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).
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).
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 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).
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).
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 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).
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).
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).
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 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).
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).
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date revised: 16 January 2008
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