Drosophila Crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres

Drosophila Crumbs (Crb) is required for apical-basal polarity and is an apical determinant in embryonic epithelia. Here, we describe properties of Crb that control the position and integrity of the photoreceptor adherens junction and photosensitive organ, or rhabdomere. In contrast to normal photoreceptor adherens junctions and rhabdomeres, which span the depth of the retina, adherens junctions and rhabdomeres of Crb-deficient photoreceptors initially accumulate at the top of the retina and fail to maintain their integrity as they stretch to the retinal floor. We show that Crb controls localization of the adherens junction through its intracellular domain containing a putative binding site for a protein 4.1 superfamily protein (FERM). Although loss of Crb or overexpression of the FERM binding domain causes mislocalization of adherens junctions, they do not result in a significant loss of photoreceptor polarity. Mutations in CRB1, a human homologue of crb, are associated with photoreceptor degeneration in retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis (LCA). The intracellular domain of CRB1 behaves similarly to its Drosophila counterpart when overexpressed in the fly eye. Our studies may provide clues for mechanisms of photoreceptor degeneration in RP12 and LCA.     

Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity.

The polarized architecture of epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical one comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, the cytoplasmic domain of which recruits the PDZ-protein Discs Lost into the complex. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Here we show that stardust provides an essential component of the SAC. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of its cytoplasmic tail. We introduce two different Stardust proteins here: one MAGUK protein, characterized by a PDZ domain, an SH3 domain and a guanylate kinase domain; and a second isoform comprising only the guanylate kinase domain. The Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity.     

Localization of apical epithelial determinants by the basolateral PDZ protein Scribble

The generation of membrane domains with distinct protein constituents is a hallmark of cell polarization. In epithelia, segregation of membrane proteins into apical and basolateral compartments is critical for cell morphology, tissue physiology and cell signalling. Drosophila proteins that confer apical membrane identity have been found, but the mechanisms that restrict these determinants to the apical cell surface are unknown. Here we show that a laterally localized protein is required for the apical confinement of polarity determinants. Mutations in Drosophila scribble (scrib), which encodes a multi-PDZ (PSD-95, Discs-large and ZO-1) and leucine-rich-repeat protein, cause aberrant cell shapes and loss of the monolayer organization of embryonic epithelia. Scrib is localized to the epithelial septate junction, the analogue of the vertebrate tight junction, at the boundary of the apical and basolateral cell surfaces. Loss of scrib function results in the misdistribution of apical proteins and adherens junctions to the basolateral cell surface, but basolateral protein localization remains intact. These phenotypes can be accounted for by mislocalization of the apical determinant Crumbs. Our results show that the lateral domain of epithelia, particularly the septate junction, functions in restricting apical membrane identity and correctly placing adherens junctions.     

Differential positioning of adherens junctions is associated with initiation of epithelial folding

During tissue morphogenesis, simple epithelial sheets undergo folding to form complex structures. The prevailing model underlying epithelial folding involves cell shape changes driven by myosin-dependent apical constriction. Here we describe an alternative mechanism that requires differential positioning of adherens junctions controlled by modulation of epithelial apical-basal polarity. Using live embryo imaging, we show that before the initiation of dorsal transverse folds during Drosophila gastrulation, adherens junctions shift basally in the initiating cells, but maintain their original subapical positioning in the neighbouring cells. Junctional positioning in the dorsal epithelium depends on the polarity proteins Bazooka and Par-1. In particular, the basal shift that occurs in the initiating cells is associated with a progressive decrease in Par-1 levels. We show that uniform reduction of the activity of Bazooka or Par-1 results in uniform apical or lateral positioning of junctions and in each case dorsal fold initiation is abolished. In addition, an increase in the Bazooka/Par-1 ratio causes formation of ectopic dorsal folds. The basal shift of junctions not only alters the apical shape of the initiating cells, but also forces the lateral membrane of the adjacent cells to bend towards the initiating cells, thereby facilitating tissue deformation. Our data thus establish a direct link between modification of epithelial polarity and initiation of epithelial folding.     

