A Conserved Functional Domain of Drosophila Coracle Is Required for Localization at the Septate Junction and Has Membrane-organizing Activity

The protein 4.1 superfamily is comprised of a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins. Coracle, a Drosophila protein 4.1 homologue, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells. Using in vitro mutagenesis, we demonstrate that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos. Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. Furthermore, the localization of Coracle and the transmembrane protein Neurexin to the septate junction display an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members.     

Drosophila coracle, a Member of the Protein 4.1 Superfamily, Has Essential Structural Functions in the Septate Junctions and Developmental Functions in Embryonic and Adult Epithelial Cells

Although extensively studied biochemically, members of the Protein 4.1 superfamily have not been as well characterized genetically. Studies of coracle, a Drosophila Protein 4.1 homologue, provide an opportunity to examine the genetic functions of this gene family. coracle was originally identified as a dominant suppressor of Egfr^Elp, a hypermorphic form of the Drosophila Epidermal growth factor receptor gene. In this article, we present a phenotypic analysis of coracle, one of the first for a member of the Protein 4.1 superfamily. Screens for new coracle alleles confirm the null coracle phenotype of embryonic lethality and failure in dorsal closure, and they identify additional defects in the embryonic epidermis and salivary glands. Hypomorphic coracle alleles reveal functions in many imaginal tissues. Analysis of coracle mutant cells indicates that Coracle is a necessary structural component of the septate junction required for the maintenance of the transepithelial barrier but is not necessary for apical–basal polarity, epithelial integrity, or cytoskeletal integrity. In addition, coracle phenotypes suggest a specific role in cell signaling events. Finally, complementation analysis provides information regarding the functional organization of Coracle and possibly other Protein 4.1 superfamily members. These studies provide insights into a range of in vivo functions for coracle in developing embryos and adults.     

The claudin-like megatrachea is essential in septate junctions for the epithelial barrier function in Drosophila

Vertebrate claudin proteins are integral components of tight junctions, which function as paracellular diffusion barriers in epithelia. We identified Megatrachea (Mega), a Drosophila transmembrane protein homologous to claudins, and show that it acts in septate junctions, the corresponding structure of invertebrates. Our analysis revealed that Mega has transepithelial barrier function similar to the claudins. Also, Mega is necessary for normal tracheal cell morphogenesis but not for apicobasal polarity or epithelial integrity. In addition, we present evidence that Mega is essential for localization of the septate junction protein complex Coracle/Neurexin. The results indicate that claudin-like proteins are functionally conserved between vertebrates and Drosophila.     

The neurofibromatosis-2 homologue, Merlin, and the tumor suppressor expanded function together in Drosophila to regulate cell proliferation and differentiation

Neurofibromatosis-2 is an inherited disorder characterized by the development of benign schwannomas and other Schwann-cell-derived tumors associated with the central nervous system. The Neurofibromatosis-2 tumor suppressor gene encodes Merlin, a member of the Protein 4.1 superfamily most closely related to Ezrin, Radixin and Moesin. This discovery suggested a novel function for Protein 4.1 family members in the regulation of cell proliferation; proteins in this family were previously thought to function primarily to link transmembrane proteins to underlying cortical actin. To understand the basic cellular functions of Merlin, we are investigating a Drosophila Neurofibromatosis-2 homologue, Merlin. Loss of Merlin function in Drosophila results in hyperplasia of the affected tissue without significant disruptions in differentiation. Similar phenotypes have been observed for mutations in another Protein 4.1 superfamily member in Drosophila, expanded. Because of the phenotypic and structural similarities between Merlin and expanded, we asked whether Merlin and Expanded function together to regulate cell proliferation. In this study, we demonstrate that recessive loss of function of either Merlin or expanded can dominantly enhance the phenotypes associated with mutations in the other. Consistent with this genetic interaction, we determined that Merlin and Expanded colocalize in Drosophila tissues and cells, and physically interact through a conserved N-terminal region of Expanded, characteristic of the Protein 4.1 family, and the C-terminal domain of Merlin. Loss of function of both Merlin and expanded in clones revealed that these proteins function to regulate differentiation in addition to proliferation in Drosophila. Further genetic analyses suggest a role for Merlin and Expanded specifically in Decapentaplegic-mediated differentiation events. These results indicate that Merlin and Expanded function together to regulate proliferation and differentiation, and have implications for understanding the functions of other Protein 4.1 superfamily members.     

