The Drosophila Claudin Kune-kune Is Required for Septate Junction Organization and Tracheal Tube Size Control

The vertebrate tight junction is a critical claudin-based cell–cell junction that functions to prevent free paracellular diffusion between epithelial cells. In Drosophila, this barrier is provided by the septate junction, which, despite being ultrastructurally distinct from the vertebrate tight junction, also contains the claudin-family proteins Megatrachea and Sinuous. Here we identify a third Drosophila claudin, Kune-kune, that localizes to septate junctions and is required for junction organization and paracellular barrier function, but not for apical-basal polarity. In the tracheal system, septate junctions have a barrier-independent function that promotes lumenal secretion of Vermiform and Serpentine, extracellular matrix modifier proteins that are required to restrict tube length. As with Sinuous and Megatrachea, loss of Kune-kune prevents this secretion and results in overly elongated tubes. Embryos lacking all three characterized claudins have tracheal phenotypes similar to any single mutant, indicating that these claudins act in the same pathway controlling tracheal tube length. However, we find that there are distinct requirements for these claudins in epithelial septate junction formation. Megatrachea is predominantly required for correct localization of septate junction components, while Sinuous is predominantly required for maintaining normal levels of septate junction proteins. Kune-kune is required for both localization and levels. Double- and triple-mutant combinations of Sinuous and Megatrachea with Kune-kune resemble the Kune-kune single mutant, suggesting that Kune-kune has a more central role in septate junction formation than either Sinuous or Megatrachea.EPITHELIA are essential for separating physiologically distinct body compartments and regulating trafficking between them. For proper function, it is imperative that epithelia maintain effective barriers against free paracellular diffusion. To this end, epithelial cells contain occluding junctions, which regulate paracellular permeability. In vertebrates, this is accomplished by tight junctions (TJ), structures that are characterized by regions of close membrane apposition between adjacent cells known as “kissing points” (Tsukita and Furuse 2002). While the TJ is made up of at least 40 different components (Schneeberger and Lynch 2004), the core proteins responsible for the paracellular barrier are the claudins (Angelow et al. 2008).Claudins are four-transmembrane domain proteins that form homo- and heterophilic interactions within the same cell (Furuse et al. 1999; Blasig et al. 2006) and with claudins in adjacent cells (Furuse et al. 1999), thereby establishing the paracellular seal. There are 24 members of the claudin family in mammals, many of which display distinct, tissue-specific expression patterns (Kiuchi-Saishin et al. 2002; Angelow et al. 2008). Mutations in several claudins can cause significant paracellular permeability defects in mice. For example, mutations in claudin-14 increase TJ permeability in the organ of Corti and cause deafness (Ben-Yosef et al. 2003), while loss of claudin-1 compromises epidermal barrier function (Furuse et al. 2002).In Drosophila, primary (ectodermally derived) epithelia lack discernable TJs and instead use pleated septate junctions (SJ) for the paracellular barrier (Baumgartner et al. 1996; Lamb et al. 1998; Genova and Fehon 2003; Paul et al. 2003). However, despite sharing a common barrier function, vertebrate TJs and invertebrate SJs differ in several ways. While vertebrate TJs are positioned apical to adherens junctions (AJ) and contain conserved apical polarity proteins, SJs are basal to AJs and contain conserved basolateral polarity proteins (reviewed in Tepass 2003; Wu and Beitel 2004). In addition, SJs do not contain kissing points, but rather ladder-like septa that span the intermembrane space (Lane and Swales 1982; Tepass and Hartenstein 1994).Beyond their general epithelial barrier function, SJs are also required for several tissue-specific processes. Glial cells, for example, ensheath nerve fibers and use SJs to maintain the blood–brain barrier (Auld et al. 1995; Baumgartner et al. 1996; Schwabe et al. 2005). In the embryonic tracheal system, SJs are required for the apical secretion of the lumenal matrix modifying proteins, Vermiform (Verm) and Serpentine (Serp), which act through undefined pathways to restrict tube length (Wang et al. 2006). This secretory pathway appears to be specific for Verm and Serp, since other apical proteins are secreted normally in SJ mutants. SJ proteins have also been shown to play a role in morphogenesis of the heart tube, even though this tissue lacks typical SJ septa (Yi et al. 2008).Although SJs have clear differences from vertebrate TJs, SJs contain at least two claudins, Megatrachea (Mega) and Sinuous (Sinu), both of which are required for the paracellular barrier (Behr et al. 2003; Wu et al. 2004; Stork et al. 2008). In this article, we identify a third claudin, Kune-kune (Kune), that is an integral SJ protein. Like the other claudins, Kune is required for maintaining epithelial paracellular barrier and tracheal tube size control and is not required for apical-basal polarity. We also find that, of all three characterized claudins, Kune has a more severe SJ phenotype, suggesting that it is a more central player in SJ organization and function than previously characterized Drosophila claudins.     