Adherens junctions inhibit asymmetric division in the Drosophila epithelium

Asymmetric division is a fundamental mechanism for generating cellular diversity. In the central nervous system of Drosophila, neural progenitor cells called neuroblasts undergo asymmetric division along the apical-basal cellular axis. Neuroblasts originate from neuroepithelial cells, which are polarized along the apical-basal axis and divide symmetrically along the planar axis. The asymmetry of neuroblasts might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. Here we show that by disrupting adherens junctions we can convert the symmetric epithelial division into asymmetric division. We further confirm that the adenomatous polyposis coli (APC) tumour suppressor protein is recruited to adherens junctions, and demonstrate that both APC and microtubule-associated EB1 homologues are required for the symmetric epithelial division along the planar axis. Our results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions.     

Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity

Two prominent characteristics of epithelial cells, apical-basal polarity and a highly ordered cytoskeleton, depend on the existence of precisely localized protein complexes associated with the apical plasma membrane, and on a separate machinery that regulates the spatial order of actin assembly. ERM (ezrin, radixin, moesin) proteins have been proposed to link transmembrane proteins to the actin cytoskeleton in the apical domain, suggesting a structural role in epithelial cells, and they have been implicated in signalling pathways. Here, we show that the sole Drosophila ERM protein Moesin functions to promote cortical actin assembly and apical-basal polarity. As a result, cells lacking Moesin lose epithelial characteristics and adopt invasive migratory behaviour. Our data demonstrate that Moesin facilitates epithelial morphology not by providing an essential structural function, but rather by antagonizing activity of the small GTPase Rho. Thus, Moesin functions in maintaining epithelial integrity by regulating cell-signalling events that affect actin organization and polarity. Furthermore, our results show that there is negative feedback between ERM activation and activity of the Rho pathway.     

Bazooka recruits Inscuteable to orient asymmetric cell divisions in Drosophila neuroblasts

Asymmetric cell divisions can be generated by the segregation of determinants into one of the two daughter cells. In Drosophila, neuroblasts divide asymmetrically along the apical-basal axis shortly after their delamination from the neuroectodermal epithelium. Several proteins, including Numb and Miranda, segregate into the basal daughter cell and are needed for the determination of its correct cell fate. Both the apical-basal orientation of the mitotic spindle and the localization of Numb and Miranda to the basal cell cortex are directed by Inscuteable, a protein that localizes to the apical cell cortex before and during neuroblast mitosis. Here we show that the apical localizaton of Inscuteable requires Bazooka, a protein containing a PDZ domain that is essential for apical-basal polarity in epithelial cells. Bazooka localizes with Inscuteable in neuroblasts and binds to the Inscuteable localization domain in vitro and in vivo. In embryos lacking both maternal and zygotic bazooka function, Inscuteable no longer localizes asymmetrically in neuroblasts and is instead uniformly distributed in the cytoplasm. Mitotic spindles in neuroblasts are misoriented in these embryos, and the proteins Numb and Miranda fail to localize asymmetrically in metaphase. Our results suggest that direct binding to Bazooka mediates the asymmetric localization of Inscuteable and connects the asymmetric division of neuroblasts to the axis of epithelial apical-basal polarity.     

Bazooka provides an apical cue for Inscuteable localization in Drosophila neuroblasts

Asymmetric cell division generates daughter cells with different developmental fates from progenitor cells that contain localized determinants. During this division, the asymmetric localization of cell-fate determinants and the orientation of the mitotic spindle must be precisely coordinated. In Drosophila neuroblasts, inscuteable controls both spindle orientation and the asymmetric localization of the cell-fate determinants Prospero and Numb. Inscuteable itself is localized in an apical cortical crescent and thus reflects the intrinsic asymmetry of the neuroblast. Here we show that localization of Inscuteable depends on Bazooka, a protein containing three PDZ domains with overall sequence similarity to Par-3 of Caenorhabditis elegans. Bazooka and Inscuteable form a complex that also contains Staufen, a protein responsible for the asymmetric localization of prospero messenger RNA. We propose that, after delamination of the neuroblast from the neuroepithelium, Bazooka provides an asymmetric cue in the apical cytocortex that is required to anchor Inscuteable. As Bazooka is also responsible for the maintenance of apical-basal polarity in epithelial tissues, it may be the missing link between epithelial polarity and neuroblast polarity.     

