A critical step for postsynaptic F‐actin organization: Regulation of Baz/Par‐3 localization by aPKC and PTEN

Actin remodeling has emerged as a critical process during synapse development and plasticity. Thus, understanding the regulatory mechanisms controlling actin organization at synapses is exceedingly important. Here, we used the highly plastic Drosophila neuromuscular junction (NMJ) to understand mechanisms of actin remodeling at postsynaptic sites. Previous studies have suggested that the actin‐binding proteins Spectrin and Coracle play a critical role in NMJ development and the anchoring of glutamate receptors most likely through actin regulation. Here, we show that an additional determinant of actin organization at the postsynaptic region is the PDZ protein Baz/Par‐3. Decreasing Baz levels in postsynaptic muscles has dramatic consequences for the size of F‐actin and spectrin domains at the postsynaptic region. In turn, proper localization of Baz at this site depends on both phosphorylation and dephosphorylation events. Baz phosphorylation by its binding partner, atypical protein kinase C (aPKC), is required for normal Baz targeting to the postsynaptic region. However, the retention of Baz at this site depends on its dephosphorylation mediated by the lipid and protein phosphatase PTEN. Misregulation of the phosphorylation state of Baz by genetic alterations in PTEN or aPKC activity has detrimental consequences for postsynaptic F‐actin and spectrin localization, synaptic growth, and receptor localization. Our results provide a novel mechanism of postsynaptic actin regulation through Baz, governed by the antagonistic actions of aPKC and PTEN. Given the conservation of these proteins from worms to mammals, these results are likely to provide new insight into actin organization pathways. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Antagonistic functions of Par-1 kinase and protein phosphatase 2A are required for localization of Bazooka and photoreceptor morphogenesis in Drosophila

Establishment and maintenance of apical basal cell polarity are essential for epithelial morphogenesis and have been studied extensively using the Drosophila eye as a model system. Bazooka (Baz), a component of the Par-6 complex, plays important roles in cell polarity in diverse cell types including the photoreceptor cells. In ovarian follicle cells, localization of Baz at the apical region is regulated by Par-1 protein kinase. In contrast, Baz in photoreceptor cells is targeted to adherens junctions (AJs). To examine the regulatory pathways responsible for Baz localization in photoreceptor cells, we studied the effects of Par-1 on Baz localization in the pupal retina. Loss of Par-1 impairs the maintenance of AJ markers including Baz and apical polarity proteins of photoreceptor cells but not the establishment of cell polarity. In contrast, overexpression of Par-1 or Baz causes severe mislocalization of junctional and apical markers, resulting in abnormal cell polarity. However, flies with similar overexpression of kinase-inactive mutant Par-1 or unphosphorylatable mutant Baz protein show relatively normal photoreceptor development. These results suggest that dephosphorylation of Baz at the Par-1 phosphorylation sites is essential for proper Baz localization. We also show that the inhibition of protein phosphatase 2A (PP2A) mimics the polarity defects caused by Par-1 overexpression. Furthermore, Par-1 gain-of-function phenotypes are strongly enhanced by reduced PP2A function. Thus, we propose that antagonism between PP2A and Par-1 plays a key role in Baz localization at AJ in photoreceptor morphogenesis.     

A novel function for the PAR complex in subcellular morphogenesis of tracheal terminal cells in Drosophila melanogaster

The processes that generate cellular morphology are not well understood. To investigate this problem, we use Drosophila melanogaster tracheal terminal cells, which undergo two distinct morphogenetic processes: subcellular branching morphogenesis and subcellular apical lumen formation. Here we show these processes are regulated by components of the PAR-polarity complex. This complex, composed of the proteins Par-6, Bazooka (Par-3), aPKC, and Cdc42, is best known for roles in asymmetric cell division and apical/basal polarity. We find Par-6, Bazooka, and aPKC, as well as known interactions between them, are required for subcellular branch initiation, but not for branch outgrowth. By analysis of single and double mutants, and isolation of two novel alleles of Par-6, one of which specifically truncates the Par-6 PDZ domain, we conclude that dynamic interactions between apical PAR-complex members control the branching pattern of terminal cells. These data suggest that canonical apical PAR-complex activity is required for subcellular branching morphogenesis. In addition, we find the PAR proteins are downstream of the FGF pathway that controls terminal cell branching. In contrast, we find that while Par-6 and aPKC are both required for subcellular lumen formation, neither Bazooka nor a direct interaction between Par-6 and aPKC is needed for this process. Thus a novel, noncanonical role for the polarity proteins Par-6 and aPKC is used in formation of this subcellular apical compartment. Our results demonstrate that proteins from the PAR complex can be deployed independently within a single cell to control two different morphogenetic processes.     