Transient apical polarization of Gliotactin and Coracle is required for parallel alignment of wing hairs in Drosophila

In Drosophila, wing hairs are aligned in a distally oriented, parallel array. The frizzled pathway determines proximal-distal cell polarity in the wing; however, in frizzled pathway mutants, wing hairs remain parallel. How wing hairs align has not been determined. We have demonstrated a novel role for the septate junction proteins Gliotactin (Gli) and Coracle (Cora) in this process. Prior to prehair extension, Gli and Cora were restricted to basolateral membranes. During pupal prehair development, Gli and Cora transiently formed apical ribbons oriented from the distal wing tip to the proximal hinge. These ribbons were aligned beneath prehair bases and persisted for several hours. During this time, Gli was lost entirely from the basolateral domain. A Gliotactin mutation altered the apical polarization Gli and Cora and induced defects in hair alignment in pupal and adult stages. Genetic and cell biological assays demonstrated that Gli and Cora function to align hairs independently of frizzled. Taken together, our results indicate that Gli and Cora function as the first-identified members of a long-predicted, frizzled-independent parallel alignment mechanism. We propose a model whereby the apical polarization of Gli and Cora functions to stabilize and align prehairs relative to anterior-posterior cell boundaries during pupal wing development.     

Band 4.1 proteins regulate integrin-dependent cell spreading

Integrins link the extracellular matrix (ECM) to the cytoskeleton to control cell behaviors including adhesion, spreading and migration. Band 4.1 proteins contain 4.1, ezrin, radixin, moesin (FERM) domains that likely mediate signaling events and cytoskeletal reorganization via integrins. However, the mechanisms by which Band 4.1 proteins and integrins are functionally interconnected remain enigmatic. Here we have investigated roles for Band 4.1 proteins in integrin-mediated cell spreading using primary astrocytes as a model system. We demonstrate that Proteins 4.1B and 4.1G show dynamic patterns of sub-cellular localization in astrocytes spreading on fibronectin. During early stages of cell spreading Proteins 4.1B and 4.1G are enriched in ECM adhesion sites but become more diffusely localized at later stages of spreading. Combinatorial inactivation of Protein 4.1B and 4.1G expression leads to impaired astrocyte spreading. Furthermore, in exogenous expression systems we show that the isolated Protein 4.1 FERM domain significantly enhances integrin-mediated cell spreading. Protein 4.1B is dispensable for reactive astrogliosis in experimental models of cortical injury, likely due to functional compensation by related Protein 4.1 family members. Collectively, these findings reveal that Band 4.1 proteins are important intracellular components for integrin-mediated cell spreading.     

Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily

The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R) is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca2+. Non-erythroid cells express the 4.1R homologues: 4.1G (general type), 4.1B (brain type), and 4.1N (neuron type), and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1G, 4.1N and 4.1B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH) proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as focal adhesion kinase (FAK), non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication.     

Neuroglian,Gliotactin, and the Na+/K+ ATPase are essential for septate junction function in Drosophila

One essential function of epithelia is to form a barrier between the apical and basolateral surfaces of the epithelium. In vertebrate epithelia, the tight junction is the primary barrier to paracellular flow across epithelia, whereas in invertebrate epithelia, the septate junction (SJ) provides this function. In this study, we identify new proteins that are required for a functional paracellular barrier in Drosophila. In addition to the previously known components Coracle (COR) and Neurexin (NRX), we show that four other proteins, Gliotactin, Neuroglian (NRG), and both the alpha and beta subunits of the Na+/K+ ATPase, are required for formation of the paracellular barrier. In contrast to previous reports, we demonstrate that the Na pump is not localized basolaterally in epithelial cells, but instead is concentrated at the SJ. Data from immunoprecipitation and somatic mosaic studies suggest that COR, NRX, NRG, and the Na+/K+ ATPase form an interdependent complex. Furthermore, the observation that NRG, a Drosophila homologue of vertebrate neurofascin, is an SJ component is consistent with the notion that the invertebrate SJ is homologous to the vertebrate paranodal SJ. These findings have implications not only for invertebrate epithelia and barrier functions, but also for understanding of neuron-glial interactions in the mammalian nervous system.     

