Regulation of cell polarity during epithelial morphogenesis

Epithelial cells have an apical surface facing a lumen or outside of the organism, and a basolateral surface facing other cells and extracellular matrix. The identity of the apical surface is determined by phosphatidylinositol 4,5-bisphosphate, while phosphatidylinositol 3,4,5-trisphophosphate determines the identity of the basolateral surface. The Par3/Par6/atypical protein kinase C complex, as well as the Crumbs and Scribble complexes, controls epithelial polarity. Par4 and AMP kinase regulate polarity during conditions of energy depletion. Lumens are formed in hollow cysts and tubules by fusions of apical vesicles, such as the vacuolar apical compartment, with the plasma membrane. The polarity of individual cells is oriented and coordinated with other cells by interactions with the extracellular matrix.     

DaPKC-dependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila

Both in Drosophila and vertebrate epithelial cells, the establishment of apicobasal polarity requires the apically localized, membrane-associated Par-3-Par-6-aPKC protein complex. In Drosophila, this complex colocalizes with the Crumbs-Stardust (Sdt)-Pals1-associated TJ protein (Patj) complex. Genetic and molecular analyses suggest a functional relationship between them. We show, by overexpression of a kinase-dead Drosophila atypical PKC (DaPKC), the requirement for the kinase activity of DaPKC to maintain the position of apical determinants and to restrict the localization of basolateral ones. We demonstrate a novel physical interaction between the apical complexes, via direct binding of DaPKC to both Crb and Patj, and identify Crumbs as a phosphorylation target of DaPKC. This phosphorylation of Crumbs is functionally significant. Thus, a nonphosphorylatable Crumbs protein behaves in vivo as a dominant negative. Moreover, the phenotypic effect of overexpressing wild-type Crumbs is suppressed by reducing DaPKC activity. These results provide a mechanistic framework for the functional interaction between the Par-3-Par-6-aPKC and Crumbs-Sdt-Patj complexes based in the posttranslational modification of Crb by DaPKC.     

Role of the polarity protein Scribble for podocyte differentiation and maintenance

The kidney filter represents a unique assembly of podocyte epithelial cells that tightly enwrap the glomerular capillaries with their complex foot process network. While deficiency of the polarity proteins Crumbs and aPKC result in impaired podocyte foot process architecture, the function of basolateral polarity proteins for podocyte differentiation and maintenance remained unclear. Here we report, that Scribble is expressed in developing podocytes, where it translocates from the lateral aspects of immature podocytes to the basal cell membrane and foot processes of mature podocytes. Immunogold electron microscopy reveals membrane associated localisation of Scribble predominantly at the basolateral site of foot processes. To further study the role of Scribble for podocyte differentiation Scribble(flox/flox) mice were generated by introducing loxP-sites into the Scribble introns 1 and 8 and these mice were crossed to NPHS2.Cre mice and Cre deleter mice. Podocyte-specific Scribble knockout mice develop normally and display no histological, ultrastructural or clinical abnormalities up to 12 months of age. In addition, no increased susceptibility to glomerular stress could be detected in these mice. In contrast, constitutive Scribble knockout animals die during embryonic development indicating the fundamental importance of Scribble for embryogenesis. Like in podocyte-specific Scribble knockout mice, the development of podocyte foot processes and the slit diaphragm was unaffected in kidney cultures from constitutive Scribble knockout animals. In summary these results indicate that basolateral polarity signaling via Scribble is dispensable for podocyte function, highlighting the unique feature of podocyte development with its significant apical membrane expansions being dominated by apical polarity complexes rather than by basolateral polarity signaling.     

Trafficking of Crumbs3 during Cytokinesis Is Crucial for Lumen Formation

Although lumen generation has been extensively studied through so-called cyst-formation assays in Madin-Darby canine kidney (MDCK) cells, an underlying mechanism that leads to the initial appearance of a solitary lumen remains elusive. Lumen formation is thought to take place at early stages in aggregates containing only a few cells. Evolutionarily conserved polarity protein complexes, namely the Crumbs, Par, and Scribble complexes, establish apicobasal polarity in epithelial cells, and interference with their function impairs the regulated formation of solitary epithelial lumina. Here, we demonstrate that MDCK cells form solitary lumina during their first cell division. Before mitosis, Crumbs3a becomes internalized and concentrated in Rab11-positive recycling endosomes. These compartments become partitioned in both daughter cells and are delivered to the site of cytokinesis, thus forming the first apical membrane, which will eventually form a lumen. Endosome trafficking in this context appears to depend on the mitotic spindle apparatus and midzone microtubules. Furthermore, we show that this early lumen formation is regulated by the apical polarity complexes because Crumbs3 assists in the recruitment of aPKC to the forming apical membrane and interference with their function can lead to the formation of a no-lumen or multiple-lumen phenotype at the two-cell stage.     

