C. elegans PAR-6

The generation of asymmetry in the one-cell embryo of Caenorhabditis elegans is necessary to establish the anterior-posterior axis and to ensure the proper identity of early blastomeres. Maternal-effect lethal mutations with a partitioning defective phenotype (par) have identified several genes involved in this process. A new gene, par-6, has been identified that acts in conjunction with other par genes to properly localize cytoplasmic components in the early embryo. The early phenotypes of par-6 embryos include the generation of equal-sized blastomeres, improper localization of P granules and SKN-1 protein, and abnormal second division cleavage patterns. Overall, this phenotype is very similar to that caused by mutations in a previously described gene, par-3. The probable basis for this similarity is revealed by genetic and immunolocalization results; par-6 acts through par-3 by localizing or maintaining the PAR-3 protein at the cell periphery. In addition, loss-of-function par-6 mutations are found to act as dominant bypass suppressors of loss-of-function mutations in par-2 (Watts, 1996).

The par genes are required to establish polarity in the Caenorhabditis elegans embryo. Mutations in two of these genes, par-3 and par-6, exhibit similar phenotypes. A third gene, pkc-3, gives a similar phenotype when the protein is depleted by RNA interference. PAR-3 and PKC-3 protein are colocalized to the anterior periphery of asymmetrically dividing cells of the germline lineage. The peripheral localizations of both proteins depend on the activity of par-6. The molecular cloning of par-6 is reported as well as the immunolocalization of PAR-6 protein. par-6 encodes a PDZ-domain-containing protein and has homologs in mammals and flies. Moreover, PAR-6 colocalizes with PAR-3 and par-3 and pkc-3 activity are required for the peripheral localization of PAR-6. The localization of both PAR-3 and PAR-6 proteins is affected identically by mutations in the par-2, par-4 (see Drosophila Lkb1) and par-5 genes. The co-dependence of PAR-3, PAR-6 and PKC-3 for peripheral localization and the overlap in their distributions led to a proposal that they act in a protein complex (Hung, 1999).

Recent analyses in mammalian cells show that activated Cdc42 (see Drosophila Cdc42)forms a complex with homologs of the C. elegans PAR-3, PAR-6, and PKC-3 proteins. In C. elegans, mutation or RNAi of these three genes causes similar polarity defects, including the disruption of spindle orientation in two-cell embryos. To determine whether the activity of this complex is required for the cdc-42(RNAi) polarity phenotypes, spindle orientation was scored in par-3; cdc-42(RNAi) two-cell embryos in which par-3 activity was eliminated either by mutation or RNAi. The spindles in both cells in par-3; cdc-42(RNAi) embryos are usually longitudinal, like in par-3 mutants, showing that par-3(+) activity inhibits nuclear-centrosome rotation in cdc-42(RNAi) embryos (Kay, 2001).

par-3(+) inhibition of nuclear-centrosome complex rotation correlates with the localization of PAR-3 protein in wild-type and par mutant embryos; therefore, it was asked whether cdc-42(RNAi) affects PAR-3 localization. In wild-type one-cell embryos, PAR-3 is at the anterior cortex. In two-cell embryos, PAR-3 is found around the entire periphery of the anterior cell, which divides transversely, while PAR-3 is restricted to the anterior cortex of the posterior cell, which divides longitudinally. In cdc-42(RNAi) one-cell embryos, PAR-3 is often localized as in wild type, but is also observed to be uniform, scattered throughout the cortex. In two-cell cdc-42(RNAi) embryos, PAR-3 is variable and often detected in cortical patches. Importantly, the A-P distribution of PAR-3 between the blastomeres is variable; patches of PAR-3 cortical staining could favor either end of the embryo, be scattered throughout the entire circumference of both blastomeres, or be concentrated between cells. In wild-type embryos, PAR-3 colocalizes with PAR-6, and in mammalian cells, activated Cdc42 binds PAR-6 homologs. Therefore, it was asked whether cdc-42(RNAi) similarly disrupts the cortical patterning of PAR-6. Wild-type and cdc-42(RNAi) embryos were stained with an anti-PAR-6 antibody and patchy PAR-6 localization was observed around the cortex of blastomeres in the cdc-42(RNAi) zygotes and early embryos. cdc-42 is therefore required to pattern the localization of both components of this conserved complex (Kay, 2001).

In wild type, the cortical localization of the PAR-3/PAR-6 complex correlates with the inhibition of spindle rotation; blastomeres with PAR-3 all around the cortex have transverse spindles, while blastomeres either lacking or with asymmetrically localized cortical PAR-3 have longitudinal spindles. Interestingly, in 26 cdc-42(RNAi) two-cell embryos costained with an anti-PAR-3 antibody to detect the complex and with an anti-tubulin antibody to determine spindle orientations, no significant correlation between PAR-3 localization or levels and spindle orientation was noted. Among the 17 embryos in which PAR-3 was mislocalized, spindle orientation correlates in only seven embryos. This indicates that when cdc-42 activity is reduced, PAR-3 localization is not sufficient to inhibit nuclear-centrosome rotation (Kay, 2001).

Previous observations suggest that mutual antagonistic interactions between PAR-2 and PAR-3 contribute to embryonic patterning. Superficially, cdc-42(+) appears to be required for this interaction, given that in cdc-42(RNAi) embryos, the PAR-2 and PAR-3 domains overlap. Nevertheless, since reducing par-2 function in cdc-42(RNAi) embryos largely restores PAR-3 localization to a wild-type pattern, par-2(+) must disrupt PAR-3 localization in cdc-42(RNAi) embryos. Likewise, reducing cdc-42 function in par-2 embryos restores normal PAR-3 patterning, indicating that cdc-42(+) stimulates cortical PAR-3 localization in par-2 mutants. Since reducing or eliminating these activities in parallel does not greatly disturb the PAR-3 localization pattern, it seems likely that the spatial coordination of their opposing activities functions to ensure a sharp boundary of PAR-3 along the A-P axis (Kay, 2001).

The mutual suppression between cdc-42(RNAi) and par-2 could be interpreted in several ways. cdc-42(RNAi) suppression of par-2 may be mechanistically similar to the suppression of par-2 by par-6 heterozygosity. Specifically, par-2(it5); par-6/+ hermaphrodites produce many viable progeny. It has been proposed that wild-type par-2 may only be required to inhibit posterior PAR-3 localization when cortical PAR-3 levels are high. Given that PAR-3 is still present at higher anterior than posterior levels in par-2 mutant zygotes, any perturbation that reduces cortical PAR-3 levels may be sufficient to reestablish a significant A-P asymmetry. Because par-6 encodes a PDZ domain protein that is required for cortical PAR-3 localization, heterozygosity for par-6 may have a direct effect on cortical PAR-3 levels. Recent studies indicate that mammalian PAR-6 homologs contain a Cdc42/Rac1 binding (CRIB) motif, which when bound to activated Cdc42 stimulates the interactions between PAR-6 and both PAR-3 and PKC-3. Thus, PAR-6 appears to be either a direct regulator of, or effector for, Cdc42 activity. In C. elegans, cdc-42(RNAi) disrupts the asymmetric cortical localization of PAR-6. Because the localization of both PAR-3 and PAR-6 appears patchy in cdc-42(RNAi) embryos, it is suggested that in C. elegans, CDC-42 acts as a membrane-associated protein that is important for the assembly of the cortical PAR complex. In addition, the assembly or maintenance of this complex may be important for the segregation of PAR-2 and PAR-3 to discrete A-P cortical domains. If viable and fertile, a cdc-42 null mutation may, like par-6, eliminate the cortical localization of PAR-3 and cause a par-3-like phenotype (Kay, 2001).

This genetic analysis of the regulatory relationships between cdc-42 and the par genes indicates that Cdc42 organizes embryonic polarity by controlling the localization and activity of the PAR proteins. Analyses of par-3; cdc-42(RNAi) double mutants suggest that cdc-42 controls par-3 activity. Consistent with this, cdc-42(RNAi) disrupts the asymmetric localization of both PAR-3 and PAR-6, further suggesting that in the C. elegans embryo, CDC-42 controls the asymmetric localization of the PAR-3/PAR-6 complex to establish or maintain embryonic polarity. Although previous genetic analysis indicates that PAR-3 localization in the posterior of the embryo is inhibited by par-2(+), cdc-42(+) appears to act independently of par-2 in the cortical patterning of PAR-3. In mammalian cells, activated Cdc42 interacts with PAR-6, suggesting that the genetic interactions in C. elegans may reflect a conserved interaction between the PAR-3/PAR-6 polarity-promoting complex and Cdc42. Such an association appears to simultaneously provide the missing links between these known mediators of cellular polarity; that is, identifying both a regulator of the PARs and effectors for Cdc42. In the C. elegans embryo, Cdc42 could provide either a primary polarity cue for the asymmetric localization of the presumed PAR-3/PAR-6/PKC-3 complex, or it may function secondarily to its distribution. In mammalian epithelial cells, overexpression of an activated form of Cdc42 causes the mislocalization of PAR-3, suggesting that Cdc42 can provide a primary localization cue for PAR-3. It would be interesting to determine whether activated CDC-42 has a similar ability in C. elegans embryos. If so, then the A-P localization of GTP-bound CDC-42 (activated) could provide the primary cue for the A-P localization of the PAR proteins (Kay, 2001).

