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.     

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

BACKGROUND: 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. RESULTS: We show that mammalian Lgl competes 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 the previous observation that the complex containing PAR-3 promotes it. CONCLUSIONS: 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.     

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.     

The Drosophila PAR-1 spacer domain is required for lateral membrane association and for polarization of follicular epithelial cells

The Ser/Thr kinases of the PAR-1/MARK/Kin1 family are conserved regulators of polarity in epithelial and non-epithelial cells . Drosophila PAR-1 localizes laterally in the follicular epithelium of the ovary , where it has been shown to function at two distinct levels: It stabilizes the cytoskeleton and it regulates apical-basal polarity by directly inhibiting lateral assembly of the apical aPKC/Bazooka/PAR-6 complex . However, it has been unclear how lateral localization of Drosophila PAR-1 is achieved and whether this localization contributes to epithelial polarity in vivo. Here we show that, through its spacer domain, Drosophila PAR-1 accumulates on the lateral plasma membrane (PM) in cells of the follicular epithelium (FE). Rescue experiments indicate that in FE cells PAR-1 kinase activity is essential for all the described functions of PAR-1. In contrast, the spacer domain of PAR-1 is required for apical-basal polarity and growth control but is dispensable for microtubule (MT) stabilization. Our data indicate that the spacer domain of PAR-1 is required for lateral PM localization of PAR-1 kinase and for development of a polarized FE.     

The positioning and segregation of apical cues during epithelial polarity establishment in Drosophila

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

PAR-6 is required for junction formation but not apicobasal polarization in C. elegans embryonic epithelial cells

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. Here, we use a targeted protein degradation strategy to remove both maternal and zygotic PAR-6 from C. elegans embryos before epithelial cells are born. We find 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.     

Role of the PAR-3-KIF3 complex in the establishment of neuronal polarity

Neurons polarize to form elaborate multiple dendrites and one long axon. The establishment and maintenance of axon/dendrite polarity are fundamentally important for neurons. Recent studies have demonstrated that the polarity complex PAR-3-PAR-6-atypical protein kinase C (aPKC) is involved in polarity determination in many tissues and cells. The function of the PAR-3-PAR-6-aPKC protein complex depends on its subcellular localization in polarized cells. PAR-3 accumulates at the tip of growing axons in cultured rat hippocampal neurons, but the molecular mechanism of this localization remains unknown. Here we identify a direct interaction between PAR-3 and KIF3A, a plus-end-directed microtubule motor protein, and show that aPKC can associate with KIF3A through its interaction with PAR-3. The expression of dominant-negative PAR-3 and KIF3A fragments that disrupt PAR-3-KIF3A binding inhibited the accumulation of PAR-3 and aPKC at the tip of the neurites and abolished neuronal polarity. These results suggest that PAR-3 is transported to the distal tip of the axon by KIF3A and that the proper localization of PAR-3 is required to establish neuronal polarity.     

Meiotic maturation induces animal-vegetal asymmetric distribution of aPKC and ASIP/PAR-3 in Xenopus oocytes

The asymmetric distribution of cellular components is an important clue for understanding cell fate decision during embryonic patterning and cell functioning after differentiation. In C. elegans embryos, PAR-3 and aPKC form a complex that colocalizes to the anterior periphery of the one-cell embryo, and are indispensable for anterior-posterior polarity that is formed prior to asymmetric cell division. In mammals, ASIP (PAR-3 homologue) and aPKCgamma form a complex and colocalize to the epithelial tight junctions, which play critical roles in epithelial cell polarity. Although the mechanism by which PAR-3/ASIP and aPKC regulate cell polarization remains to be clarified, evolutionary conservation of the PAR-3/ASIP-aPKC complex suggests their general role in cell polarity organization. Here, we show the presence of the protein complex in Xenopus laevis. In epithelial cells, XASIP and XaPKC colocalize to the cell-cell contact region. To our surprise, they also colocalize to the animal hemisphere of mature oocytes, whereas they localize uniformly in immature oocytes. Moreover, hormonal stimulation of immature oocytes results in a change in the distribution of XaPKC 2-3 hours after the completion of germinal vesicle breakdown, which requires the kinase activity of aPKC. These results suggest that meiotic maturation induces the animal-vegetal asymmetry of aPKC.     

Rho-kinase phosphorylates PAR-3 and disrupts PAR complex formation

A polarity complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events. PAR-3 directly interacts with Tiam1/Taim2 (STEF), Rac1-specific guanine nucleotide exchange factors, and forms a complex with aPKC-PAR-6-Cdc42*GTP, leading to Rac1 activation. RhoA antagonizes Rac1 in certain types of cells. However, the relationship between RhoA and the PAR complex remains elusive. We found here that Rho-kinase/ROCK/ROK, the effector of RhoA, phosphorylated PAR-3 at Thr833 and thereby disrupted its interaction with aPKC and PAR-6, but not with Tiam2. Phosphorylated PAR-3 was observed in the leading edge, and in central and rear portions of migrating cells having front-rear polarity. Knockdown of PAR-3 by small interfering RNA (siRNA) impaired cell migration, front-rear polarization, and PAR-3-mediated Rac1 activation, which were recovered with siRNA-resistant PAR-3, but not with the phospho-mimic PAR-3 mutant. We propose that RhoA/Rho-kinase inhibits PAR complex formation through PAR-3 phosphorylation, resulting in Rac1 inactivation.     