Leptin Regulation of Synaptic Function at Hippocampal TA-CA1 and SC-CA1 Synapses: Implications for Health and Disease

Growing evidence indicates that the endocrine hormone leptin regulates hippocampal synaptic function in addition to its established role as a hypothalamic satiety signal. Indeed, numerous studies show that leptin facilitates the cellular events that underlie hippocampal learning and memory including activity-dependent synaptic plasticity and glutamate receptor trafficking, indicating that leptin may be a potential cognitive enhancer. Although there has been extensive investigation into the modulatory role of leptin at hippocampal Schaffer collateral (SC)-CA1 synapses, recent evidence indicates that leptin also potently regulates excitatory synaptic transmission at the anatomically distinct temporoammonic (TA) input to hippocampal CA1 neurons. The cellular mechanisms underlying activity-dependent synaptic plasticity at TA-CA1 synapses differ from those at SC-CA1 synapses and the TA input is implicated in spatial and episodic memory formation. Furthermore, the TA input is an early target for neurodegeneration in Alzheimer’s disease (AD) and aberrant leptin function is linked to AD. Here, we review the evidence that leptin regulates hippocampal synaptic function at both SC- and TA-CA1 synapses and discuss the consequences for neurodegenerative disorders like AD.

     

Par-1 and PP2A: Yin-Yang of Bazooka Localization

Apical basal cell polarity is a fundamental feature of all epithelial cells. Identification of the genes involved in the polarization of epithelial cells has begun to reveal the mechanisms underlying the establishment and maintenance of cell polarity. An important issue is to understand the molecular basis for localization of cell polarity proteins in the context of the developing organism. Bazooka (Baz, Drosophila homolog of Par-3) plays a crucial role in organizing cell polarity in several different tissues. In the ovarian follicle epithelium, Par-1 protein kinase regulates Baz localization to the apical cell cortex by excluding phosphorylated Baz from the lateral region. In photoreceptor cells of retinal epithelium, Baz is targeted to the adherens junction (AJ) instead of the apical domain. Our study suggests that in photoreceptors, Par-1 blocks the localization of Baz to AJ whereas protein phosphatase 2A (PP2A) promotes Baz localization by antagonizing the Par-1 effects. In this extra view, we provide a brief overview and perspective of our findings on the antagonistic function of Par-1 and PP2A in Baz localization during photoreceptor morphogenesis.     

Par-1 and PP2A: Yin-Yang of Bazooka localization

Apical basal cell polarity is a fundamental feature of all epithelial cells. Identification of the genes involved in the polarization of epithelial cells has begun to reveal the mechanisms underlying the establishment and maintenance of cell polarity. An important issue is to understand the molecular basis for localization of cell polarity proteins in the context of the developing organism. Bazooka (Baz, Drosophila homolog of Par-3) plays a crucial role in organizing cell polarity in several different tissues. In the ovarian follicle epithelium, Par-1 protein kinase regulates Baz localization to the apical cell cortex by excluding phosphorylated Baz from the lateral region. In photoreceptor cells of retinal epithelium, Baz is targeted to the adherens junction (AJ) instead of the apical domain. Our study suggests that in photoreceptors, Par-1 blocks the localization of Baz to AJ whereas protein phosphatase 2A (PP2A) promotes Baz localization by antagonizing the Par-1 effects. In this extra view, we provide a brief overview and perspective of our findings on the antagonistic function of Par-1 and PP2A in Baz localization during photoreceptor morphogenesis.     

AhR activation protects intestinal epithelial barrier function through regulation of Par-6

The Par complex (Par-6/Par-3/aPKC) plays a key role in the maintenance of the intestinal barrier function through the regulation of epithelial junction formation. The aryl hydrocarbon receptor (AhR) has been shown to be an important regulator for intestinal homeostasis. In this study, we investigated the role of the AhR activation on the regulation of Par complex. AhR activation by 6-formylindolo (3,2-b) carbazole (FICZ) represses the abnormal expression of the Par complex in a mouse model of dextran sulphate sodium (DSS)-induced colitis. In T84 cells, overexpression of Par-6 causes intestinal barrier dysfunction. Lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction and increase in Par-6 expression was prevented by AhR activation. However, FICZ did not alter the expression of Par-3 or aPKC. Furthermore, AhR activation alleviated LPS-induced increase of Par-6 through repressing the expression of activating protein-2γ (Ap-2γ). These results reveal the protective effects of AhR activation on LPS induced disruption of intestinal epithelial barrier function through suppressing the expression of Par-6 expression. Our findings provide novel insights into the protective role of AhR in intestinal barrier function.     