Gap junction channel protein innexin 2 is essential for epithelial morphogenesis in the Drosophila embryo

Direct communication of neighboring cells by gap junction channels is essential for the development of tissues and organs in the body. Whereas vertebrate gap junctions are composed of members of the connexin family of transmembrane proteins, in invertebrates gap junctions consist of Innexin channel proteins. Innexins display very low sequence homology to connexins. In addition, very little is known about their cellular role during developmental processes. In this report, we examined the function and the distribution of Drosophila Innexin 2 protein in embryonic epithelia. Both loss-of-function and gain-of-function innexin 2 mutants display severe developmental defects due to cell death and a failure of proper epithelial morphogenesis. Furthermore, immunohistochemical analyses using antibodies against the Innexins 1 and 2 indicate that the distribution of Innexin gap junction proteins to specific membrane domains is regulated by tissue specific factors. Finally, biochemical interaction studies together with genetic loss- and gain-of-function experiments provide evidence that Innexin 2 interacts with core proteins of adherens and septate junctions. This is the first study, to our knowledge, of cellular distribution and protein-protein interactions of an Innexin gap junctional channel protein in the developing epithelia of Drosophila.     

The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions.

Mutations of the lethal(1)discs large-1 (dlg) tumor suppressor gene of Drosophila cause neoplastic overgrowth of the imaginal discs. Sequencing of a near full-length cDNA predicts a protein containing a domain homologous to yeast guanylate kinase and a region homologous to SH3, a putative regulatory motif in nonreceptor protein tyrosine kinases and other signal transduction proteins. Immunofluorescence analysis using antibodies directed against fusion peptides shows that the dlg gene product is localized in an apical belt of the lateral cell membrane, at the position of the septate junction. The results suggest that a signal transduction process involving guanine nucleotides occurs at the septate junction and is necessary for cell proliferation control in Drosophila epithelia.     

Epithelial septate junction assembly relies on melanotransferrin iron binding and endocytosis in Drosophila

Iron is an essential element in many biological processes. In vertebrates, serum transferrin is the major supplier of iron to tissues, but the function of additional transferrin-like proteins remains poorly understood. Melanotransferrin (MTf) is a phylogenetically conserved, iron-binding epithelial protein. Elevated MTf levels have been implicated in melanoma pathogenesis. Here, we present a functional analysis of MTf in Drosophila melanogaster. Similarly to its human homologue, Drosophila MTf is a lipid-modified, iron-binding protein attached to epithelial cell membranes, and is a component of the septate junctions that form the paracellular permeability barrier in epithelial tissues. We demonstrate that septate junction assembly during epithelial maturation relies on endocytosis and apicolateral recycling of iron-bound MTf. Mouse MTf complements the defects of Drosophila MTf mutants. Drosophila provides the first genetic model for the functional dissection of MTf in epithelial junction assembly and morphogenesis.     

Drosophila Yurt is a new protein-4.1-like protein required for epithelial morphogenesis

Proteins of the 4.1 family play a key role in the integrity of the cytoskeleton and in epithelial tissue movement, as shown by the disruption of the actin cytoskeleton in human erythrocytes caused by genetic loss of protein 4.1, and the failure of epithelial tissue migration during Drosophila embryogenesis caused by genetic loss of the 4.1 homolog Coracle. Here we report the genetic characterization of Yurt, a novel protein 4.1 family member in Drosophila that is associated with the plasma membrane of epithelial cells. Homozygous loss-of-function mutations in the yurt gene cause failure of germ-band retraction, dorsal closure, and head involution, associated with degeneration of the amnioserosa and followed by embryonic lethality. A mammalian homolog of Yurt is up-regulated in metastatic melanoma cells. These novel cytoskeletal proteins appear to play important roles in epithelial cell movements and in the morphogenetic tissue changes that depend on them.     