Generation of asymmetry in the one-cell embryo of C. elegans establishes the anterior-posterior axis (A-P), and is necessary for the proper identity of early blastomeres. Conserved PAR proteins are asymmetrically distributed and are required for the generation of this early asymmetry. The small G protein Cdc42 is a key regulator of polarity in other systems, and recently it has been shown to interact with the mammalian homolog of PAR-6. The function of Cdc42 in C. elegans had not yet been investigated, however. C. elegans cdc-42 plays an essential role in the polarity of the one-cell embryo and the proper localization of PAR proteins. Inhibition of cdc-42 using RNA interference results in embryos with a phenotype that is nearly identical to par-3, par-6, and pkc-3 mutants, and asymmetric localization of these and other PAR proteins is lost. CDC-42 physically interacts with PAR-6 in a yeast two-hybrid system, consistent with data on the interaction of human homologs. It is concluded that CDC-42 acts in concert with the PAR proteins to control the polarity of the C. elegans embryo, and the interaction of CDC-42 and the PAR-3/PAR-6/PKC-3 complex has been evolutionarily conserved as a functional unit (Gotta, 2001).

Phenotypic and two-hybrid data suggest that CDC-42 might activate the PAR-3/PAR-6/PKC-3 complex through interaction with PAR-6. CDC-42 appears to be necessary for the activity of the complex as well as for its correct localization. It is also possible that the PAR-3/PAR-6/PKC-3 complex has a role in activating CDC-42, since its initial anterior localization seems CDC-42 independent. One way that CDC-42 and the PAR-3/PAR-6/PKC-3 complex might direct polarity is through the regulation of the actin cytoskeleton. In par-3 mutants and in cdc-42(RNAi) embryos, enrichment of actin at the anterior of early embryos is lost. Further, cosuppression of par-2 and cdc-42(RNAi) mutant phenotypes suggests that CDC-42 and PAR-2 have counterbalancing, antagonistic activities. Because PAR-2 has a RING finger, a motif that has been proposed to be involved in ubiquitin-mediated protein degradation, CDC-42 might normally activate a protein that is a target of PAR-2. Future biochemical and in vivo studies should help to reveal the nature of these interactions and identify downstream targets (Gotta, 2001 and references therein).

Polarization of the C. elegans zygote along the anterior-posterior axis depends on cortically enriched (PAR) and cytoplasmic (MEX-5/6) proteins, which function together to localize determinants (e.g. PIE-1) in response to a polarizing cue associated with the sperm asters. Using time-lapse microscopy and GFP fusions, the localization dynamics of PAR-2, PAR-6, MEX-5, MEX-6 and PIE-1 were studied in wild-type and mutant embryos. These studies reveal that polarization involves two genetically and temporally distinct phases. During the first phase (establishment), the sperm asters at one end of the embryo exclude the PAR-3/PAR-6/PKC3 complex from the nearby cortex, allowing the ring finger protein PAR-2 to accumulate in an expanding `posterior' domain. Onset of the establishment phase involves the non-muscle myosin NMY-2 and the 14-3-3 protein PAR-5. The kinase PAR-1 and the CCCH finger proteins MEX-5 and MEX-6 also function during the establishment phase in a feedback loop to regulate growth of the posterior domain. The second phase begins after pronuclear meeting, when the sperm asters begin to invade the anterior. During this phase (maintenance), PAR-2 maintains anterior-posterior polarity by excluding the PAR-3/PAR-6/PKC3 complex from the posterior. These findings provide a model for how PAR and MEX proteins convert a transient asymmetry into a stably polarized axis (Cuenca, 2003).

Previous studies have implicated the sperm-derived MTOC as the most likely source for the spatial cue that initially polarizes the zygote. Time-lapse analysis supports this view. Formation of the MTOC correlates temporally and spatially with the earliest evidences of polarity: (1) cessation of ruffling, (2) enrichment of GFP:PAR-2, and (3) loss of GFP:PAR-6 in the posterior cortex. The data also demonstrates that the primary effect of the polarizing cue is to clear the PAR-3/PAR-6/PKC-3 complex from the posterior cortex. This effect does not require PAR-2. In contrast, restriction of PAR-2 to the posterior requires PAR-6, PAR-3 and PKC-3, suggesting that PAR-2 does not sense the polarity cue directly but instead responds to local displacement of the anterior complex (Cuenca, 2003).

The establishment phase requires the class II non-muscle myosin, NMY-2: nmy-2(RNAi) prevents PAR-6 (and presumably associated PAR-3 and PKC-3) from sensing the polarity cue, causing it to remain uniformly distributed throughout the cortex. In NMY-2-depleted embryos, PAR-2 is prevented from accumulating at the cortex by PAR-6 (and/or its partners). This 'default' state of PAR-6 on/PAR-2 off is also observed in mutants lacking sperm asters and in mutants where the MTOC detaches from the cortex prematurely. These observations suggest that the initial symmetry-breaking event involves signaling between the MTOC and the actin cytoskeleton. Consistent with this view, one of the earliest signs of polarization is cessation of ruffling in the cortex nearest the MTOC. Cessation of ruffling correlates with MTOC formation, but does not appear to require PAR activity (cessation of ruffling was observed in all par mutants examined in this study). These observations suggest that modification of the actin cytoskeleton may be an obligatory step before the onset of PAR asymmetry. It is proposed that signaling from the MTOC modifies the actin cytoskeleton locally, which causes the PAR-3/PAR-6/PKC-3 complex to become destabilized, allowing PAR-2 to accumulate in its place (Cuenca, 2003).

The establishment phase also requires the 14-3-3 protein PAR-5. In its absence, PAR-6 responds only weakly, if at all, to the polarity cue and PAR-2 is no longer excluded from the cortex by the PAR-3/PAR-6/PKC-3 complex. Cessation of ruffling in the posterior, however, still occurs in par-5(RNAi) embryos, suggesting that PAR-5 is not required for the initial MTOC/actin cytoskeleton interaction. Although this interpretation is complicated by the fact that residual PAR-5 activity may persist in par-5(RNAi) embryos, it is proposed that PAR-5 functions primarily by regulating the ability of the PAR-3/PAR-6/PKC-3 complex to (1) exclude PAR-2 and (2) respond to changes in the cytoskeleton. The presence of a potential 14-3-3 binding motif in PAR-3 is consistent with the possibility that PAR-5 regulates the PAR-3/PAR-6/PKC-3 complex by binding to it directly (Cuenca, 2003).

Surprisingly, it was found that the predominantly cytoplasmic MEX-5 and MEX-6 also play a role during the establishment phase. In the absence of MEX-5 and MEX-6, the posterior domain occasionally does not form (15%-30% of embryos), and frequently (50% or more of embryos) is slow to reach its final configuration. These observations indicate that, although MEX-5 and MEX-6 are not absolutely required for PAR localization in the zygote, they do play a role in ensuring a robust response by the PAR-3/PAR-6/PKC-3 complex to the MTOC/actin cytoskeleton signal (Cuenca, 2003).

This aspect of MEX-5/6 function is negatively regulated by PAR-1. In par-1 mutants, MEX-5 and MEX-6 cause the posterior domain to extend further towards the anterior during the establishment phase. Since PAR-1 itself becomes enriched in the posterior domain, one attractive possibility is that PAR-1 and MEX-5/6 participate in a feedback loop that limits expansion of the posterior domain. It is proposed that at the beginning of the establishment phase, MEX-5 and MEX-6 levels are high throughout the zygote and help clear the PAR-3/PAR-6/PKC-3 complex from the region nearest the sperm asters. This clearing allows PAR-2 and PAR-1 to accumulate on the cortex, which in turn reduces MEX-5/6 activity and/or levels in the surrounding cytoplasm. Eventually, MEX-5/6 levels become too low to fuel further expansion of the posterior domain. It is not yet known whether the partial penetrance of the mex-5(-);mex-6(-) phenotype is due to redundancy with other factors, or is indicative of a minor role for the feedback loop in regulating PAR asymmetry (Cuenca, 2003).

The finding that the sperm-derived MTOCs play a role in initiating polarity raised the question of how polarity is maintained after pronuclear meeting, when the pronuclei/centrosome complex rotates and microtubules invade the anterior end of the embryo. In the absence of PAR-2, PAR-3 and PAR-6 and PKC-3 can become asymmetric before pronuclear meeting, but return into the posterior domain afterwards. This finding demonstrates two points: (1) the PAR-6/PAR-3/PKC-3 complex no longer responds to the MTOC-dependent cue after pronuclear meeting, and (2) PAR-2 is required after pronuclear meeting, but not earlier, to exclude the PAR-6/PAR-3/PKC-3 complex from the posterior. It is proposed that pronuclear meeting (and/or the end of prophase) triggers a change in the cytoskeleton, or in the PAR-6/PAR-3/PKC-3 complex, that turns off the MTOC-dependent polarity signal, or the ability to respond to it. From that point on, PAR-2 becomes essential to keep PAR-6/PAR-3/PKC-3 out of the posterior cortex. It is intriguing that PAR-6 briefly localizes to nuclei at pronuclear meeting, raising the possibility that it becomes modified at that time (Cuenca, 2003).

