Bazooka and PAR-6 are required with PAR-1 for the maintenance of oocyte fate in Drosophila

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 [1-4]. 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 [5, 6]. 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 [7, 8]. Here, we show that 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, as the localization of PAR-1 does not require Bazooka or PAR-6 in Drosophila, as it does in the worm.     

The Drosophila homolog of C. elegans PAR-1 organizes the oocyte cytoskeleton and directs oskar mRNA localization to the posterior pole

In C. elegans, the PAR-1 kinase is localized to the posterior of the zygote and is required for anterior-posterior axis formation. Here, we report that a Drosophila PAR-1 homolog localizes to the posterior of the oocyte with oskar mRNA. Furthermore, par-1 mutants show a novel polarity phenotype in which bicoid mRNA accumulates normally at the anterior, but oskar mRNA is redirected to the center of the oocyte, resulting in embryonic patterning defects. These phenotypes arise from a disorganization of the oocyte microtubule cytoskeleton, consistent with reports that mammalian PAR-1 homologs regulate microtubule dynamics. Thus, Drosophila PAR-1 may remodel the oocyte microtubule network to define the posterior as the site for oskar localization. These results identify a molecular parallel between anterior-posterior polarization in Drosophila and C. elegans.     

Drosophila PAR-1 and 14-3-3 inhibit Bazooka/PAR-3 to establish complementary cortical domains in polarized cells

PAR-1 kinases are required for polarity in diverse cell types, such as epithelial cells, where they localize laterally. PAR-1 activity is believed to be transduced by binding of 14-3-3 proteins to its phosphorylated substrates, but the relevant targets are unknown. We show that PAR-1 phosphorylates Bazooka/PAR-3 on two conserved serines to generate 14-3-3 binding sites. This inhibits formation of the Bazooka/PAR-6/aPKC complex by blocking Bazooka oligomerization and binding to aPKC. In epithelia, this complex localizes apically and defines the apical membrane, whereas Bazooka lacking PAR-1 phosphorylation/14-3-3 binding sites forms ectopic lateral complexes. Lateral exclusion by PAR-1/14-3-3 cooperates with apical anchoring by Crumbs/Stardust to restrict Bazooka localization, and loss of both pathways disrupts epithelial polarity. PAR-1 also excludes Bazooka from the posterior of the oocyte, and disruption of this regulation causes anterior-posterior polarity defects. Thus, antagonism of Bazooka by PAR-1/14-3-3 may represent a general mechanism for establishing complementary cortical domains in polarized cells.     

Drosophila anterior-posterior polarity requires actin-dependent PAR-1 recruitment to the oocyte posterior

The Drosophila anterior-posterior axis is established at stage 7 of oogenesis when the posterior follicle cells signal to polarize the oocyte microtubule cytoskeleton. This requires the conserved PAR-1 kinase, which can be detected at the posterior of the oocyte in immunostainings from stage 9. However, this localization depends on Oskar localization, which requires the earlier PAR-1-dependent microtubule reorganization, indicating that Oskar-associated PAR-1 cannot establish oocyte polarity. Here we analyze the function of the different PAR-1 isoforms and find that only PAR-1 N1 isoforms can completely rescue the oocyte polarity phenotype. Furthermore, PAR-1 N1 is recruited to the posterior cortex of the oocyte at stage 7 in response to the polarizing follicle cell signal, and this requires actin, but not microtubules. This suggests that posterior PAR-1 N1 polarizes the microtubule cytoskeleton. PAR-1 N1 localization is mediated by a cortical targeting domain and a conserved anterior-lateral exclusion signal in its C-terminal linker domain. PAR-1 is also required for the polarization of the C. elegans zygote and is recruited to the posterior cortex in an actin-dependent manner. Our results therefore identify a molecular parallel between axis formation in Drosophila and C. elegans and make Drosophila PAR-1 N1 the earliest known marker for the polarization of the oocyte.     

The Caenorhabditis elegans par-5 gene encodes a 14-3-3 protein required for cellular asymmetry in the early embryo.