The Protein 4.1 family: Hub proteins in animals for organizing membrane proteins

Proteins of the 4.1 family are characteristic of eumetazoan organisms. Invertebrates contain single 4.1 genes and the Drosophila model suggests that 4.1 is essential for animal life. Vertebrates have four paralogues, known as 4.1R, 4.1N, 4.1G and 4.1B, which are additionally duplicated in the ray-finned fish. Protein 4.1R was the first to be discovered: it is a major mammalian erythrocyte cytoskeletal protein, essential to the mechanochemical properties of red cell membranes because it promotes the interaction between spectrin and actin in the membrane cytoskeleton. 4.1R also binds certain phospholipids and is required for the stable cell surface accumulation of a number of erythrocyte transmembrane proteins that span multiple functional classes; these include cell adhesion molecules, transporters and a chemokine receptor. The vertebrate 4.1 proteins are expressed in most tissues, and they are required for the correct cell surface accumulation of a very wide variety of membrane proteins including G-Protein coupled receptors, voltage-gated and ligand-gated channels, as well as the classes identified in erythrocytes. Indeed, such large numbers of protein interactions have been mapped for mammalian 4.1 proteins, most especially 4.1R, that it appears that they can act as hubs for membrane protein organization. The range of critical interactions of 4.1 proteins is reflected in disease relationships that include hereditary anaemias, tumour suppression, control of heartbeat and nervous system function. The 4.1 proteins are defined by their domain structure: apart from the spectrin/actin-binding domain they have FERM and FERM-adjacent domains and a unique C-terminal domain. Both the FERM and C-terminal domains can bind transmembrane proteins, thus they have the potential to be cross-linkers for membrane proteins. The activity of the FERM domain is subject to multiple modes of regulation via binding of regulatory ligands, phosphorylation of the FERM associated domain and differential mRNA splicing. Finally, the spectrum of interactions of the 4.1 proteins overlaps with that of another membrane-cytoskeleton linker, ankyrin. Both ankyrin and 4.1 link to the actin cytoskeleton via spectrin, and we hypothesize that differential regulation of 4.1 proteins and ankyrins allows highly selective control of cell surface protein accumulation and, hence, function. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé     

The Na+/K+ ATPase is required for septate junction function and epithelial tube-size control in the Drosophila tracheal system

Although the correct architecture of epithelial tubes is crucial for the function of organs such as the lung, kidney and vascular system, little is known about the molecular mechanisms that control tube size. We show that mutations in the ATPalpha alpha and nrv2 beta subunits of the Na+/K+ ATPase cause Drosophila tracheal tubes to have increased lengths and expanded diameters. ATPalpha and nrv2 mutations also disrupt stable formation of septate junctions, structures with some functional and molecular similarities to vertebrate tight junctions. The Nrv2 beta subunit isoforms have unique tube size and junctional functions because Nrv2, but not other Drosophila Na+/K+ ATPase beta subunits, can rescue nrv2 mutant phenotypes. Mutations in known septate junctions genes cause the same tracheal tube-size defects as ATPalpha and nrv2 mutations, indicating that septate junctions have a previously unidentified role in epithelial tube-size control. Double mutant analyses suggest that tube-size control by septate junctions is mediated by at least two discernable pathways, although the paracellular diffusion barrier function does not appear to involved because tube-size control and diffusion barrier function are genetically separable. Together, our results demonstrate that specific isoforms of the Na+/K+ ATPase play a crucial role in septate junction function and that septate junctions have multiple distinct functions that regulate paracellular transport and epithelial tube size.     

Muscle dystroglycan organizes the postsynapse and regulates presynaptic neurotransmitter release at the Drosophila neuromuscular junction

Background

The Dystrophin-glycoprotein complex (DGC) comprises dystrophin, dystroglycan, sarcoglycan, dystrobrevin and syntrophin subunits. In muscle fibers, it is thought to provide an essential mechanical link between the intracellular cytoskeleton and the extracellular matrix and to protect the sarcolemma during muscle contraction. Mutations affecting the DGC cause muscular dystrophies. Most members of the DGC are also concentrated at the neuromuscular junction (NMJ), where their deficiency is often associated with NMJ structural defects. Hence, synaptic dysfunction may also intervene in the pathology of dystrophic muscles. Dystroglycan is a central component of the DGC because it establishes a link between the extracellular matrix and Dystrophin. In this study, we focused on the synaptic role of Dystroglycan (Dg) in Drosophila.