The existence of distinct establishment and maintenance phases is also supported by the observation that cdc-42 is required after prophase, but not earlier, for PAR-3, PAR-6 and PKC-3 asymmetry. Analysis of GFP:PAR-6 dynamics in par-1(RNAi) embryos suggests that PAR-1 also contributes to maintenance of PAR asymmetry after pronuclear meeting. How PAR-2, CDC-42 and PAR-1 function together to maintain the balance between anterior and posterior PAR domains remains to be determined (Cuenca, 2003).

PAR proteins distribute asymmetrically across the anterior-posterior axis of the 1-cell-stage C. elegans embryo, and function to establish subsequent anterior-posterior asymmetries. By the end of the 4-cell stage, anteriorly localized PAR proteins, such as PAR-3 and PAR-6, redistribute to the outer, apical surfaces of cells, whereas posteriorly localized PAR proteins, such as PAR-1 and PAR-2, redistribute to the inner, basolateral surfaces. Because PAR proteins are provided maternally, distinguishing apicobasal from earlier anterior-posterior functions requires a method that selectively prevents PAR activity after the 1-cell stage. In the present study hybrid PAR proteins were generated that are targeted for degradation after the 1-cell stage. Embryos containing the hybrid PAR proteins had normal anterior-posterior polarity, but show defects in apicobasal asymmetries associated with gastrulation. Ectopic separations appear between lateral surfaces of cells that are normally tightly adherent; cells that ingress during gastrulation fail to accumulate nonmuscle myosin at their apical surfaces, and ingression is slowed. Thus, PAR proteins function in both apicobasal and anterior-posterior asymmetry during the first few cell cycles of embryogenesis (Nance, 2003).

During the 4-cell stage of embryogenesis, the PAR proteins undergo a dramatic redistribution along the apicobasal axis. The results indicate that recruitment of PAR-3 to the apical cortex is a key step in this redistribution, analogous to previous observations on the role of PAR-3 at the 1-cell stage. PAR-3 localization to the apical cortex occurs independently of PAR-6 and PAR-2. Moreover, PAR-3 localization is crucial for recruiting PAR-6 and PKC-3 to the apical cortex, and restricting PAR-2 to basolateral surfaces. Localization of PAR-3 to the apical cortex is not sufficient for the colocalization of PAR-6 and PKC-3: PAR-6 does not colocalize with apical PAR-3 in pkc-3(RNAi) embryos, and PKC-3 does not colocalize with apical PAR-3 in par-6(ZF1) embryos, that is, in embryos in which PAR-6 is degraded after the first division. Thus both PAR-6 and PKC-3 must be present for either protein to associate with apical PAR-3. Biochemical studies of PAR-3, PAR-6 and PKC-3 homologs in mammalian cells have shown that these proteins can bind to one another directly, indicating that interactions between all three proteins might be necessary to stabilize a complex with apical PAR-3 (Nance, 2003).

C. elegans embryos have at least three distinct periods in which the PAR-3 complex must distinguish different cell surfaces. At the 1-cell stage, PAR-3 associates with the anterior surface, and at the 4-cell stage, PAR-3 associates with the apical surface. In late embryogenesis PAR-3 is localized asymmetrically in epithelial cells, and the apicobasal axis of the internal epithelia is inverted with respect to that of earlier embryonic cells. These localization patterns appear to be specified de novo during each period. Disruption of PAR asymmetry at the 1-cell stage by mutations in par-2 does not prevent apical localization of PAR-3 after the 4-cell stage. Similarly, the absence of the PAR-3 complex between the 4-400-cell stages in par-3(ZF1) embryos does not prevent the subsequent apical localization of PAR-3 during organogenesis (Nance, 2003).

The molecular cues used to localize the PAR-3 complex remain to be identified and, at some level, these are likely to vary. For example, sperm position and cell contacts specify polarity at the 1- and 4-cell stages, respectively. Although the mechanism of PAR localization has not been studied extensively in the epithelial cells of C. elegans, genetic studies in Drosophila have identified homologs of proteins in the C. elegans PAR-3 complex that regulate apicobasal polarity in epithelial cells. E-cadherin-mediated cell adhesion is required for apical PAR-3 complex localization in Drosophila epithelial cells, whereas HMR-1/E-cadherin is not essential for PAR-3 complex asymmetry at either the 1-cell or 4-cell stage in C. elegans. Apical localization of the PAR-3 complex in Drosophila epithelia is antagonized by a basolateral complex of proteins that includes Discs large and Scribble. The C. elegans homologs of the latter proteins, DLG-1/Discs large and LET-413/Scribble, are expressed in epithelial cells, and depletion of these proteins causes epithelial defects. However, these proteins do not appear to function in apicobasal polarity of early embryonic cells because they are either not expressed in the early embryo (DLG-1) or are not required for apical localization of PAR-3. Thus, identifying the molecular basis of cell-contact-dependent PAR localization remains an important goal for future studies on apicobasal PAR asymmetry (Nance, 2003).

Epithelial cells perform important roles in the formation and function of organs and the genesis of many solid tumors. A distinguishing feature of epithelial cells is their apicobasal polarity and the presence of apical junctions that link cells together. The interacting proteins Par-6 (a PDZ and CRIB domain protein) and aPKC (an atypical protein kinase C) localize apically in fly and mammalian epithelial cells and are important for apicobasal polarity and junction formation. Caenorhabditis elegans PAR-6 and PKC-3/aPKC also localize apically in epithelial cells, but a role for these proteins in polarizing epithelial cells or forming junctions has not been described. This study used a targeted protein degradation strategy to remove both maternal and zygotic PAR-6 from C. elegans embryos before epithelial cells are born. It was found that PKC-3 does not localize asymmetrically in epithelial cells lacking PAR-6, apical junctions are fragmented, and epithelial cells lose adhesion with one another. Surprisingly, junction proteins still localize apically, indicating that PAR-6 and asymmetric PKC-3 are not needed for epithelial cells to polarize. Thus, whereas the role of PAR-6 in junction formation appears to be widely conserved, PAR-6-independent mechanisms can be used to polarize epithelial cells (Totong, 2007).

C. elegans embryonic polarity requires the asymmetrically distributed proteins PAR-3, PAR-6 and PKC-3. The rho family GTPase CDC-42 regulates the activities of these proteins in mammals, flies and worms. To clarify its mode of action in C. elegans, the interaction between PAR-6 and CDC-42 was disrupted in vivo, and also the distribution of GFP-tagged CDC-42 was determined in the early embryo. Mutant PAR-6 proteins unable to interact with CDC-42 accumulate asymmetrically, at a reduced level, but this asymmetry is not maintained during the first division. Constitutively active GFP::CDC-42 becomes enriched in the anterior during the first cell cycle in a domain that overlaps with PAR-6. The asymmetry is dependent on PAR-2, PAR-5 and PAR-6. Furthermore, it was found that overexpression of constitutively active GFP::CDC-42 increased the size of the anterior domain. It is concluded that the CDC-42 interaction with PAR-6 is not required for the initial establishment of asymmetry but is required for maximal cortical accumulation of PAR-6 and to maintain its asymmetry (Aceto, 2006).

In C. elegans one-cell embryos, polarity is conventionally defined along the anteroposterior axis by the segregation of partitioning-defective (PAR) proteins into anterior (PAR-3, PAR-6) and posterior (PAR-1, PAR-2) cortical domains. The establishment of PAR asymmetry is coupled with acto-myosin cytoskeleton rearrangements. The small GTPases RHO-1 and CDC-42 are key players in cytoskeletal remodeling and cell polarity in a number of different systems. This study investigated he roles of these two GTPases and the RhoGEF ECT-2 in polarity establishment in C. elegans embryos. CDC-42 is shown to be required to remove PAR-2 from the cortex at the end of meiosis and to localize PAR-6 to the cortex. By contrast, RHO-1 activity is required to facilitate the segregation of CDC-42 and PAR-6 to the anterior. Loss of RHO-1 activity causes defects in the early organization of the myosin cytoskeleton but does not inhibit segregation of myosin to the anterior. It is therefore proposed that RHO-1 couples the polarization of the acto-myosin cytoskeleton with the proper segregation of CDC-42, which, in turn, localizes PAR-6 to the anterior cortex (Schonegg, 2006).

