Par6-aPKC-Lgl complex at the apical cortex of the neuroblast (Rolls, 2003).

Sequential roles of Cdc42, Par-6, aPKC, and Lgl in the establishment of epithelial polarity during Drosophila embryogenesis

How epithelial cells subdivide their plasma membrane into an apical and a basolateral domain is largely unclear. In Drosophila embryos, epithelial cells are generated from a syncytium during cellularization. Polarity is established shortly after cellularization when Par-6 and the atypical protein kinase C concentrate on the apical side of the newly formed cells. Apical localization of Par-6 requires its interaction with activated Cdc42 and dominant-active or dominant-negative Cdc42 disrupt epithelial polarity, suggesting that activation of this GTPase is crucial for the establishment of epithelial polarity. Maintenance of Par-6 localization requires the cytoskeletal protein Lgl. Genetic and biochemical experiments suggest that phosphorylation by aPKC inactivates Lgl on the apical side. On the basolateral side, Lgl is active and excludes Par-6 from the cell cortex, suggesting that complementary cortical domains are maintained by mutual inhibition of aPKC and Lgl on opposite sides of an epithelial cell (Hutterer, 2004).

These results describe the first steps of a molecular pathway that leads to the establishment of polarity in epithelial cells of the Drosophila ectoderm. The Par-6 protein localizes to the apical cell cortex by binding to Cdc42. Par-6 recruits Bazooka and aPKC and is essential for establishment of the apical domain. Maintenance of Par-6 localization requires Lgl, a substrate of aPKC. Phosphorylation by aPKC inactivates Lgl at the apical cell cortex and restricts Lgl to the basolateral cortex to establish the basolateral domain (Hutterer, 2004).

Apical localization of Par-6 is a key event in the establishment of epithelial polarity. How is Par-6 recruited to the apical cell cortex? In C. elegans, the proteins Par-3, Par-6, and aPKC are localized to the anterior cell cortex before and during the first cell division. Their asymmetric localization is initiated by interaction of the sperm aster with the overlying cell cortex that excludes Par-6 from the posterior cell cortex. During Drosophila cellularization, centrosomes are located apically and it is therefore unlikely that a similar cortical microtubule interaction is responsible for the apical localization of Par-6 (Hutterer, 2004).

Although a distinct apical domain with sharp boundaries is established in epithelial cells only after cellularization, elegant membrane tracer experiments have revealed a subdivision of the plasma membrane into distinct regions already during cellularization. Are these membrane compartments prefiguring the future apical and basolateral domains and is Par-6 localizing apically by recognizing a preformed membrane domain? The first membrane domain is the furrow canal at the tip of the ingrowing cellularization front that is marked by Patj. This domain disintegrates after cellularization and is therefore unlikely to participate in Par-6 localization. During later stages, new membrane is preferentially inserted apically, then apicolaterally. At these stages, newly inserted membrane displaces the pre-existing membrane toward both the apical and basolateral side, indicating that a distinct apical membrane compartment is not established by the end of cellularization. It is therefore unlikely that Par-6 recognizes a preformed apical membrane compartment although these experiments do not rule out a more general role of the vesicle transport machinery in Par-6 localization (Hutterer, 2004).

The results indicate that Par-6 needs to bind to activated Cdc42 in order to localize apically. Since cdc42 mutants cannot be analyzed at this stage, a conserved proline in the CRIB domain was mutated to generate a Par-6 version that no longer binds Cdc42. The structure of the Par-6 Cdc42 complex shows that this residue comes to lie in a hydrophobic groove of the Cdc42 molecule. This may explain why it can be replaced by alanine without affecting Cdc42 binding. When it is deleted, however, one of the adjacent highly charged amino acids will occupy the position of the proline. This could strongly inhibit interaction with the hydrophobic pocket and eliminate binding to Cdc42 both in vertebrates and in flies. Since both Lgl and aPKC still bind Par-6-DeltaP and the protein is expressed at almost wild-type levels from the endogenous promoter in an otherwise null mutant background, par-6-DeltaP embryos are specifically defective in binding of Cdc42 to the Par-6/aPKC complex (Hutterer, 2004).

How does activated Cdc42 localize Par-6? Cdc42 might be required for association of an unidentified Par-6 binding partner that is essential for apical localization of the protein. The conformation of Par-6 changes upon binding to Cdc42, and this could affect interactions with other proteins. However, aPKC and Lgl are the only proteins identified in the Par-6 complex, and their interaction does not depend upon Cdc42 binding. In vertebrates, Par-6 interacts with the Stardust homolog Pals1, and this interaction is regulated by Cdc42. Stardust acts together with its binding partner Crumbs, but apical protein localization is initiated correctly in crumbs mutants. Therefore, it is unlikely that Stardust binding to Par-6 is critical for the initial apical localization of Par-6. It is more likely that Cdc42 activation provides an instructive cue for Par-6 localization. Cdc42 could be preferentially activated on the apical side, for example by localization of an exchange factor, and this could recruit Par-6 to the apical cell cortex. This hypothesis is supported by the ectopic patches of Par-6, which are observed after overexpression of constitutively active Cdc42. Asymmetric activation of Cdc42 is known to polarize other cell types. In yeast, the exchange factor Cdc24 is localized to the incipient bud site. This locally activates Cdc42 and polarizes the actin cytoskeleton toward the site. In migrating neutrophils, Cdc42 is locally activated in response to a chemoattractant gradient by the exchange factor PIXalpha. A clear Drosophila ortholog of PIXalpha exists, but whether it is involved in epithelial polarity remains to be determined (Hutterer, 2004).

Maintenance of Par-6 localization requires the cytoskeletal protein Lgl. Lgl acts at the basolateral cortex where it inhibits cortical localization of Par-6. How Lgl excludes Par-6 from the cortex is unclear, but it is remarkable that in other tissues, Lgl actually promotes cortical protein localization. In MDCK cells, Lgl was suggested to regulate basolateral exocytosis and it could recruit a Par-6 antagonizing factor to the basolateral plasma membrane. Since Lgl and Bazooka binding to Par-6 seem to be mutually exclusive, Lgl could also inactivate the Par protein complex by displacing Bazooka. To perform its role in epithelial polarity, Lgl needs to be phosphorylated by aPKC. This modification has been shown to inactivate the protein and release it from its association with membranes and the cytoskeleton. These results suggest that in epithelial cells, apically localized aPKC phosphorylates Lgl to displace the protein from the apical cell cortex. A simple model is proposed in which mutual inhibition between Par-6/aPKC on the apical and Lgl on the basolateral cell cortex maintains epithelial polarity. This model is in agreement with previous studies that demonstrate negative genetic interactions between lgl and proteins that localize to the apical domain. Furthermore, it provides a molecular explanation for the recently described suppression of the lgl mutant epithelial polarity phenotype by reduction of aPKC levels. Negative interactions between the apical and basolateral domains of epithelial cells have been described before. In the Drosophila follicular epithelium, Bazooka is phosphorylated and inhibited by Par-1, a protein kinase located on the basolateral domain, thus restricting the Par protein complex to the apical domain (Hutterer, 2004).

The proteins Par-6, Bazooka, and aPKC localize to the apical cell cortex of both neuroblasts and epithelial cells, but the mechanism of apical localization seems to be different in the two cell types. In epithelial cells, Lgl is required for maintaining Par proteins at the apical cell cortex, while Par protein localization in neuroblasts is Lgl independent. Expression of nonphosphorylatable Lgl disrupts asymmetric cell division in neuroblasts but is without effect in epithelial cells. In addition, overexpression of dominant-active or -negative Cdc42 disrupts epithelial polarity but has no effect on neuroblast division. What is the basis for these differences (Hutterer, 2004)?

Epithelial cells rely on adherens junctions for maintaining distinct membrane compartments. Such junctions are absent from neuroblasts, and in fact, distinct membrane compartments do not seem to exist. Instead, Par protein localization in neuroblasts requires a protein called Inscuteable that is recruited apically by binding to Bazooka and aPKC and activates heterotrimeric G proteins through an adaptor molecule called Pins. Both Inscuteable and G proteins are essential for maintaining Par protein localization in neuroblasts but not epithelial cells. It is possible that a feedback loop operates downstream of the G proteins to maintain polarity in the absence of diffusion barriers and cellular junctions. Mechanistic differences in the way Par proteins localize are also observed between species. In C. elegans, neither Lgl nor G proteins are required for Par-3 or Par-6 localization. Instead, a Ring finger protein called Par-2 maintains Par-3 and Par-6 at the anterior pole. Cdc42 plays a role, but only in maintenance and not establishment of polarity. Clearly, key players are missing that might help in an understanding of these mechanistic differences (Hutterer, 2004).

Cdc42 binds vertebrate Par-6. Both proteins are implicated in polarizing vertebrate epithelial cells, and their conserved interaction suggests that they achieve this via a conserved mechanism. Although in vertebrates both proteins primarily act on tight junctions, the role of Cdc42 in localizing the Par proteins seems conserved since overexpression of an activated form inhibits the localization of Par-3 to tight junctions in MDCK cells. However, current experiments do not confirm a previously demonstrated role of Cdc42 in activating Par-6-associated aPKC in vitro. Unlike in vertebrates, aPKC is shown to be equally active - at least toward Lgl - when bound to a form of Par-6 that does not interact with Cdc42. Whether species-specific differences or the different experimental setups are responsible for this apparent discrepancy remains unclear. Besides their function in polarity, the Par proteins are involved in proliferation control of vertebrate epithelial cells. Par-6 cooperates with Cdc42 in transforming cells, suggesting a role in oncogenic transformation. In Drosophila, Cdc42, Lgl, and Bazooka were shown to cooperate with activated ras in the formation of metastatic tumors. It can be anticipated that the powerful tool of Drosophila genetics will help to identify other components of this pathway that might clarify its role in carcinogenesis (Hutterer, 2004).

Fragile X protein functions with Lgl and the PAR complex in flies and mice

Fragile X syndrome, the most common form of inherited mental retardation, is caused by loss of function for the Fragile X Mental Retardation 1 gene (FMR1). FMR1 protein (FMRP) has specific mRNA targets and is thought to be involved in their transport to subsynaptic sites as well as translation regulation. A saturating genetic screen of the Drosophila autosomal genome was used to identify functional partners of dFmr1. Nineteen mutations were recovered in the tumor suppressor lethal (2) giant larvae (dlgl) gene and 90 mutations at other loci. dlgl encodes a cytoskeletal protein involved in cellular polarity and cytoplasmic transport and is regulated by the PAR complex through phosphorylation. Direct evidence is provided for a Fmrp/Lgl/mRNA complex, which functions in neural development in flies and is developmentally regulated in mice. The data suggest that Lgl may regulate Fmrp/mRNA sorting, transport, and anchoring via the PAR complex (Zarnescu, 2005).

This study reports the identification of Lgl as a functional partner of the Fragile X protein, Fmrp. Lgl forms a large macromolecular complex with Fmrp, which is developmentally regulated and modulates the architecture of the neuromuscular junction in the fly. At the cellular level, Lgl and Fmrp are temporally and spatially coexpressed during development and colocalize in granules in the soma and the developing neurites of mouse cultured cells. Fractionation experiments show that Fmrp and Lgl comigrate with Golgi membrane-associated complexes. Furthermore, the Fmrp/Lgl complex contains a subset of mRNAs and interacts physically and genetically with the PAR complex, an essential component of the cellular polarization pathway. These results suggest that Lgl functions with Fmrp to regulate a subset of target mRNAs during synaptic development and/or function. It is proposed that Lgl may regulate Fmrp/mRNA containing RNPs by (1) sorting at the Golgi, (2) transport in neurites, and (3) anchoring at specific membrane domains, such as subsynaptic sites. The neurite transport function may involve molecular motors such as myosin II and kinesin, previously shown to associate with Lgl and Fmrp, respectively. The anchoring mechanism may involve the PAR complex, which has a demonstrated role in defining membrane domains and has recently been shown to generate asymmetry in the C. elegans embryo by stabilizing RNPs at the posterior pole (Zarnescu, 2005).

The data demonstrate that Fmrp and Lgl form a functional complex in living neurons, and this is conserved in flies and mice. In the mouse brain, mFmrp associates with mLgl preferentially at a time of increased synaptogenesis, demonstrating a developmentally regulated interaction between Lgl and Fmrp. The data suggest that dLgl acts to regulate a subset of dFmr1-associated mRNAs with some encoding circadian regulated molecules (CG3348 and CG9681) and some encoding secreted or transmembrane proteins (CG6136, CG4101, CG9681) among others. dLgl also associates with mRNA independent of dFmr1; thus, it is formally possible that dLgl interacts with other RNA binding proteins, which remain to be determined (Zarnescu, 2005).

Fmrp has been implicated in the translational regulation of specific target mRNAs, perhaps via the RNAi pathway. Also, it has been proposed that Fmrp is involved in the transport and localization of mRNAs: cellular fractionation and immunolocalization data revealed the association of Fmrp-containing complexes with molecular motors such as kinesin and myosin V. Lgl functions in cellular polarity via regulating myosin motor activity and/or vesicle transport and has been shown to regulate polarized delivery by sorting at the Golgi. Taken together, these concepts suggest that Lgl may act as a scaffold for Fmrp granules, possibly at the Golgi, and perhaps aids in carrying specific mRNA targets to sites of locally controlled translation (such as subsynaptic sites) (Zarnescu, 2005).

It is also possible that Lgl anchors Fmrp at specific membrane domains such as synapses, perhaps via the PAR complex. Lgl function is regulated by the PAR complex, specifically via phosphorylation by aPKC-zeta and by direct binding to PAR6. The genetic interaction data suggest that PAR6 and Baz antagonize dFmr1 function, which is in accordance with previously published work showing that the PAR complex inhibits dlgl and with data that demonstrate that dlgl functions cooperatively with dFmr1. aPKC-zeta was shown to antagonize most dLgl functions with the exception of its role in regulating neuroblast apical size and the data are be consistent with such reports. Loss of function for aPKC-zeta suppresses gain of function sev:dFmr1 as well as the loss-of-function phenotype of dFmr1 at the NMJs. Taken together, these data suggest a dynamic relationship between the various members of the complex. One possible interpretation of the results is that aPKC/PAR can act on Fmrp directly or via Lgl depending on the developmental and/or cellular context. Furthermore, this is consistent with sucrose fractionation experiments, which suggest the existence of at least two complexes comprising dFmr1/PAR proteins and dFmr1/dLgl/PAR proteins (Zarnescu, 2005).

The PAR complex not only functions in cell polarity, but also at the synapse, where it is believed to function in synaptic tagging. Synaptic tags have been proposed to transiently mark a synapse after activation in a way that will translate the local events into persistent functional changes (such as long-term depression), processes in which Fmrp is also thought to act. Thus, the Fmrp/Lgl/PAR complex may act in synaptic plasticity linking synaptic input to the remodeling of the cytoskeleton and mediating required translational changes (Zarnescu, 2005).

Cdc42 regulates the Par-6 PDZ domain through an allosteric CRIB-PDZ transition

Regulation of protein interaction domains is required for cellular signaling dynamics. This study shows that the PDZ protein interaction domain from the cell polarity protein Par-6 is regulated by the Rho GTPase Cdc42. Cdc42 binds to a CRIB domain adjacent to the PDZ domain, increasing the affinity of the Par-6 PDZ for its carboxy-terminal ligand by approximately 13-fold. Par-6 PDZ regulation is required for function; mutational disruption of Cdc42-Par-6 PDZ coupling leads to inactivation of Par-6 in polarized MDCK epithelial cells. Structural analysis reveals that the free PDZ domain has several deviations from the canonical PDZ conformation that account for its low ligand affinity. Regulation results from a Cdc42-induced conformational transition in the CRIB-PDZ module that causes the PDZ to assume a canonical, high-affinity PDZ conformation. The coupled CRIB and PDZ architecture of Par-6 reveals how simple binding domains can be combined to yield complex regulation (Peterson, 2004).

This study describes a set of Cdc42-dependent and -independent interactions for the Par-6 CRIB-PDZ module involving the known ligands Par-3 and Pals1/Sdt and a carboxy-terminal ligand identified in a peptide library screen. The functional relevance of Cdc42 regulation of carboxy-terminal ligand binding by Par-6 was established in polarized epithelial cells. The intrinsic low affinity of the Par-6 PDZ domain was found to arise from structural deviations from the canonical PDZ conformation, both in the peptide binding pocket and in regions that contact Cdc42. Binding of Cdc42 causes an allosteric transition in the CRIB-PDZ, which leads to a typical PDZ conformation that binds C-terminal ligand with high affinity. A simple thermodynamic cycle model indicates that binding of Cdc42 or carboxy-terminal peptide should induce the Par-6 PDZ allosteric transition, which was verified by X-ray crystallography (Peterson, 2006).

Although protein interaction domains must be regulated to yield the complex signaling dynamics observed in cells, little is known about the mechanisms underlying this regulation. PDZ domains are very common in metazoans and are also found in many bacteria and yeast. Individual PDZ domains have been shown to bind to carboxy-terminal ligands with affinities ranging from high nanomolar to low micromolar. Like most isolated domains, PDZ domains typically bind their ligands in a constitutive manner. The Par-6 PDZ differs in this respect, with low intrinsic affinity for ligand that can be increased into the low micromolar range upon Cdc42 binding. This may be a common regulatory mechanism used by protein interaction domains. However, as ligand screens are often performed with isolated domains, ligands regulated in this manner are likely to be missed. As with the Par-6 PDZ domain, ligand binding may only be observed with the proper set of intra- and/or intermolecular ligands (Peterson, 2006).

The different affinities of free and Cdc42-bound Par-6 for carboxy-terminal ligand result from an allosteric transition in the PDZ domain. Analysis of multiple sequence alignments and double mutant cycles has suggested that energetic pathways within PDZ domains may support allostery. In this study, a pathway of physical connectivity was identified that runs between the two PDZ helices. This pathway is consistent with the conformational changes that occur in the Par-6 PDZ upon Cdc42 binding as it connects the Cdc42 and carboxy-terminal ligand binding sites. The energetic coupling in PDZ domains, along with the Par-6 PDZ results discussed in this study, suggest that allostery may be a general feature of the PDZ domain family (Peterson, 2006).

Allostery appears to be a common mechanism used by Cdc42 effectors to translate binding into changes in activity. Rho GTPases, including Cdc42, are found in many different cellular systems and, as such, utilize a diverse array of effectors. Over 60 targets have been identified for the prototypical Rho GTPases, Rho, Rac, and Cdc42. Besides Par-6, Cdc42 also regulates several widely varying classes of proteins, including kinases (p21 activated kinase, PAK) and actin regulatory molecules (Wiskott-Aldrich syndrome protein, WASP). Although each of these effectors has an entirely different domain structure, each contains a CRIB motif for binding to Cdc42 (Peterson, 2006).