Par6b Regulates the Dynamics of Apicobasal Polarity during Development of the Stratified Xenopus Epidermis

During early vertebrate development, epithelial cells establish and maintain apicobasal polarity, failure of which can cause developmental defects or cancer metastasis. This process has been mostly studied in simple epithelia that have only one layer of cells, but is poorly understood in stratified epithelia. In this paper we address the role of the polarity protein Partitioning defective-6 homolog beta (Par6b) in the developing stratified epidermis of Xenopus laevis. At the blastula stage, animal blastomeres divide perpendicularly to the apicobasal axis to generate partially polarized superficial cells and non-polarized deep cells. Both cell populations modify their apicobasal polarity during the gastrula stage, before differentiating into the superficial and deep layers of epidermis. Early differentiation of the epidermis is normal in Par6b-depleted embryos; however, epidermal cells dissociate and detach from embryos at the tailbud stage. Par6b-depleted epidermal cells exhibit a significant reduction in basolaterally localized E-cadherin. Examination of the apical marker Crumbs homolog 3 (Crb3) and the basolateral marker Lethal giant larvae 2 (Lgl2) after Par6b depletion reveals that Par6b cell-autonomously regulates the dynamics of apicobasal polarity in both superficial and deep epidermal layers. Par6b is required to maintain the “basolateral” state in both epidermal layers, which explains the reduction of basolateral adhesion complexes and epidermal cells shedding.     

Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells

Par3 is a PDZ protein important for the formation of junctional complexes in epithelial cells. We have identified an additional role for Par3 in membrane biogenesis. Although Par3 was not required for maintaining polarized apical or basolateral membrane domains, at the apical surface, Par3 was absolutely essential for the growth and elongation of the primary cilium. The activity reflected its ability to interact with kinesin-2, the microtubule motor responsible for anterograde transport of intraflagellar transport particles to the tip of the growing cilium. The Par3 binding partners Par6 and atypical protein kinase C interacted with the ciliary membrane component Crumbs3 and we show that the PDZ binding motif of Crumbs3 was necessary for its targeting to the ciliary membrane. Thus, the Par complex likely serves as an adaptor that couples the vectorial movement of at least a subset of membrane proteins to microtubule-dependent transport during ciliogenesis.     

Direct interaction of two polarity complexes implicated in epithelial tight junction assembly

Tight junctions help establish polarity in mammalian epithelia by forming a physical barrier that separates apical and basolateral membranes. Two evolutionarily conserved multi-protein complexes, Crumbs (Crb)-PALS1 (Stardust)-PATJ (DiscsLost) and Cdc42-Par6-Par3-atypical protein kinase C (aPKC), have been implicated in the assembly of tight junctions and in polarization of Drosophila melanogaster epithelia. Here we identify a biochemical and functional link between these two complexes that is mediated by Par6 and PALS1 (proteins associated with Lin7). The interaction between Par6 and PALS1 is direct, requires the amino terminus of PALS1 and the PDZ domain of Par6, and is regulated by Cdc42-GTP. The transmembrane protein Crb can recruit wild-type Par6, but not Par6 with a mutated PDZ domain, to the cell surface. Expression of dominant-negative PALS1-associated tight junction protein (PATJ) in MDCK cells results in mis-localization of PALS1, members of the Par3-Par6-aPKC complex and the tight junction marker, ZO-1. Similarly, overexpression of Par6 in MDCK cells inhibits localization of PALS1 to the tight junction. Our data highlight a previously unrecognized link between protein complexes that are essential for epithelial polarity and formation of tight junctions.     