Drosophila 14-3-3/PAR-5 is an essential mediator of PAR-1 function in axis formation

PAR-1 kinases are required to determine the anterior-posterior (A-P) axis in C. elegans and Drosophila, but little is known about their molecular function. We identified 14-3-3 proteins as Drosophila PAR-1 interactors and show that PAR-1 binds a domain of 14-3-3 distinct from the phosphoserine binding pocket. PAR-1 kinases phosphorylate proteins to generate 14-3-3 binding sites and may therefore directly deliver 14-3-3 to these targets. 14-3-3 mutants display identical phenotypes to par-1 mutants in oocyte determination and the polarization of the A-P axis. Together, these results indicate that PAR-1's function is mediated by the binding of 14-3-3 to its substrates. The C. elegans 14-3-3 protein, PAR-5, is also required for A-P polarization, suggesting that this is a conserved mechanism by which PAR-1 establishes cellular asymmetries.     

Direct association of Bazooka/PAR-3 with the lipid phosphatase PTEN reveals a link between the PAR/aPKC complex and phosphoinositide signaling

Cell polarity in Drosophila epithelia, oocytes and neuroblasts is controlled by the evolutionarily conserved PAR/aPKC complex, which consists of the serine-threonine protein kinase aPKC and the PDZ-domain proteins Bazooka (Baz) and PAR-6. The PAR/aPKC complex is required for the separation of apical and basolateral plasma membrane domains, for the asymmetric localization of cell fate determinants and for the proper orientation of the mitotic spindle. How the complex exerts these different functions is not known. We show that the lipid phosphatase PTEN directly binds to Baz in vitro and in vivo, and colocalizes with Baz in the apical cortex of epithelia and neuroblasts. PTEN is an important regulator of phosphoinositide turnover that antagonizes the activity of PI3-kinase. We show that Pten mutant ovaries and embryos lacking maternal and zygotic Pten function display phenotypes consistent with a function for PTEN in the organization of the actin cytoskeleton. In freshly laid eggs, the germ plasm determinants oskar mRNA and Vasa are not localized properly to the posterior cytocortex and pole cells do not form. In addition, the actin-dependent posterior movement of nuclei during early cleavage divisions does not occur and the synchrony of nuclear divisions at syncytial blastoderm stages is lost. Pten mutant embryos also show severe defects during cellularization. Our data provide evidence for a link between the PAR/aPKC complex, the actin cytoskeleton and PI3-kinase signaling mediated by PTEN.     

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.     

Phosphorylation-dependent binding of 14-3-3 to the polarity protein Par3 regulates cell polarity in mammalian epithelia

The mammalian homologs of the C. elegans partitioning-defective (Par) proteins have been demonstrated to be necessary for establishment of cell polarity. In mammalian epithelia, the Par3/Par6/aPKC polarity complex is localized to the tight junction and regulates its formation and positioning with respect to basolateral and apical membrane domains. Here we demonstrate a previously undescribed phosphorylation-dependent interaction between a mammalian homolog of the C. elegans polarity protein Par5, 14-3-3, and the tight junction-associated protein Par3. We identify phosphorylated serine 144 as a site of 14-3-3 binding. Expression of a Par3 mutant that contains serine 144 mutated to alanine (S144A) results in defects in epithelial cell polarity. In addition, overexpression of 14-3-3zeta results in a severe disruption of polarity, whereas overexpression of a 14-3-3 mutant that is defective in binding to phosphoproteins has no effect on cell polarity. Together, these data suggest a novel, phosphorylation-dependent mechanism that regulates the function of the Par3/Par6/aPKC polarity complex through 14-3-3 binding.     

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

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

PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiam1

A polarity complex of PAR-3, PAR-6 and atypical protein kinase C (aPKC) functions in various cell-polarization events, including neuron specification. The small GTPase Cdc42 binds to PAR-6 and regulates cell polarity. However, little is known about the downstream signals of the Cdc42-PAR protein complex. Here, we found that PAR-3 directly interacted with STEF/Tiam1, which are Rac-specific guanine nucleotide-exchange factors, and that STEF formed a complex with PAR-3-aPKC-PAR-6-Cdc42-GTP. Cdc42 induces lamellipodia in a Rac-dependent manner in N1E-115 neuroblastoma cells. Disruption of Cdc42-PAR-6 or PAR-3-STEF binding inhibited Cdc42-induced lamellipodia but not filopodia. The isolated STEF-binding PAR-3 fragment was sufficient to induce lamellipodia independently of Cdc42 and PAR-6. PAR-3 is required for Cdc42-induced Rac activation, but is not essential for lamellipodia formation itself. In cultured hippocampal neurons, STEF accumulated at the tip of the growing axon and colocalized with PAR-3. The spatio-temporal activation and signalling of Cdc42-PAR-6-PAR-3-STEF/Tiam1-Rac seem to be involved in neurite growth and axon specification. We propose that the PAR-6-PAR-3 complex mediates Cdc42-induced Rac activation by means of STEF/Tiam1, and that this process seems to be required for the establishment of neuronal polarity.     