Recent studies have begun to uncover the molecular mechanism by which binding to the CRIB motif in PAK and WASP is translated into activity modulation. In these proteins, the CRIB is part of a larger module that regulates the protein's kinase or Arp2/3 activating domains, respectively. In both cases, residues carboxy-terminal to the CRIB motif form an intramolecular interaction that inactivates the protein. Cdc42 binding to the CRIB leads to a conformational transition in the regulatory module that is incompatible with the intramolecular interaction leading to activation (Peterson, 2006).

The mechanism of Par-6 regulation by Cdc42 shows that the CRIB is a versatile motif that can be coupled to diverse domains to regulate effector function. Rather than the specialized regulatory module found in PAK and N-WASP, the Par-6 CRIB is coupled to a PDZ protein interaction domain. However, in all cases, the CRIB motif transmits binding information to the adjacent domain through allosteric changes. While binding of Cdc42 to PAK and N-WASP leads to a decrease in affinity of the adjacent domain for their intramolecular ligands, Cdc42 binding to Par-6 leads to an increase in affinity for an intermolecular PDZ ligand (Peterson, 2006).

Many diverse systems require Cdc42 and Par-6 activity for cell polarity. The molecular mechanism by which polarity is controlled by these signaling molecules is poorly understood. One function of Cdc42 in cell polarity appears to be regulation of aPKC kinase activity through binding to Par-6. Although aPKC has a conserved carboxy-terminal sequence (-SLEDCV-COOH) with similarities to the Par-6 PDZ ligand identified it this study, it does not bind to the Par-6 PDZ domain. As such, it is expected that Cdc42 regulates formation of an unidentified Par-6 complex (Peterson, 2006).

The Cdc42-Par-6 interaction may also play a role in localization of Par-6, aPKC, and their ligands as inactivation of Cdc42 leads to symmetrical localization of these proteins. Cooperative binding of Cdc42 and Par-6 PDZ ligands would then allow for correct spatial and temporal activation. This may be an essential feature of Par-6 regulation as localization is a defining feature of cell polarization. Future work will be directed at further exploring the role of Cdc42-Par-6 PDZ ligand coupling in cell polarity (Peterson, 2006).

Computer modelling in combination with in vitro studies reveals similar binding affinities of Drosophila Crumbs for the PDZ domains of Stardust and DmPar-6

Formation of multiprotein complexes is a common theme to pattern a cell, thereby generating spatially and functionally distinct entities at specialised regions. Central components of these complexes are scaffold proteins, which contain several protein-protein interaction domains and provide a platform to recruit a variety of additional components. There is increasing evidence that protein complexes are dynamic structures and that their components can undergo various interactions depending on the cellular context. However, little is known so far about the factors regulating this behaviour. One evolutionarily conserved protein complex, which can be found both in Drosophila and mammalian epithelial cells, is composed of the transmembrane protein Crumbs/Crb3 and the scaffolding proteins Stardust/Pals1 and DPATJ/PATJ, respectively, and localises apically to the zonula adherens. In vitro analysis shows that, similar as in vertebrates, the single PDZ domain of Drosophila Par-6 can bind to the four C-terminal amino acids (ERLI) of the transmembrane protein Crumbs. To further evaluate the binding capability of Crumbs to Par-6 and the MAGUK protein Stardust, analysis of the PDZ structural database and modelling of the interactions between the C-terminus of Crumbs and the PDZ domains of these two proteins were performed. The results suggest that both PDZ domains bind Crumbs with similar affinities. These data are supported by quantitative yeast two-hybrid interactions. In vivo analysis performed in cell cultures and in the Drosophila embryo show that the cytoplasmic domain of Crumbs can recruit Par-6 and DaPKC to the plasma membrane. The data presented here are discussed with respect to possible dynamic interactions between these proteins (Kempkens, 2006).

Homology modelling and energy calculations indicate that the PDZ domains of Sdt and Par-6 both show a high affinity for Crb. According to a classification of PDZ domains, the PDZ domains of Par-6 and Sdt fall into different categories: n (neutral) – h (hydrophic) for Par-6 and Sp (small and polar) – h (hydrophobic) for Sdt. The distinction is based on the nature of the amino acids in two critical positions, those immediately following the second β-strand and at the beginning of the second α-helix. However, this classification, based exclusively on the amino acids at two positions, does not adequately describe the complexity of the PDZ family and is not sufficient to predict the specificity of binding. This is borne out by the results presented in this study (Kempkens, 2006).

The crystal structure used as a template in this study (D. melanogaster Par-6, 1RZX.PDB) includes a canonical type I ligand (ESLV), and this can easily be replaced in the structure by the C-terminal tetrapeptide of Crb (ERLI). Although better binding of the Crb C-terminus to Par-6 than to Sdt is predicted, this conclusion should be treated with caution, since the predicted structure of the Crb-Sdt complex is based solely on homology modelling. The analysis of interactions between Crb and PDZ domains showed minor van der Waals clashes (<0.3 kcal/mol), as well as strong polar and hydrophobic ligand/PDZ-domain interactions. An important component of the interaction is electrostatic. This could favour fast association (Kon) and dissociation rates (Koff), which would facilitate flexible regulation of complex formation. These values, which are slightly better for Par-6, could account for the affinity and specificity observed in the experimental interaction studies. The predicted Kd for interaction between the domains considered in this study fall in the usual range for experimental determinations of PDZ-ligand binding affinities reported by many authors. The theoretical determination of binding interactions by FoldX has been tested for many domain-peptide interactions (i.e. SH3, SH2, PDZ, Ras-Rab, etc.), is validated by available experimental data, and shows a high degree of accuracy. In this sense, the theoretical cut-off for non-binders can be reasonably approximated to that used for experimental procedures, normally 1E−4M. Thus, all putative ligands with interaction energies below 1E−4M are considered to be non-binders (Kempkens, 2006).

It has been shown that the PDZ domain of vertebrate Par-6 can bind not only to the C-terminus of CRB3, but also to the C-terminus of the N-type Ca2+ channel and the C-terminus of neurexin, and that the PDZ domains of vertebrate and Drosophila Par-6 proteins can interact with an internal region in the N-terminal portion of Pals1/Sdt. These data support the view that PDZ domains are promiscuous and can bind different ligands, thanks to the plasticity of the carboxylate-binding loop. This conformational flexibility can be modulated by interaction with other proteins. For example, binding of Cdc42 to Par-6 significantly enhances its affinity for the C-terminal ligand, while it has no effect on the interaction with the internal binding sequence of Pals1. To what extent other components facilitate loop rearrangement to accommodate internal ligands to the Par-6 PDZ binding pocket in vivo remains to be determined. Interestingly, although the PDZ domains of both Par-6 and Sdt bind to the C-terminus of Crb, only the PDZ domain of Par-6 interacts with the internal sequence in Sdt (Kempkens, 2006).

Since the binding affinities of the PDZ domains of Sdt and Par-6 for the C-terminus of Crb are in the same range, and all three proteins are expressed in the same cells, other factors must be considered that might influence their interactions. These could include, for example, (1) temporal differences in expression, (2) protein modifications, and (3) interactions with other factors. Par-6 is present in the embryo before Sdt and Crb appear, since it is expressed maternally. In the genetic hierarchy controlling cell polarity and ZA assembly, components of the Baz/Par-6/DaPKC complex appear to form the top tier, with Baz acting very early during cellularisation to establish the apical domain in an adherens junction-independent manner. At this stage, Par-6 is diffusely distributed in the cytoplasm. It becomes localised apically only after cellularisation is complete. Localisation of Par-6 requires an interaction with the monomeric GTPase Cdc42, which, like its vertebrate homologue, binds Par-6 via a conserved CRIB (Cdc42/Rac interactive binding) domain. Thus, overexpression of a dominant-negative form of Cdc42 prevents apical localisation of Par-6, and a mutant version of Par-6 that cannot interact with Cdc42 (Par-6-ΔP) remains diffusely distributed in the cytoplasm (Kempkens, 2006).

Binding of the PDZ domain of Sdt to the C-terminus of Crb may also depend on additional contacts mediated by the other interaction domains of Sdt. Sdt is a member of the MAGUK protein family, with two L27 domains, one SH3, one PDZ and one guanylate kinase (GUK) domain. The L27 domains of Pals1 and Sdt bind to PATJ/DPATJ and mLin-7/DLin-7. No partner(s) for the SH3 and GUK domains of Sdt have been described so far. Recently, a direct interaction was shown to occur between the SH3/Hook domain of MPP5/Pals1 and the GUK domain of another MAGUK family member, MPP4, in mouse photoreceptor cells. Furthermore, yeast two-hybrid experiments have uncovered an interaction between the SH3 and the Hook/GUK domains of Sdt. In other members of the MAGUK family, such as human Discs large (hDlg), CASK and p55, these two domains participate in intra- as well as intermolecular interactions, with the former being preferred, at least in vitro. Currently, the data do not allow a decision as to whether the observed SH3-Hook/GUK interaction is intra- or intermolecular, nor to what extent this might influence the capacity of the PDZ domain to bind to Crb in vivo. Similarly, at present it is only possible to speculate on the possible influence on the binding specificity/activity of the phosphorylation of the intracellular domain of Crb, which has been demonstrated by in vitro assays, but also occurs under certain in vivo conditions. Future experiments will contribute to the elucidation of the dynamic interactions among these proteins within the cell and help in the understanding of how they establish and maintain the polarised phenotype (Kempkens, 2006).

Cdc42 acts downstream of Bazooka to regulate neuroblast polarity through Par-6 aPKC

Cdc42 recruits Par-6-aPKC to establish cell polarity from worms to mammals. Although Cdc42 is reported to have no function in Drosophila neuroblasts, a model for cell polarity and asymmetric cell division, this study shows that Cdc42 colocalizes with Par-6-aPKC at the apical cortex in a Bazooka-dependent manner, and is required for Par-6-aPKC localization. Loss of Cdc42 disrupts neuroblast polarity: cdc42 mutant neuroblasts have cytoplasmic Par-6-aPKC, and this phenotype is mimicked by neuroblast-specific expression of a dominant-negative Cdc42 protein or a Par-6 protein that lacks Cdc42-binding ability. Conversely, expression of constitutively active Cdc42 leads to ectopic Par-6-aPKC localization and corresponding cell polarity defects. Bazooka remains apically enriched in cdc42 mutants. Robust Cdc42 localization requires Par-6, indicating the presence of feedback in this pathway. In addition to regulating Par-6-aPKC localization, Cdc42 increases aPKC activity by relieving Par-6 inhibition. It is concluded that Cdc42 regulates aPKC localization and activity downstream of Bazooka, thereby directing neuroblast cell polarity and asymmetric cell division (Atwood, 2007).

Little is currently known about how the Par complex is localized or regulated in Drosophila neuroblasts, despite the importance of this complex for neuroblast polarity, asymmetric cell division and progenitor self-renewal. This study shows that Cdc42 plays an essential role in regulating neuroblast cell polarity and asymmetric cell division. Baz localizes Cdc42 to the apical cortex where it recruits Par-6-aPKC, leading to polarization of cortical kinase activity that is essential for directing neuroblast cell polarity, asymmetric cell division, and sibling cell fate (Atwood, 2007).

Asymmetric aPKC kinase activity is essential for the restriction of components such as Mira and Numb to the basal cortex. The aPKC substrates Lgl and Numb are thought to establish basal polarity either by antagonizing activity of myosin II or by direct displacement from the cortex. This study found that Cdc42 recruits Par-6-aPKC to the apical cortex and that Cdc42 relieves Par-6 inhibition of aPKC kinase activity. In the absence of Cdc42, aPKC is delocalized and has reduced activity, resulting in uniform cortical Mira. Expression of Cdc42-DN leads to cortical overlap of inactive Par-6-aPKC and Mira indicating the importance of Cdc42-dependent activation of aPKC kinase activity. Expression of Cdc42-CA leads to cortical aPKC that displaces Mira from the cortex, presumably because Lgl is phosphorylated at the entire cell cortex. This is similar to what is seen when a membrane-targeted aPKC is expressed (Atwood, 2007).

Baz, Par-6 and aPKC have been considered to be part of a single complex (the Par complex). This study found that, when Cdc42 function is perturbed, Par-6 and aPKC localization is disrupted but Baz is unaffected. Why is Baz unable to recruit Par-6-aPKC in the absence of Cdc42? One explanation is that Cdc42 modulates the Par-6-Baz interaction, although Cdc42 has no direct effect on Par-6-Baz affinity. Alternatively, Baz might only be transiently associated with the Par-6-aPKC complex (e.g. as an enzyme-substrate complex); this is consistent with the observation that Baz does not colocalize with Par-6-aPKC in Drosophila embryonic epithelia and its localization is not dependent on either protein. How does Baz recruit Cdc42 to the apical cortex? Like other Rho GTPases, Cdc42 is lipid modified (prenylated), which is sufficient for cortical localization. Baz is known to bind GDP-exchange factors (GEFs), which may induce accumulation of activated Cdc42 at the apical cortex (Atwood, 2007).

The requirement of Par-6 for robust Cdc42 apical enrichment suggests that positive feedback exists in this pathway, a signaling pathway property that is also found in polarized neutrophils. More work is required to test the role of feedback in neuroblast polarity but one attractive model is that Baz establishes an initial polarity landmark at the apical cortex in response to external cues, which leads to localized Par-6-aPKC activity through Cdc42. Phosphorylation of Baz by aPKC might further increase asymmetric Cdc42 activation, perhaps by increased GEF association, thereby reinforcing cell polarity. Such a mechanism could generate the robust polarity observed in neuroblasts and might explain why expression of dominant Cdc42 mutants late in embryogenesis does not lead to significant defects in polarity (Atwood, 2007).

This study argues that Cdc42 functions downstream of Baz. Cdc42 is required for Baz-Par-6-aPKC localization in C. elegans embryos and mammalian neural progenitors. In C. elegans embryos, RNA interference of cdc42 disrupts Par-6 localization, whereas PAR-3 localization is slightly perturbed. In this case, Cdc42 is required for the maintenance but not establishment of PAR-3-Par-6 asymmetry; however, other proteins have been shown to localize Par complex members independently of Cdc42. Conditional deletion of cdc42 in the mouse brain causes significant Par-3 localization defects, although this may be caused by the loss of adherens junctions. More work will be required in these systems to determine if the pathway that has been proposed is conserved (Atwood, 2007).

This study has identified at least two functions of Cdc42 in neuroblasts: first, to recruit Par-6-aPKC to the apical cortex by direct interaction with its CRIB domain and, second, to promote aPKC activity by relieving Par-6 repression. aPKC activity is required to partition Mira and associated differentiation factors into the basal GMC; this ensures maintenance of the apical neuroblast fate as well as the generation of differentiated neurons. Polarized Cdc42 activity may also have a third independent function in promoting physically asymmetric cell division, because uniform cortical localization of active Cdc42 leads to same-size sibling cells. Loss of active Cdc42 at the cortex by overexpression of Cdc42-DN still results in asymmetric cell division, suggesting that other factors also regulate cell-size asymmetry, such as Lgl and Pins. In conclusion, these data show that Cdc42 is essential for the establishment of neuroblast cell polarity and asymmetric cell division, and defines its role in recruiting and regulating Par-6-aPKC function. These findings now allow Drosophila neuroblasts to be used as a model system for investigating the regulation and function of Cdc42 in cell polarity, asymmetric cell division and neural stem cell self-renewal (Atwood, 2007).

Polarity proteins and Rho GTPases cooperate to spatially organise epithelial actin-based protrusions

Different actin-filament-based structures co-exist in many cells. This study characterises dynamic actin-based protrusions that form at distinct positions within columnar epithelial cells in the Drosophila pupal notum, focusing on basal filopodia and sheet-like intermediate-level protrusions that extend between surrounding epithelial cells. Using a genetic analysis, it was found that the form and distribution of these actin-filament-based structures depends on the activities of apical polarity determinants, not on basal integrin signalling. Bazooka/Par3 acts upstream of the RacGEF Sif/TIAM1 to limit filopodia to the basal domain, whereas Cdc42, aPKC and Par6 are required for normal protrusion morphology and dynamics. Downstream of these polarity regulators, Sif/TIAM1, Rac, SCAR and Arp2/3 complexes catalyse actin nucleation to generate lamellipodia and filopodia, whose form depends on the level of Rac activation. Taken together, these data reveal a role for Baz/Par3 in the establishment of an intercellular gradient of Rac inhibition, from apical to basal, and an intimate association between different apically concentrated Par proteins and Rho-family GTPases in the regulation of the distribution and structure of the polarised epithelial actin cytoskeleton (Georgiou, 2010).

Although many studies have used the segregation of apical, junctional and basolateral markers as a model of epithelial polarity, and a number of studies have reported the existence of cell protrusions in the notum and other epithelia, these structures and the genes regulating their formation have not been characterised in detail. This study used Neuralized-Gal4 to express GFP-fusion proteins in isolated epithelial cells to reveal the dynamic shape of cells within the dorsal thorax of the fly during pupal development. Using this method, distinct populations of protrusions were characterised based on their form, dynamics and location within the basolateral domain of columnar epithelial cells. The analysis reveals dynamic protrusions at three distinct locations within the epithelial cell: apical microvillus-like structures, intermediate-level sheet-like protrusions and basal-level lamellipodia and filopodia. Importantly, although these are all dependent on continued actin filament dynamics, these populations of protrusions rely on different gene activities for their formation (Georgiou, 2010).

Cdc42, Rac, SCAR/WAVE and the Arp2/3 complex are required for the formation of basal lamellipodia and filopodia, but not for the formation of the apical microvillus-like structures. This analysis also confirms that HSPC300 should be considered to be a functional component of the SCAR complex. Moreover, the SCAR and Arp2/3 complexes are required to induce the formation of both lamellipodia and filopodia in this system. Although many studies have suggested that Rac activates the SCAR complex to induce branched Arp2/3-dependent actin nucleation that underlies lamellipodial formation, whereas Cdc42 is required to induce filopodial formation, this analysis suggests that the macroscopic form of the protrusion in a tissue context is not dictated by the nucleator used. In this, the current results are in line with several recent studies in cell culture. Instead, the macroscopic structure generated depends on the local level of Rac activity, with high levels of Rac driving filopodial formation and low levels leading to lamellipodial formation. Since the forces required to distort the membrane to generate finger-like protrusions are likely to be greater than those required to generate the equivalent section of a sheet-like protrusion, protrusion morphology might be a product of a force balance between membrane tension, extracellular confinement and local actin-filament formation. Since wild-type cells have a graded distribution of protrusions, with lamellipodia predominating apically and filopodia basally, wild-type cell morphology might reflect a gradient in the level of Rac activation, from high basal levels to low apical levels (Georgiou, 2010).