The PAR proteins have an essential and conserved function in establishing polarity in many cell types and organisms. However, their key upstream regulators remain to be identified. In C. elegans, regulators of the PAR proteins can be identified by their ability to suppress the lethality of par-2 mutant embryos. This study shows that a loss of function mutant in a Nanos homolog nos-3 suppresses the lethality of par-2 mutants by regulating PAR-6 protein levels. The suppression requires the activity of the sex determination genes fem-1/2/3 and of the cullin cul-2. FEM-1 is a substrate-specific adaptor for a CUL-2-based ubiquitin ligase (CBCFEM-1). Interestingly, CUL-2 is required for the regulation of PAR-6 levels and that PAR-6 physically interacts with FEM-1. These data strongly suggest that PAR-6 levels are regulated by the CBCFEM-1 ubiquitin ligase thereby uncovering a novel role for the FEM proteins and cullin-dependent degradation in regulating PAR proteins and polarity processes (Pacquelet, 2007).

Cortical flows powered by asymmetrical contraction transport PAR proteins to establish and maintain anterior-posterior polarity in the early C. elegans embryo

The C. elegans PAR proteins PAR-3, PAR-6, and PKC-3 are asymmetrically localized and have essential roles in cell polarity. The one-cell C. elegans embryo contains a dynamic and contractile actomyosin network that appears to be destabilized near the point of sperm entry. This asymmetry initiates a flow of cortical nonmuscle myosin (NMY-2) and F-actin toward the opposite, future anterior, pole. PAR-3, PAR-6, and PKC-3, as well as non-PAR proteins that associate with the cytoskeleton, appear to be transported to the anterior by this cortical flow. In turn, PAR-3, PAR-6, and PKC-3 modulate cortical actomyosin dynamics and promote cortical flow. PAR-2, which localizes to the posterior cortex, inhibits NMY-2 from accumulating at the posterior cortex during flow, thus maintaining asymmetry by preventing inappropriate, posterior-directed flows. Similar actomyosin flows accompany the establishment of PAR asymmetries that form after the one-cell stage, suggesting that actomyosin-mediated cortical flows have a general role in PAR asymmetry (Munro, 2004).

It has been proposed how a local modulation of global cortical contractility could produce tension gradients in the cortex that would drive flows of cortical cytoplasm away from regions of low cortical tension and toward regions of high cortical tension. More specifically, it has been hypothesized that cortical flows in the one-cell C. elegans embryo might be caused by a local relaxation of cortical tension through an interaction between the sperm MTOC and the cortex. The results support this idea and reveal the cytoskeletal basis for contractile force generation and cortical flow: prior to the appearance of a distinct sperm pronucleus, F-actin and NMY-2 constitute a dynamic network of short-lived foci that form through transient local, myosin-dependent, contractions of the cortical meshwork. The association of surface invaginations with groups of foci and interfoci and the evident continuity of the actomyosin meshwork implies that neighboring foci are mechanically coupled to form a network of tensioned elements. Prior to appearance of the sperm MTOC, local contractions associated with focus formation drive small transient displacements of foci, but the network is globally stable. Appearance of the sperm MTOC is associated with an immediate, local cessation of focus formation, reflecting an apparent diminution of contractility or weakening of the otherwise symmetrical actomyosin network. The resulting global tension imbalance should cause an immediate and collective flow of remaining foci toward one another and away from the sperm MTOC as the entire meshwork contracts asymmetrically toward the opposite, anterior pole. Measurements of focus movements before and after appearance of the sperm MTOC confirm this prediction, and the loss of convergent flow in embryos lacking the regulatory myosin light chain MLC-4 confirms the contractile basis for these flows (Munro, 2004).

How do flows continue once initiated? The observations reveal a continuous local cycle of focus assembly/contraction followed by disassembly. The local contractions that produce each individual focus are short-lived, but at any time during flow, these contractions are distributed throughout the anterior cap and coupled to one another to form a continuously tensioned network. Thus the cortex appears to be a self-renewing contractile engine that continues to generate tension even as it contracts, rather than a pre-tensioned network that contracts once to release stored tension. Likewise, the persistent absence of foci in the posterior clear zone, and the constant flow speeds of filaments away from this zone, suggest a continued absence of contractility near the sperm MTOC. Thus, sustained, asymmetrical contractile force generation appears to sustain a continued cortical flow (Munro, 2004).

Previous studies suggest that factors closely associated with the sperm MTOC supply the cue that initiates cortical flow in the one-cell embryo, although the relative importance of the centrosome itself and the sperm astral microtubules remains controversial. The observations that focus dynamics remain unchanged near the newly formed sperm pronucleus, and that cortical flows never initiate in embryos depleted of essential centrosomal components (which also lack a sperm aster), support this hypothesis. Furthermore, they suggest that the sperm cue acts by modulating the actomyosin contractility cycle to produce a local reduction of cortical tension. Discovering the nature of this modulation is an important future goal (Munro, 2004).

NMY-2::GFP foci, PAR-6::GFP puncta, and yolk granules each move toward the anterior pole at the same velocity, indicating that all are components of the same cortical flow. The observation that the rear boundary of the anterior PAR-6 cap moves at the same speed as this flow suggests that cortical transport is the dominant mechanism for establishing an anterior PAR domain. Several lines of evidence suggest that the other members of the anterior PAR complex, PAR-3 and PKC-3, localize through the same cortical flow. PAR-3, PAR-6, and PKC-3 colocalize extensively in coimmunostaining experiments, and analysis of fixed embryos shows coincident distributions of NMY-2 and endogenous PAR-3. PAR-3 and PKC-3 are both essential for the cortical localization of PAR-6, and homologs of these proteins can complex in vitro. PAR-3 appears to provide the critical link that enables PAR-6 and PKC-3 to associate with the cortex, since it is the only member of the complex that can localize cortically in the absence of the others. PAR-3 is unlikely to bind NMY-2 directly since it remains associated with the cortex when NMY-2 is depleted from the embryo. However, PAR-3 cortical localization may involve actin or actin binding proteins since depleting cortical F-actin prevents PAR-3 from associating with the cortex (Munro, 2004).

The non-PAR proteins HMR-1/E-Cadherin, HMP-2/ß-catenin, and LAD-1/L1CAM each localize to the anterior pole of the embryo during cortical flow. None of these proteins have a known role in early embryonic polarity, but they or their homologs in other animals can associate indirectly with the cortical actin cytoskeleton. Thus cortical flow at the one-cell stage may be a feature common to all proteins associated with the actomyosin cytoskeleton (Munro, 2004).

Coordinate flows of NMY-2 and PAR-6 are associated with multiple examples of cortical PAR asymmetry in the early C. elegans embryo. This suggests that asymmetrical contractile flows underlie each asymmetry, although the cues that initiate these flows must at some level be different. As the sperm MTOC appears to be the cue for anterior/posterior polarity at the one-cell stage, centrosomes might function in subsequent anterior/posterior polarity for the lineage of cells that produce the germline; after the first cell division, there is a migration of the nucleus and centrosomes toward the posterior cortex of the germline precursor that precedes cortical flows of both yolk granules and NMY-2 and PAR-6. However, all other early embryonic cells exhibit apicobasal PAR asymmetry, and in these cells there is no obvious relationship between centrosomes/microtubules and the apicobasal axis. Instead, experiments on isolated cells suggest that contacts between cells determine the apicobasal axis. Thus there are likely to be multiple cues that cells can exploit to generate cortical flows, and it will be of interest to see how these converge on the actomyosin cytoskeleton (Munro, 2004).

The fact that depleting embryos of the anterior PAR proteins alters actomyosin dynamics and attenuates cortical flow implies that these proteins are not simply passive cargo transported by an independent flow. It suggests rather that they actively modulate cortical dynamics to promote cortical flow and thus their own transport. The net force acting on the NMY-2 foci must be the sum of active (e.g., contractile) forces that tend to move foci and passive (e.g., viscous and elastic) forces that resist these movements. The deep local furrowing observed in par-3 mutants argues against a simple decrease in contractile force generation in par-3 mutant embryos. An alternative possibility is that the anterior PAR complex could modulate cortical elements that passively resist focus movement, for example by modulating crosslinks within the actomyosin network itself or by modulating other cortical structures that interact mechanically with the actomyosin network to resist its deformation (Munro, 2004).

Studies in several labs suggest that the anterior PAR complex inhibits the association of PAR-2 with the cortex and that depletion of the anterior PAR complex from the posterior cortex removes this inhibition and allows PAR-2 to accumulate there. PAR-2 in turn is required to maintain the anterior localization of anterior PAR proteins after pseudocleavage in one-cell embryos, but the mechanism by which it acts is unknown. The gap observed between anterior and posterior PAR domains after pseudocleavage makes it unlikely that PAR-2 acts directly upon members of the anterior PAR complex. Instead, the observations suggest that the loss of anterior PAR asymmetry in par-2 (RNAi) embryos is caused by an aberrant flow of NMY-2::GFP and PAR-6::GFP toward the posterior pole that results from the ectopic return of NMY-2 to the posterior cortex after pseudocleavage. Thus it is proposed that PAR-2 contributes indirectly to maintaining the anterior localization of the anterior PAR complex, by inhibiting recruitment of NMY-2 to the posterior cortex, and thus by preventing inappropriate, NMY-2-driven, flows to the posterior. The same inhibition may also contribute to maintenance of anterior PAR asymmetries by promoting normal anterior-directed flows after pseudocleavage. However, PAR-2 does not appear to be highly conserved in animal evolution. Thus other mechanisms, including interactions among PAR-1, PAR-5, and the anterior PAR complex, or between PAR-1 and NMY-2, must also operate in animals to maintain PAR asymmetries once they are established (Munro, 2004).