Interaction of Par-6 and Crumbs complexes is essential for photoreceptor morphogenesis in Drosophila

Apicobasal cell polarity is crucial for morphogenesis of photoreceptor rhabdomeres and adherens junctions (AJs) in the Drosophila eye. Crumbs (Crb) is specifically localized to the apical membrane of photoreceptors, providing a positional cue for the organization of rhabdomeres and AJs. We show that the Crb complex consisting of Crb, Stardust (Sdt) and Discs-lost (Dlt) colocalizes with another protein complex containing Par-6 and atypical protein kinase C (aPKC) in the rhabdomere stalk of photoreceptors. Loss of each component of the Crb complex causes age-dependent mislocalization of Par-6 complex proteins, and ectopic expression of Crb intracellular domain is sufficient to recruit the Par-6 complex. We also show that the absence of Par-6 complex proteins results in severe mislocalization and loss of Crb complex. We further demonstrate that Dlt directly binds to Par-6, providing a molecular basis for the mutual dependence of the two complexes. These results suggest that the interaction of Crb and Par-6 complexes is required for the organization and maintenance of apical membranes and AJs of photoreceptors.     

Apicobasal polarity in the kidney

The apicobasal polarization of epithelia is critical for many aspects of kidney function. Over the last decade there have been major advances in our understanding of the mechanisms that underlie this polarity. Critical to this understanding has been the identification of protein complexes on the apical and basolateral sides of epithelial cells that act in a mutually antagonistic manner to define these domains. Concomitant with the creation of apical and basolateral domains is the formation of highly specialized cell-cell junctions including adherens junctions and tight junctions. Recent research points to variability in the polarity and junctional complexes amongst different species and between different cell types of the kidney. Defects in apicobasal polarity are prominent in several disorders including acute renal failure and polycystic kidney disease.     

Crumbs3 Is Essential for Proper Epithelial Development and Viability

First identified in Drosophila, the Crumbs (Crb) proteins are important in epithelial polarity, apical membrane formation, and tight junction (TJ) assembly. The conserved Crb intracellular region includes a FERM (band 4.1/ezrin/radixin/moesin) binding domain (FBD) whose mammalian binding partners are not well understood and a PDZ binding motif that interacts with mammalian Pals1 (protein associated with lin seven) (also known as MPP5). Pals1 binds Patj (Pals1-associated tight-junction protein), a multi-PDZ-domain protein that associates with many tight junction proteins. The Crb complex also binds the conserved Par3/Par6/atypical protein kinase C (aPKC) polarity cassette that restricts migration of basolateral proteins through phosphorylation. Here, we describe a Crb3 knockout mouse that demonstrates extensive defects in epithelial morphogenesis. The mice die shortly after birth, with cystic kidneys and proteinaceous debris throughout the lungs. The intestines display villus fusion, apical membrane blebs, and disrupted microvilli. These intestinal defects phenocopy those of Ezrin knockout mice, and we demonstrate an interaction between Crumbs3 and ezrin. Taken together, our data indicate that Crumbs3 is crucial for epithelial morphogenesis and plays a role in linking the apical membrane to the underlying ezrin-containing cytoskeleton.     

The WD40 protein Morg1 facilitates Par6–aPKC binding to Crb3 for apical identity in epithelial cells

Formation of apico-basal polarity in epithelial cells is crucial for both morphogenesis (e.g., cyst formation) and function (e.g., tight junction development). Atypical protein kinase C (aPKC), complexed with Par6, is considered to translocate to the apical membrane and function in epithelial cell polarization. However, the mechanism for translocation of the Par6–aPKC complex has remained largely unknown. Here, we show that the WD40 protein Morg1 (mitogen-activated protein kinase organizer 1) directly binds to Par6 and thus facilitates apical targeting of Par6–aPKC in Madin-Darby canine kidney epithelial cells. Morg1 also interacts with the apical transmembrane protein Crumbs3 to promote Par6–aPKC binding to Crumbs3, which is reinforced with the apically localized small GTPase Cdc42. Depletion of Morg1 disrupted both tight junction development in monolayer culture and cyst formation in three-dimensional culture; apico-basal polarity was notably restored by forced targeting of aPKC to the apical surface. Thus, Par6–aPKC recruitment to the premature apical membrane appears to be required for definition of apical identity of epithelial cells.     