Within this system, Cdc42-Par6-aPKC and Baz/Par3 appear to have antagonistic roles in the formation of basolateral protrusions. Cdc42-Par6-aPKC is required for actin filament formation and protrusion dynamics, whereas Baz/Par3 ensures the separation of basal and intermediate protrusions by limiting the extent of basal filopodia along the apical-basal axis. In this, the current analysis adds to the growing body of evidence that Baz/Par3 and Par6-aPKC have distinct molecular targets. Moreover, the data confirm that Par6-aPKC act together with the Rho-family GTPase Cdc42. Significantly, the loss of Baz/Par3 phenocopies gain-of-function mutations in Rac and the overexpression of the Rac-GEF Sif/TIAM1, a Par3-interacting protein. Baz/Par3 might therefore serve as a cell-intrinsic cue to polarise the dynamic actin cytoskeleton along the epithelial apical-basal axis, giving epithelial cells their characteristic polarised morphology (Georgiou, 2010).

Baz/Par3 has previously been implicated in the restriction of actin polymerisation to specific subcompartments within a cell, allowing for the formation of distinct populations of protrusions. This has been studied most extensively in hippocampal neurons, in which Par3 was shown to interact with TIAM1 to regulate the activation of Rac within distinct domains of the cell during axon specification and dendritic spine morphogenesis. Indeed, it has been suggested that the formation of a Cdc42-Par6-Par3-TIAM1-Rac1 complex is required to establish neuronal polarity. The current study suggests that Baz/Par3 acts in a similar fashion in the morphogenesis and positioning of dynamic protrusions in epithelia. However, this analysis reveals an antagonistic relationship between Sif/TIAM1 and Baz/Par3 in protrusion formation. Baz/Par3 might sequester Sif/TIAM1 to prevent its association with Rac. Furthermore, because the loss of Cdc42, Par6 or aPKC results in the loss of basolateral protrusions and a marked reduction in the GFP:Moe reporter (a phenotype that can be rescued by the coexpression of RacV12 or Sif) Cdc42, Par6 and aPKC are probably required for the basal activation of Rac in epithelial cells in the Drosophila notum. Thus, signals from apically concentrated polarity determinants appear to be communicated and translated into local protrusion formation within the basolateral domain. Whether this occurs through the diffusion of an apically localised regulator or via long-range transmission of polarity information e.g. via microtubules, will be an important area of future research. An intriguing correlation is the largely apical localisation of Baz and its proximity to intermediate-level sheet-like protrusions. This would suggest a possible gradient of Baz/Par3-mediated Rac inhibition, allowing sheet-like protrusions at an intermediate level and restricting filopodial protrusions to the very base of the cell. Since Baz/Par3 has been shown to localise PTEN to apical junctions, it is possible that Baz recruits PTEN, which acts on PtdIns(3,4,5)P3 to generate a PtdIns(3,4,5)P3 gradient from high levels basally to low levels apically. PIP3 could then act to aid in the recruitment and activation of Rac at the membrane (Georgiou, 2010).

Taken together, these data demonstrate that different components of the apical determinants of cell polarity act in conjunction with the Rho-family GTPases Cdc42 and Rac to regulate the positioning of lamellipodial and filopodial protrusions over the entire span of the apical-basal cell axis. Significantly, in this tissue context, Rac, SCAR and Arp2/3 complexes promote the formation of both lamellipodia and filopodia, whose structure appears to depend on the level of Rac activation (Georgiou, 2010).

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

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

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

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

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

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

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

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

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

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

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

Drosophila Cip4 and WASp define a branch of the Cdc42-Par6-aPKC pathway regulating E-Cadherin endocytosis

Integral to the function and morphology of the epithelium is the lattice of cell-cell junctions known as adherens junctions (AJs). AJ stability and plasticity relies on E-Cadherin exocytosis and endocytosis. A mechanism regulating E-Cadherin (E-Cad) exocytosis to the AJs has implicated proteins of the exocyst complex, but mechanisms regulating E-Cad endocytosis from the AJs remain less well understood. This study shows that Cdc42, Par6, or aPKC loss of function is accompanied by the accumulation of apical E-Cad intracellular punctate structures and the disruption of AJs in Drosophila epithelial cells. These punctate structures derive from large and malformed endocytic vesicles that emanate from the AJs; a phenotype that is also observed upon blocking vesicle scission in dynamin mutant cells. The Drosophila Cdc42-interacting protein 4 (Cip4) is a Cdc42 effector that interacts with Dynamin and the Arp2/3 activator WASp in Drosophila. Accordingly, Cip4, WASp, or Arp2/3 loss of function also results in defective E-Cadherin endocytosis. Altogether These results show that Cdc42 functions with Par6 and aPKC to regulate E-Cad endocytosis and define Cip4 and WASp as regulators of the early E-Cad endocytic events in epithelial tissue (Leibfried, 2008).

Cdc42 has been implicated in the regulation of polarity establishment in the early Drosophila embryo. The function was shown to be dependent upon the interaction of Cdc42 with the Baz-Par6-aPKC complex that promotes the exclusion of Lgl through Lgl phosphorylation by aPKC. However, the role of Cdc42 in epithelial tissue is unlikely to depend only on its regulation of aPKC because aPKC was shown to be dispensable for apico-basal polarity establishment in the Drosophila embryo. The role of Cdc42 in mammalian epithelial cells has so far been examined by the expression of constitutively active and dominant-negative forms of Cdc42, and such an examination has led to conflicting results in establishing the exact role of Cdc42 in apico-basal polarity maintenance. Nonetheless, they point toward an important role of Cdc42 in the regulation of polarized trafficking. The possible role of Cdc42 in polarized trafficking in epithelial cells was further strengthened by the identification of Cdc42 and the Par complex as regulators of endocytosis in both mammalian cells and C. elegans. Nevertheless, the precise role of Cdc42 and the Par complex in the regulation of endocytosis has remained poorly understood except in migrating cells in which the Par complex was shown to inhibit integrin endocytosis via Numb (Leibfried, 2008).

Cdc42 and its effector Drosophila Cip4 have been found to regulate E-Cad endocytosis and that their loss of function is associated with the formation of long tubular endocytic structures similar to what is observed upon blocking Dynamin function. It is therefore proposed that in Drosophila epithelial cells, Cdc42 controls the early steps of E-Cad endocytosis via Cip4. Because Cdc42, aPKC, and Par6 loss of function are associated with similar defects in E-Cad and Cip4 localization, a simple model is favored, in which the loss of aPKC or Par6 activity disrupts Cdc42 localization or activity and in turn prevents Cip4 function (Leibfried, 2008).

The identified role of PCH family of protein stems in part from the biochemical analysis of Toca-1 as a regulator of actin polymerization. Toca-1 is necessary to activate actin polymerization and actin comet formation downstream of PIP2 and Cdc42 in a WASp-dependent manner (Ho, 2004). On the basis of elegant biochemical assays, Toca-1 was further shown to be necessary to alleviate the WIP inhibitory activity on WASp, in order to allow efficient Arp2/3 activation by WASp (Ho, 2004). Toca-1 was proposed to play an essential role in the fine spatial and temporal regulation of actin polymerization in both cell migration and vesicle movement. Cip4 has been implicated in microtubule organizing center (MTOC) polarization in immune natural killer cells (Banerjee, 2007), a process in which Cdc42 and the Par complex are also involved. Importantly, because Cip4 was shown to bind microtubules, the interaction between Cdc42 and Cip4 might indicate that Cip4 might also be an effector of Cdc42-Par complex in the regulation of MTOC polarization (Leibfried, 2008).

In mammalian cells, regulation of endocytic-vesicle formation has been proposed to be dependent upon both branched actin-filament formation and Dynamin. The role of WASp and Arp2/3 in the regulation of E-Cad endocytosis may therefore indicate that Cip4, which is also known to form dimers, can promote vesicle scission by recruiting Dynamin and promoting actin polymerization via WASp. Therefore, it is proposed that Cip4 and WASp act as a link between Cdc42-Par6-aPKC and the early endocytic machinery to regulate E-Cadherin endocytosis in epithelial cells (Leibfried, 2008).

Protein phosphatase 2A negatively regulates aPKC signaling by modulating phosphorylation of Par-6 in Drosophila neuroblast asymmetric divisions

Drosophila neural stem cells or neuroblasts undergo typical asymmetric cell division. An evolutionally conserved protein complex, comprising atypical protein kinase C (aPKC), Bazooka (Par-3) and Par-6, organizes cell polarity to direct these asymmetric divisions. Aurora-A (AurA) is a key molecule that links the divisions to the cell cycle. Upon its activation in metaphase, AurA phosphorylates Par-6 and activates aPKC signaling, triggering the asymmetric organization of neuroblasts. Little is known, however, about how such a positive regulatory cue is counteracted to coordinate aPKC signaling with other cellular processes. During a mutational screen using the Drosophila compound eye, microtubule star (mts), which encodes a catalytic subunit of protein phosphatase 2A (PP2A), was identified as a negative regulator for aPKC signaling. Impairment of mts function causes defects in neuroblast divisions, as observed in lethal (2) giant larvae (lgl) mutants. mts genetically interacts with par-6 and lgl in a cooperative manner in asymmetric neuroblast division. Furthermore, Mts tightly associates with Par-6 and dephosphorylates AurA-phosphorylated Par-6. This genetic and biochemical evidence indicates that PP2A suppresses aPKC signaling by promoting Par-6 dephosphorylation in neuroblasts, which uncovers a novel balancing mechanism for aPKC signaling in the regulation of asymmetric cell division (Ogawa, 2009).

Polarity is a fundamental characteristic of cells and underlies a variety of cellular processes involved in the development and homeostasis of living organisms. In epithelial cells, which consist of the apical and basolateral membrane domains, cell polarity creates distinct subcellular compartments to arrange the cells into a well-ordered structure. In asymmetric cell division, cell polarity is coupled with mitosis. Cell polarity creates two subcellular domains with distinct characteristics in the mitotic mother cell and coordinates the mitotic spindle with the polarity axis to allow the two daughter cells to be distinct. Because these cell polarity events are tightly linked to other elementary processes such as the cell cycle and mitotic events, cell polarity is finely controlled to coordinate with those cellular processes (Ogawa, 2009).

Drosophila neural-stem-like cells, or neuroblasts, undergo typical asymmetric divisions, providing an excellent model for the study of how cell polarity is controlled. Neuroblasts repeatedly divide into a large, self-renewing daughter (the neuroblast itself) and a smaller, differentiating daughter [the ganglion mother cell (GMC)]. Cell fate determinants, such as Prospero, Brain tumor (Brat) and Numb, are segregated to the GMC. The localization of these determinants and the coordination with mitotic spindle orientation are controlled by the apically localized protein complexes -- the aPKC-Par complex and the Pins complex -- which are mutually linked by Inscuteable (Insc). The aPKC-Par complex consists of atypical protein kinase C (aPKC), Bazooka (Baz) and Par-6 and is primarily involved in organizing cell polarity and the asymmetric distribution of the cell fate determinants along the axis of polarity. The Pins complex, which consists of Partner of Inscuteable (Pins), Locomotion defects (Loco) and Gαi, determines the orientation of the mitotic spindle relative to the cell polarity axis (Ogawa, 2009).

aPKC is a key enzyme involved in establishment of neuroblast polarity and definition of the apical cortex. A tumor suppressor protein, Lethal (2) giant larvae (Lgl), is thought to antagonize aPKC as an inhibitory substrate. Although aPKC binds to non-phosphorylated Lgl, Lgl that is phosphorylated by aPKC dissociates from it and is released from the cell cortex. In the absence of aPKC, the entire cortex becomes basal, and Miranda, an adaptor protein for Prospero and Brat, distributes uniformly throughout the cortex. However, loss of Lgl results in uniform activation of aPKC in the cortex just as if the entire cortex were apical. Consequently, Miranda misdistributes into the cytoplasm and concentrates on mitotic spindles (Ogawa, 2009).

The apical complex and the basal determinants dynamically change their localization as the cell cycle progresses. The apical complex accumulates at the apical cortex during late interphase, retains its apical localization during metaphase, and then initiates expansion through the cortex in anaphase. Miranda and its cargos are temporally found in the apical cortex in late interphase and, after spreading into the cytoplasm at the onset of mitosis, form the basal crescent that is complementary to the localization of the apical complex during metaphase. At late anaphase onwards, they are restricted to the GMC compartment, which is separated by the contractile ring from the neuroblast compartment (Ogawa, 2009).

It was recently shown that the mitotic kinase Aurora-A (AurA) has an important role in linking the cell cycle to the asymmetric cell division of neuroblasts and sensory organ precursors (SOPs) by phosphorylating Par-6. When AurA is inactive, aPKC binds to unphosphorylated Par-6 and Lgl and remains inactive. Phosphorylation of Par-6 by AurA blocks the interaction of Par-6 with aPKC, which in turn leads to activation of aPKC. Activated aPKC then phosphorylates Lgl to replace it with Baz. The Par complex that has recruited Baz is able to phosphorylate Numb, leading to an exclusion of Numb from the apical domain. Because AurA becomes active in the mitotic phase, it is able to synchronize aPKC activation with the entry into mitosis. Given the role of AurA as a positive regulator of aPKC signaling, it is also likely that dephosphorylation negatively regulates this signaling pathway (Ogawa, 2009).

The serine/threonine phosphatases are grouped into four major classes based on their sensitivity to inhibitors and requirement for divalent cations: protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), protein phosphatase 2B (PP2B) and protein phosphatase 2C (PP2C). PP2A holoenzyme functions as a heterotrimeric complex comprising a catalytic C subunit [Microtubule star (Mts) in Drosophila], a scaffolding A subunit (PP2A-29B in Drosophila) and a regulatory B-subunit [Twins (Tws), Widerborst (Wdb) and PP2A-B' in Drosophila]. The A-subunit can serve as a linker between the C- and B-subunits, and the B-subunit can influence the enzymatic activity and substrate specificity of the holoenzyme. In a genetic screen using the Drosophila compound eye, mts was identified as an enhancer of the aPKC-induced eye phenotype. The genetic and biochemical evidence indicating that Mts suppresses aPKC activity by enhancing dephosphorylation of Par-6 in neuroblasts uncovered an antagonistic role of PP2A in the aPKC signaling pathway (Ogawa, 2009).

The Drosophila compound eye is composed of repetitive ommatidia that contain epithelial retinal cells. Because of the crystal-like arrangement of ommatidia in the compound eye, it is sensitive to defects in epithelial polarity and therefore ideal for use in mutational screens for components involved in epithelial polarity. A modifier screen was undertaken under a sensitized background to look for mutations that affected epithelial polarity. When the membrane-tethered aPKC (aPKCCAAX) is expressed by GMR-GAL4, it becomes expressed in all differentiated retinal cells, the apicobasal polarity of the retinal cells is severely impaired, and the compound eye becomes small and rough. Kinase activity of aPKCCAAX is essential for inducing this eye phenotype, because the kinase-dead version, in which Lys293 is mutated to Trp (K293W), does not alter the eye morphology. Using this system, mutants were screened that modify the aPKCCAAX-induced eye phenotype among lethal mutants available from stock centers, and mts was identified as a phenotypic enhancer. When the GMR-GAL4/UAS-aPKCCAAX fly was crossed to the mts02496 or mtsXE-2258 mutant fly, a smaller and rougher eye was observed, suggesting that Mts acts as an antagonist for the aPKC signaling pathway (Ogawa, 2009).

The mts gene is expressed ubiquitously during embryogenesis and its protein product localizes to the cytoplasm in neuroblasts as well as in epithelial cells. Because a large amount of mts mRNA is maternally supplied, zygotic mts mutant embryos do not show significant defects with regard to cell polarity, and germline clones do not produce an egg. Therefore loss-of-function phenotypes were examined in neuroblasts by overexpressing a dominant-negative mutant of Mts (dnMts) (Hannus, 2002), which lacks the N-terminal region of the phosphatase domain. In wild-type neuroblasts, the protein complex containing aPKC, Par-6 and Baz localizes to the apical cortex and directs Miranda to the basal cortex at metaphase. In the dnMts-expressing embryos, the apical complex localizes to the apical cortex, and its distribution is broader than normal. By contrast, localization of Miranda is severely affected, and it is distributed less asymmetrically along the cell cortex and into the cytoplasm, where it is concentrated on the mitotic spindles. This phenotype resembles that of lgl, raising the possibility that Mts functions in the same pathway as Lgl (Ogawa, 2009).

This study shows that PP2A functions as a negative regulator of the aPKC signaling pathway in Drosophila neuroblasts. Although several studies have suggested that PP2A negatively regulates aPKC signaling in mammalian culture cells, the critical target(s) of PP2A is unknown in these studies. The substrates of aPKC, which include Lgl and aPKC itself, can also be substrates for PP2A. However, none of them has been molecularly identified as a target to be dephosphorylated by PP2A. This study has identified Par-6 as a direct target of PP2A, which has its catalytic subunit encoded by the mts gene. Par-6 is known to be phosphorylated by AurA to trigger aPKC activation when neuroblasts and SOPs enter mitosis. Biochemical and genetic evidence reveals that Mts dephosphorylates Par-6 to suppress the aPKC pathway, suggesting an antagonistic role for Mts against AurA in the regulation of cell polarity that is governed by aPKC signaling (Ogawa, 2009).

Co-immunoprecipitation assays of overexpressed Par-6, aPKC or Lgl with Mts in S2 cells indicated that all these molecules can form a complex either directly or indirectly. Among these, the association of Mts with aPKC and Lgl is relatively weaker than the association with Par-6, although a previous study suggested that PP2A associates with aPKC to suppress its kinase activity in mammalian cultured cells (Nunbhakdi-Craig, 2002). In the current study results indicate that, in S2 cells, Par-6 most efficiently forms a complex with Mts. Consistently, the in vitro dephosphorylation assay showed that PP2A effectively dephosphorylates AurA-phosphorylated Par-6 but not the auto-phosphorylated PKCzeta or the PKCzeta-phosphorylated Lgl. It is inferred from these results that Par-6 is a direct target of Mts. Substrate specificity of PP2A is greatly influenced by the B-subunit incorporated into the holoenzyme. Thus, differences in the affinity with Mts among the three tested molecules in co-immunoprecipitation assays might, therefore, partly reflect the B-subunit(s) that is expressed in S2 cells endogenously. The Drosophila genome contains three genes for the B-subunit: tws, wdb and PP2A-B', all of which are ubiquitously expressed during embryogenesis, as is mts (Ogawa, 2009).