Rap1 and Canoe/afadin are essential for establishment of apical–basal polarity in the Drosophila embryo

The establishment and maintenance of apical–basal cell polarity is critical for assembling epithelia and maintaining organ architecture. Drosophila embryos provide a superb model. In the current view, apically positioned Bazooka/Par3 is the initial polarity cue as cells form during cellularization. Bazooka then helps to position both adherens junctions and atypical protein kinase C (aPKC). Although a polarized cytoskeleton is critical for Bazooka positioning, proteins mediating this remained unknown. We found that the small GTPase Rap1 and the actin-junctional linker Canoe/afadin are essential for polarity establishment, as both adherens junctions and Bazooka are mispositioned in their absence. Rap1 and Canoe do not simply organize the cytoskeleton, as actin and microtubules become properly polarized in their absence. Canoe can recruit Bazooka when ectopically expressed, but they do not obligatorily colocalize. Rap1 and Canoe play continuing roles in Bazooka localization during gastrulation, but other polarity cues partially restore apical Bazooka in the absence of Rap1 or Canoe. We next tested the current linear model for polarity establishment. Both Bazooka and aPKC regulate Canoe localization despite being “downstream” of Canoe. Further, Rap1, Bazooka, and aPKC, but not Canoe, regulate columnar cell shape. These data reshape our view, suggesting that polarity establishment is regulated by a protein network rather than a linear pathway.     

Similar requirements for CDC-42 and the PAR-3/PAR-6/PKC-3 complex in diverse cell types

During animal development, a complex of Par3, Par6 and atypical protein kinase C (aPKC) plays a central role in cell polarisation. The small G protein Cdc42 also functions in cell polarity and has been shown in some cases to act by regulating the Par3 complex. However, it is not yet known whether Cdc42 and the Par3 complex widely function together in development or whether they have independent functions. For example, many studies have implicated Cdc42 in cell migrations, but the Par3 complex has only been little studied, with conflicting results. Here we examine the requirements for CDC-42 and the PAR-3/PAR-6/PKC-3 complex in a range of different developmental events. We found similar requirements in all tissues examined, including polarised growth of vulval precursors and seam cells, migrations of neuroblasts and axons, and the development of the somatic gonad. We also propose a novel role for primordial germ cells in mediating coalescence of the Caenorhabditis elegans gonad. These results indicate that CDC-42 and the PAR-3/PAR-6/aPKC complex function together in diverse cell types.     

Caenorhabditis elegans screen reveals role of PAR-5 in RAB-11-recycling endosome positioning and apicobasal cell polarity

Apically enriched Rab11-positive recycling endosomes (Rab11-REs) are important for establishing and maintaining epithelial polarity. Yet, little is known about the molecules controlling trafficking of Rab11-REs in an epithelium in vivo. Here, we report a genome-wide, image-based RNA interference screen for regulators of Rab11-RE positioning and transport of an apical membrane protein (PEPT-1) in C. elegans intestine. Among the 356 screen hits was the 14-3-3 and partitioning defective protein PAR-5, which we found to be specifically required for Rab11-RE positioning and apicobasal polarity maintenance. Depletion of PAR-5 induced abnormal clustering of Rab11-REs to ectopic sites at the basolateral cortex containing F-actin and other apical domain components. This phenotype required key regulators of F-actin dynamics and polarity, such as Rho GTPases (RHO-1 and the Rac1 orthologue CED-10) and apical PAR?proteins. Our data suggest that PAR-5 acts as a regulatory hub for a polarity-maintaining network required for apicobasal asymmetry of F-actin and proper Rab11-RE positioning.     

Self-association of PAR-3-mediated by the conserved N-terminal domain contributes to the development of epithelial tight junctions

PAR-3 is a scaffold-like PDZ-containing protein that forms a complex with PAR-6 and atypical protein kinase C (PAR-3-atypical protein kinase C-PAR-6 complex) and contributes to the establishment of cell polarity in a wide variety of biological contexts. In mammalian epithelial cells, it localizes to tight junctions, the most apical end of epithelial cell-cell junctions, and contributes to the formation of functional tight junctions. However, the mechanism by which PAR-3 localizes to tight junctions and contributes to their formation remains to be clarified. Here we show that the N-terminal conserved region, CR1-(1-86), and the sequence 937-1,024 are required for its recruitment to the most apical side of the cell-cell contact region in epithelial Madin-Darby canine kidney cells. We also show that CR1 self-associates to form an oligomeric complex in vivo and in vitro. Further, overexpression of CR1 in Madin-Darby canine kidney cells disturbs the distribution of atypical protein kinase C and PAR-6 as well as PAR-3 and delays the formation of functional tight junctions. These results support the notion that the CR1-mediated self-association of the PAR-3-containing protein complex plays a role during the formation of functional tight junctions.     

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.