Positive feedback and mutual antagonism combine to polarize crumbs in the Drosophila follicle cell epithelium

Epithelial tissues are composed of polarized cells with distinct apical and basolateral membrane domains. In the Drosophila ovarian follicle cell epithelium, apical membranes are specified by Crumbs (Crb), Stardust (Sdt), and the aPKC-Par6-cdc42 complex. Basolateral membranes are specified by Lethal giant larvae (Lgl), Discs large (Dlg), and Scribble (Scrib). Apical and basolateral determinants are known to act in a mutually antagonistic fashion, but it remains unclear how this interaction generates polarity. We have built a computer model of apicobasal polarity that suggests that the combination of positive feedback among apical determinants plus mutual antagonism between apical and basal determinants is essential for polarization. In agreement with this model, in vivo experiments define a positive feedback loop in which Crb self-recruits via Crb-Crb extracellular domain interactions, recruitment of Sdt-aPKC-Par6-cdc42, aPKC phosphorylation of Crb, and recruitment of Expanded (Ex) and Kibra (Kib) to prevent endocytic removal of Crb from the plasma membrane. Lgl antagonizes the operation of this feedback loop, explaining why apical determinants do not normally spread into the basolateral domain. Once Crb is removed from the plasma membrane, it undergoes recycling via Rab11 endosomes. Our results provide a dynamic model for understanding how epithelial polarity is maintained in Drosophila follicle cells.     

Comprehensive proteomic analysis of human Par protein complexes reveals an interconnected protein network

The polarization of eukaryotic cells is controlled by the concerted activities of asymmetrically localized proteins. The PAR proteins, first identified in Caenorhabditis elegans, are common regulators of cell polarity conserved from nematode and flies to man. However, little is known about the molecular mechanisms by which these proteins and protein complexes establish cell polarity in mammals. We have mapped multiprotein complexes formed around the putative human Par orthologs MARK4 (microtubule-associated protein/microtubule affinity-regulating kinase 4) (Par-1), Par-3, LKB1 (Par-4), 14-3-3zeta and eta (Par-5), Par-6a, -b, -c, and PKClambda (PKC3). We employed a proteomic approach comprising tandem affinity purification (TAP) of protein complexes from cultured cells and protein sequencing by tandem mass spectrometry. From these data we constructed a highly interconnected protein network consisting of three core complex "modules" formed around MARK4 (Par-1), Par-3.Par-6, and LKB1 (Par-4). The network confirms most previously reported interactions. In addition we identified more than 50 novel interactors, some of which, like the 14-3-3 phospho-protein scaffolds, occur in more than one distinct complex. We demonstrate that the complex formation between LKB1.Par-4, PAPK, and Mo25 results in the translocation of LKB1 from the nucleus to the cytoplasm and to tight junctions and show that the LKB1 complex may activate MARKs, which are known to introduce 14-3-3 binding sites into several substrates. Our findings suggest co-regulation and/or signaling events between the distinct Par complexes and provide a basis for further elucidation of the molecular mechanisms that govern cell polarity.     

aPKC, Crumbs3 and Lgl2 control apicobasal polarity in early vertebrate development

In early vertebrate development, apicobasally polarised blastomeres divide to produce inner non-polarised cells and outer polarised cells that follow different fates. How the polarity of these early blastomeres is established is not known. We have examined the role of Crumbs3, Lgl2 and the apical aPKC in the polarisation of frog blastomeres. Lgl2 localises to the basolateral membrane of blastomeres, while Crumbs3 localises to the apical and basolateral membranes. Overexpression aPKC and Crumbs3 expands the apical domain at the expense of the basolateral and repositions tight junctions in the new apical-basolateral interface. Loss of aPKC function causes loss of apical markers and redirects basolateral markers ectopically to the apical membrane. Cell polarity and tight junctions, but not cell adhesion, are lost and outer polarised cells become inner-like apolar cells. Overexpression of Xenopus Lgl2 phenocopies the aPKC knockout, suggesting that Lgl2 and aPKC act antagonistically. This was confirmed by showing that aPKC and Lgl2 can inhibit the localisation of each other and that Lgl2 rescues the apicalisation caused by aPKC. We conclude that an instrumental antagonistic interaction between aPKC and Lgl2 defines apicobasal polarity in early vertebrate development.     