At present, it is not clear which of these three is used for targeting Par-6. Among them, tws mutants often show bristle duplications that are due to defective cell fate decisions of the SOP, as lgl4/lglts3 flies show. Since this lgl4/lglts3 phenotype is enhanced by mts, mts is also likely to be involved in the same pathway. Furthermore, a recent study demonstrated that Tws, together with Mts, is included in the aPKC complex to regulate the asymmetric cell division of larval neuroblasts (Chabu, 2009). These results suggest that Mts uses Tws to target Par-6 in the asymmetric cell divisions of SOPs as well as of neuroblasts (Ogawa, 2009).

Par-6 is an essential cofactor for aPKC activity, and it is known to keep aPKC inactive in the absence of AurA-dependent phosphorylation of Par-6 in neuroblasts. The complete deprivation of Par-6 results in the uniform distribution of Miranda into the cell cortex, which is reminiscent of the aPKC mutant phenotype. Thus, Par-6 is required to produce functional aPKC, and its kinase activity becomes active only when Par-6 is phosphorylated by AurA. par-6 heterozygotes (par-6δ226/+) do not show any defect in Miranda localization during the embryonic stage, which indicates that one copy of par-6 in addition to the maternal supply is sufficient to support normal aPKC function. When mts is further inactivated under this condition (par-6δ226/+, mtsXE-2258/mts02496), some neuroblasts exhibit an lgl-like phenotype in Miranda localization, which is indicative of aPKC hyperactivation and unlike the par-6 loss-of-function mutant. This result suggests that Par-6, because of its hyper-phosphorylation, becomes unable to restrict aPKC activity within the normal range. Thus, a probable normal function of Mts is to promote the inhibitory function of Par-6 on aPKC without affecting its function as an essential subunit of the aPKC complex (Ogawa, 2009).

Whereas AurA seems to be active only during the mitotic phase in cell-cycling cells, mitotically inactive or interphase epithelial cells exhibit concrete apicobasal polarity. How do those cells activate aPKC signaling even though AurA is inactive? This apparent paradox raises several possibilities. Par-6 phosphorylation is required for aPKC activation in epithelial cells but might be mediated by kinase(s) other than AurA. Alternatively, aPKC might be activated by mechanisms other than the phosphorylation of Par-6. Indeed, it has been reported that the active form of Cdc42 binds to the CRIB domain of Par-6 to relieve its inhibitory effect on aPKC, leading to the activation of aPKC (Ogawa, 2009).

Although obvious defects are not detected in the epithelial cells of zygotic mts mutant embryos, Oogenesis clones of mts show dramatic defects in their epithelial polarity. This follicle cell phenotype is different from that caused by hyperactivation of aPKC, as observed in the lgl mutant, suggesting that the action of Mts is mechanistically different in the maintenance of follicle cell polarity from that observed in neuroblasts. In photoreceptor cells, Mts operates antagonistically against Par-1 kinase, which restricts Baz to the adherens junctions. It is also known that Par-1 phosphorylates Baz directly to inhibit its incorporation into the apical aPKC complex, thereby restricting Baz to the apical domain in follicle cells. These data raise the possibility that Mts antagonizes Par-1 in Baz localization in follicle cells by inhibiting Par-1-dependent Baz phosphorylation. If this is the case, Mts positively regulates the aPKC pathway in follicle cells and photoreceptor cells, unlike the situation in neuroblasts. To date, there is no report for Par-1 function in Drosophila neuroblasts. Further study is necessary to test whether a similar antagonism between Mts and Par-1 has a role in the regulation of neuroblast polarity (Ogawa, 2009).

AurA-mediated Par-6 phosphorylation is a key step in initiating the asymmetric segregation of the cell fate determinants in the neuroblast cell cycle. Once Par-6 is phosphorylated, aPKC will be continuously activated during the mitotic phase. The apical domain would overwhelm the entire cortex unless an antagonistic reaction occurred. PP2A will be able to balance AurA in Par-6 phosphorylation during mitosis. Thus, PP2A, together with the antagonistic ligand Lgl, might have a role in maintaining aPKC activity at an appropriate level to create both apical and basal domains in the cortex during mitosis. Although both Mts and Lgl negatively regulate aPKC signaling, Mts operates on aPKC activity by directly regulating the cell-signaling cascade, whereas Lgl does so through the direct physical association as a substrate. Therefore, they are different in their mechanisms of action (Ogawa, 2009).

When neuroblasts complete cell cleavage, the basal membrane is largely segregated into the GMC, and the entire cell cortex of neuroblasts appears to become apical. It is therefore necessary to repolarize in order to make the apical and basal domains in the cell cortex for the onset of the next cell cycle. To do so, Par-6 phosphorylation must be removed before entering the next cell cycle, to reset the configuration of the apical complex. A model is proposed in which Mts actively dephosphorylates Par-6 to reset the membrane polarity after the completion of each division cycle. In this context, it will be important to examine whether Mts function depends on the cell-cycle stage in neuroblasts (Ogawa, 2009).

In eukaryotes, serine/threonine phosphatases are categorized into four major groups: PP1, PP2A, PP2B and PP2C. Recent studies have shown that PP1α affects Par-3 activity through the regulation of a phosphorylation-dependent interaction of Par-3 with 14-3-3 or PKCzeta. This study also identified a Pp1-87B mutation as an enhancer of the aPKCCAAX-induced eye phenotype in the genetic screen and found defects in localization of Miranda as well as in epithelial cell polarity in the Pp1-87B mutant. Furthermore, it has been reported that protein phosphatase 4 (PP4), which is a PP2A family member, regulates Miranda localization in Drosophila neuroblasts, although the direct substrate of PP4 is not yet clear. Thus, other classes of phosphatases in addition to PP2A are involved in the regulation of cell polarity in various cellular contexts. Further delineation of phosphatase functions and the crosstalk between phosphatases should help lead to an understanding of the global control of cellular processes regulated by cell polarity (Ogawa, 2009).

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

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

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

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

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

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

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

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

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

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

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

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

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

Bazooka inhibits aPKC to limit antagonism of actomyosin networks during amnioserosa apical constriction

Cell shape changes drive tissue morphogenesis during animal development. An important example is the apical cell constriction that initiates tissue internalisation. Apical constriction can occur through a phase of cyclic assembly and disassembly of apicomedial actomyosin networks, followed by stabilisation of these networks. Delayed negative-feedback mechanisms typically underlie cyclic behaviour, but the mechanisms regulating cyclic actomyosin networks remain obscure, as do mechanisms that transform overall network behaviour. This study shows that a known inhibitor of apicomedial actomyosin networks in Drosophila amnioserosa cells, the Par-6-aPKC complex, is recruited to the apicomedial domain by actomyosin networks during dorsal closure of the embryo. This finding establishes an actomyosin-aPKC negative-feedback loop in the system. Additionally, aPKC was found to recruit Bazooka to the apicomedial domain, and phosphorylates Bazooka for a dynamic interaction. Remarkably, stabilising aPKC-Bazooka interactions can inhibit the antagonism of actomyosin by aPKC, suggesting that Bazooka acts as an aPKC inhibitor, and providing a possible mechanism for delaying the actomyosin-aPKC negative-feedback loop. These data also implicate an increasing degree of Par-6-aPKC-Bazooka interactions as dorsal closure progresses, potentially explaining a developmental transition in actomyosin behaviour from cyclic to persistent networks. This later impact of aPKC inhibition is supported by mathematical modelling of the system. Overall, this work illustrates how shifting chemical signals can tune actomyosin network behaviour during development (David, 2013).

These data outline a regulatory circuit for guiding amnioserosa apical constriction. The circuit controls both the localisation and activity of its components. In terms of protein localisation, it was found that amnioserosa actomyosin networks recruit the Par proteins to the apicomedial domain. Although Par protein puncta are not continually dependent on the actomyosin networks, their numbers build over developmental time, apparently owing to the cumulative effect of multiple rounds of actomyosin network assembly. The networks appear to impact aPKC directly, and in turn, aPKC recruits Baz to the apical domain. This recruitment depends on the C-terminal aPKC-binding region of Baz, which aPKC phosphorylates for a dynamic relationship with Baz in the apical domain of amnioserosa cells (David, 2013).

Par-6-aPKC activity inhibits amnioserosa actomyosin networks (David, 2010), and the recruitment of aPKC by the networks implicates a negative-feedback loop. As delayed negative feedback tied to a continual input signal can produce an oscillatory output, the actomyosin-aPKC negative-feedback loop might explain how aPKC regulates actomyosin network assembly-disassembly cycles (David, 2010). However, apical populations of Par-6-aPKC puncta are not fully recruited and fully removed with each actomyosin cycle, suggesting additional mechanisms. Importantly, Par-6-aPKC activity can be tempered by Baz. Thus, aPKC inhibition by Baz might delay the actomyosin-aPKC negative-feedback loop during early DC, promoting the actomyosin assembly-disassembly cycles. As DC proceeds, the additive effects of actomyosin assembly-disassembly cycles could increase apical Par protein levels; additionally, the gradual apical constriction of the cells decreases their apical surface areas and could thus increase apical surface Par protein concentrations. It is proposed that a gradual increase to apicomedial aPKC-Baz interactions inhibits aPKC and thus leads to the stabilisation of actomyosin networks. Simulations indicate that this transition in network behaviour can occur abruptly following incremental reductions to myosin inhibition during earlier DC. It is proposed that Baz acts as a competitive inhibitor to reduce aPKC phosphorylation of cytoskeletal regulators. This idea is consistent with reports of Par-3 inhibiting aPKC in kinase assays in vitro. However, Baz is also known to promote aPKC localisation in the epidermis and amnioserosa. Thus, Baz appears to both promote and inhibit aPKC activity, potentially forming a paradoxical circuit (or incoherent feed-forward loop) in which Baz and aPKC promote each other's recruitment, and in which Baz competitively inhibits aPKC activity. Significantly, Baz has multiple binding sites for the Par-6-aPKC complex [Par-6 binds Baz PDZ1; aPKC binds Baz PDZ2-3; aPKC binds the Baz C-terminal aPKC-binding region], suggesting cooperative binding and that Baz interactions with the Par-6-aPKC complex are stronger than those between the Par-6-aPKC complex and its cytoskeleton targets. Notably, this study found that Baz apical surface levels are ~66% lower than those of Par-6, suggesting that the inhibitory effect of Baz must be dynamic; Baz cannot simply sequester all Par-6-aPKC complexes by outnumbering them. The inhibitory effect must also depend on phosphatases because aPKC interactions with Baz are weakened following phosphorylation (Morais-de-Sá, 2010). Baz/Par-3 is known to be regulated by Protein phosphatase 1 and Protein phosphatase 2A with Protein phosphatase 1 de-phosphorylating the aPKC phosphorylation site of Par-3. Thus, Baz may act as a strong and dynamic inhibitor of Par-6-aPKC to buffer and eventually overcome the actomyosin-aPKC negative-feedback loop (David, 2013).

A crucial unknown is the identity of the cytoskeletal target(s) of aPKC. Cytoskeletal targets of aPKC have been identified but have not been examined during amnioserosa apical constriction. In mammalian cells, Par-6-aPKC can phosphorylate Smurf1, an E3 ubiquitin ligase, in turn leading to RhoA degradation in cellular protrusions (Wang, 2003). During dendritic spine morphogenesis, Par-6-aPKC acts though p190RhoGAP to inhibit RhoA (Zhang, 2008). As well, aPKC phosphorylation of Rho kinase leads to its cortical dissociation in mammalian cell culture (Ishiuchi, 2011), and apparently during salivary gland tubulogenesis in Drosophila (Röper, 2012). Of note, the persistent Par-6-aPKC puncta could actively downregulate actomyosin activity, or prolong the lull between actomyosin activations, or do both. Another question is how actomyosin networks recruit aPKC. The recruitment of Par proteins by actomyosin networks has been documented during Drosophila cellularisation and C. elegans one-cell polarisation, and Baz and aPKC have been shown to co-immunoprecipitate with myosin regulatory light chain from Drosophila egg chambers, but specific linkages have yet to be identified. Defining further components of the actomyosin-aPKC negative-feedback loop will be crucial for understanding its regulation and its effects on actomyosin network dynamics. In particular, despite identifying a potential delay mechanism for the loop, it is unclear how the loop and the delay mechanism could translate into oscillatory network behaviour. Perhaps the cytoskeletal target(s) of aPKC are co-recruited with the assembling networks, which in combination with the buffering effect of Baz, could delay their phosphorylation by aPKC. It is also possible that the clustering of Par protein puncta with each network assembly event could somehow modify the Baz buffering effect (David, 2013).

Another unanswered question is the influence of circumferential anchors for Baz or Par-6-aPKC, as weakening of these anchors could contribute to apicomedial Par protein accumulation over DC. Echinoid (Ed), a transmembrane AJ-associated protein that can directly bind Baz, is normally lost from the amnioserosa during DC. It is hypothesised that this loss might promote the loss of Baz from AJs and its apicomedial accumulation. However, ectopic expression of Ed in the amnioserosa leading to circumferential Ed levels higher than those seen in the epidermis had no apparent effect on apicomedial Baz localisation. Thus, differences in Ed expression alone cannot account for the differential localisation of Par proteins between the amnioserosa and epidermis. It is possible that the effects of actomyosin can overpower ectopic Ed, or that other changes to the apical circumference of amnioserosa cells are involved. More generally, other Par protein interaction partners should be considered. For example, Baz and Stardust also interact and, together with Crumbs and Patj, they form the apical Crumbs complex (Tepass, 2012). Recent results suggest Patj can activate myosin by suppressing myosin light chain phosphatase. Intriguingly, amnioserosa BazS980A apical surface puncta also recruit Patj, suggesting that this pathway might contribute to myosin activity as well (David, 2013).

In summary, the data argue that the differential regulation of amnioserosa actomyosin networks by Baz and Par-6-aPKC can be explained by a single pathway in which Baz inhibits Par-6-aPKC antagonism of the cytoskeletal networks. It was also found that the actomyosin networks recruit aPKC, forming a negative-feedback loop. It is proposed that the inhibition of aPKC by Baz delays the negative feedback at earlier DC for cycling actomyosin networks, and with increased inhibition of aPKC by later DC, the actomyosin networks persist. These findings provide an example of how chemical signalling, and changes to this signalling, can modify the behaviour of actomyosin networks during embryo development (David, 2013).

Slmb antagonises the aPKC/Par-6 complex to control oocyte and epithelial polarity

The Drosophila anterior-posterior axis is specified when the posterior follicle cells signal to polarise the oocyte, leading to the anterior/lateral localisation of the Par-6/aPKC complex and the posterior recruitment of Par-1, which induces a microtubule reorganisation that localises bicoid and oskar mRNAs. This study shows that oocyte polarity requires Slmb, the substrate specificity subunit of the SCF E3 ubiquitin ligase that targets proteins for degradation. The Par-6/aPKC complex is ectopically localised to the posterior of slmb mutant oocytes, and Par-1 and oskar mRNA are mislocalised. Slmb appears to play a related role in epithelial follicle cells, as large slmb mutant clones disrupt epithelial organisation, whereas small clones show an expansion of the apical domain, with increased accumulation of apical polarity factors at the apical cortex. The levels of aPKC and Par-6 are significantly increased in slmb mutants, whereas Baz is slightly reduced. Thus, Slmb may induce the polarisation of the anterior-posterior axis of the oocyte by targeting the Par-6/aPKC complex for degradation at the oocyte posterior. Consistent with this, overexpression of the aPKC antagonist Lgl strongly rescues the polarity defects of slmb mutant germline clones. The role of Slmb in oocyte polarity raises an intriguing parallel with C. elegans axis formation, in which PAR-2 excludes the anterior PAR complex from the posterior cortex to induce polarity, but its function can be substituted by overexpressing Lgl (Morais-de-Sa, 2014).

Very little is known about how the posterior follicle cells signal to polarise the AP axis of the oocyte, except that signalling is disrupted when the germline is mutant for components of the exon junction complex, such as Mago nashi. The current results reveal that Slmb also plays an essential role in this pathway, where it acts to establish the complementary cortical domains of Baz/Par-6/aPKC and Par-1. Although Slmb might act in a variety of ways to establish this asymmetry, the observation that it regulates the levels of the Par-6/aPKC complex suggests a simple model in which Slmb directly or indirectly targets a component of the complex for degradation at the posterior of the oocyte. Since aPKC phosphorylates Par-1 to exclude the latter from the cortex, the degradation of aPKC would allow the posterior recruitment of Par-1, which would then maintain polarity by phosphorylating and antagonising Baz. Indeed, this might explain the observation that Par-6 is excluded from the posterior cortex before Baz. The polarisation of the oocyte therefore appears to occur in two phases. During the initiation phase, Slmb removes the Par-6/aPKC complex from the posterior cortex to allow the recruitment of Par-1. Par-1 then maintains and reinforces this asymmetry by phosphorylating Baz to exclude it from the posterior cortex, thereby removing the cortical anchor for the Par-6/aPKC complex (Morais-de-Sa, 2014).

Slmb is usually recruited to its targets by binding to phosphorylated residues that lie 9-14 amino acids downstream from the ubiquitylated lysine. Although both aPKC and Par-6 contain several sequences that could serve as atypical Slmb binding sites, neither contains a classic Slmb-dependent degron sequence. It is therefore unclear whether the SCFSlmb complex directly ubiquitylates either protein to target it for degradation or whether it targets another, unknown component of the complex that is required for the stability of Par-6 and aPKC. Nevertheless, this model leads to the prediction that the polarising signal from the follicle cells will induce the activation of a kinase that phosphorylates a Slmb substrate at the posterior of the oocyte, thereby triggering the local degradation of the Par-6/aPKC complex (Morais-de-Sa, 2014).

The demonstration that Slmb is required for the exclusion of the Par-6/aPKC complex from the posterior of the Drosophila oocyte raises interesting parallels with AP axis formation in C. elegans. Although Drosophila does not have an equivalent of the main symmetry-breaking step in the worm, in which a contraction of the actomyosin cortex removes the anterior PAR proteins from the posterior, the function of Slmb is analogous to that of PAR-2 in the alternative polarity induction pathway. Both proteins act to remove the Par-6/aPKC complex from the posterior cortex to allow the posterior recruitment of Par-1, which then reinforces polarity by excluding Baz/PAR-3 by phosphorylation. Furthermore, the polarity phenotypes of both slmb and par-2 mutants can be rescued by the overexpression of Lgl. Slmb and PAR-2 act by different mechanisms, since the former is a subunit of the SCF ubiquitin ligase complex and promotes the degradation of the Par-6/aPKC complex, whereas the latter functions by recruiting PAR-1. Nevertheless, it is intriguing that PAR-2 contains a RING finger domain that is typically found in ubiquitin ligases, suggesting that it might have lost this activity during evolution (Morais-de-Sa, 2014).