The establishment of anterior-posterior polarity in the Caenorhabditis elegans embryo requires the activity of the maternally expressed par genes. We report the identification and analysis of a new par gene, par-5. We show that par-5 is required for asynchrony and asymmetry in the first embryonic cell divisions, normal pseudocleavage, normal cleavage spindle orientation at the two-cell stage, and localization of P granules and MEX-5 during the first and subsequent cell cycles. Furthermore, par-5 activity is required in the first cell cycle for the asymmetric cortical localization of PAR-1 and PAR-2 to the posterior, and PAR-3, PAR-6, and PKC-3 to the anterior. When PAR-5 is reduced by mutation or by RNA interference, these proteins spread around the cortex of the one-cell embryo and partially overlap. We have shown by sequence analysis of par-5 mutants and by RNA interference that the par-5 gene is the same as the ftt-1 gene, and encodes a 14-3-3 protein. The PAR-5 14-3-3 protein is present in gonads, oocytes, and early embryos, but is not asymmetrically distributed. Our analysis indicates that the par-5 14-3-3 gene plays a crucial role in the early events leading to polarization of the C. elegans zygote.     

CDC-42 controls early cell polarity and spindle orientation in C. elegans.

BACKGROUND: 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. RESULTS: Here, we show that 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. We further show that C. elegans CDC-42 physically interacts with PAR-6 in a yeast two-hybrid system, consistent with data on the interaction of human homologs. CONCLUSIONS: Our results show that CDC-42 acts in concert with the PAR proteins to control the polarity of the C. elegans embryo, and provide evidence that the interaction of CDC-42 and the PAR-3/PAR-6/PKC-3 complex has been evolutionarily conserved as a functional unit.     

The C. elegans homolog of Drosophila Lethal giant larvae functions redundantly with PAR-2 to maintain polarity in the early embryo

Polarity is essential for generating cell diversity. The one-cell C. elegans embryo serves as a model for studying the establishment and maintenance of polarity. In the early embryo, a myosin II-dependent contraction of the cortical meshwork asymmetrically distributes the highly conserved PDZ proteins PAR-3 and PAR-6, as well as an atypical protein kinase C (PKC-3), to the anterior. The RING-finger protein PAR-2 becomes enriched on the posterior cortex and prevents these three proteins from returning to the posterior. In addition to the PAR proteins, other proteins are required for polarity in many metazoans. One example is the conserved Drosophila tumor-suppressor protein Lethal giant larvae (Lgl). In Drosophila and mammals, Lgl contributes to the maintenance of cell polarity and plays a role in asymmetric cell division. We have found that the C. elegans homolog of Lgl, LGL-1, has a role in polarity but is not essential. It localizes asymmetrically to the posterior of the early embryo in a PKC-3-dependent manner, and functions redundantly with PAR-2 to maintain polarity. Furthermore, overexpression of LGL-1 is sufficient to rescue loss of PAR-2 function. LGL-1 negatively regulates the accumulation of myosin (NMY-2) on the posterior cortex, representing a possible mechanism by which LGL-1 might contribute to polarity maintenance.     

A conserved oligomerization domain in drosophila Bazooka/PAR-3 is important for apical localization and epithelial polarity

The PAR-3/PAR-6/aPKC complex is required to establish polarity in many different cell types, including the C. elegans zygote and epithelial and neuronal cells in Drosophila and mammals. In each context, the components of this complex display a mutually dependent asymmetric cortical localization. PAR-6 is a direct effector of Rho family GTPases and binds to and regulates aPKC. Mammalian PAR-3 (mPar3) can associate with transmembrane proteins and may link the complex to the membrane, but this can account for only part of the requirement for this protein in the complex. Here we investigate the function of a novel conserved domain, CR1, of PAR-3 using computational, biochemical, and genetic approaches. Sequence-structure comparison by FUGUE predicts that CR1 has the same structural fold as a bacterial oligomerization domain. We show that CR1 of the Drosophila homolog, Bazooka (BAZ), mediates oligomerization in vitro and in vivo. Furthermore, deletion of CR1 disrupts BAZ localization in both epithelial cells and the germline and strongly impairs BAZ function in epithelial polarity. These results indicate that this domain is important for the localization and activity of the PAR-3/PAR6/aPKC complex and define a new role for PAR-3 in assembling higher order protein complexes.     