The role of the actomyosin cytoskeleton in coordination of tissue growth during Drosophila oogenesis

The Drosophila egg chamber is an organ composed of a somatic epithelium that covers a germline cyst. After egg-chamber formation, the germline cells grow rapidly without dividing while the surface of the epithelium expands by cell proliferation [1, 2]. The mechanisms that coordinate growth and morphogenesis of the two tissues are not known. Here we identify a role for the actomyosin cytoskeleton in this process. We show that myosin activity is restricted to the epithelium's apical surface, which is facing the growing cyst. We demonstrate that the epithelium collapses in the absence of myosin activity and show that the force that deforms the epithelium originates from the growing cyst. Thus, myosin activity maintains epithelial shape by balancing the force emanating from cyst growth. Further, our data indicate that cyst growth induces cell division in the epithelium. In addition, we show how apical restriction of myosin activity is controlled. Myosin is activated at the apical cortex by localized Rho kinase and inhibited at the basolateral cortex by PP1beta9C. In addition, our data indicate that active myosin is apically anchored by the Baz/Par-6/aPKC complex.     

Partitioning-defective protein 6 (Par-6) activates atypical protein kinase C (aPKC) by pseudosubstrate displacement

Atypical protein kinase C (aPKC) controls cell polarity by modulating substrate cortical localization. Aberrant aPKC activity disrupts polarity, yet the mechanisms that control aPKC remain poorly understood. We used a reconstituted system with purified components and a cultured cell cortical displacement assay to investigate aPKC regulation. We find that aPKC is autoinhibited by two domains within its NH(2)-terminal regulatory half, a pseudosubstrate motif that occupies the kinase active site, and a C1 domain that assists in this process. The Par complex member Par-6, previously thought to inhibit aPKC, is a potent activator of aPKC in our assays. Par-6 and aPKC interact via PB1 domain heterodimerization, and this interaction activates aPKC by displacing the pseudosubstrate, although full activity requires the Par-6 CRIB-PDZ domains. We propose that, along with its previously described roles in controlling aPKC localization, Par-6 allosterically activates aPKC to allow for high spatial and temporal control of substrate phosphorylation and polarization.     

Positional cloning of heart and soul reveals multiple roles for PKC lambda in zebrafish organogenesis

BACKGROUND: The Par-3/Par-6/aPKC complex is a key regulator of cell polarity in a number of systems. In Drosophila, this complex acts at the zonula adherens (adherens junctions) to establish epithelial polarity and helps to orient the mitotic spindle during asymmetric neuroblast divisions. In MDCKII cells, this complex localizes to the zonula occludens (tight junctions) and appears to regulate epithelial polarity. However, the in vivo role of this complex during vertebrate embryogenesis is not known, due to the lack of relevant mutations. RESULTS: We have positionally cloned the zebrafish heart and soul (has) mutation, which affects the morphogenesis of several embryonic tissues, and show that it encodes atypical protein kinase C lambda (aPKC lambda). We find that loss of aPKC lambda affects the formation and maintenance of the zonula adherens in the polarized epithelia of the retina, neural tube, and digestive tract, leading to novel phenotypes, such as the formation of multiple lumens in the developing intestine. In addition, has mutants display defects in gut looping and endodermal organ morphogenesis that appear to be independent of the defects in epithelial polarity. Finally, we show that loss of aPKC lambda leads to defects in spindle orientation during progenitor cell divisions in the neural retina. CONCLUSIONS: Our results show that aPKC lambda is required for the formation and maintenance of the zonula adherens during early epithelial development in vertebrates and demonstrate a previously undescribed yet critical role for this protein in organ morphogenesis. Furthermore, our studies identify the first genetic locus regulating the orientation of cell division in vertebrates.     

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.     