PAR-3 mediates the initial clustering and apical localization of junction and polarity proteins during C. elegans intestinal epithelial cell polarization: PAR-3 and PAR-6 function sequentially to position and mature apical junctions

The apicobasal polarity of epithelial cells is critical for organ morphogenesis and function, and loss of polarity can promote tumorigenesis. Most epithelial cells form when precursor cells receive a polarization cue, develop distinct apical and basolateral domains and assemble junctions near their apical surface. The scaffolding protein PAR-3 regulates epithelial cell polarity, but its cellular role in the transition from precursor cell to polarized epithelial cell has not been determined in vivo. This study used a targeted protein-degradation strategy to remove PAR-3 from C. elegans embryos and examine its cellular role as intestinal precursor cells become polarized epithelial cells. At initial stages of polarization, PAR-3 accumulates in cortical foci that contain E-cadherin, other adherens junction proteins, and the polarity proteins PAR-6 and PKC-3. Using live imaging, PAR-3 foci were shown to move apically and cluster, and it was shown that PAR-3 is required to assemble E-cadherin into foci and for foci to accumulate at the apical surface. It is proposed that PAR-3 facilitates polarization by promoting the initial clustering of junction and polarity proteins that then travel and accumulate apically. Unexpectedly, superficial epidermal cells form apical junctions in the absence of PAR-3, and PAR-6 was shown to have a PAR-3-independent role in these cells to promote apical junction maturation. These findings indicate that PAR-3 and PAR-6 function sequentially to position and mature apical junctions, and that the requirement for PAR-3 can vary in different types of epithelial cells (Achilleos, 2010).

Vertebrate PAR-6 homologs

The par genes are required to establish polarity in the Caenorhabditis elegans embryo. The Xenopus homolog of C. elegans PAR-6 (XPAR-6) has been identified. XPAR-6 is a protein of 377 amino acids with one PDZ domain that is involved in mediating protein-protein interactions. It shares 59% and 58% amino acid identity with the mouse and Drosophila PAR-6, respectively, and 54% overall identity with C. elegans PAR-6. Xpar-6 is expressed both maternally and zygotically. Xpar-6 is first detected in the animal half of the egg, and this pattern of expression persists into the cleavage and blastula stages. At the gastrula stage, the message is detected in the animal pole area and in a broad domain of the ventral region, but is excluded from the dorsal region. With the onset of neurulation, the localized expression of Xpar-6 becomes more obvious, leading to it being enriched in the dorsolateral region along the lateral edges of neural plate and anterior presumptive head region surrounding the anterior border of neural plate. At late tailbud stage, Xpar-6 transcripts show localized expression throughout the head, labeling the branchial arches, eyes, otic vesicles and brain, while more posteriorly, Xpar-6 labels the somites, pronephros, tail tip and proctodeum. Therefore, this analysis suggests that Xpar-6 has a regionalized pattern of expression during Xenopus early embryogenesis (Choi, 2000).

PAR-6, Atypical protein kinase C and Rac/Cdc42

PAR (partitioning-defective) proteins, which were first identified in the nematode Caenorhabditis elegans, are essential for asymmetric cell division and polarized growth, whereas Cdc42 mediates establishment of cell polarity. An unexpected link between these two systems is described. A family of mammalian Par6 proteins have been identified that are similar to the C. elegans PDZ-domain protein PAR-6. Par6 forms a complex with Cdc42-GTP, with a human homolog of the multi-PDZ protein PAR-3 and with the regulatory domains of atypical protein kinase C (PKC) proteins. This assembly is implicated in the formation of normal tight junctions at epithelial cell-cell contacts. Thus, Par6 is a key adaptor that links Cdc42 and atypical PKCs to Par3 (Joberty, 2000).

Cellular asymmetry is critical for the development of multicellular organisms. Homologs of proteins necessary for asymmetric cell division in Caenorhabditis elegans associate with each other in mammalian cells and tissues. mPAR-3 and mPAR-6 exhibit similar expression patterns and subcellular distributions in the CNS and associate through their PDZ (PSD-95/Dlg/ZO-1) domains. mPAR-6 binds to Cdc42/Rac1 GTPases, and mPAR-3 and mPAR-6 bind independently to atypical protein kinase C (aPKC) isoforms. In vitro, mPAR-3 acts as a substrate and an inhibitor of aPKC. It is concluded that mPAR-3 and mPAR-6 have a scaffolding function, coordinating the activities of several signaling proteins that are implicated in mammalian cell polarity (Lin, 2000).

A novel effector of Rac and Cdc42, hPar-6, has been identified that is the human homolog of a cell-polarity determinant in C. elegans. hPar-6 contains a PDZ domain and a Cdc42/Rac interactive binding (CRIB) motif, and interacts with Rac1 and Cdc42 in a GTP-dependent manner. hPar-6 also binds directly to an atypical protein kinase C isoform, PKC, and forms a stable ternary complex with either Rac1 or Cdc42 and PKC. This association results in stimulation of PKC kinase activity. Moreover, hPar-6 potentiates cell transformation by Rac1/Cdc42 and its interaction with Rac1/Cdc42 is essential for this effect. Cell transformation by hPar-6 involves a PKC-dependent pathway distinct from the pathway mediated by Raf (Qui, 2000).

Many direct targets of Rac1 and Cdc42 have been identified, but none has been shown to have a direct role in cell transformation by Rac1 and Cdc42. hPar-6 is a novel effector of Rac1 and Cdc42 that promotes PKCzeta-dependent transformation by both GTPases. Although it has been suggested that PAK1 may also contribute to transformation by Rac1 in Rat1 fibroblasts, PAK1 does not enhance transformation by activated Raf or activated Rac1 in NIH-3T3 cells, and studies using effector domain mutants indicate that interaction of PAK1 with Rac1 does not correlate with cell-cycle progression or transformation. Thus, hPar-6 appears to be the first effector shown to directly mediate transformation by Rac1 and Cdc42. The identification of PKCzeta as a downstream effector of hPar-6 represents the first elucidation of a signaling pathway linking Rac1/Cdc42 to cell transformation. A model is presented depicting two separate pathways downstream of Ras that lead to cell polarity and growth control: these pathways can contribute to cell transformation. One pathway is comprised of Rac/Cdc42, hPar-6 and PKCzeta, and the other is mediated by Raf, MEK and MAP kinase (Qui, 2000).

The mechanism by which hPar-6 regulates the kinase activity of PKCzeta is currently under investigation. Subcellular targeting by interaction with specific proteins provides an attractive mechanism for PKC isozyme-specific regulation. It is possible that hPar-6 and PKCzeta are translocated by Rac1 or Cdc42 to the membrane, where PKCzeta could interact with an activator. One candidate activator is the phosphatidylinositol 3-kinase (PI3-kinase) target PDK1, since PDK1 and PKCzeta associate in vivo via their catalytic domains, and both PI3-kinase and PDK1 stimulate PKCzeta activity. Consistent with this model, it has been demonstrated that PI3-kinase can act as a link between Ras and Rac in transformation and that membrane-targeted PKCzeta is constitutively active. The observation that hPar-6 alone exhibits little, if any, transforming activity is also consistent with the membrane-targeting model. It should also be noted that although overexpression of hPar-6 alone (i.e., in the absence of Rac1[G12V]) is sufficient to activate PKCzeta kinase activity, overexpression of hPar-6 and PKCzeta only marginally promotes focus formation, suggesting that activated Rac1 is necessary to target PKCzeta to substrates involved in transformation. However, the possibility that Rac1 activates some other pathway that is also necessary for transformation cannot be ruled out. In addition to being activated by hPar-6, PKCzeta might in turn phosphorylate hPar-6. In this regard, it should be noted that there is a putative PKCzeta-phosphorylation site in mammalian Par-6 (Qui, 2000).

The mechanism underlying transformation by hPar-6 and PKCzeta is not yet clear. Stimulation of cell proliferation and inhibition of apoptosis are, however, important characteristics of cell transformation. In this regard, it has been shown that Rac1 and Cdc42 induce cyclin D1 transcription and accumulation, phosphorylation and inactivation of the tumor suppressor protein Rb, and activation of the transcription factor E2F. Inactivation of Rb may be necessary for Rac1/Cdc42 stimulation of cell proliferation, and it is possible that hPar-6 and PKCzeta have a role in this pathway. In addition, Ras, Rac1, Cdc42 and PKCzeta are all able to activate the transcription factor NF-kappaB. NF-kappaB activation is associated with mitogenesis, anti-apoptotic activity and cell transformation. Thus, the hpar6-PKCzeta pathway might mediate NF-kappaB activation, and thereby contribute to cell transformation by Rac1 and Ras. Another possibility is that the hpar6-PKCzeta pathway may mediate growth control by Rac1/Cdc42 by inducing downregulation of the pro-apoptotic protein Par-4 (prostate apoptosis response-4; unrelated to the C. elegans Par gene product). Par-4 interacts with PKCzeta and overexpression of PKCzeta downregulates Par-4, an event that appears important for Ras transformation and tumor progression. Thus, cyclin D1, Rb, E2F, NF-kappaB and Par-4 all warrant further investigation as possible downstream targets of the hpar6-PKCzeta pathway (Qui, 2000).