Methodology/Principal Findings

We show that Dg was concentrated postsynaptically at the glutamatergic NMJ, where, like in vertebrates, it controls the concentration of synaptic Laminin and Dystrophin homologues. We also found that synaptic Dg controlled the amount of postsynaptic 4.1 protein Coracle and alpha-Spectrin, as well as the relative subunit composition of glutamate receptors. In addition, both Dystrophin and Coracle were required for normal Dg concentration at the synapse. In electrophysiological recordings, loss of postsynaptic Dg did not affect postsynaptic response, but, surprisingly, led to a decrease in glutamate release from the presynaptic site.

Conclusion/Significance

Altogether, our study illustrates a conservation of DGC composition and interactions between Drosophila and vertebrates at the synapse, highlights new proteins associated with this complex and suggests an unsuspected trans-synaptic function of Dg.     

Expanded functions in the apical cell domain to regulate the growth rate of imaginal discs

The Drosophila expanded (ex) gene encodes a product (Ex) that shares homology with the Protein 4.1 family of proteins, many of which are enriched at specific lateral cell junctions and the apical cellular domain. Ex colocalizes with actin in the apical domain of imaginal disc epithelial cells, where it partially overlaps the distribution of phosphotyrosine (PY)-containing proteins. This suggests that Ex is present in or associated with adherens junctions. Genetic studies show that Ex is necessary for proper regulation of final cell number in adult wings and for the formation of eyes, distal leg, and distal antennal segments. We have generated mitotic clones that lack Ex using the twin spot technique, and demonstrated that the primary function of Ex is to regulate cell proliferation. Overexpressing Ex protein results in a decrease in final cell number in wings, suggesting a direct relationship between Ex function and proliferation rate. Dev. Genet. 20:103–110, 1997. © 1997 Wiley-Liss, Inc.     

Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia

The Discs large (Dlg) protein of Drosophila is the prototypic member of a growing family of proteins termed membrane-associated guanylate kinase homologs (MAGUKs). The MAGUKs are composed of a series of peptide domains that include one or three DHR/PDZs, an SH3, and a region homologous to guanylate kinase (GUK). We have previously shown that the product of this gene, the Dlg protein, is localized at the septate junctions between epithelial cells, and that mutations in the gene cause neoplastic overgrowth of the imaginal discs. The dlg locus is therefore defined as a tumor suppressor gene. In this paper, we show that the Dlg protein is localized on the cytoplasmic face of the septate junction and is required for the maintenance of this structure. It is also required for proper organization of the cytoskeleton, for the differential localization of membrane proteins, and for apicobasal polarity of epithelial cells. However, these other functions can be uncoupled from Dlg's role as a tumor suppressor since mutations in two domains of the protein, the SH3 and GUK, cause loss of normal cell proliferation control without affecting the other functions of the protein. These results suggest that, besides regulating cellular proliferation, the Dlg protein is a critical component of the septate junctions and is required for maintaining apicobasal polarity in Drosophila epithelium.     

Nak regulates Dlg basal localization in Drosophila salivary gland cells

Protein trafficking is highly regulated in polarized cells. During development, how the trafficking of cell junctional proteins is regulated for cell specialization is largely unknown. In the maturation of Drosophila larval salivary glands (SGs), the Dlg protein is essential for septate junction formation. We show that Dlg was enriched in the apical membrane domain of proximal cells and localized basolaterally in distal mature cells. The transition of Dlg distribution was disrupted in nak mutants. Nak associated with the AP-2 subunit α-Ada and the AP-1 subunit AP-1γ. In SG cells disrupting AP-1 and AP-2 activities, Dlg was enriched in the apical membrane. Therefore, Nak regulates the transition of Dlg distribution likely through endocytosis of Dlg from the apical membrane domain and transcytosis of Dlg to the basolateral membrane domain during the maturation of SGs development.