A polarity complex of mPar-6 and atypical PKC binds,phosphorylates and regulates mammalian Lgl

The evolutionarily conserved proteins Par-6, atypical protein kinase C (aPKC), Cdc42 and Par-3 associate to regulate cell polarity and asymmetric cell division, but the downstream targets of this complex are largely unknown. Here we identify direct physiological interactions between mammalian aPKC, murine Par-6C (mPar-6C) and Mlgl, the mammalian orthologue of the Drosophila melanogaster tumour suppressor Lethal (2) giant larvae. In cultured cell lines and in mouse brain, aPKC, mPar-6C and Mlgl form a multiprotein complex in which Mlgl is targeted for phosphorylation on conserved serine residues. These phosphorylation sites are important for embryonic fibroblasts to polarize correctly in response to wounding and may regulate the ability of Mlgl to direct protein trafficking. Our data provide a direct physical and regulatory link between proteins of distinct polarity complexes, identify Mlgl as a functional substrate for aPKC in cell polarization and indicate that aPKC is directed to cell polarity substrates through a network of protein-protein interactions.     

Magi Is Associated with the Par Complex and Functions Antagonistically with Bazooka to Regulate the Apical Polarity Complex

The mammalian MAGI proteins play important roles in the maintenance of adherens and tight junctions. The MAGI family of proteins contains modular domains such as WW and PDZ domains necessary for scaffolding of membrane receptors and intracellular signaling components. Loss of MAGI leads to reduced junction stability while overexpression of MAGI can lead to increased adhesion and stabilization of epithelial morphology. However, how Magi regulates junction assembly in epithelia is largely unknown. We investigated the single Drosophila homologue of Magi to study the in vivo role of Magi in epithelial development. Magi is localized at the adherens junction and forms a complex with the polarity proteins, Par3/Bazooka and aPKC. We generated a Magi null mutant and found that Magi null mutants were viable with no detectable morphological defects even though the Magi protein is highly conserved with vertebrate Magi homologues. However, overexpression of Magi resulted in the displacement of Baz/Par3 and aPKC and lead to an increase in the level of PIP3. Interestingly, we found that Magi and Baz functioned in an antagonistic manner to regulate the localization of the apical polarity complex. Maintaining the balance between the level of Magi and Baz is an important determinant of the levels and localization of apical polarity complex.     

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.     

Stepwise polarisation of the Drosophila follicular epithelium

The function of epithelial tissues is dependent on their polarised architecture, and loss of cell polarity is a hallmark of various diseases. Here we analyse cell polarisation in the follicular epithelium of Drosophila, an epithelium that arises by a mesenchymal-epithelial transition. Although many epithelia are formed by mesenchymal precursors, it is unclear how they polarise. Here we show how lateral, apical, and adherens junction proteins act stepwise to establish polarity in the follicular epithelium. Polarisation starts with the formation of adherens junctions, whose positioning is controlled by combined activities of Par-3, β-catenin, and Discs large. Subsequently, Par-6 and aPKC localise to the apical membrane in a Par-3-dependent manner. Apical membrane specification continues by the accumulation of the Crumbs complex, which is controlled by Par-3, Par-6, and aPKC. Thus, our data elucidate the genetic mechanisms leading to the stepwise polarisation of an epithelium with a mesenchymal origin.     

The Par-3 NTD adopts a PB1-like structure required for Par-3 oligomerization and membrane localization

The evolutionarily conserved Par-3/Par-6/aPKC complex is essential for the establishment and maintenance of polarity of a wide range of cells. Both Par-3 and Par-6 are PDZ domain containing scaffold proteins capable of binding to polarity regulatory proteins. In addition to three PDZ domains, Par-3 also contains a conserved N-terminal oligomerization domain (NTD) that is essential for proper subapical membrane localization and consequently the functions of Par-3. The molecular basis of NTD-mediated Par-3 membrane localization is poorly understood. Here, we describe the structure of a monomeric form of the Par-3 NTD. Unexpectedly, the domain adopts a PB1-like fold with both type-I and type-II structural features. The Par-3 NTD oligomerizes into helical filaments via front-to-back interactions. We further demonstrate that the NTD-mediated membrane localization of Par-3 in MDCK cells is solely attributed to its oligomerization capacity. The data presented in this study suggest that the Par-3 NTD is likely to facilitate the assembly of higher-order Par-3/Par-6/aPKC complex with increased avidities in targeting the complex to the subapical membrane domain and in binding to other polarity-regulating proteins.     