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.     

Anterior and intermediate pituitary tissues express claudin 4 in follicle stellate cells and claudins 2 and 5 in endothelial cells

Follicle-stellate cells are pituitary non-granular cells that are arranged between secretory cells or organized in follicles with small lumens. Cells from the follicles exhibit the typical phenotype of a transporting epithelium, including apical microvilli with a cilium and tight junctions. Freeze-fracture electron microscopy images show that the tight junctions consist of 5–7 anastomosing strands and that cultured follicle-stellate cells develop a trans-epithelial electrical resistance characteristic of “tight” epithelia. Here, we investigate the molecular composition of the tight junction from follicle stellate cells. We found that the rat anterior pituitary lobe expresses mRNAs for claudins 2, 4 and 5; the proteins of all these claudins are observed in the anterior lobe, whereas the intermediate lobe expresses claudins 2 and 5 and the posterior lobe contains only claudin 5. Follicle-stellate cells, identified by their protein marker S100β, expresses claudin 4 in the apical membrane, in co-localization with dipeptidyl-peptidase and near acetylated β-tubulin. Claudin 4 partially co-localizes with E-cadherin, indicating that a fraction of the protein is located in the basolateral domain. Follicle-stellate-enriched cell cultures develop patches of polygonal cells expressing claudin 4 and E-cadherin, encircled by extensive monolayers of fusiform cells. Claudin 2 stains specifically blood vessels, identified by claudin 5 and VE-cadherin labels. Thus, follicles in the anterior pituitary consist of “tight” epithelia that can carry out intense vectorial transport, together with a high cation movement in blood vessels, possibly related to the ion requirements of excitable secretory cells for hormone secretion.     

Claudins--key pieces in the tight junction puzzle

Tight junctions restrict the flow of ions and aqueous molecules between cells by forming a selective barrier to the paracellular pathway. Permeability of the tight junction barrier is determined by a class of transmembrane proteins known as claudins. The relationship between claudins and paracellular permeability is complex and determined not only by the profile of claudin expression but also by the arrangement of claudins and other proteins into tight junction strands. This review summarizes progress in understanding how claudins are assembled into tight junctions and how they interact with other tight junction proteins.     

Clostridium perfringens Enterotoxin Interacts with Claudins via Electrostatic Attraction

Clostridium perfringens enterotoxin (CPE), a causative agent of food poisoning, is a pore-forming toxin disrupting the selective permeability of the plasma membrane of target cells, resulting in cell death. We previously identified claudin as the cell surface receptor for CPE. Claudin, a component of tight junctions, is a tetratransmembrane protein and constitutes a large family of more than 20 members, not all of which serve as the receptor for CPE. The mechanism by which the toxin distinguishes the sensitive claudins is unknown. In this study, we localized the region of claudin responsible for interaction with CPE to the C-terminal part of the second extracellular loop and found that the isoelectric point of this region in sensitive claudins was higher than insensitive claudins. Amino acid substitutions to lower the pI resulted in reduced sensitivity to CPE among sensitive claudins, whereas substitutions to raise the pI endowed CPE-insensitive claudins with sensitivity. The steric structure of the claudin-binding domain of CPE reveals an acidic cleft surrounded by Tyr³⁰⁶, Tyr³¹⁰, Tyr³¹², and Leu³¹⁵, which were reported to be essential for interaction with the sensitive claudins. These results imply that an electrostatic attraction between the basic claudin region and the acidic CPE cleft is involved in their interaction.     