In situ hybridization using a Par-6 probe has shown that there is a high maternal contribution in early embryos, and there is ubiquitous expression throughout embryogenesis with slightly elevated expression levels in the gut. In contrast to inscuteable and bazooka, no asymmetric localization of Par-6 RNA is detected. To determine expression of the Par-6 protein, a Par-6 peptide antibody was generated and used it to stain Drosophila embryos. Par-6 protein is present in all cells, but staining is more intense in epithelial tissues including the developing epidermis, foregut, hindgut, salivary glands, Malpighian tubules and the tracheal system (Petronczki, 2001).

Par-6 and synaptic plasticity

The Baz/Par-3-Par-6-aPKC complex is an evolutionarily conserved cassette critical for the development of polarity in epithelial cells, neuroblasts, and oocytes. aPKC is also implicated in long-term synaptic plasticity in mammals and the persistence of memory in flies, suggesting a synaptic function for this cassette. At Drosophila glutamatergic synapses, aPKC controls the formation and structure of synapses by regulating microtubule (MT) dynamics. At the presynapse, aPKC regulates the stability of MTs by promoting the association of the MAP1B-related protein Futsch to MTs. At the postsynapse, aPKC regulates the synaptic cytoskeleton by controlling the extent of Actin-rich and MT-rich areas. In addition, Baz and Par-6 are also expressed at synapses and their synaptic localization depends on aPKC activity. These findings establish a novel role for this complex during synapse development and provide a cellular context for understanding the role of aPKC in synaptic plasticity and memory (Ruiz-Canada, 2004).

During expansion of the NMJ, parent boutons located at the distal end of a branch give rise to new synaptic boutons by budding. New buds separate from parent boutons by the formation of a neck, and NMJ branches extend by neck elongation and bouton enlargement. Throughout this process, the postsynaptic membrane and underlying cytoskeleton impose a barrier to presynaptic extension, since synaptic boutons and their buds are completely surrounded by the muscle cell membrane and underlying cytoskeleton. During branch elongation, a presynaptic signal may induce the retraction of the postsynaptic cytoskeleton barrier. It is proposed that changes in both the pre- and postsynaptic cytoskeleton during branch elongation mediate these events and that these processes are regulated by aPKC with the collaboration of Baz and Par-6 in both locales (Ruiz-Canada, 2004).

The results show that changes in aPKC activity affect both postsynaptic MT and Actin domains. Based on the lack of aPKC within the Actin domain and the enrichment of aPKC at the MT domain, a primary action of aPKC in muscle cells might be through MTs that surround the peribouton area. Alternatively, the effect of aPKC activity on muscle MTs may arise as a consequence of changes in Baz and Par-6 in the Actin-rich peribouton area, which is spatially segregated from postsynaptic MTs (Ruiz-Canada, 2004).

An interesting finding is that both an increase and decrease in aPKC activity, either pre- or post-synaptically, result in reduction of NMJ expansion. This may reflect the possibility that the pre- and post-synaptic cytoskeleton antagonize one another during NMJ expansion and that an asymmetric perturbation of the cytoskeleton in each cell prevents normal synaptic growth. An alternative or additional possibility is that aPKC is asymmetrically regulated at the pre- and postsynaptic cell, being activated in one cell and inhibited in the other. In this regard, it was noteworthy that while increasing aPKC activity increases the stability of presynaptic microtubules, increasing aPKC postsynaptically results in microtubules that appeared to retract from the junctional area (Ruiz-Canada, 2004).

These studies indicate that Baz and Par-6 are colocalized with aPKC, although this colocalization is only partial. Further, a decrease in Baz or Par-6 gene dosage has been shown to alter NMJ growth and the genes interact genetically with aPKC. That all three proteins coimmunoprecipitate supports the notion that they exist in a tripartite complex. However, it is also likely that at different regions of the NMJ, the composition of the complex is reduced to aPKC-Par-6 or Baz-Par-6. This is suggested by the colocalization studies showing that only Par-6 and aPKC are concentrated at the MT bundle and that only Par-6 and Baz are concentrated at the peribouton area (Ruiz-Canada, 2004).

Baz and Par-6 are localized to the Actin/Spectrin peribouton area, and loss of Baz in dapkc mutants or baz4/+ mutants decreases peribouton Spectrin localization, suggesting that Baz regulates the Actin/Spectrin network. In epithelial cells, Baz is required for the maintenance of the zonula adherens, an Actin belt that encircles the cell just below its apical face. At the NMJ, Baz may similarly contribute to the maintenance of the Actin-rich domain (Ruiz-Canada, 2004).

The composition of the Baz/Par-6/aPKC complex is likely to be regulated by the kinase activity of aPKC; expressing PKM increased the amount of Baz associated to the complex. Mammalian Par-6 is known to bind to both aPKC and Baz at distinct sites, and Par-6 activates aPKC when bound to activated Cdc42 and Rac1. Mammalian Baz/Par-3 is also known to bind to both aPKC and Par-6 at distinct sites, but in contrast to Par-6, Baz inhibits aPKC activity. This inhibition can be suppressed by aPKC-dependent Baz phosphorylation at a highly conserved protein region, and this phosphorylation promotes the dissociation of Baz and aPKC. At the NMJ, it was found that increasing PKM, which lacks the Par-6 binding site, increases the binding between Par-6 and Baz, suggesting that Baz phosphorylation may promote the association between Baz and Par-6. A potential scenario is that Baz and aPKC may exist as an inactive complex at the muscle cortex. Phosphorylation of Baz dissociates the complex and phosphorylated Baz may accumulate at the peribouton region. In agreement with this model, it was found that overexpressing PKM postsynaptically results in an expansion of the peribouton area and increased accumulation of Baz at this area (Ruiz-Canada, 2004).

Electrophysiological studies show that aPKC activity also influences synaptic efficacy. This may result from cytoskeletal changes, which may alter the localization of synaptic proteins, such as GluRs. Indeed, changes in aPKC activity were found to affect both GluR levels or distribution and mEJP amplitude. Many synaptic receptors are anchored to the Actin submembrane matrix. For example, the scaffolding protein DLG, which is responsible for the clustering of Shaker K+ channels and the cell adhesion molecule FasII at the peribouton area, depends on normal Spectrin levels for proper localization at this area. Similarly, in mammals, the DLG homolog SAP97 binds to band 4.1, which is anchored at the Actin/Spectrin network, and NMDA receptors bind alpha-Actinin, an Actin binding protein. Therefore, the changes in GluR levels and distribution found in dapkc mutants may result from alterations in the postsynaptic Actin network (Ruiz-Canada, 2004).

Despite the changes in mEJP amplitude, synaptic junction efficacy (represented by quantal content) was decreased in both aPKC gain- and loss-of-function mutants. This is in contrast to other mutants that affect synaptic transmission in which quantal content is maintained despite changes in postsynaptic sensitivity. For example, reduction of GluR at the postsynapse results in an increase in the amount of neurotransmitter release at Drosophila NMJs. The results raise the possibility that aPKC may be affecting the mechanism that controls retrograde regulation of neurotransmitter release (Ruiz-Canada, 2004).

In addition to changes in quantal content and mEJP amplitude, a reduction was also observed in mEJP frequency. Changes in the frequency of mEJP may arise from a decrease in the probability of release or in the number of release sites. At the NMJ, the reduction in mEJP frequency may reflect the reduction in bouton number observed in dapkc mutants (Ruiz-Canada, 2004).

In the mammalian hippocampus, atypical PKMzeta is necessary and sufficient for LTP maintenance. In flies, overexpression of PKMzeta enhances memory in a Pavlovian olfactory learning paradigm. Moreover, aPKC inhibition using a kinase dead dominant-negative or chelerythrine treatment, which specifically inhibits the catalytic domain of aPKC, diminishes memory without affecting learning. Although these studies suggest that aPKC is involved in functional plasticity of synapses, the cellular mechanism for this effect is unknown (Ruiz-Canada, 2004).

Recent studies suggest that morphological modifications of dendritic spines accompany synapse plasticity, and therefore, changes in spine structure might be at the core of learning and adaptive mechanisms. Spines are particularly enriched in Actin, and interfering with the Actin cytoskeleton inhibits spine motility. Further, many members of the postsynaptic complex, including NMDA receptors, CaMKII, PSD-95, SPAR, and Shank associate with F-Actin through Actin binding proteins. MTs, in contrast, localize to dendritic shafts and are believed to constitute a more stable component. This partitioning between MT and microfilament domains, however, is reminiscent of these domains in growth cones, where Actin and MT dynamics are highly interdependent and ultimately responsible for growth cone dynamics. Similarly, in these studies it has been shown that interfering with normal MT dynamics though modifications in aPKC activity has important consequences for the arrangement of the Actin-rich peribouton area and the normal localization of GluRs. Therefore, although the influence of MTs in spine structure has received less attention, it may be the case that spine architecture is ultimately defined by an interplay between Actin- and MT-rich domains (Ruiz-Canada, 2004).

These studies demonstrate that changes in MT organization are an essential aspect of synapse development and that the aPKC/Baz/Par-6 complex plays an important role in their regulation. In addition, the results show that at the postsynaptic cell, changes in aPKC activity result in dramatic changes in both the MT and Actin networks. Commensurate with the behavioral and electrophysiological studies in which increasing aPKC activity enhanced LTP and memory maintenance, it was found that increases and decreases in aPKC activity inversely regulated the synaptic cytoskeleton. These observations raise the attractive possibility that aPKC regulates synapse plasticity, at least in part, by affecting the organization of the synaptic cytoskeleton (Ruiz-Canada, 2004).

The positioning and segregation of apical cues during epithelial polarity establishment in Drosophila: PAR-6 is recruited to the extreme apical region and maintains Baz and AJs

Cell polarity is critical for epithelial structure and function. Adherens junctions (AJs) often direct this polarity, but it has been found that Bazooka (Baz) acts upstream of AJs as epithelial polarity is first established in Drosophila. This prompted an investigation into how Baz is positioned and how downstream polarity is elaborated. Surprisingly, it was found that Baz localizes to an apical domain below (basally to) its typical binding partners atypical protein kinase C (aPKC) and partitioning defective (PAR)-6 as the Drosophila epithelium first forms. In fact, Baz positioning is independent of aPKC and PAR-6 relying instead on cytoskeletal cues, including an apical scaffold and dynein-mediated basal-to-apical transport. AJ assembly is closely coupled to Baz positioning, whereas aPKC and PAR-6 are positioned separately. This forms a stratified apical domain with Baz and AJs localizing basally to aPKC and PAR-6, and specific mechanisms were identified that keep these proteins apart. These results reveal key steps in the assembly of the apical domain in Drosophila (Harris, 2005).

These results frame a model of apical domain assembly during epithelial polarity establishment in Drosophila. During cellularization, Baz acts as a primary polarity landmark that positions AJs and aPKC. Baz, itself, is positioned by two cues (an apical scaffold and dynein-mediated transport). Baz recruits and colocalizes with AJ proteins in a subapical region while helping direct aPKC to the extreme apical region. During gastrulation, a third cue becomes important for Baz and AJ positioning. At this stage, aPKC becomes required for maintaining Baz and AJs. PAR-6 is also recruited to the extreme apical region and maintains Baz and AJs. Although Baz can interact with aPKC and PAR-6 at this stage, Crb blocks these interactions. It is proposed that this interaction network establishes a robust, stratified apical domain from the earliest stages of epithelial development (Harris, 2005).

AJs are often key polarity landmarks. However, Baz positioning is AJ independent at the time that epithelial polarity is first established in Drosophila. Here, Baz appears to act as a primary polarity landmark, but what cues position Baz? The data indicate that Baz is initially positioned by cytoskeletal cues that support an apical Baz-binding scaffold and mediate basal-to-apical Baz transport. The apical scaffold is saturable. Its function requires actin; Baz becomes basally mislocalized after actin disruption. However, since Baz overlaps only the basal reaches of the apical actin network, it is unlikely that Baz simply binds actin. Interestingly, Baz remains largely membrane associated when actin is disrupted. One caveat is that there is some residual actin. However, the same treatment dissociates APC2 from the cortex. Actin is also required for PAR-3 cortical association in C. elegans one-cell embryos. During Drosophila cellularization, it is speculated that Baz may have other cortical anchors and that actin may control their distribution -- of course, actin is critical for many cellular processes and could play other roles in positioning Baz. It will be important to identify the apical scaffold for Baz (Harris, 2005).

Baz positioning also requires the minus-end-directed MT motor dynein. Live imaging of BazGFP revealed basal-to-apical translocation of BazGFP puncta during cellularization. Baz-GFP that diffuses to ectopic basal positions appears to engage a preexisting, dynein-based, basal-to-apical transport system. Such a system transports Golgi vesicles apically during cellularization. Baz-dynein associations appear to cease once dynein brings Baz to the apical region, where Baz presumably docks with its apical scaffold. Although BazGFP puncta move slower than in vitro dynein velocity measurements, dynein-mediated lipid droplet movements have similar speeds during Drosophila cellularization. In vivo, BazGFP puncta may be slowed because they form large cortical complexes. Indeed, DE-Cad, aPKC, and PAR-6 associate with these puncta and Baz oligomerization may promote complex assembly. Further supporting a role for dynein, endogenous Baz is positioned near MT minus ends in WT embryos, but mislocalizes basally in dhc64Cm/z mutants. dhc64C mutations also enhance the baz mutant embryonic phenotype. This is the first report of dynein positioning Baz or its homologues (Harris, 2005).

Analysis of dynein mutants also revealed a third mechanism that can reposition Baz apically during gastrulation. Perhaps the apical Baz-binding scaffold is strengthened during this stage. Alternatively, a distinct polarizing mechanism may be activated, or aPKC and PAR-6 may be involved. Having three Baz positioning mechanisms may ensure proper Baz localization for regulating downstream polarity (Harris, 2005).

Baz acts upstream of AJs as epithelial polarity is first established in Drosophila. The following model is proposed in which AJ assembly may be coupled to Baz positioning. During cellularization, AJ proteins accumulate in both apical and basal junctions. Basal junctions form transiently near the base of each invaginating furrow. Baz is not required for basal junctions, but is required for recruiting AJ proteins into apical junctions. Apical Baz may provide a landmark for apical AJ assembly (Harris, 2005).

The data also suggest that Baz may be involved in ferrying DE-Cad to the apical domain via dynein-mediated transport. Dynein is required for correct apical positioning of both Baz and DE-Cad, and their colocalization in ectopic basal complexes in dhc64Cm/z mutants suggests they may normally be transported to the apical domain together. Indeed, Baz can form complexes with DE-Cad and Arm. Although most endogenous Baz is apical during WT cellularization, its basal mislocalization in dhc64Cm/z mutants suggests that some Baz may normally move basally. In fact, excess BazGFP displaced from the apical domain preferentially accumulates at basal junctions. It is hypothesized that some Baz may normally interact transiently with basal junctions. From there, it may help ferry AJ proteins apically via dynein-mediated transport. MT motors have been implicated in AJ assembly. For example, dynein interacts with ß-catenin and may tether MTs to AJs assembling between PtK2 cells. Kinesin transports AJ proteins to nascent AJs in cell culture, and the mitotic kinesin-like protein 1 is required for apical targeting of AJs and other cues in C. elegans epithelia. It will be important to see if these targeting mechanisms have commonalities with AJ positioning in Drosophila, and if Baz homologues are involved (Harris, 2005).

Finally, it is hypothesized that the third Baz-AJ positioning mechanism revealed in dhc64Cm/z mutants might be related to the normal maturation/stabilization of AJs at gastrulation. At this stage, precursory spot AJs fuse into continuous belt junctions around the top of each cell. In mammalian cell culture, aPKC is required for such AJ maturation. Similarly, aPKC is required for proper AJ and Baz positioning during Drosophila gastrulation, as has been shown for PAR-6. Considering aPKC and PAR-6 are positioned apically as dhc64Cm/z mutants gastrulate, they might recruit Baz and AJs apically in this context as well (Harris, 2005).

Based on their shared roles in polarity in C. elegans, characterized physical interactions, and colocalization in mammalian cells, Baz, aPKC, and PAR-6 are thought to function, at least in some cases, as an obligate tripartite complex. The data suggest that the bulk of cortical Baz and aPKC/PAR-6 do not form obligate complexes during epithelial development in Drosophila. Instead, aPKC and PAR-6 localize to an apical region above Baz and AJs, and are positioned there by distinct mechanisms. Baz/PAR-3 also segregates from aPKC and PAR-6 in other cell types. In C. elegans one-cell embryos, PAR-3, aPKC, and PAR-6 each localize in clusters on the anterior cortex, but these different clusters have limited colocalization (60%-85% fail to colocalize. aPKC and PAR-6 colocalize without PAR-3 at the leading edge of migrating mammalian astrocytes. In Drosophila photoreceptors, Baz colocalizes with AJs below aPKC, PAR-6, and Crb. Even in polarized MDCK cells, aPKC and PAR-6 show some segregation above PAR-3, and although they mainly colocalize at tight junctions, mammalian PAR-3 can regulate tight junction assembly independently of aPKC and PAR-6. Thus, in many contexts interactions between Baz/PAR-3, aPKC, and PAR-6 are dynamic and/or regulated (Harris, 2005).

Baz (PAR-3), aPKC, and PAR-6 often recruit each other to the cortex, but the assembly pathways vary. In C. elegans, one-cell embryos, PAR-3, aPKC, and PAR-6 are mutually dependent for their cortical recruitment. However, in Drosophila neuroblasts, Baz can be positioned without aPKC and PAR-6. Similarly, apical Baz is positioned without aPKC and PAR-6 during Drosophila cellularization. In contrast, apical aPKC recruitment requires Baz, whereas PAR-6 is largely nonpolarized at this stage. Given the lack of extensive colocalization of Baz and aPKC in WT embryos, Baz may control aPKC positioning indirectly, perhaps regulating binding to a separate apical scaffold. Alternately, cortical recruitment might involve cytoplasmic Baz-aPKC complexes. Apical PAR-6 accumulates at gastrulation, and this appears partially Baz independent. Indeed, cdc42 recruits PAR-6 at this stage, and at the same time aPKC and PAR-6 become required for maintaining apical Baz. Thus, although Baz is first positioned independently of aPKC and PAR-6, these cues soon develop complex interdependencies (Harris, 2005).

Although Baz can directly bind both aPKC and PAR-6, at least two mechanisms keep them apart. During cellularization, Baz colocalizes with aPKC and PAR-6 when overexpressed, but normally it localizes with AJs below aPKC and PAR-6. This normal segregation may thus involve competition with other binding partners. After cellularization, Crb also becomes important for segregating Baz and AJs from aPKC and PAR-6. These segregation mechanisms help form a stratified apical domain from the earliest stages of epithelial development (Harris, 2005).