Polarity is a fundamental feature of all eukaryotic cells. Rac, Cdc42, Par-6 and atypical PKCs appear to be conserved in diverse metazoans, including Drosophila, C. elegans, Xenopus, mouse and humans. The CRIB motif of Par-6 is also conserved, suggesting that it interacts with Rac and/or Cdc42 in these different species. In C. elegans, inhibition of Cdc42 function by RNA-mediated gene interference (RNAi) produces defects in cell polarity similar to those observed in par and pkc-3 mutants, while in mammalian cells, Par-6 is localized to tight junctions, together with atypical PKC and ASIP, the mammalian homolog of Par-3. Moreover in C. elegans, Par-6 interacts with Par-3, and in Drosophila the Par-3 homolog has an important role in the asymmetric cleavage of epithelial cells and neuroblasts. Taken together, these observations suggest that Rac or Cdc42, Par-6, atypical PKC, and perhaps Par-3, constitute a conserved pathway that regulates cell polarity. As hPar-6 and PKCzeta mediate cell transformation by Rac1 and Cdc42, there may be a link between cell-polarity signaling and growth control: aberrant cell-polarity signaling could lead to oncogenic transformation. In the light of the important roles of Rac1/Cdc42 in Ras-induced transformation, hPar-6 and PKCzeta could represent potential targets for anti-cancer therapeutics (Qui, 2000).

A mammalian homolog of the PDZ domain containing C. elegans protein PAR-6 binds to the Rho family member Cdc42. PAR-6 contains a PDZ domain and in C. elegans it has been shown to be crucial for the asymmetric cleavage and establishment of cell polarity during the first cell divisions in the growing embryo. Mammalian PAR-6 interacts with Cdc42 and Rac1 both in the yeast two-hybrid system and in in vitro binding assays. Co-immunoprecipitation experiments, employing transiently transfected Cos-1 cells, further confirm that Cdc42 and Rac1 are physiological binding partners for PAR-6. In epithelial Madin-Darby canine kidney cells (MDCK), endogenous PAR-6 is present in the tight junctions, as judged from its co-localization with the tight junction protein ZO-1, however, PAR-6 is also detected in the cell nucleus. Stimulation of MDCK cells with scatter factor/hepatocyte growth factor induces a loss of PAR-6 from the areas of cell-cell contacts in conformity with their progressive breakdown. In C. elegans PAR-6 co-localizes with PAR-3 and has been suggested to form a direct complex. In agreement with earlier studies, mammalian PAR-3 is present in tight junctions of MDCK cells but, in contrast to PAR-6, the protein could not be detected in the nucleus. Furthermore, co-immunoprecipitation experiments, employing Cos-1 cells, demonstrate that mammalian PAR-6 and PAR-3 form a direct complex. These findings, together with the reported roles of PAR-6 and PAR-3 in C. elegans, suggest that Cdc42 and Rac1 and PAR-6/PAR-3 are involved in the establishment of cell polarity in epithelial cells (Johansson, 2000).

During early C. elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3. Together with the fact that aPKC and a mammalian PAR-3 homolog, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells, this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. The overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na(+),K(+)-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. aPKC associates not only with ASIP/PAR-3, but also with a mammalian homolog of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3-PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and that aPKC plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells (Suzuki, 2001).

Epithelial cells display apical-basal polarity, and the apical surface is segregated from the basolateral membranes by a barrier called the tight junction (TJ). TJs are constructed from transmembrane proteins that form cell-cell contacts -- claudins, occludin, and junctional adhesion molecule (JAM) -- plus peripheral proteins such as ZO-1. The Par proteins (partitioning-defective) Par3 and Par6, plus atypical protein kinase C (aPKC) function in the formation or maintenance of TJs and more generally in metazoan cell polarity establishment. Par6 contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain and binds the small GTPase Cdc42. Par6 inhibits TJ assembly in MDCK II epithelial cells after their disruption by Ca2+ depletion but does not inhibit adherens junction (AJ) formation. Transepithelial resistance and paracellular diffusion assays have confirmed that assembly of functional TJs is delayed by Par6 overexpression. Strikingly, the isolated, N-terminal fragment of PKCzeta, which binds Par6, also inhibits TJ assembly. Activated Cdc42 can disrupt TJs , but neither a dominant-negative Cdc42 mutant nor the CRIB domain of gammaPAK (p21-activated kinase), which inhibits Cdc42 function, observably inhibit TJ formation. These results suggest that Cdc42 and Par6 negatively regulate TJ assembly in mammalian epithelial cells (Gao, 2002).

A model is proposed in which Par6 exists in an equilibrium between an inactive and an active state. The active state binds to the PDZ1 domain of Par3 and may block the interaction of Par3 with junctional adhesion molecule (JAM), thereby preventing TJ assembly. The Par3-JAM interaction is proposed to be reversible, but recruitment of further components stabilizes the TJ, so that junctions will eventually form even in the presence of inhibitory Par6. Activation is induced by binding either Cdc42-GTP or the regulatory domain of aPKC. Activation can be artificially induced by removing the N-terminal domain of Par6 (which binds aPKC). This model accounts for the inhibitory effects of the overexpression of wild-type Par6 (which increases the concentration of the active form), of the DeltaN mutant (constitutively active), and of the PKCzeta (1-126) fragment (which will bind to and activate endogenous Par6) and for the reduced potency of Par6BdeltaPro136 (defective in Cdc42 binding). It also accounts for the inhibitory effects of Cdc42 (Q61L) (which will activate endogenous Par6) and of the N-terminal region of Par3 (which will compete with endogenous Par3 for JAM binding). Whether this model is indeed correct will require further investigation (Gao, 2002).

Maximal activation of NADPH oxidase requires formation of a complex between the p40phox and p67phox subunits via association of their PB1 domains. The crystal structure has been determined of the p40phox/p67phox PB1 heterodimer; the structure reveals that both domains have a β grasp topology and that they bind in a front-to-back arrangement through conserved electrostatic interactions between an acidic OPCA motif [the short sequence motif present in some PB1 domains, that previously has been referred to as the octicosapeptide repeat (OPR), PC motif (phox and cdc24p), and the AID motif (atypical protein kinase C-interaction domain)] on p40phox and basic residues in p67phox. The structure enabled the identification of residues critical for heterodimerization among other members of the PB1 domain family, including the atypical protein kinase Cζ (PKCζ) and its partners Par6 and p62 (ZIP, sequestosome). Both Par6 and p62 use their basic 'back' to interact with the OPCA motif on the 'front' of the PKCζ. Besides heterodimeric interactions, some PB1 domains, like the p62 PB1, can make homotypic front-to-back arrays (Wilson, 2003).

In order to resolve which interfaces p62, Par6, and PKCζ actually use in formation of heterodimeric complexes, site-specific mutants of these proteins were constructed. Using GST pull-down binding assays, it was found that PKCζ interacts with both Par6 and p62 only when it has a wild-type OPCA motif on its front. Mutation D62A/D66A in the OPCA motif of PKCζ abolishes binding to both Par6 and p62 PB1 domains. The same mutation affects PKCζ function in vivo. In contrast, a point mutation of a basic residue in the PKCζ 'back' (equivalent to Lys 355p67) has no influence on binding to wild-type PB1 domains from p62 and Par6. This suggests that PKCζ uses its acidic front to interact with the basic back of Par6 and p62. Consistent with this notion, mutation of a single basic residue at the back of either Par6 or p62 PB1 domains eliminates interaction with wild-type PKCζ, whereas mutations of the acidic cluster at the front of these adaptors have no impact on binding to PKCζ. These results suggest that binding of the adaptor proteins p62 and Par6 to PKCζ is mutually exclusive. Indeed, this is confirmed in direct competition experiments (Wilson, 2003).

Cdc42 is a small GTPase that is required for cell polarity establishment in eukaryotes as diverse as budding yeast and mammals. Par6 is also implicated in metazoan cell polarity establishment and asymmetric cell divisions. Cdc42.GTP interacts with proteins that contain a conserved sequence called a CRIB motif. Uniquely, Par6 possesses a semi-CRIB motif that is not sufficient for binding to Cdc42. An adjacent PDZ domain is also necessary and is required for biological effects of Par6. The crystal structure of a complex between Cdc42 and the Par6 GTPase-binding domain is reported in this study. The semi-CRIB motif forms a beta-strand that inserts between the four strands of Cdc42 and the three strands of the PDZ domain to form a continuous eight-stranded sheet. Cdc42 induces a conformational change in Par6, detectable by fluorescence resonance energy transfer spectroscopy. Nuclear magnetic resonance studies indicate that the semi-CRIB motif of Par6 is at least partially structured by the PDZ domain. The structure highlights a novel role for a PDZ domain as a structural scaffold (Garrard, 2003).