A role for Drosophila LKB1 in anterior-posterior axis formation and epithelial polarity

The PAR-4 and PAR-1 kinases are necessary for the formation of the anterior-posterior (A-P) axis in Caenorhabditis elegans. PAR-1 is also required for A-P axis determination in Drosophila. Here we show that the Drosophila par-4 homologue, lkb1, is required for the early A-P polarity of the oocyte, and for the repolarization of the oocyte cytoskeleton that defines the embryonic A-P axis. LKB1 is phosphorylated by PAR-1 in vitro, and overexpression of LKB1 partially rescues the par-1 phenotype. These two kinases therefore function in a conserved pathway for axis formation in flies and worms. lkb1 mutant clones also disrupt apical-basal epithelial polarity, suggesting a general role in cell polarization. The human homologue, LKB1, is mutated in Peutz-Jeghers syndrome and is regulated by prenylation and by phosphorylation by protein kinase A. We show that protein kinase A phosphorylates Drosophila LKB1 on a conserved site that is important for its activity. Thus, Drosophila and human LKB1 may be functional homologues, suggesting that loss of cell polarity may contribute to tumour formation in individuals with Peutz-Jeghers syndrome.     

Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts.

Establishing cellular polarity is critical for tissue organization and function. Initially discovered in the landmark genetic screen for Drosophila developmental mutants, bazooka, crumbs, shotgun and stardust mutants exhibit severe disruption in apicobasal polarity in embryonic epithelia, resulting in multilayered epithelia, tissue disintegration, and defects in cuticle formation. Here we report that stardust encodes single PDZ domain MAGUK (membrane-associated guanylate kinase) proteins that are expressed in all primary embryonic epithelia from the onset of gastrulation. Stardust colocalizes with Crumbs at the apicolateral boundary, although their expression patterns in sensory organs differ. Stardust binds to the carboxy terminus of Crumbs in vitro, and Stardust and Crumbs are mutually dependent in their stability, localization and function in controlling the apicobasal polarity of epithelial cells. However, for the subset of ectodermal cells that delaminate and form neuroblasts, their polarity requires the function of Bazooka, but not of Stardust or Crumbs.     

PTK7/CCK-4 is a novel regulator of planar cell polarity in vertebrates

In addition to the apical-basal polarity pathway operating in epithelial cells, a planar cell polarity (PCP) pathway establishes polarity within the plane of epithelial tissues and is conserved from Drosophila to mammals. In Drosophila, a 'core' group of PCP genes including frizzled (fz), flamingo/starry night, dishevelled (dsh), Van Gogh/strabismus and prickle, function to regulate wing hair, bristle and ommatidial polarity. In vertebrates, the PCP pathway regulates convergent extension movements and neural tube closure, as well as the orientation of stereociliary bundles of sensory hair cells in the inner ear. Here we show that a mutation in the mouse protein tyrosine kinase 7 (PTK7) gene, which encodes an evolutionarily conserved transmembrane protein with tyrosine kinase homology, disrupts neural tube closure and stereociliary bundle orientation, and shows genetic interactions with a mutation in the mouse Van Gogh homologue vangl2. We also show that PTK7 is dynamically localized during hair cell polarization, and that the Xenopus homologue of PTK7 is required for neural convergent extension and neural tube closure. These results identify PTK7 as a novel regulator of PCP in vertebrates.     