A stratified apical domain may strengthen the boundary between the apical and basolateral domains. This boundary forms via reciprocal antagonism between polarity cues. For example, aPKC phosphorylates and excludes Lethal giant larvae (Lgl) from the apical domain in Drosophila epithelia and Lgl appears to repel PAR-6 from the basolateral domain. The Crb and Dlg complexes also have mutual antagonism. It is proposed that the subapical Baz-AJ region may insulate the apical and basolateral domains. For example, it may inhibit active aPKC from moving basally. Indeed, PAR-3 binding can block mammalian aPKC kinase activity. The Baz-AJ subapical region could also block basolateral cues, since AJs are required to segregate Dlg. In this way, the Baz-AJ subapical region could help define a distinct apical-basolateral boundary (Harris, 2005).

To conclude, Baz appears to be a primary epithelial polarity landmark in Drosophila. It is positioned by multiple mechanisms, including an apical scaffold and dynein-mediated transport, and organizes a stratified apical domain, in which it colocalizes with AJs below its typical partners aPKC and PAR-6 (Harris, 2005).

The PAR complex regulates pulsed actomyosin contractions during amnioserosa apical constriction in Drosophila

Apical constriction is a major mechanism underlying tissue internalization during development. This cell constriction typically requires actomyosin contractility. Thus, understanding apical constriction requires characterization of the mechanics and regulation of actomyosin assemblies. This study analyzed the relationship between myosin and the polarity regulators Par-6, aPKC and Bazooka (Par-3) (the PAR complex) during amnioserosa apical constriction at Drosophila dorsal closure. The PAR complex and myosin accumulate at the apical surface domain of amnioserosa cells at dorsal closure, the PAR complex forming a patch of puncta and myosin forming an associated network. Genetic interactions indicate that the PAR complex supports myosin activity during dorsal closure, as well as during other steps of embryogenesis. It was found that actomyosin contractility in amnioserosa cells is based on the repeated assembly and disassembly of apical actomyosin networks, with each assembly event driving constriction of the apical domain. As the networks assemble they translocate across the apical patch of PAR proteins, which persist at the apical domain. Loss- and gain-of-function studies show that different PAR complex components regulate distinct phases of the actomyosin assembly/disassembly cycle: Bazooka promotes the duration of actomyosin pulses and Par-6/aPKC promotes the lull time between pulses. These results identify the mechanics of actomyosin contractility that drive amnioserosa apical constriction and how specific steps of the contractile mechanism are regulated by the PAR complex (David, 2010).

The repeated assembly and disassembly of apical actomyosin networks is an integral part of amnioserosa tissue morphogenesis during DC. Restricting myosin to the amnioserosa alone is sufficient for amnioserosa apical constriction and overall DC. Dynamic apical myosin has been described in the amnioserosa. This study defined these dynamics as repeated assembly and disassembly cycles of actomyosin networks. Moreover, assembly and disassembly are linked to apical constriction and relaxation, respectively. This is consistent with laser ablation studies showing that the apical surfaces of amnioserosa cells maintain tension across the tissue. Moreover, AJ live imaging has revealed general pulsing of amnioserosa cells from germband retraction through DC. The pulsing actomyosin networks arise with this same developmental timing. A 230±76 second periodicity of cortical pulsing has been described at DC, similar to that of the pulsing actomyosin networks. This study found that increased network durations and decreased lull times with amnioserosa-targeted Baz overexpression coincide with faster DC, as compared with amnioserosa-targeted Par-6 plus aPKC-CAAX overexpression, which increases lull times. It is concluded that the pulsing actomyosin networks mediate the constriction of individual amnioserosa cells and that this contributes to DC (David, 2010).

Remarkably, a single amnioserosa apical constriction event is followed by an almost equal relaxation. However, over many constrictions the cells progressively reduce their apical surface area. This suggests that ratcheting mechanisms incrementally harness the constrictions for overall tissue change. Intracellular and extracellular ratchets are possible. Cells of the Drosophila ventral furrow also display pulsed contractions of apical actomyosin networks as they apically constrict. However, there is minimal relaxation after each constriction. Instead, residual myosin filaments are retained between pulses, and may act as intracellular ratchets to harness the pulsed contractions. By contrast, residual myosin filaments were rarely observed between actomyosin pulses in amnioserosa cells, possibly explaining their relaxation after each cell constriction. It has been proposed that the leading edge actomyosin cable of the surrounding epidermis acts as an extracellular ratchet to harness amnioserosa contractility. However, the ability of myosin expression in the amnioserosa alone to drive DC suggests that other mechanisms contribute. Indeed, DC is a robust process with redundant contributions from both amnioserosa and epidermis. At later stages, filopodia-based epidermal zippering at the canthi could provide another extracellular ratchet. In addition, each amnioserosa cell has a persistent circumferential actin belt that might act as an intracellular ratchet, and other uncharacterized processes, such as membrane trafficking or basal activities, could also contribute (David, 2010).

Actomyosin activity also appears to be linked between cells. The networks display preferential D-V movement, and a network in one cell appears to promote network formation in neighbors. Overall amnioserosa cell shape changes are also coordinated between neighbors. Moreover, myosin activity in isolated amnioserosa cells can elicit cortical myosin accumulation in neighboring epidermal cells. It is speculated that feedback from epidermal cells might orient the D-V movement of amnioserosa actomyosin networks. Interestingly, amnioserosa cells also preferentially contract along the D-V axis. Although the actomyosin networks move in this direction, it is unlikely that they are solely responsible for the directional cell shape changes -- the networks affect the cell circumference both along the axis of their trajectory and perpendicular to it, and, as discussed, both effects are transient. Thus, forces from the epidermis might be needed for the biased D-V amnioserosa cell contraction, and they might also direct the D-V movement of amnioserosa actomyosin networks to facilitate DC (David, 2010).

As the actomyosin networks assemble and disassemble, they translocate across a persistent PAR protein patch. These transient associations and lack of specific colocalization between the actomyosin networks and the PAR proteins argue against PAR proteins being integral parts of the actomyosin networks. However, the current results show that the PAR proteins regulate the networks. Genetic interaction tests indicate that Baz, Par-6 and aPKC support myosin activity for proper DC. Strikingly, the live imaging revealed that Baz and Par-6/aPKC regulate distinct phases of the myosin assembly/disassembly cycle. Together, the loss-of-function and gain-of-function studies show that Baz promotes network durations, whereas Par-6 and aPKC promote lull times between pulses. Baz overexpression also decreased lull times, which could result indirectly from increased network durations or from more direct inhibition of the lull phase. Importantly, overexpression experiments indicate that the effects occur specifically in amnioserosa cells, and analyses of cell polarity and AJs indicate that the PAR proteins have relatively direct effects on the actomyosin networks. However, it remains possible that the PAR proteins have additional functions in the amnioserosa (David, 2010).

A number of molecular interactions must control PAR protein activity in the apical domain of amnioserosa cells. The PAR proteins often, but not exclusively, colocalize in amnioserosa cells, suggesting a dynamic relationship consistent with separate Baz and Par-6/aPKC functions. They also show colocalization with Crb, an apical transmembrane protein at the core of the Crb polarity complex. Interestingly, Crb is known to regulate DC, and Par-6 and aPKC can bind Crb complex components. Thus, Crb might be one anchor for PAR proteins at the apical surface of amnioserosa cells (David, 2010).

Molecular mechanisms connecting PAR proteins to myosin and actin have been implicated in a number of studies. For example, aPKC phosphorylates and inhibits mammalian myosin IIB, although these sites are not present in Drosophila Myosin II (Zipper). Par-6/aPKC also inhibits Rho by activating the ubiquitin ligase Smurf1 in mammalian cells. Additionally, Baz and aPKC immunoprecipitate with Sqh from Drosophila egg chambers. Analogous to amnioserosa morphogenesis, mammalian Par-3 and Par-6/aPKC regulate distinct aspects of cell shape change through different cytoskeletal regulators during dendritic spine morphogenesis: Par-3 inhibits cell protrusions by inhibiting Rac through sequestering the RacGEF Tiam1, whereas Par-6/aPKC promotes protrusions by inhibiting Rho via p190 RhoGAP (David, 2010 and references therein).

Amnioserosa cell apical constriction has similarities to endoderm precursor cell apical constriction during C. elegans gastrulation. Here, myosin activity drives cell ingression. Similar to in amnioserosa cells, the PAR complex and myosin accumulate at the center of the apical surface of these cells and of earlier cells as well. However, these C. elegans actomyosin networks do not appear to undergo full assembly/disassembly cycles and instead progressively accumulate or display continual network flows. Interestingly, apical myosin enrichment requires PAR-3 in C. elegans endodermal precursor cells. Apical myosin enrichment also requires Baz, Par-6 and aPKC in Drosophila egg chamber follicle cells. These results suggest that the PAR complex initiates actomyosin network assembly, contrasting with the amnioserosa, in which networks can assemble without detectable Baz and are inhibited by Par-6/aPKC. Perhaps, actomyosin networks with full assembly/disassembly cycles are regulated distinctly. In the one-cell C. elegans embryo, PAR protein puncta move with a multifaceted cortical myosin network to the embryo anterior. Each facet of the network assembles and disassembles with durations similar to those of the amnioserosa actomyosin networks. The network can also form without the PAR proteins, but the overall flow of the network fails with loss of PAR-3, PAR-6 or aPKC. It would be interesting to test whether PAR-3, PAR-6 and aPKC have distinct effects on the individual facets of these networks (David, 2010).

What triggers actomyosin network assembly in amnioserosa cells? It appears to be independent of Baz, and must overcome Par-6/aPKC inhibition. The Rho pathway triggers actomyosin contractility in many contexts. However, amnioserosa-targeted expression of dominant-negative Rho does not appear to block DC. Alternatively, actin assembly might trigger the networks. Actin networks appear larger and last longer than myosin networks as both start forming during germband retraction. This suggests that actin might organize these networks during germband retraction and possibly DC. Intriguingly, Rac inhibition disrupts DC and reduces amnioserosa actin levels. The trigger might also involve intercellular forces from networks in neighboring cells (David, 2010).

How is the actomyosin assembly/disassembly periodicity regulated? Since more than one network per cell is rarely observed, network assembly might require disassembly of the existing network. Disassembly might begin a cascade that ultimately triggers formation of the next network. For cycling, assembly might likewise elicit disassembly. The data indicate that the PAR proteins are important elements of the regulatory network that is involved. Once a network is triggered, Baz prolongs it, but as the network persists, trigger and maintenance signals must be overcome for network disassembly. With disassembly, Par-6/aPKC activity appears to inhibit new assembly, promoting lull times. With time, this Par-6/aPKC activity must diminish and/or be overwhelmed by the trigger mechanism for new network assembly to occur. Identifying trigger and feedback mechanisms within this cycle will be key for understanding how pulsed actomyosin contractions are regulated in the amnioserosa (David, 2010).

Retromer controls epithelial cell polarity by trafficking the apical determinant Crumbs

The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localization. Using a newly established, liposome-based system described in this study, Crb was determined to be an interaction partner and cargo of the retromer complex (See Retromer-mediated sorting). Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals. Loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localization of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. These data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localization. A novel function is also shown for retromer in maintaining epithelial cell polarity (Pocha, 2011).

This study aimed to identify factors that interact with the cytoplasmic domain of the type I transmembrane protein Crumbs (Crb) and are involved in its trafficking. A strategy was devised to present the Crb cytoplasmic tail on liposomes, a method uniquely suited to recruit and identify coats, because it mimics the native configuration of a receptor tail at the membrane/cytosol interface (Pocha, 2011).

Proteoliposomes have been used successfully to identify coat complexes and their accessory proteins; however, these studies were restricted to short, chemically synthesized peptides, which severely limited the length of the cytoplasmic tail. To overcome this, this study redesigned the recruitment assay enabling the use of tails expressed and purified from E. coli. A bacterial expression plasmid was designed containing an N-terminal tandem affinity tag followed by a tobacco etch virus (TEV) protease cleavage site and a single cysteine for the chemical coupling to liposomes, to which the cytoplasmic tail of mouse Crb2 (amino acids R1246 to I1282) was fused (Pocha, 2011).

Because the levels of many transmembrane proteins are regulated by sorting decisions in the early (sorting) endosome, phosphatidylinositol 3-phosphate, the predominant inositol phospholipid of early endosomes, was incorporated into proteoliposomes to selectively enrich endosomal trafficking proteins. These proteoliposomes were used for recruiting cytosolic coat components and other interactors from brain extract], followed by protein identification by tandem mass spectrometry (MS/MS). Crb2 was chosen, because it is the predominant Crb gene expressed in the vertebrate brain. Importantly, the tails of all Crb proteins are highly conserved, suggesting that their trafficking mechanisms may also be conserved. Mass spectroscopic analysis confirmed that large amounts of Crb2 (∼600 MS2 spectra) were coupled onto the liposomes. The most abundant protein isolated (as determined by MS2 spectra) with an established role in the recognition and trafficking of transmembrane cargoes was the retromer subunit Vps35. In addition, Vps26B was identified. Western blotting confirmed the presence of Vps35 in our Crb2 recruitment reactions and showed it to be highly enriched relative to two independent controls (Pocha, 2011).

The mammalian retromer is composed of a cargo recognition subcomplex containing Vps35, Vps26, and Vps29 and a membrane interacting subcomplex consisting of SNX1/SNX2 and SNX5/SNX6 heterodimers. Because both Vps35 and Vps26 are crucial for cargo recognition and binding, the recruitment data suggest that Crb2 is a retromer cargo (Pocha, 2011).

To probe the hypothesis that Crb is a retromer cargo, internalization assays were performed by overexpressing Flag-hCrb2 in HeLa cells and analyzing the uptake of anti-Flag antibodies, visualizing compartments through which Crb2 traffics. Previous studies using the classical retromer cargo, the cation-independent mannose-6-phosphate receptor (ciMPR), have shown that retromer subunits and cargo decorate tubules that emanate from endosomes and travel toward the trans-Golgi network (TGN). This study observed colocalization of Crb2 with Vps35 on intracellular vesicles and tubules as well as an overlap with ciMPR- and galactosyltransferase (GalT) label. These data suggest that in HeLa cells, Crb2 travels in retromer-decorated tubules and can traffic via the TGN. However, it should be noted that it does not accumulate there like other retromer cargoes (e.g., ciMPR). Instead, Crb2 appears to undergo rapid transport back to the plasma membrane. RNA interference (RNAi) suppression of Vps35 in HeLa cells displays enhanced localization of Crb2 in lysosomal structures positive for Lamp-I, a phenotype described previously for other retromer cargoes. These data are all in line with Crb being a potential retromer cargo (Pocha, 2011).

To study the functional interaction between Crb and retromer in Drosophila, a previously generated null allele of Vps35, Vps35MH20 was used. As a result of strong maternal contribution, animals homozygous for this allele reach the third larval instar, allowing analysis of Crb in homozygous mutants. Because retromer is required for the retrieval of receptors from endosomes and thus the prevention of their lysosomal degradation, total Crb levels were analyzed and found to be reduced in Vps35MH20 heterozygote third-instar larvae compared to stage-matched wild-type (WT) larvae and dramatically reduced in Vps35MH20 homozygotes. Analysis of the mRNA levels of Crb showed that loss of Vps35 has very little effect on crb transcripts, suggesting that the dramatic reduction in Crb protein that was seem is due to posttranscriptional regulation of Crb by Vps35 (Pocha, 2011).

This led to an investigation of Crb at a cellular level. For this, two different epithelia, wing discs of third-instar larvae and the follicle epithelium, were chosen. Clones of Vps35MH20 mutant cells in wing disc epithelia, labeled with GFP using the mosaic analysis with a repressible cell marker (MARCM) system, were induced by heat shock-Flp at early larval stages. Crb localizes to the subapical region of wing disc epithelial cells. In agreement with results from western blot analysis, Crb staining is decreased in Vps35MH20 clones. Quantification of the fluorescence intensity in the clone and in surrounding tissues revealed that there is an ∼50% reduction in Crb signal within Vps35MH20 clones. The wing discs of Vps35MH20 homozygous animals are small and show variable morphological defects, presumably as a result of defective Wingless secretion. Analysis of Crb localization (by immunofluorescence) and protein levels (by western blotting) in Vps35MH20 hetero- and homozygous wing discs corroborated the data that were obtained using Vps35MH20 clones and larval lysate, respectively (Pocha, 2011).

The stability of the cargo-selective retromer subcomplex is dependent on the presence of all of its components [8 and 16]. To show that the loss of Crb is due to loss of retromer function rather than just the loss of Vps35, the effect was compared of Vps26 and Vps35 knockdown in the posterior compartment of the wing disc using engrailed-Gal4 to drive UAS-Vps26RNAi and UAS-Vps35RNAi. Hedgehog expression, which is unperturbed by loss of retromer, served to label the posterior compartment. Expression of either RNAi construct resulted in a clear reduction of Crb staining in the posterior compartment (∼50% reduction in fluorescence). Expression of engrailed-Gal4 alone had no effect on Crb. From these data, it is concluded that the retromer cargo recognition subcomplex is required for the maintenance of Crb levels (Pocha, 2011).

To further analyze the relation between Crb and retromer, the follicular epithelium, which surrounds the germline cysts of the Drosophila ovary, was examined. Previous work has identified key roles for Crb in polarization of the follicular epithelium. Crb localizes to the entire apical membrane of the follicle epithelial cells, with very little detectable in the cytoplasm. Vps35MH20 clones show strong reduction in Crb staining and protein loss from the apical membrane. Interestingly, although Crb staining at the apical membrane is strongly reduced, it is not detected at increased levels within the cytoplasm, suggesting that Crb is not merely mislocalized but reduced at the protein level, as shown in larvae. The cytoplasmic domain of Crb organizes an apical, membrane-associated protein complex by recruiting the scaffolding proteins Stardust (Sdt), DPATJ, and DLin-7. Therefore, the apical localization of Sdt in the follicular epithelium was assessed, and at was found to be heavily reduced in Vps35MH20 clones. Probing whole larval lysates from third-instar WT and Vps35MH20 hetero- and homozygotes for Sdt confirmed that at the protein level, like Crb, Sdt shows a dose dependence on Vps35. Thus, retromer function in maintaining Crb levels and function is conserved between wing and follicle epithelia (Pocha, 2011).