MEX-5 asymmetry in one-cell C. elegans embryos requires PAR-4- and PAR-1-dependent phosphorylation

Anteroposterior polarity in early C. elegans embryos is required for the specification of somatic and germline lineages, and is initiated by a sperm-induced reorganization of the cortical cytoskeleton and PAR polarity proteins. Through mechanisms that are not understood, the kinases PAR-1 and PAR-4, and other PAR proteins cause the cytoplasmic zinc finger protein MEX-5 to accumulate asymmetrically in the anterior half of the one-cell embryo. We show that MEX-5 asymmetry requires neither vectorial transport to the anterior, nor protein degradation in the posterior. MEX-5 has a restricted mobility before fertilization and in the anterior of one-cell embryos. However, MEX-5 mobility in the posterior increases as asymmetry develops, presumably allowing accumulation in the anterior. The MEX-5 zinc fingers and a small, C-terminal domain are essential for asymmetry; the zinc fingers restrict MEX-5 mobility, and the C-terminal domain is required for the increase in posterior mobility. We show that a crucial residue in the C-terminus, Ser 458, is phosphorylated in vivo. PAR-1 and PAR-4 kinase activities are required for the phosphorylation of S458, providing a link between PAR polarity proteins and the cytoplasmic asymmetry of MEX-5.     

C. elegans PAR-3 and PAR-6 are required for apicobasal asymmetries associated with cell adhesion and gastrulation

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 we generated hybrid PAR proteins that are targeted for degradation after the 1-cell stage. Embryos containing the hybrid PAR proteins had normal anterior-posterior polarity, but showed defects in apicobasal asymmetries associated with gastrulation. Ectopic separations appeared between lateral surfaces of cells that are normally tightly adherent, cells that ingress during gastrulation failed to accumulate nonmuscle myosin at their apical surfaces and ingression was slowed. Thus, PAR proteins function in both apicobasal and anterior-posterior asymmetry during the first few cell cycles of embryogenesis.     

Binding to PKC-3, but not to PAR-3 or to a conventional PDZ domain ligand, is required for PAR-6 function in C. elegans

PAR-6 is a conserved protein important for establishment and maintenance of cell polarity in a variety of metazoans. PAR-6 proteins function together with PAR-3, aPKC and CDC-42. Mechanistic details of their interactions, however, are not fully understood. We studied the biochemical interactions between C. elegans PAR-6 and its binding partners and tested the requirements of these interactions in living worms. We show that PB1 domain-mediated binding of PAR-6 to PKC-3 is necessary for polarity establishment and PAR-6 cortical localization in C. elegans embryos. We also show that binding of PAR-6 and PAR-3 is mediated in vitro by a novel type of PDZ-PDZ interaction; the βC strand of PAR-6 PDZ binds the βD strand of PAR-3 PDZ1. However, this interaction is dispensable in vivo for PAR-6 function throughout the life of C. elegans. Mutations that specifically abolish conventional ligand binding to the PAR-6 PDZ domain also failed to affect PAR-6 function in vivo. We conclude that PAR-6 binding to PKC-3, but not to PAR-3 nor to a conventional PDZ ligand, is required for PAR-6 cortical localization and function in C. elegans.     

Distinct PAR-1 proteins function in different branches of Wnt signaling during vertebrate development

The kinase PAR-1 plays conserved roles in cell polarity. PAR-1 has also been implicated in axis establishment in C. elegans and Drosophila and in Wnt signaling, but its role in vertebrate development is unclear. Here we report that PAR-1 has two distinct and essential roles in axial development in Xenopus mediated by different PAR-1 isoforms. Depletion of PAR-1A or PAR-1BX causes dorsoanterior deficits, reduced Spemann organizer gene expression, and inhibition of canonical Wnt-beta-catenin signaling. By contrast, PAR-1BY depletion inhibits cell movements and localization of Dishevelled protein to the cell cortex, processes associated with noncanonical Wnt signaling. PAR-1 phosphorylation sites in Dishevelled are required for this translocation, but not for canonical Wnt signaling. We conclude that PAR-1BY is required in the PCP branch and mediates Dsh membrane localization while PAR-1A and PAR-1BX are essential for canonical signaling to beta-catenin, possibly via targets other than Dishevelled.     

aPKC acts upstream of PAR-1b in both the establishment and maintenance of mammalian epithelial polarity