Regulation of Lethal giant larvae by Dishevelled

The establishment of polarity in many cell types depends on Lgl, the tumour suppressor product of lethal giant larvae, which is involved in basolateral protein targeting. The conserved complex of Par3, Par6 and atypical protein kinase C phosphorylates and inactivates Lgl at the apical surface; however, the signalling mechanisms that coordinate cell polarization in development are not well defined. Here we show that a vertebrate homologue of Lgl associates with Dishevelled, an essential mediator of Wnt signalling, and that Dishevelled regulates the localization of Lgl in Xenopus ectoderm and Drosophila follicular epithelium. We show that both Lgl and Dsh are required for normal apical-basal polarity of Xenopus ectodermal cells. In addition, we show that the Wnt receptor Frizzled 8, but not Frizzled 7, causes Lgl to dissociate from the cortex with the concomitant loss of its activity in vivo. These findings suggest a molecular basis for the regulation of cell polarity by Frizzled and Dishevelled.     

Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis

Axis formation occurs in plants, as in animals, during early embryogenesis. However, the underlying mechanism is not known. Here we show that the first manifestation of the apical-basal axis in plants, the asymmetric division of the zygote, produces a basal cell that transports and an apical cell that responds to the signalling molecule auxin. This apical-basal auxin activity gradient triggers the specification of apical embryo structures and is actively maintained by a novel component of auxin efflux, PIN7, which is located apically in the basal cell. Later, the developmentally regulated reversal of PIN7 and onset of PIN1 polar localization reorganize the auxin gradient for specification of the basal root pole. An analysis of pin quadruple mutants identifies PIN-dependent transport as an essential part of the mechanism for embryo axis formation. Our results indicate how the establishment of cell polarity, polar auxin efflux and local auxin response result in apical-basal axis formation of the embryo, and thus determine the axiality of the adult plant.     

Basement membrane polarizes lectin binding sites of Drosophila larval fat body cells

Vertebrate epithelial cells in monolayers are asymmetrical in that their apical and basal membranes differ in morphology and function. That this cell polarity depends on the presence of tight junctions can be demonstrated by labelling one surface of a cell monolayer in culture with fluorescent lectins and lipid probes, and subsequently observing whether the labels disperse to the opposite cell surfaces. Here we report on a differential distribution of binding sites for the lectin wheat germ agglutinin (WGA) on the cells of Drosophila melanogaster larval fat body, and show that the pattern is correlated with the structural association between the cell surfaces and their overlying basement membrane.     

Clathrin is a key regulator of basolateral polarity

Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells.     

An H+-ATPase in opposite plasma membrane domains in kidney epithelial cell subpopulations

Vectorial solute transport by epithelia requires the polarized insertion of transport proteins into apical or basolateral plasmalemmal domains. In the specialized intercalated cells of the kidney collecting duct, the selective placement of an apical plasma membrane proton-pumping ATPase (H+-ATPase) and of a basolateral membrane anion-exchange protein results in transepithelial proton secretion. It is currently believed that amino-acid sequences of membrane proteins contain critical signalling regions involved in sorting these proteins to specific membrane domains. Recently, it was proposed that intercalated cells can reverse their direction of proton secretion under different acid-base conditions by redirecting proton pumps from apical to basolateral membranes, and anion exchangers from basolateral to apical membranes. But others have found that antibodies raised against the red cell anion-exchange protein (Band 3) only labelled intercalated cells at the basolateral plasma membrane, providing evidence against the model of polarity reversal. In this report, we have examined directly the distribution of proton pumps in kidney intercalated cells using specific polyclonal antibodies against subunits of a bovine kidney medullary H+-ATPase. We find that some cortical collecting duct intercalated cells have apical plasma membrane proton pumps, whereas others have basolateral pumps. This is the first direct demonstration of neighbouring epithelial cells maintaining opposite polarities of a transport protein. Thus, either subtle structural differences exist between proton pumps located at opposite poles of the cell, or factors other than protein sequence determine the polarity of H+-ATPase insertion.     