Interestingly, in some Vps35MH20 clones, the strict monolayer structure of the epithelium is disrupted and the tissue appears multilayered, an indication of polarity defects and characteristic of loss of Crb at early stages of follicle development, whereas loss at later stages results only in the mislocalization of other polarity proteins, without affecting tissue integrity. Multilayering was observed in 19% of Vps35MH20 clones in follicles between stages 7 and 10 and did not appear to be dependent on clone size or position. Given that various links between Crb and Notch have been reported, tests were performed to see whether the multilayering phenotype observed in the follicle epithelium upon loss of Vps35MH20 could be the result of defective Notch signaling. The expression of Notch and Hindsight, a transcription factor downstream of Notch signaling that represses proliferation in the follicle epithelium, were examined in Vps35MH20 mutant clones. Both showed wild-type expression, suggesting that Notch signaling is not affected by loss of retromer, similar to previous findings in the wing disc (Pocha, 2011).

To test whether the loss of Crb in retromer mutants is due to missorting of Crb to the lysosome, follicles harboring Vps35MH20 clones were incubated in leupeptin, a potent inhibitor of lysosomal proteases. After a 3 hr incubation, a dramatic accumulation of Crb was observed in punctae within the cytoplasm of Vps35MH20 cells, a phenomenon that was not seen in WT tissue or in follicles containing Vps35MH20 clones that were incubated in control medium lacking leupeptin. Additionally, colocalization of these intracellular Crb punctae with LysoTracker was observed. Together with the reduction of Crb protein levels and constant crb mRNA levels in Vps35MH20 larvae and tissue, these data strongly suggest that retromer ablation leads to lysosomal degradation of Crb, as observed for other retromer cargoes (Pocha, 2011).

To test whether retromer functions after endocytosis of Crb, internalization of Crb from the plasma membrane was blocked by expression of a dominant-negative construct of shibire (dynamin) or by incubating follicles in dynasore, a dynamin inhibitor. In Vps35MH20 clones, this resulted in the accumulation of Crb at the plasma membrane, confirming that retromer is indeed transporting Crb after internalization from the plasma membrane (Pocha, 2011).

Crb is required, together with atypical protein kinase C (aPKC), to restrict Bazooka/Par3 to the zonula adherens, an adhesion belt at the apex of epithelial cells, in the follicle epithelium, and in photoreceptor cells, thus excluding it from the apical membrane and specifying the border between apical and lateral domains. In previous studies, it was shown that the localization of aPKC and Par6 was dependent on Crb. To test whether loss of retromer phenocopies the loss of Crb, aPKC and Par6 localization were examined in follicles containing Vps35MH20 clones. Indeed, the level of both proteins is reduced at the apical surface in Vps35MH20 clones. Interestingly, unlike Crb and the Crb complex member Sdt, Par6 and aPKC protein levels are not reduced in Vps35MH20 mutant larvae. Therefore, it is likely that the loss of Par6 and aPKC from the apical membrane of Vps35MH20 clones in the follicle epithelium is due to loss of cell polarity in the absence of Crb rather than loss of the proteins themselves (Pocha, 2011).

To test this, Crb was overexpressed in Vps35MH20 clones. Because overexpression of Crb causes defects in epithelial cell polarity, Crb overexpression was induced using GABFc204 Gal4, a follicle epithelium-specific driver that starts expression late in follicle development (stage 8). Thereby, it was possible to rescue the apical localization of Par6 and Sdt. This rescue did not appear to be dependent on clone size or location. From these data, it is concluded that the loss of polarity observed in retromer mutant clones is the direct result of loss of Crb (Pocha, 2011).

The identification of Crb as a retromer cargo confirms the hypothesis that one crucial step in the regulation of Crb occurs at the early (sorting) endosome and, importantly, fills a gap in the current understanding of Crb trafficking. Previous reports showed that transport of Crb to the plasma membrane is reliant on Rab11, the exocyst and Cdc42 in Drosophila embryonic epithelia. Internalization of Crb from the plasma membrane into endosomes is mediated by the syntaxin Avalanche and Rab5. This study has shown that retromer is responsible for sorting Crb away from the degradative pathway and into a recycling one, thus allowing a high level of control over the amount of cellular Crb, previously shown to be vital for maintaining epithelial polarity and integrity, as demonstrated by numerous loss- and gain-of-function studies. Interestingly, retromer was previously shown to play a role in the apical delivery of the polymeric immunoglobulin receptor (pIgR) in Madin-Darby canine kidney cells. However, as for Crb, it remains unclear whether this transport occurs via the TGN, via recycling endosomes, or through alternative pathways. The exact trafficking itinerary of Crb following recycling by retromer remains unclear and may depend upon the purpose of Crb recycling (Pocha, 2011).

Which function of Crb is the prime target of retromer-driven retrieval? Is this a Crb level-sensing mechanism, in which retromer regulates the amount of protein at the plasma membrane, which is crucial for cell homeostasis? To date, all known functions of Crb require an intact Crb complex. By controlling the recycling of Crb and thereby its level at the plasma membrane, retromer could define the amount of Crb available for complex formation. Alternatively, it is tempting to speculate that Crb, much like Wntless (Wls), acts as a transport receptor and that apical delivery of its (yet to be identified) ligand or many ligands is the main purpose of its recycling to the TGN. These are fascinating hypotheses that will be the focus of future research (Pocha, 2011).

Effects of mutation or Deletion

The apical localization of Par-6 in epithelial cells and neuroblasts indicates that it has a role in cell polarity. To analyse its function, a P-element inserted 3.5-kilobases (kb) upstream of the Par-6 transcriptional start site was identified and imprecise excision of this transposon was used to generate deletions of the Par-6 gene. Three independent deletions, Par-6Delta426, Par-6Delta219 and Par-6Delta226, removed the start codon and the first 26, 38 or 121 amino acids of Par-6, respectively. The largest deletion, Par-6Delta226, could be rescued to complete viability and fertility by a genomic fragment containing the Par-6 locus and was chosen for further analysis. No protein could be detected in embryos from germline clones homozygous for this deletion indicating that it represents a null or strong loss of function allele (Petronczki, 2001).

Homozygous Par-6 mutants are late embryonic or early larval lethal. Around 25% (n = 102) of Par-6Delta226 mutant embryos fail to hatch, and identical results were obtained for the two other alleles. Cuticle preparations of these dead embryos reveal large holes at random positions. Similar holes are detected in bazooka mutant embryos and are indicative of a defect in epithelial polarity. To determine whether the late phenotype of Par-6 mutants is caused by the strong maternal contribution, germline clones were generated that lack both maternal and zygotic Par-6 (called Par-6GLC embryos). Only a small number of eggs could be recovered from Par-6 mutant germline clones. This might indicate a function of Par-6 during oogenesis, even though no reproducible dorsal-ventral or anterior-posterior defects were detected in Par-6GLC embryos (Petronczki, 2001).

Par-6GLC mutants are embryonic lethal, but early development, including cellularization and morphological changes during gastrulation, are normal in these embryos. During stage 10 of embryonic development, however, epithelial cells have lost their regular arrangement, and after germband retraction they frequently undergo apoptosis. Cell outlines in control and Par-6GLC embryos were visualized by staining for alpha-spectrin. Epithelial cells are rectangular and formed a regular monolayer in control embryos, but are round and irregularly arranged in Par-6GLC embryos. Armadillo protein is apically localized and concentrated at adherens junctions in wild-type epithelial cells, but completely loses its apical localization in Par-6GLC mutant cells. Similarly, apically localized Bazooka protein redistributes to the cytoplasm in these mutants. It is concluded that epithelial apical-basal polarity is lost during embryonic development in Par-6GLC mutants (Petronczki, 2001).

Par-6 is also 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. 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).

The anterior-posterior axis of C. elegans is defined by the asymmetric division of the one-cell zygote, and this is controlled by the PAR proteins, including PAR-3 and PAR-6, which form a complex at the anterior of the cell, and PAR-1, which localizes at the posterior. PAR-1 plays a similar role in axis formation in Drosophila: the protein localizes to the posterior of the oocyte and is necessary for the localization of the posterior and germline determinants. PAR-1 has recently been shown to have an earlier function in oogenesis, where it is required for the maintenance of oocyte fate and the posterior localization of oocyte-specific markers. The homologs of PAR-3 (Bazooka) and PAR-6 are also required to maintain oocyte fate. Germline clones of mutants in either gene give rise to egg chambers that develop 16 nurse cells and no oocyte. Furthermore, oocyte-specific factors, such as Orb protein and the centrosomes, still localize to one cell but fail to move from the anterior to the posterior cortex. Thus, PAR-1, Bazooka, and PAR-6 are required for the earliest polarity in the oocyte, providing the first example in Drosophila where the three homologs function in the same process. Although these PAR proteins therefore seem to play a conserved role in early anterior-posterior polarity in C. elegans and Drosophila, the relationships between them are different, since the localization of PAR-1 does not require Bazooka or PAR-6 in Drosophila, as it does in the worm (Huynh, 2001).

PAR-6 has been shown to localize to the same protein complex as PAR-3 in C. elegans, Drosophila, and mammalian cells and is essential both for the localization and the function of this complex. In Drosophila, Bazooka and PAR-6 colocalize to the apical side of the embryonic ectoderm, where they are necessary for the maintenance of epithelial polarity, and both proteins are also inherited by the neuroblasts when they delaminate and are required for the basal localization of cell fate determinants during their asymmetric divisions. To test if Drosophila PAR-6 also functions with Bazooka during oogenesis, germline clones were generated of the par-6Delta226 allele, which is a deletion of the promoter, the start codon, and the first 121 amino acids of the protein and is therefore a strong loss of function mutation if not a null. The majority of mutant egg chambers appear small, oval-shaped, and contain 16 polyploid nurse cells and no oocyte, indicating that PAR-6 is also required for oocyte determination. Furthermore, Orb and the centrosomes accumulate in one cell at the posterior of the cyst, although with a slight delay compared to wild-type. Both remain at the anterior of the oocyte, however, and fail to translocate to the posterior pole. Thus, the loss of PAR-6 from the germline gives an identical phenotype to Bazooka and PAR-1. As is the case for bazooka germline clones, some of the par-6 mutant egg chambers escape the early arrest and go on to produce normal eggs. When the females are scored 2 days after eclosion, half of the egg chambers form a normal oocyte, and about a quarter still do so after 10 days. This increase in the penetrance of the phenotype with age shows that PAR-6 protein perdures for many days after the clones are produced. Consistent with this, PAR-6 appears to be unusually stable in the embryo; the protein can be detected throughout embryogenesis in zygotic par-6 null embryos, at levels that are only slightly lower than in wild-type. However, the continued presence of escapers after 10 days suggests that PAR-6 may not be essential for oocyte determination in all cases and that there may be redundant pathways that can partially compensate for its absence (Huynh, 2001).

During the asymmetric divisions of the neuroblasts, the Bazooka/PAR-6 complex recruits Inscuteable to the apical side of the cell, where it plays a role in directing the basal localization of Miranda protein. Germline clones of null mutants in inscuteable or miranda cause no visible defects in oocyte determination or the posterior localization of Orb, however, and give rise to normal eggs that can be fertilized. Furthermore, neither protein shows any asymmetric localization in early egg chambers. Thus, some of the downstream effectors of early oocyte and neuroblast polarity are different, despite the similar roles of Baz and PAR-6 in the two processes (Huynh, 2001).

To investigate the relationships between Bazooka, PAR-6, and PAR-1 during oocyte determination, their localizations were analyzed in both wild-type and mutant germaria. In region 2a to region 3 of the germarium, Bazooka localizes around the ring canals, in a ring that is about twice the diameter of that formed by actin. This localization is very similar to that of the adherens junction components Shotgun (E-cadherin) and Armadillo. A double staining was therefore performed for Arm and Baz. Although Arm localizes to these rings before Bazooka in early region 2a, the two proteins colocalize from the middle of region 2a until region 3, when they both disappear. Bazooka also colocalizes with Shotgun and Armadillo in the zonula adherens of the embryonic epithelium, which provides a boundary between the apical and basolateral membrane domains. This raises the possibility that the Shotgun, Armadillo, and Bazooka rings in the germarium perform a similar function by marking the separation between an anterior and a posterior domain within the oocyte. It is unclear whether PAR-6 also localizes to these rings, since none of the available antibodies give any significant staining that disappears in par-6 null germline clones (Huynh, 2001).

In C. elegans, the PAR-3/PAR-6 complex is required for the posterior localization of PAR-1. This is not the case during Drosophila oogenesis, however, since PAR-1 shows a wild-type localization to the fusome in baz and par-6 germline clones. Furthermore, the localization of Bazooka around the ring canals does not require PAR-6, since it is unaffected in mutant germline clones. This is in marked contrast to both the C. elegans zygote and Drosophila neuroblasts and epithelia, where the localizations of PAR-3/Baz and PAR-6 depend on each other. Bazooka and PAR-6 also localize to the apical sides of the somatic follicle cells of the egg chamber, and mutants in either gene disrupt the localization of both proteins and cause the cells to overproliferate and lose their apical-basal polarity. Thus, the relationship between Bazooka and PAR-6 is different in the germline and the somatic follicle cells, where they appear to have a similar role to that described in other epithelia (Huynh, 2001).

These results show that PAR-1, Bazooka, and PAR-6 act in the same step in oocyte determination, providing the first example in Drosophila where these three homologs of C. elegans PAR proteins participate in the same process. Furthermore, mutants in all three genes disrupt the movement of oocyte-specific proteins and the centrosomes from the anterior to the posterior of the oocyte, which is the earliest visible sign of polarity within the oocyte. Given the role of these PAR proteins in other systems, it seems very likely that their primary function in the germarium is in the anterior-posterior polarization of the oocyte, and that the failure to maintain oocyte fate is a consequence of this defect (Huynh, 2001).

It is intriguing that this very early anterior-posterior polarity of the Drosophila oocyte requires three of the PAR proteins that mediate the anterior-posterior polarization of the first cell division in C. elegans. Although this suggests that these proteins act in a conserved pathway for generating cell polarity in these two systems, the relationships between the localizations of these proteins are quite different in the Drosophila oocyte and C. elegans zygote. Thus, at least some aspects of their function are not conserved, and it will therefore be interesting to determine whether the downstream pathways that generate other cellular asymmetries in response to this polarity are related (Huynh, 2001).

Par-3/Baz, Par-6, and aPKC are evolutionarily conserved regulators of cell polarity, and overexpression experiments implicate them as axon determinants in vertebrate hippocampal neurons. Their mutant and overexpression phenotypes were examined in Drosophila melanogaster. Mutants neurons have normal axon and dendrite morphology and remodel axons correctly in metamorphosis, and overexpression does not affect axon or dendrite specification. Baz/Par-6/aPKC are therefore not essential for axon specification in Drosophila (Rolls, 2004).

Therefore, Drosophila Baz, Par-6 and aPKC are not required for axon specification in vivo, and their overexpression has no effect on axon specification or outgrowth. In contrast, overexpression of Par-3 or Par-6 in cultured mammalian hippocampal neurons results in multiple axon-like processes, leading to the hypothesis that these proteins are axon determinants. How can these apparently paradoxical results be reconciled? One possibility is that vertebrate neurons require Par-complex proteins for axon specification, whereas Drosophila neurons do not. If this is the case, it would be interesting to learn how different molecular pathways in mammals and flies generate the same functional subcellular domain (the axon). Another possibility is that neither fly nor vertebrate neurons use Par proteins to specify axon identity in vivo; cultured hippocampal neurons are separated from normal external polarity cues and may use a different mechanism for axon specification. Polarity cues from surrounding cells may also inhibit neurons in vivo from changing polarity in response to extra Par-3 or Par-6, explaining the different effects of overexpressing these proteins in Drosophila and in hippocampal neurons. A third possibility is that the overexpression experiments, where proteins are present at higher-than-normal levels, do not reflect the in vivo functions of the proteins. Loss-of-function and overexpression experiments that examine vertebrate neurons in vivo or in slice preparations will be crucial for fully understanding the role of Par complex proteins in vertebrate axon specification (Rolls, 2004).

Requirement for Par-6 and Bazooka in Drosophila border cell migration

Polarized epithelial cells convert into migratory invasive cells during a number of developmental processes, as well as when tumors metastasize. Much has been learned recently concerning the molecules and mechanisms that are responsible for generating and maintaining epithelial cell polarity. However, less is known about what becomes of epithelial polarity proteins when various cell types become migratory and invasive. This study reports the localization of several apical epithelial proteins, Par-6, Par-3/Bazooka and aPKC, during border cell migration in the Drosophila ovary. All of these proteins remain asymmetrically distributed throughout migration. Moreover, depletion of either Par-6 or Par-3/Bazooka by RNAi results in disorganization of the border cell cluster and impaired migration. The distributions of several transmembrane proteins required for migration were abnormal following Par-6 or Par-3/Bazooka downregulation, possibly accounting for the migration defects. Taken together, these results indicate that cells need not lose apical/basal polarity in order to invade neighboring tissues and in some cases even require such polarity for proper motility (Pinheiro, 2004).

Therefore, border cells retain an asymmetric distribution of the apical epithelial proteins Baz, Par-6 and aPKC throughout their migration, raising the question as to why. One possibility could be that these proteins contribute to the cells' direction-sensing mechanism. However, neither Par-6 nor Baz localized asymmetrically with respect to the direction of migration, making this possibility seem less likely. In premigratory border cells, the apical domain is oriented towards the nurse cells and the direction of migration. However, once the cells separate from the epithelium, the side of the cluster with the highest levels of Baz, Par-6 and aPKC was found to be roughly orthogonal to the direction of migration. These findings are consistent with observations regarding the distribution of Crumbs, another apical marker, and suggest that early in migration the entire cluster rotates so that the leading edge is roughly perpendicular to the apical domain (Pinheiro, 2004).

A second possibility is that maintaining some aspects of epithelial polarity during migration eliminates the need to re-establish polarity de novo when the border cells reach the oocyte. While possible, this hypothesis is difficult to test and cannot be the only function for Par-6 and Baz in border cells, since these proteins are also required during migration (Pinheiro, 2004).

A third possibility is that cellular asymmetry is retained during border cell migration in order to achieve the proper asymmetries in the distributions of other proteins. Consistent with this proposal, the normally asymmetric accumulations of E-cadherin and ßps-integrin within border cells are dramatically altered in cells depleted of Baz or Par-6. Loss of E-cadherin from border cells has been shown to inhibit migration, and misdistribution of E-cadherin at border cell/nurse cell boundaries correlates with a migration defect. The defects in the distributions of E-cadherin and other membrane-associated proteins in border cells either depleted of, or overexpressing Par-6 and Baz, may collectively lead to the observed migration defect (Pinheiro, 2004).