Formation of a Bazooka–Stardust complex is essential for plasma membrane polarity in epithelia

Apical–basal polarity in Drosophila melanogaster epithelia depends on several evolutionarily conserved proteins that have been assigned to two distinct protein complexes: the Bazooka (Baz)–PAR-6 (partitioning defective 6)–atypical protein kinase C (aPKC) complex and the Crumbs (Crb)–Stardust (Sdt) complex. These proteins operate in a functional hierarchy, in which Baz is required for the proper subcellular localization of all other proteins. We investigated how these proteins interact and how this interaction is regulated. We show that Baz recruits Sdt to the plasma membrane by direct interaction between the Postsynaptic density 95/Discs large/Zonula occludens 1 (PDZ) domain of Sdt and a region of Baz that contains a phosphorylation site for aPKC. Phosphorylation of Baz causes the dissociation of the Baz–Sdt complex. Overexpression of a nonphosphorylatable version of Baz blocks the dissociation of Sdt from Baz, causing phenotypes very similar to those of crb and sdt mutations. Our findings provide a molecular mechanism for the phosphorylation-dependent interaction between the Baz–PAR-3 and Crb complexes during the establishment of epithelial polarity.     

The polarity-inducing kinase Par-1 controls Xenopus gastrulation in cooperation with 14-3-3 and aPKC

Par (partitioning-defective) genes were originally identified in Caenorhabditis elegans as determinants of anterior/posterior polarity. However, neither their function in vertebrate development nor their action mechanism has been fully addressed. Here we show that two members of Par proteins, 14-3-3 (Par-5) and atypical PKC (aPKC), regulate the serine/threonine kinase Par-1 to control Xenopus gastrulation. We find first that Xenopus Par-1 (xPar-1) is essential for gastrulation but not for cell fate specification during early embryonic development. We then find that xPar-1 binds to 14-3-3 in an aPKC-dependent manner. Our analyses identify two aPKC phosphorylation sites in xPar-1, which are essential for 14-3-3 binding and for proper gastrulation movements. The aPKC phosphorylation-dependent binding of xPar-1 to 14-3-3 does not markedly affect the kinase activity of xPar-1, but induces relocation of xPar-1 from the plasma membranes to the cytoplasm. Finally, we show that Xenopus aPKC and its binding partner Xenopus Par-6 are also essential for gastrulation. Thus, our results identify a requirement of Par proteins for Xenopus gastrulation and reveal a novel interrelationship within Par proteins that may provide a general mechanism for spatial control of Par-1.     

The fusome and microtubules enrich Par-1 in the oocyte,where it effects polarization in conjunction with Par-3, BicD,Egl, and dynein

After its specification, the Drosophila oocyte undergoes a critical polarization event that involves a reorganization of the microtubules (MT) and relocalization of the determinant Orb within the oocyte. This polarization requires Par-1 kinase and the PDZ-containing Par-3 homolog, Bazooka (Baz). Par-1 has been observed on the fusome, which degenerates before the onset of oocyte polarization. How Par-1 acts to polarize the oocyte has been unclear. Here we show that Par-1 becomes restricted to the oocyte in a MT-dependent fashion after disappearance of the fusome. At the time of polarization, the kinase itself and the determinant BicaudalD (BicD) are relocalized from the anterior to the posterior of the oocyte. Par-1 and BicD are interdependent and require MT and the minus end-directed motor Dynein for their relocalization. We show that baz is required for Par-1 relocalization within the oocyte and that the distributions of Baz and Par-1 in the Drosophila oocyte are complementary and strikingly reminiscent of the two PAR proteins in the C. elegans embryo. We propose that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where, in conjunction with BicD, Egalitarian (Egl), and Dynein, it acts on the MT cytoskeleton to effect polarization.     

Atypical PKC phosphorylates PAR-1 kinases to regulate localization and activity

The establishment and maintenance of cellular polarity are essential biological processes that must be maintained throughout the lifetime of eukaryotic organisms. The Par-1 protein kinases are key polarity determinants that have been conserved throughout evolution. Par-1 directs anterior-posterior asymmetry in the one-cell C. elegans embryo and the Drosophila oocyte. In mammalian cells, Par-1 may regulate epithelial cell polarity. Relevant substrates of Par-1 in these pathways are just being identified, but it is not yet known how Par-1 itself is regulated. Here, we demonstrate that human Par-1b (hPar-1b) interacts with and is negatively regulated by atypical PKC. hPar-1b is phosphorylated by aPKC on threonine 595, a residue conserved in Par-1 orthologs in mammals, worms, and flies. The equivalent site in hPar-1a, T564, is phosphorylated in vivo and by aPKC in vitro. Importantly, phosphorylation of hPar-1b on T595 negatively regulates the kinase activity and plasma membrane localization of hPar-1b in vivo. This study establishes a novel functional link between two central determinants of cellular polarity, aPKC and Par-1, and suggests a model by which aPKC may regulate Par-1 in polarized cells.