BACKGROUND: aPKC and PAR-1 are required for cell polarity in various contexts. In mammalian epithelial cells, aPKC localizes at tight junctions (TJs) and plays an indispensable role in the development of asymmetric intercellular junctions essential for the establishment and maintenance of apicobasal polarity. On the other hand, one of the mammalian PAR-1 kinases, PAR-1b/EMK1/MARK2, localizes to the lateral membrane in a complimentary manner with aPKC, but little is known about its role in apicobasal polarity of epithelial cells as well as its functional relationship with aPKC. RESULTS: We demonstrate that PAR-1b is essential for the asymmetric development of membrane domains of polarized MDCK cells. Nonetheless, it is not required for the junctional localization of aPKC nor the formation of TJs, suggesting that PAR-1b works downstream of aPKC during epithelial cell polarization. On the other hand, aPKC phosphorylates threonine 595 of PAR-1b and enhances its binding with 14-3-3/PAR-5. In polarized MDCK cells, T595 phosphorylation and 14-3-3 binding are observed only in the soluble form of PAR-1b, and okadaic acid treatment induces T595-dependent dissociation of PAR-1b from the lateral membrane. Furthermore, T595A mutation induces not only PAR-1b leakage into the apical membrane, but also abnormal development of membrane domains. These results suggest that in polarized epithelial cells, aPKC phosphorylates PAR-1b at TJs, and in cooperation with 14-3-3, promotes the dissociation of PAR-1b from the lateral membrane to regulate PAR-1b activity for the membrane domain development. CONCLUSIONS: These results suggest that mammalian aPKC functions upstream of PAR-1b in both the establishment and maintenance of epithelial cell polarity.     

PAR-3 is required for epithelial cell polarity in the distal spermatheca of C. elegans

PAR-3 is localized asymmetrically in epithelial cells in a variety of animals from Caenorhabditis elegans to mammals. Although C. elegans PAR-3 is known to act in early blastomeres to polarize the embryo, a role for PAR-3 in epithelial cells of C. elegans has not been established. Using RNA interference to deplete PAR-3 in developing larvae, we discovered a requirement for PAR-3 in spermathecal development. Spermathecal precursor cells are born during larval development and differentiate into an epithelium that forms a tube for the storage of sperm. Eggs must enter the spermatheca to complete ovulation. PAR-3-depleted worms exhibit defects in ovulation. Consistent with this phenotype, PAR-3 is transiently expressed and localized asymmetrically in the developing somatic gonad, including the spermathecal precursor cells of L4 larvae. We found that the defect in ovulation can be partially suppressed by a mutation in IPP-5, an inositol polyphosphate 5-phosphatase, indicating that one effect of PAR-3 depletion is disruption of signaling between oocyte and spermatheca. Microscopy revealed that the distribution of AJM-1, an apical junction marker, and apical microfilaments are severely affected in the distal spermatheca of PAR-3-depleted worms. We propose that PAR-3 activity is required for the proper polarization of spermathecal cells and that defective ovulation results from defective distal spermathecal development.     

Depletion of the co-chaperone CDC-37 reveals two modes of PAR-6 cortical association in C. elegans embryos

PAR proteins play roles in the establishment and maintenance of polarity in many different cell types in metazoans. In C. elegans, polarity established in the one-cell embryo determines the anteroposterior axis of the developing animal and is essential to set the identities of the early blastomeres. PAR-1 and PAR-2 colocalize at the posterior cortex of the embryo. PAR-3, PAR-6 and PKC-3 (aPKC) colocalize at the anterior cortex of the embryo. A process of mutual exclusion maintains the anterior and posterior protein domains. We present results indicating that a homolog of the Hsp90 co-chaperone Cdc37 plays a role in dynamic interactions among the PAR proteins. We show that CDC-37 is required for the establishment phase of embryonic polarity; that CDC-37 reduction allows PAR-3-independent cortical accumulation of PAR-6 and PKC-3; and that CDC-37 is required for the mutual exclusion of the anterior and posterior group PAR proteins. Our results indicate that CDC-37 acts in part by maintaining PKC-3 levels and in part by influencing the activity or levels of other client proteins. Loss of the activities of these client proteins reveals that there are two sites for PAR-6 cortical association, one dependent on CDC-42 and not associated with PAR-3, and the other independent of CDC-42 and co-localizing with PAR-3. We propose that, in wild-type embryos, CDC-37-mediated inhibition of the CDC-42-dependent binding site and PAR-3-mediated release of this inhibition provide a key mechanism for the anterior accumulation of PAR-6.     