A large number of mosaic egg chambers containing clones mutant for par-6 or baz were examined and delays were observed in border cell migration as well as defects in cohesion within the cluster. It has been reported that mosaic clones of baz show a lack of adhesion within the border cell cluster but no migratory defects. It is likely that this difference is due to clone size and/or protein perdurance, since only large clones in which the majority of the border cells were mutant, showed defects in border cell migration. Consistent with this, RNAi-mediated reduction of Par-6 and Baz in the border cells results in delayed migration, suggesting that the strongest migration defects are observed only when all the border cells lack Par-6 or Baz. This is not unusual. Mutations in slbo, jing, stat92E and shotgun, which encode E-cadherin, exhibit similar behavior such that clusters containing some wild-type cells can migrate. These findings seem to indicate that wild-type cells can 'drag' a few mutant cells, but when the number of migration-defective cells exceeds the number of migration-competent cells, migration slows or stops (Pinheiro, 2004).

RNAi-mediated reduction of Par-6 and Baz in the polar cells, in addition to the outer border cells, exacerbates the defects caused by expression in the migratory cells alone. This suggests that polar cells contribute to organizing the cluster. Cohesion of the cluster may be necessary in order for the migratory cells to receive continuous activation of the JAK/STAT pathway during migration. Consistent with this, in those clusters that split, those cells that remain attached to the polar cells migrate further than the cells that become detached. Polar cells require the migratory cells to reach the oocyte because they are not motile themselves, but the migratory cells also appear to need the polar cells in order to sustain their motility. This mutual requirement may serve to ensure that the migratory cells do not run off without the polar cells, since the polar cells are required at the oocyte surface to form the pore in the micropyle through which a sperm enters at fertilization (Pinheiro, 2004).

The observations presented in this study demonstrate that Par-6 and Par-3/Baz are distributed asymmetrically in migrating border cells, suggesting that not all epithelial polarity is lost when these epithelial cells become motile. In spite of this, the morphology of the border cells, particularly at the border cell/nurse cell interface, can appear fibroblast-like. This interface must support protrusive behavior and dynamic adhesion, so that the cells can move along the nurse cells, while they simultaneously remain firmly attached to each other and to the polar cells. Therefore, migrating border cells possess both epithelial and mesenchymal characteristics (Pinheiro, 2004).

It is proposed that the Par-3/Par-6/aPKC complex functions in these cells, as it does in an epithelium or in asymmetrically dividing neuroblasts, to maintain distinct protein distributions and functional domains in different parts of the cell. In the case of the border cells, three important domains are the interfaces between border cells and nurse cells, between border cells and polar cells and between adjacent border cells. Such distinct domains may be present in other types of cells that maintain contacts with an intact epithelium while they migrate, such as motile keratinocytes at a wound edge or leading endothelial cells during angiogenesis. Tumor cells that metastasize in groups or 'nests' may also possess both epithelial and mesenchymal characteristics. Thus the Par-3/Par-6/aPKC complex may contribute to the invasiveness of other cell populations as well (Pinheiro, 2004).

Bazooka is required for polarisation of the Drosophila anterior-posterior axis

The Drosophila anterior-posterior (AP) axis is determined by the polarisation of the stage 9 oocyte and the subsequent localisation of bicoid and oskar mRNAs to opposite poles of the cell. Oocyte polarity has been proposed to depend on the same PAR proteins that generate AP polarity in C. elegans, with a complex of Bazooka (Baz; Par-3), Par-6 and aPKC marking the anterior and lateral cortex, and Par-1 defining the posterior. The function of the Baz complex in oocyte polarity has remained unclear, however, because although baz-null mutants block oocyte determination, egg chambers that escape this early arrest usually develop normal polarity at stage 9. This study characterised a baz allele that produces a penetrant polarity phenotype at stage 9 without affecting oocyte determination, demonstrating that Baz is essential for axis formation. The dynamics of Baz, Par-6 and Par-1 localisation in the oocyte indicate that the axis is not polarised by a cortical contraction as in C. elegans, and instead suggest that repolarisation of the oocyte is triggered by posterior inactivation of aPKC or activation of Par-1. This initial asymmetry is then reinforced by mutual inhibition between the anterior Baz complex and posterior Par-1 and Lgl. Finally, it was shown that mutation of the aPKC phosphorylation site in Par-1 results in the uniform cortical localisation of Par-1 and the loss of cortical microtubules. Since non-phosphorylatable Par-1 is epistatic to uninhibitable Baz, Par-1 seems to function downstream of the other PAR proteins to polarize the oocyte microtubule cytoskeleton (Doerflinger, 2010).

The baz358-12 allele causes a fully penetrant defect in the localisation of bcd and osk mRNAs and in the positioning of the oocyte nucleus and gurken mRNA, providing the first demonstration that Baz is required for the polarisation of the Drosophila AP and dorsal-ventral axes. This raises the question of why baz-null mutant germline clones that escape the block in early oogenesis sometimes develop into eggs with normal polarity. Although it is formally possible that Baz is not absolutely essential for oocyte polarity and that the baz358-12 allele has a dominant-negative effect, this seems very unlikely. First, baz358-12 behaves like a typical hypomorphic mutation as it is recessive and fails to complement the lethality of baz-null alleles. Second, nearly half of the escapers from baz-null germline clones show similar polarity defects to baz358-12 at stage 9, indicating that this is a loss-of-function phenotype. Thus, it seems more likely that whatever allows a few of the null germline clones to escape the early-arrest phenotype also allows some of them to escape the polarity defect at stage 9. For example, other polarity pathways might be activated in baz-null mutant germaria that can partially compensate for the loss of Baz in both oocyte determination and axis formation (Doerflinger, 2010).

The observation that baz358-12 does not cause any defects in the initial polarisation of the oocyte, although it is essential for the AP polarisation at stage 9, indicates that there must be some differences in the functions of Baz at each stage. During early oogenesis, Baz localises in a ring around each ring canal at the anterior of the oocyte and shows perfect colocalisation with DE-cadherin (Shotgun - FlyBase) and Armadillo. Since the PDZ domains of Baz have been shown to interact with Armadillo, it might be recruited to the anterior rings through this interaction, which should still occur normally in the baz358-12 mutant. By contrast, the truncated Baz protein does not localise to the cortex of the oocyte at stages 7-9, indicating that the C-terminal region is necessary for its cortical recruitment at this stage. The only identified domain in this region is CR3, which binds to the kinase domain of aPKC. However, a point mutation in CR3 that disrupts its interaction with aPKC has no effect on the cortical localisation of Baz at stage 9. There must therefore be another domain in the C-terminal region of Baz that is required for its recruitment to the oocyte cortex (Doerflinger, 2010).

Another important difference between the initial polarisation of the oocyte and the repolarisation at mid-oogenesis is the relationship between the PAR proteins. During early oogenesis, the localisation of Baz is unchanged by loss of Par-1 and vice versa. By contrast, Baz and Par-1 show mutually exclusive localisations at stage 9, with Par-1 spreading around the lateral cortex in baz mutants, and Baz and Par-6 localising to the posterior in par-1 mutants. Baz is required to recruit Par-6 to the cortex in mid-oogenesis, as Par-6 disappears from the anterior cortex in baz358-12 clones and localises to the posterior with BazS151A S1085A-GFP. Thus, Baz, Par-6 and presumably also aPKC form a complex in the stage 9 oocyte, making the arrangement of PAR proteins much more similar to that in the C. elegans zygote, with Baz (PAR-3), Par-6 and aPKC defining the anterior and lateral cortex and Par-1 the posterior. As in C. elegans, these complementary localisations are also maintained by mutual antagonism between the anterior and posterior PAR proteins. It has been shown that Par-1 phosphorylates Baz to exclude it from the posterior. This study shows that mutation of the conserved aPKC site in the Par-1 linker region leads to the mislocalisation of Par-1 around the anterior and lateral cortex, strongly suggesting that aPKC phosphorylates this site to restrict Par-1 to the posterior (Doerflinger, 2010).

Although the final pattern of PAR proteins in the stage 9 Drosophila oocyte is similar to that in the C. elegans zygote, this pattern develops over a much longer period of time and in a different way. Baz-GFP is enriched at the posterior of the oocyte at the beginning of stage 7 and gradually spreads anteriorly during the succeeding 12 hours, before finally disappearing from the posterior at stage 9. Since Par-1 appears at the posterior early in stage 7, Baz and Par-1 overlap at the posterior for some considerable time. By contrast, Par-6-Cherry starts to disappear from the posterior during stage 7, and already shows a complementary pattern to Par-1 at stage 8. This raises the question of why Par-6, which is recruited to the cortex by Baz, disappears more rapidly from the posterior. Although this might mean that they are excluded by different mechanisms, both Par-6 and Baz localise to the posterior in par-1 mutants and in BazS151A S1085A-GFP-expressing oocytes, indicating that their exclusion depends on the phosphorylation of Baz by Par-1. Thus, Par-1 phosphorylation might first release Par-6 from Baz, and then more gradually displace Baz from the cortex. The phosphorylation of serine 1085 of Baz by Par-1 disrupts the interaction of Baz with aPKC and this might be sufficient to release the Par-6-aPKC complex. However, Par-6 also binds directly to the PDZ domains of Baz, and the phosphorylation of serine 1085 alone would not be expected to interfere with this interaction. Thus, Par-1 might also act in some other way to release Par-6, perhaps by promoting the posterior recruitment of Lgl, as the latter is known to inhibit the interaction of Par-6-aPKC with Baz in neuroblasts (Doerflinger, 2010).

The gradual evolution of PAR protein localisation during stages 7-9 argues against the idea that the oocyte is polarised by a cortical contraction, as in C. elegans, and no any evidence has been observed for cortical movements of the actin cytoskeleton. This raises the question of how this asymmetry arises. Two possible scenarios for how the polarising signal from the posterior follicle cells triggers PAR protein asymmetry can be invisioned. First, the initial cue could remove or inactivate aPKC and Par-6 at the posterior, which would then allow Par-1 to localise there because aPKC is no longer present or able to exclude it. Although aPKC can be inhibited at the posterior by Lgl, this seems unlikely to provide the cue because Lgl localises to the posterior after Par-1 and is not essential for oocyte polarity. Alternatively, the initial asymmetry could be generated by the posterior recruitment and activation of Par-1. Work in mammals has shown that LKB1 (STK11) phosphorylates the activation loop of PAR-1 (MARK2) to turn on its kinase activity, and this is likely to be case in Drosophila as well, as lkb1 mutants exhibit a very similar phenotype to par-1 mutants. LKB1 activity is regulated by protein kinase A (PKA), which is required for the transduction of the polarising follicle cell signal in the oocyte. Thus, it is possible that the initial asymmetry is generated by a kinase cascade at the posterior of the oocyte, consisting of PKA, which activates LKB1, which activates Par-1 (Doerflinger, 2010).

Once the PAR polarity has been established, it must somehow polarise the oocyte microtubule cytoskeleton to direct the localisation of bcd and osk mRNAs. The epistasis experiment suggests that Par-1 provides the primary output from the PAR system, as uniformly distributed Par-1 makes the whole cortex behave like the posterior cortex regardless of whether Baz is also uniformly distributed or not. Based on the par-1 loss- and gain-of-function phenotypes in the oocyte and follicle cells, Par-1 might act to stabilise microtubule plus ends at the cortex and to inhibit the nucleation or anchoring of microtubule minus ends (Doerflinger, 2010).

One key remaining question is the identity of the Par-1 substrates that mediate its effect on microtubule organisation. In addition to Baz, Par-1 has also been shown to phosphorylate Exuperantia and Ensconsin in the oocyte to regulate bcd mRNA localisation and the activity of Kinesin. However, neither of these targets can account for the dramatic effects of Par-1 on microtubule organisation. It has recently been claimed that Par-1 regulates the oocyte microtubule cytoskeleton by phosphorylating the microtubule-stabilising protein Tau, thereby destabilising the microtubules at the posterior of the oocyte. This conclusion was based on the observation that germline clones of tauDf(3R)MR22 produce a partially penetrant defect in the anchoring of the oocyte nucleus. However, the tauDf(3R)MR22 mutation is a 65 kb deletion that removes eight other genes as well as tau, and the phenotype could therefore be due to the loss of one of these other loci. More importantly, tau can be specifically removed without deleting any other genes by generating heterozygotes for two overlapping deficiencies, and these tau-null flies are homozygous viable and fertile and develop normally polarised oocytes. Thus, it seems highly unlikely that Tau is a relevant substrate for Par-1 in the polarisation of the oocyte. A full understanding of oocyte polarity will therefore depend on the identification of the Par-1 targets that control microtubule nucleation, anchoring and stability (Doerflinger, 2010).

Cdc42, Par6, and aPKC regulate Arp2/3-mediated endocytosis to control local adherens junction stability

By acting as a dynamic link between adjacent cells in a monolayer, adherens junctions (AJs) maintain the integrity of epithelial tissues while allowing for neighbor exchange. Although it is not currently understood how this combination of AJ stability and plasticity is achieved, junctionally associated actin filaments are likely to play a role, because actin-based structures have been implicated in AJ organization and in the regulation of junctional turnover. Through exploring the role of actin cytoskeletal regulators in the developing Drosophila notum, this study has identified a critical role for Cdc42-aPKC-Par6 in the maintenance of AJ organization. In this system, the loss or inhibition of Cdc42-aPKC-Par6 leads to junctional discontinuities, the formation of ectopic junctional structures, and defects in apical actin cytoskeletal organization. Affected cells also undergo progressive apical constriction and, frequently, delamination. Surprisingly, this Cdc42-aPKC-Par6-dependent regulation of junctional stability was found to be independent of several well-known targets of Cdc42-aPKC-Par6: Baz, Lgl, Rac, and SCAR. However, similar AJ defects are observed in wasp, arp2/3, and dynamin mutant cells, suggesting a requirement for actin-mediated endocytosis in the maintenance of junctional stability downstream of Cdc42. This was confirmed in endocytosis assays, which revealed a requirement for Cdc42, Arp2/3, and Dynamin for normal rates of E-cadherin internalization. In conclusion, by focusing on the molecular mechanisms required to maintain an epithelium, this analysis reveals a novel role for the epithelial polarity machinery, Cdc42-Par6-aPKC, in local AJ remodeling through the control of Arp2/3-dependent endocytosis (Georgiou, 2008).

This analysis of Cdc42 in Drosophila epithelial cells reveals a novel role for Cdc42, both in the regulation of the actin cytoskeleton and in AJ maintenance and stability. In this capacity, Cdc42 appears to function together with junctional Par6 and aPKC. It may appear surprising that Cdc42, aPKC, and Par6 act relatively independently of Baz (Par3) in the notum, given that Baz has been shown to act as a landmark to define the future site of E-cad localization and AJ formation in the embryo and to define neuroblast polarity upstream of Cdc42, aPKC, and Par6. However, the localization and function of Baz are distinct from those of Par6 and aPKC in many tissues. Moreover, the differences go further, because in the context of the notum, Cdc42, Par6, and aPKC are not required to maintain a polarized epithelium, and do not appear to function together with Lgl. A possible explanation for the reduced dependency of epithelial architecture in the notum on these molecules is the structural support gained from overlying apical cuticle and the basal lamina during its relatively slow development. Because of this, junctional defects in cdc42 mutant clones in this tissue are less likely to be an indirect consequence of a primary defect in apical-basal polarity. Moreover, the inherent stability of this epithelium made it possible to identify a novel role for the Cdc42-Par6-aPKC complex in the communication of polarity information across cell-cell junctions within the plane of the epithelium, something that may have been obscured by apical-basal polarity defects in studies in less stable epithelia (Georgiou, 2008).

Several lines of evidence suggest that the AJ defects seen in cdc42, aPKC, or par6 mutant cells result from defects in the internalization of junctional material, rather than from defects in the delivery of E-cadherin to the plasma membrane. First, junctional E-cadherin levels remain high in cdc42 mutant clones, enabling these cells to pull on surrounding cells. Second, in cdc42 clones, tubules and tubule-derived puncta containing E-cadherin, alpha-Catenin, and beta-Catenin remain continuous with the cell surface—as visualized with a probe for extracellular E-cadherin. This is the case, even though the vast majority of the rare, large E-cadherin puncta seen by light microscopy in the wild-type are found associated with an internalized fluid phase marker. Third, similar structures accumulate after a transient block in endocytic vesicle scission, resulting from the inhibition of Dynamin function. These data suggest that the primary defect induced by loss of Cdc42 is a defect in the endocytosis-mediated turnover of junctional material. Nevertheless, a detailed analysis of the molecular dynamics of E-cadherin-GFP at wild-type and mutant AJs is needed to quantify the contribution of Cdc42-mediated endocytosis to the normal rate of E-cadherin turnover (Georgiou, 2008).

How then do Cdc42, aPKC, and Par6 activate endocytosis to drive normal junctional turnover? Having identified a role for Cdc42 (aPKC and Par6) in apical actin organization, attention was focused on actin cytoskeletal regulators known to act downstream of Cdc42 in answering this question. Significantly, both WASp and the Arp2/3 complex (but not Rac or SCAR) were found to be required for the maintenance of a normal apical actin cytoskeleton and AJs. Because WASp is a well-established Cdc42 target, this suggested that the effect of Cdc42 on junctional endocytosis is mediated directly through WASp. The junctional phenotypes observed in WASp, however, appear weaker than those seen after the loss of Arp2/3, Cdc42, or Dynamin. This may be the result of protein perdurance. Alternatively, this observation may point to the existence of other proteins that act in parallel with WASp to stimulate Arp2/3-mediated vesicle scission downstream of Cdc42. In fact, a number of Cdc42 targets have been shown to affect actin nucleation and membrane tubulation. Nevertheless, the striking similarities between the AJ phenotypes induced by loss of Arp2/3, Dynamin, and Cdc42, together with recent data implicating Cdc42, Par6, and aPKC in the regulation of vesicle trafficking and endocytosis, provide strong evidence that the primary defect in each case is a block in actin-mediated endocytosis. On the basis of this analysis, it is suggested that the cdc42, par6, and apkc phenotype arises in the following way. First, a reduction in the activity of Cdc42-aPKC-Par6 on one side of an AJ translates into a reduction of Cdc42-aPKC-Par6 activity on the opposing side, resulting in a concomitant reduction in the activity of WASp and the Arp2/3 complex, leading to defects in Dynamin-dependent endocytosis along the entire cell-cell interface. The resulting failure to remove excess material from the ends of the AJ causes junctional spreading, as observed in electron micrographs. This leads to the formation of the discontinuous junctions, junctional extensions, and punctate surface structures visible in confocal images. Over time, this has the effect of destabilizing AJs, leading to the loss of apical material and, eventually, to cell delamination. In this view, Cdc42-Par6-aPKC regulate local Arp2/3-mediated endocytosis to maintain AJs in a state of dynamic equilibrium. Internalized junctional material can then be recycled back to the cell surface to engage in cell-cell adhesion in a well-regulated fashion. Importantly, this model predicts that the stability of AJs is intimately linked to their turnover -- a feature that makes AJs inherently plastic (Georgiou, 2008).


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par-6: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 August 2014

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