Cingulin, a new peripheral component of tight junctions

The tight junction (Zonula occludens), a belt-like region of contact between cells of polarized epithelia, serves as a selective barrier to small molecules and as a total barrier to large molecules, and is involved in the separation between lumenal and basolateral compartments of the epithelium. In the electron microscope, tight junctions show focal regions of apparent fusion between the adjoining cell membranes, and freeze-fractured membranes display an elaborate network of branching and anastomosing strands. Very little is known about the molecular composition and architecture of tight junctions. The first specific zonula occludens-associated protein, designated ZO-1, has recently been identified in mammalian epithelial and endothelial cells. Here we describe the identification and purification of a new component of this junctional complex in avian brush-border cells, which we name cingulin. Cingulin is an acidic, heat-stable protein, with a highly elongated shape. Immunofluorescence and immunoelectron microscopy of brush-border cells with anti-cingulin antibodies show that cingulin is localized in the apical zone of the terminal web, at the endofacial surfaces of the zonula occludens.     

Segregation of receptor and ligand regulates activation of epithelial growth factor receptor

Interactions between ligands and receptors are central to communication between cells and tissues. Human airway epithelia constitutively produce both a ligand, the growth factor heregulin, and its receptors--erbB2, erbB3 and erbB4 (refs 1-3). Although heregulin binding initiates cellular proliferation and differentiation, airway epithelia have a low rate of cell division. This raises the question of how ligand-receptor interactions are controlled in epithelia. Here we show that in differentiated human airway epithelia, heregulin-alpha is present exclusively in the apical membrane and the overlying airway surface liquid, physically separated from erbB2-4, which segregate to the basolateral membrane. This physical arrangement creates a ligand-receptor pair poised for activation whenever epithelial integrity is disrupted. Indeed, immediately following a mechanical injury, heregulin-alpha activates erbB2 in cells at the edge of the wound, and this process hastens restoration of epithelial integrity. Likewise, when epithelial cells are not separated into apical and basolateral membranes ('polarized'), or when tight junctions between adjacent cells are opened, heregulin-alpha activates its receptor. This mechanism of ligand-receptor segregation on either side of epithelial tight junctions may be vital for rapid restoration of integrity following injury, and hence critical for survival. This model also suggests a mechanism for abnormal receptor activation in diseases with increased epithelial permeability.     

The tight junction does not allow lipid molecules to diffuse from one epithelial cell to the next

The tight junction (zonula occludens) links epithelial cells into a monolayer by forming a continuous belt of sealing contacts around the apex of each cell. They appear in thin sections as if they were 'fusions' between the apposed plasma membranes and in freeze-fracture replicas as patterns of complementary strands and furrows. These images have led to the proposal that the core of the tight junction is formed by a hexagonal cylinder of lipids. In this model, the cytoplasmic leaflet of the apical and basolateral plasma membrane domains would be continuous, whereas the exoplasmic leaflets of the two plasma membrane domains of the same cell would be separated at the tight junction and are instead predicted to be continuous between the plasma membranes of neighbouring cells. We demonstrate here that this prediction does not hold true. An endogenous glycolipid (Forssman antigen), present in the exoplasmic leaflet of the apical membrane of MDCK strain II cells, is unable to pass to MDCK strain I cells (which lack this glycolipid) under conditions where these cells are connected by tight junctions. In addition, fluorescent lipids which have been fused into the plasma membrane of one MDCK cell do not diffuse to neighbouring cells while the tight junctions between the cells are intact.     

Plasticity of functional epithelial polarity

The fundamental characteristics that allow vectorial transport across an epithelial cell are the differential sorting and insertion of transport proteins either in the apical or the basolateral plasma membrane, and the preferential association of endocytosis and exocytosis with one or the other pole of the cell. Asymmetrical cellular structure and function, being manifestations of terminal differentiation, might be expected to be predetermined and invariant. Here we show that the polarity of transepithelial H+ transport, endocytosis and exocytosis in kidney can be reversed by environmental stimuli. The HCO3- secreting cell in the cortical collecting tubule is found to be an intercalated cell possessing a Cl-/HCO3- exchanger in the apical membrane and proton pumps in endocytic vesicles that fuse with the basolateral membrane; the H+-secreting cell in the medullary collecting tubule has these transport functions on the opposite membranes. Further, the HCO3- -secreting cell can be induced to change its functional polarity to that of the H+-secreting cell by acid-loading the animal.