Par6 and cell polarity

How a neuron becomes polarized remains an as yet unanswered question. Selection of the future axon among neurites of a cultured hippocampal neuron requires the activity of growth factor receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI 3-kinase), as well as atypical protein kinase C (aPKC). The PI 3-kinase activity, highly localized to the tip of the newly specified axon of stage 3 neurons, is essential for the proper subcellular localization of mPar3, the mammalian homolog of C. elegans polarity protein Par3. Polarized distribution of not only mPar3 but also mPar6 is important for axon formation; ectopic expression of mPar6 or mPar3, or just the N terminus of mPar3, leaves neurons with no axon specified. Thus, neuronal polarity is likely to be controlled by the mPar3/mPar6/aPKC complex and the PI 3-kinase signaling pathway, both serving evolutionarily conserved roles in specifying cell polarity (Shi, 2003).

Meiotic maturation in mammals is characterized by two asymmetric divisions, leading to the formation of two polar bodies and the female gamete. Whereas the mouse oocyte is a polarized cell, molecules implicated in the establishment of this polarity are still unknown. PAR proteins have been demonstrated to play an important role in cell polarity in many cell types, where they control spindle positioning and asymmetric distribution of determinants. Two PAR6-related proteins have distinct polarized distributions in mouse oocytes. mPARD6a is first localized on the spindle and then accumulates at the pole nearest the cortex during spindle migration. In the absence of microtubules, the chromosomes still migrate to the cortex, and mPARD6a was found associated with the chromosomes and faces the cortex. mPARD6a is the first identified protein to associate with the spindle during spindle migration and to relocalize to the chromosomes in the absence of microtubule behavior, suggesting a role in spindle migration. The other protein, mPARD6b, was found on spindle microtubules until entry into meiosis II and relocalizes to the cortex at the animal pole during metaphase II arrest. mPARD6b is the first identified protein to localize to the animal pole of the mouse oocyte and likely contributes to the polarization of the cortex (Vinot, 2004).

Nucleotide exchange factor ECT2 interacts with the polarity protein complex Par6/Par3/protein kinase Czeta (PKCzeta) and regulates PKCzeta activity

Regulation of cell polarity is an important biological event that governs diverse cell functions such as localization of embryonic determinants and establishment of tissue and organ architecture. The Rho family GTPases and the polarity complex Par6/Par3/atypical protein kinase C (PKC) play a key role in the signaling pathway, but the molecules that regulate upstream signaling are still not known. The guanine nucleotide exchange factor ECT2 (Drosophila homolog: Peeble) has been identified as an activator of the polarity complex. ECT2 interacts with Par6 as well as Par3 and PKCzeta. Coexpression of Par6 and ECT2 efficiently activates Cdc42 in vivo. Overexpression of ECT2 also stimulates the PKCzeta activity, whereas dominant-negative ECT2 inhibits the increase in PKCzeta activity stimulated by Par6. ECT2 localization was detected at sites of cell-cell contact as well as in the nucleus of MDCK cells. The expression and localization of ECT2 are regulated by calcium, which is a critical regulator of cell-cell adhesion. Together, these results suggest that ECT2 regulates the polarity complex Par6/Par3/PKCzeta and possibly plays a role in epithelial cell polarity (Liu, 2004).

Mammalian Lgl forms a protein complex with PAR-6 and aPKC independently of PAR-3 to regulate epithelial cell polarity

Epithelial cells have apicobasal polarity and an asymmetric junctional complex that provides the bases for development and tissue maintenance. In both vertebrates and invertebrates, the evolutionarily conserved protein complex, PAR-6/aPKC/PAR-3, localizes to the subapical region and plays critical roles in the establishment of a junctional complex and cell polarity. In Drosophila, another set of proteins called tumor suppressors, such as Lgl, which localize separately to the basolateral membrane domain but genetically interact with the subapical proteins, also contribute to the establishment of cell polarity. However, how physically separated proteins interact remains to be clarified. Mammalian Lgl is shown to compete for PAR-3 in forming an independent complex with PAR-6/aPKC. During cell polarization, mLgl initially colocalizes with PAR-6/aPKC at the cell-cell contact region and is phosphorylated by aPKC, followed by segregation from apical PAR-6/aPKC to the basolateral membrane after cells are polarized. Overexpression studies establish that increased amounts of the mLgl/PAR-6/aPKC complex suppress the formation of epithelial junctions; this contrasts with a previous observation that the complex containing PAR-3 promotes it.These results indicate that PAR-6/aPKC selectively interacts with either mLgl or PAR-3 under the control of aPKC activity to regulate epithelial cell polarity (Yamanaka, 2003).

Thus evidence is presented showing that the PAR-6β/aPKCλ complex interacts with either mLgl or PAR-3 in a mutually exclusive manner, forming two independent protein complexes. Notably, overexpression of mLgl-2 inhibits TJ formation; this finding is in direct contrast with the data found for PAR-3, whose overexpression, but not that of its mutant lacking the aPKC binding region, promotes TJ formation. This suggests that the two independent complexes have distinct functions in the establishment of epithelial cell polarity. This is consistent with the results of genetic studies of Drosophila in which Lgl is required for formation of the basolateral membrane domain through the inhibition of the formation of apical identity, whereas subapical Bazooka (PAR-3) is required for the formation of the apical membrane domain (Yamanaka, 2003).

In polarized epithelial cells, mLgl localizes to the lateral region, in contrast to the PAR-6β/aPKCλ/PAR-3 complex that localizes to the apical end of the lateral domain. Interestingly, mLgl-2 transiently codistributes with PAR-6β and aPKCλ during the initial phase of epithelial cell polarization, whereas PAR-3 stably codistributes with them at the apical end of the cell-cell contact region; this finding indicates that the balance between the two independent complexes changes during the initial phase of epithelial cell polarization. Further, overexpression of aPKCλ kn (kinase deficient aPKC) results in the abnormal codistribution of PAR-6β and mLgl-2 at the cell periphery; this finding suggests that aPKCλ activity is required for the segregation of PAR-6β and mLgl-2 localization during this process. Thus, the present results, as well as previous findings, led to the following working model. The cell-cell contact initially stimulates the localization of the protein complex containing PAR-6β, aPKCλ, and mLgl at the cell-cell contact region. The complex is 'inactive' for TJ formation. Once aPKCλ is activated, mLgl segregates from the PAR-6β/aPKCλ complex. This triggers the formation of the 'active' PAR-6β/aPKCλ/PAR-3 complex that promotes the formation of the epithelial junctional complex. Segregated mLgl remains in the lateral region and seems to contribute to the establishment of the basolateral membrane identity, because mLgl-1 has been reported to interact with syntaxin-4, a component of the basolateral exocytic machinery. Although the mechanism for activation of aPKCλ remains to be clarified, Cdc42 and/or Rac1 are strong candidates as activators of aPKC in MDCK cells, since the GTP-bound form of Cdc42 activates aPKCλ kinase activity through PAR-6 in vitro and cell-cell adhesion activates Cdc42 and Rac1 in epithelial cells (Yamanaka, 2003).

mLgl is phosphorylated by aPKCλ and this phosphorylation increases in response to cell-cell adhesion-mediated cell polarization. Further, a phosphomimicking mutant of mLgl-2 (3SE) fails to bind to aPKCλ. These results imply that aPKCλ-dependent phosphorylation of mLgl is involved in the regulation of its interaction with the PAR-6β/aPKCλ complex. In contrast, no difference could be detected between mLgl-2 wild-type and its 3SE mutant in their interactions with the PDZ domain of PAR-6β. In addition, overexpression of mLgl-2 mutants (3SA or 3SE) affects TJ formation similarly to that of wild-type. These results suggest the existence of another mechanism regulating the interaction of mLgl-2 with PAR-6β. Mammalian Crumbs/Stardust (Pals1) has been shown to interact with the PDZ domain of PAR-6β and this interaction is enhanced by activated Cdc42. Taken together with the present results, this suggests that the Crumbs/Pals1 complex might also be involved in the regulation of the interaction between mLgl and the PAR-6β/aPKCλ complex; the PAR-6β/aPKCλ complex, together with PAR-3, may involve the Crumbs/Pals1 complex to promote TJ formation. Thus, the dissociation of mLgl from the PAR-6β/aPKCλ complex likely triggers the interaction of the PAR-6β/aPKCλ complex with the Crumbs/Pals1 complex in addition to its interaction with PAR-3. The functional interactions proposed by this model are consistent with the results of recent genetic studies of Drosophila in which Lgl and Crumbs compete with each other to define respective membrane identity (Yamanaka, 2003).