Basolateral protein Scribble binds phosphatase PP1 to establish a signaling network maintaining apicobasal polarity

Scribble, a member of the LAP protein family, contributes to the apicobasal polarity (ABP) of epithelial cells. The LAP-unique region of these proteins, which is essential and sufficient for ABP, includes a conserved Leucine-Rich Repeat (LRR) domain. The major binding partners of this region that could regulate ABP remain unknown. Here, using proteomics, native gel electrophoresis, and site-directed mutagenesis, we show that the concave surface of LRR domain in Scribble participates in three types of mutually exclusive interactions—(i) homodimerization, serving as an auto-inhibitory mechanism; (ii) interactions with a diverse set of polarity proteins, such as Llgl1, Llgl2, EPB41L2, and EPB41L5, which produce distinct multiprotein complexes; and (iii) a direct interaction with the protein phosphatase, PP1. Analogy with the complex between PP1 and LRR domain of SDS22, a well-studied PP1 regulator, suggests that the Scibble-PP1 complex stores a latent form of PP1 in the basolateral cell cortex. Such organization may generate a dynamic signaling network wherein PP1 could be dispatched from the complex with Scribble to particular protein ligands, achieving fast dephosphorylation kinetics.     

The Scribble-Dlg-Lgl polarity module in development and cancer: from flies to man

The Scribble, Par and Crumbs modules were originally identified in the vinegar (fruit) fly, Drosophila melanogaster, as being critical regulators of apico-basal cell polarity. In the present chapter we focus on the Scribble polarity module, composed of Scribble, discs large and lethal giant larvae. Since the discovery of the role of the Scribble polarity module in apico-basal cell polarity, these proteins have also been recognized as having important roles in other forms of polarity, as well as regulation of the actin cytoskeleton, cell signalling and vesicular trafficking. In addition to these physiological roles, an important role for polarity proteins in cancer progression has also been uncovered, with loss of polarity and tissue architecture being strongly correlated with metastatic disease.     

Epithelial Cell Polarity Determinant CRB3 in Cancer Development

Cell polarity, which is defined as asymmetry in cell shape, organelle distribution and cell function, is essential in numerous biological processes, including cell growth, cell migration and invasion, molecular transport, and cell fate. Epithelial cell polarity is mainly regulated by three conserved polarity protein complexes, the Crumbs (CRB) complex, partitioning defective (PAR) complex and Scribble (SCRIB) complex. Research evidence has indicated that dysregulation of cell polarity proteins may play an important role in cancer development. Crumbs homolog 3 (CRB3), a member of the CRB complex, may act as a cancer suppressor in mouse kidney epithelium and mouse mammary epithelium. In this review, we focus on the current data available on the roles of CRB3 in cancer development.     

The Scribble and Par complexes in polarity and migration: friends or foes?

The Par complex [consisting of Bazooka (also called Par3), Par6 and aPKC] is a well-described regulator of cell polarity whose role in many aspects of cell morphogenesis is under intense investigation. Recently, another set of proteins known as the Scribble complex (consisting of Scribble, Discs large and Lethal giant larvae) has also been shown to be important in polarity regulation in several settings. Here, we describe the current status of Scribble in polarity and review evidence from various model systems that indicates an essential but context-dependent role for the Scribble and Par complexes in directed cell migration. Based on the known interactions of Scribble and Par complexes with each other and with other signalling pathways, we propose models by which Par and Scribble might interact to regulate cell migration.     

Interactions between the crumbs,lethal giant larvae and bazooka pathways in epithelial polarization

Several protein complexes that are involved in epithelial apicobasal polarity have been identified. However, the mechanism by which these complexes interact to form an integrated polarized cell morphology remains unclear. Crumbs (Crb) and Lethal giant larvae (Lgl) are components of distinct complexes that regulate epithelial polarization in Drosophila melanogaster, but may not interact directly as they localize to the apical and basolateral membrane, respectively. Nevertheless, a genetic screen identifies marked functional interactions between crb and lgl. These interactions extend to other genes within the crb (stardust, sdt) and lgl (discs large, dlg; scribble, scrib) pathways. Our findings suggest that the crb and lgl pathways function competitively to define apical and basolateral surfaces. They also suggest that in the absence of lgl pathway activity, the crb pathway is not required to maintain epithelial polarity. Moreover, we show that crb and lgl cooperate in zonula adherens formation early in development. At later stages, epithelial cells in these mutants acquire normal polarity, indicating the presence of compensatory mechanisms. We find that bazooka (baz) functions redundantly with crb/sdt to support apical polarity at mid- to late-embryogenesis. Despite regaining cell polarity, however, epithelial cells in crb and lgl pathway mutants fail to re-establish normal overall tissue architecture, indicating that the timely acquisition of polarized cell structure is essential for normal tissue organization.