Patterned gene expression directs bipolar planar polarity in Drosophila

During convergent extension in Drosophila, polarized cell movements cause the germband to narrow along the dorsal-ventral (D-V) axis and more than double in length along the anterior-posterior (A-P) axis. This tissue remodeling requires the correct patterning of gene expression along the A-P axis, perpendicular to the direction of cell movement. Here, we demonstrate that A-P patterning information results in the polarized localization of cortical proteins in intercalating cells. In particular, cell fate differences conferred by striped expression of the even-skipped and runt pair-rule genes are both necessary and sufficient to orient planar polarity. This polarity consists of an enrichment of nonmuscle myosin II at A-P cell borders and Bazooka/PAR-3 protein at the reciprocal D-V cell borders. Moreover, bazooka mutants are defective for germband extension. These results indicate that spatial patterns of gene expression coordinate planar polarity across a multicellular population through the localized distribution of proteins required for cell movement.     

The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM).

The establishment and maintenance of cellular polarity are critical for the development of multicellular organisms. PAR (partitioning-defective) proteins were identified in Caenorhabditis elegans as determinants of asymmetric cell division and polarized cell growth. Recently, vertebrate orthologues of two of these proteins, ASIP/PAR-3 and PAR-6, were found to form a signalling complex with the small GTPases Cdc42/Rac1 and with atypical protein kinase C (PKC). Here we show that ASIP/PAR-3 associates with the tight-junction-associated protein junctional adhesion molecule (JAM) in vitro and in vivo. No binding was observed with claudin-1, -4 or -5. In fibroblasts and CHO cells overexpressing JAM, endogenous ASIP is recruited to JAM at sites of cell-cell contact. Over expression of truncated JAM lacking the extracellular part disrupts ASIP/PAR-3 localization at intercellular junctions and delays ASIP/PAR-3 recruitment to newly formed cell junctions. During junction formation, JAM appears early in primordial forms of junctions. Our data suggest that the ASIP/PAR-3-aPKC complex is tethered to tight junctions via its association with JAM, indicating a potential role for JAM in the generation of cell polarity in epithelial cells.     

PAR-3 and PAR-1 inhibit LET-99 localization to generate a cortical band important for spindle positioning in Caenorhabditis elegans embryos

The conserved PAR proteins are localized in asymmetric cortical domains and are required for the polarized localization of cell fate determinants in many organisms. In Caenorhabditis elegans embryos, LET-99 and G protein signaling act downstream of the PARs to regulate spindle positioning and ensure asymmetric division. PAR-3 and PAR-2 localize LET-99 to a posterior cortical band through an unknown mechanism. Here we report that LET-99 asymmetry depends on cortically localized PAR-1 and PAR-4 but not on cytoplasmic polarity effectors. In par-1 and par-4 embryos, LET-99 accumulates at the entire posterior cortex, but remains at low levels at the anterior cortex occupied by PAR-3. Further, PAR-3 and PAR-1 have graded cortical distributions with the highest levels at the anterior and posterior poles, respectively, and the lowest levels of these proteins correlate with high LET-99 accumulation. These results suggest that PAR-3 and PAR-1 inhibit the localization of LET-99 to generate a band pattern. In addition, PAR-1 kinase activity is required for the inhibition of LET-99 localization, and PAR-1 associates with LET-99. Finally, examination of par-1 embryos suggests that the banded pattern of LET-99 is critical for normal posterior spindle displacement and to prevent spindle misorientation caused by cell shape constraints.     

Different domains of C. elegans PAR-3 are required at different times in development

Polarity is a fundamental cellular feature that is critical for generating cell diversity and maintaining organ functions during development. In C. elegans, the one-cell embryo is polarized via asymmetric localization of the PAR proteins, which in turn are required to establish the future anterior-posterior axis of the embryo. PAR-3, a conserved PDZ domain-containing protein, acts with PAR-6 and PKC-3 (atypical protein kinase; aPKC) to regulate cell polarity and junction formation in a variety of cell types. To understand how PAR-3 localizes and functions during C. elegans development, we produced targeted mutations and deletions of conserved domains of PAR-3 and examined the localization and function of the GFP-tagged proteins in C. elegans embryos and larvae. We find that CR1, the PAR-3 self-oligomerization domain, is required for PAR-3 cortical distribution and function only during early embryogenesis and that PDZ2 is required for PAR-3 to accumulate stably at the cell periphery in early embryos and at the apical surface in pharyngeal and intestinal epithelial cells. We also show that phosphorylation at S863 by PKC-3 is not essential in early embryogenesis, but is important in later development. Surprisingly neither PDZ1 nor PDZ3 are essential for localization or function. Our results indicate that the different domains and phosphorylated forms of PAR-3 can have different roles during C. elegans development.