The findings in this study suggest a notable analogy in the mechanism regulating epithelial polarity between Drosophila and mammals. This notion is supported by a recent observation in Drosophila that Lgl interacts with PAR-6 and aPKC and regulates the apicobasal polarity of Drosophila neuroblasts. In mammalian fibroblasts, mLgl-1 has been reported to form a protein complex with PAR-6α (also called PAR-6C) and aPKC and is involved in the polarized migration of wounded MEF cells. Further studies will further an understanding of the molecular mechanism underlying the establishment of cell polarity in a variety of biological contexts (Yamanaka, 2003).

Thus mammalian Lgl competes for PAR-3 in forming an independent protein complex with PAR-6 and aPKC in epithelial cells. During epithelial cell polarization, mLgl transiently colocalizes with PAR-6 and aPKC at the cell-cell contact region, and increased localization of mLgl and PAR-6 to the cell-cell contact region suppresses TJ formation. This finding contrasts with the data found for PAR-3, which promotes TJ formation and thus indicates that the balance between the two independent protein complexes regulates the establishment of epithelial cell polarity. It is also suggested that aPKC activity-mediated phosphorylation of mLgl is involved in the regulation of mLgl's interaction with PAR-6/aPKC. These findings provide new insight into the mechanism underlying the establishment of epithelial cell polarity (Yamanaka, 2003).

The PAR-6 polarity protein regulates dendritic spine morphogenesis through p190 RhoGAP and the Rho GTPase

The majority of excitatory synaptic transmission in the brain occurs at dendritic spines, which are actin-rich protrusions on the dendrites. The asymmetric nature of these structures suggests that proteins regulating cell polarity might be involved in their formation. Indeed, the polarity protein PAR-3 is required for normal spine morphogenesis. However, this function is independent of association with atypical protein kinase C (aPKC) and PAR-6. This study shows that PAR-6 together with aPKC plays a distinct but essential role in spine morphogenesis. Knockdown of PAR-6 inhibits spine morphogenesis, whereas overexpression of PAR-6 increases spine density, and these effects are mediated by aPKC. Using a FRET biosensor, it was further shown that p190 RhoGAP and RhoA act downstream of the PAR-6/aPKC complex. These results define a role for PAR-6 and aPKC in dendritic spine biogenesis and maintenance, and reveal an unexpected link between the PAR-6/aPKC complex and RhoA activity (Zhang, 2008).

Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6

Collective cell migration occurs in a range of contexts: cancer cells frequently invade in cohorts while retaining cell-cell junctions. This study shows that collective invasion by cancer cells depends on decreasing actomyosin contractility at sites of cell-cell contact. When actomyosin is not downregulated at cell-cell contacts, migrating cells lose cohesion. A molecular mechanism is provided for this downregulation. Depletion of discoidin domain receptor 1 (DDR1) blocks collective cancer-cell invasion in a range of two-dimensional, three-dimensional and 'organotypic' models. DDR1 coordinates the Par3/Par6 cell-polarity complex through its carboxy terminus, binding PDZ domains in Par3 and Par6. The DDR1-Par3/Par6 complex controls the localization of RhoE to cell-cell contacts, where it antagonizes ROCK-driven actomyosin contractility. Depletion of DDR1, Par3, Par6 or RhoE leads to increased actomyosin contactility at cell-cell contacts, a loss of cell-cell cohesion and defective collective cell invasion (Hidalgo-Carcedo, 2011).

Collective movement requires the coordination of actomyosin organization between cells. Actomyosin contractility is high around the edge of the cell cluster and low between cells. At the margin of the group, actomyosin is organized in a supracellular structure analogous to the 'purse-string' observed in epithelial wound closure. Both the elevated actomyosin levels observed around the edges of groups of invading cancer cells and 'purse-string' wound closure are dependent on Cdc42. Force is transmitted between cells through cell-cell contacts near the edge of the group. However, if force is applied uniformly around the cell margin, the cell junctions become compromised and the coordination of movement between neighbouring cells fails. Consistent with this, contact inhibition of locomotion and cell-cell repulsion are associated with increased Rho-driven actomyosin contraction function after cell-cell contact. Therefore a mechanism is required to decrease actomyosin contractility at sites of cell-cell contact. DDR1 acts in a new non-collagen-binding capacity at cell-cell contacts. The localization of DDR1 to cell-cell contacts requires E-cadherin. Once localized at cell-cell contacts, DDR1 helps to recruit Par3 and Par6; these molecules are required for efficient collective invasion. Cell polarity regulators are required for optimal migration in two-dimensional scratch/wound assays, which have some aspects of a collective nature. Moreover, Par3 and Par6 are required for collective migration of border cells in the Drosophila embryo41. The DDR1-Par3/Par6 complex then controls the localization of RhoE. RhoE may be localized through the intermediary p190ARhoGAP, which can bind both Par6 and RhoE. Consistent with this, depletion of p190ARhoGAP gave a similar phenotype to that after DDR1 or Par3/Par6 depletion. Both RhoE and p190ARhoGAP can antagonize Rho-ROCK-mediated regulation of actomyosin. The Par3-dependent suppression of actomyosin that was observe reciprocates the suppression of Par3 function by the actomyosin regulator ROCK. It is likely that the reciprocal nature of these negative interactions serves to segregate Par3 and actomyosin robustly. The DDR1-dependent mechanism that is describe most probably acts together with other proteins to decrease Rho-ROCK function at cell-cell contacts, such as p120 catenin-dependent mechanisms. This analysis has not yet allowed determination of all the components of DDR1 complexes at cell-cell contacts (Hidalgo-Carcedo, 2011).

Various regulators of cell polarity become misregulated in cancer; this has been linked to increased metastasis. It is believed that disruption of DDR1-dependent Par3 localization to cell-cell contacts might be expected to favour blood-borne metastasis. The data do not exclude a positive role for DDR1 in metastasis as a collagen receptor. Indeed, it was found that interference with DDR1 function in metastatic MTLn3 cells decreased their ability to colonize lung tissue. DDR1 expression may therefore not correlate simply with metastatic ability, but it is important to consider whether it is acting in a cell-matrix or cell-cell adhesion context: in the former it may promote single-cell cancer invasion and processes such as lung colonization; in the latter it may only promote more local and lymphatic invasion and hinder haematogenous metastasis. It is likely that DDR1 engages in different molecular complexes depending on whether it is involved in cell-cell interactions or cell-matrix interactions. For example, the data suggest that DDR1 does not associate with myosin IIa at cell-cell contacts but it has been reported to associate with myosin IIa in other contexts (Hidalgo-Carcedo, 2011).

This study described a mechanism that is required to decrease actomyosin contractility at sites of cell-cell contact. DDR1 acts in a new non-collagen-binding capacity at cell-cell contacts. DDR1 helps to recruit Par3 and Par6; this complex then controls the localization of RhoE, which can antagonize Rho-ROCK-mediated regulation of actomyosin. Thus, DDR1 functions at cell-cell contacts to keep actomyosin activity at low levels. Without this decrease in actomyosin activity, cell cohesion cannot be maintained during collective cell migration (Hidalgo-Carcedo, 2011).

The tumor suppressor Lgl1 forms discrete complexes with NMII-A, and Par6α-aPKCζ that are affected by Lgl1 phosphorylation

Non-muscle myosin IIA (NMII-A) and the tumor suppressor Lgl1 play a central role in the polarization of migrating cells. Mammalian Lgl1 interacts directly with NMII-A, inhibiting its ability to assemble into filaments in vitro. Lgl1 also regulates the cellular localization of NMII-A, the maturation of focal adhesions and cell migration. In Drosophila, phosphorylation of Lgl affects its association with the cytoskeleton. This study shows that phosphorylation of mammalian Lgl1 by aPKCζ prevents its interaction with NMII-A both in vitro and in vivo, and affects its inhibition on NMII-A filament assembly. Phosphorylation of Lgl1 affects its cellular localization and is important for the cellular organization of the acto-NMII cytoskeleton. It was further shown that Lgl1 forms two distinct complexes in vivo, Lgl1-NMIIA and Lgl1-Par6α-aPKCζ, and that the complexes formation is affected by the phosphorylation state of Lgl1. The complex Lgl1-Par6α-aPKCζ resides in the leading edge of the cell. Finally, it was shown that aPKCzeta and NMII-A compete to bind directly to Lgl1 via the same domain. These results provide new insights into the mechanism regulating the interaction between Lgl1, NMII-A, Par6α, and aPKCζ in polarized migrating cells (Dahan, 2013).

A model is proposed for the role of Lgl1-NMIIA and Lgl1-Par6α-aPKCζ in establishing front-rear polarization in migrating cells. In migrating polarized cells Lgl1 resides at the cell’s leading edge in a complex with Par6α-aPKCζ, and it is this complex which defines the leading edge of the cell. In the lamellipodium Lgl1 binds to NMII-A but not to aPKCζ, inhibiting NMII-A filament assembly. These events allow the cell to polymerize F-actin to move the cell forward. According to this model Lgl1 is absent from the rear part of the cell, allowing NMII-A to assemble into filaments to enable cell retraction (Dahan, 2013).

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

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