Dynamic changes to claudins in the uterine epithelial cells of the marsupial Sminthopsis crassicaudata (Dasyuridae) during pregnancy

The fluid that surrounds the embryo in the uterus contains important nourishing factors and secretions. To maintain the distinct microenvironment in the uterine lumen, the tight junctions between uterine epithelial cells are remodeled to decrease paracellular movement of molecules and solutes. Modifications to tight junctions between uterine epithelial cells is a common feature of pregnancy in eutherian mammals, regardless of placental type. Here we used immunofluorescence microscopy and western blot analysis to describe distributional changes to tight junctional proteins, claudin‐1, ‐3, ‐4, and ‐5, in the uterine epithelial cells of a marsupial species, Sminthopsis crassicaudata. Immunofluorescence microscopy revealed claudin‐1, ‐3, and ‐5 in the tight junctions of the uterine epithelium of S. crassicaudata during pregnancy. These specific claudins are associated with restricting passive movement of fluid between epithelial cells in eutherians. Hence, their function during pregnancy in S. crassicaudata may be to maintain the uterine luminal content surrounding developing embryos. Claudin‐4 disappears from all uterine regions of S. crassicaudata at the time of implantation, in contrast with the distribution of this claudin in some eutherian mammals. We conclude that like eutherian mammals, distributional changes to claudins in the uterine epithelial cells of S. crassicaudata are necessary to support pregnancy. However, the combination of individual claudin isoforms in the tight junctions of the uterine epithelium of S. crassicaudata differs from that of eutherian mammals. Our findings suggest that the precise permeability of the paracellular pathway of the uterine epithelium is species‐specific.     

Claudins in occluding junctions of humans and flies

The epithelial barrier is fundamental to the physiology of most metazoan organ systems. Occluding junctions, including vertebrate tight junctions and invertebrate septate junctions, contribute to the epithelial barrier function by restricting free diffusion of solutes through the paracellular route. The recent identification and characterization of claudins, which are tight junction-associated adhesion molecules, gives insight into the molecular architecture of tight junctions and their barrier-forming mechanism in vertebrates. Mice lacking the expression of various claudins, and human hereditary diseases with claudin mutations, have revealed that the claudin-based barrier function of tight junctions is indispensable in vivo. Interestingly, claudin-like molecules have recently been identified in septate junctions of Drosophila. Here, we present an overview of recent progress in claudin studies conducted in mammals and flies.     

Tight junction claudins and the kidney in sickness and in health

The epithelial cell tight junction has several functions including the control of paracellular transport between epithelial cells. Renal paracellular transport has been long recognized to exhibit unique characteristics within different segments of the nephron, functions as an important component of normal renal physiology and has been speculated to contribute to renal related pathology if functioning abnormally. The discovery of a large family of tight junction associated 4-transmembrane spanning domain proteins named claudins has advanced our understanding on how the paracellular permeability properties of tight junctions are determined. In the kidney, claudins are expressed in a nephron-specific pattern and are major determinants of the paracellular permeability of tight junctions in different nephron segments. The combination of nephron segment claudin expression patterns, inherited renal diseases, and renal epithelial cell culture models is providing important clues about how tight junction claudin molecules function in different segments of the nephron under normal and pathological conditions. This review discusses early observations of renal tubule paracellular transport and more recent information on the discovery of the claudin family of tight junction associated membrane proteins and how they relate to normal renal function as well as diseases of the human kidney.     

Claudins in the brain: Unconventional functions in neurons

Bonafide claudin proteins are functional and structural components of tight junctions and are largely responsible for barrier formation across epithelial and endothelial membranes. However, current advances in the understanding of claudin biology have revealed their unexpected functions in the brain. Apart from maintaining blood‐brain barriers in the brain, other functions of claudins in neurons and at synapses have been largely elusive and are just coming to light. In this review, we summarize the functions of claudins in the brain and their association in neuronal diseases. Further, we go on to cover some recent studies that show that claudins play signaling functions in neurons by regulating trafficking of postsynaptic receptors and controlling dendritic morphogenesis in the model organism Caenorhabditis elegans.     

Septate junctions are required for ommatidial integrity and blood-eye barrier function in Drosophila

The anatomical organization of the Drosophila ommatidia is achieved by specification and contextual placement of photoreceptors, cone and pigment cells. The photoreceptors must be sealed from high ionic concentrations of the hemolymph by a barrier to allow phototransduction. In vertebrates, a blood-retinal barrier (BRB) is established by tight junctions (TJs) present in the retinal pigment epithelium and endothelial membrane of the retinal vessels. In Drosophila ommatidia, the junctional organization and barrier formation is poorly understood. Here we report that septate junctions (SJs), the vertebrate analogs of TJs, are present in the adult ommatidia and are formed between and among the cone and pigment cells. We show that the localization of Neurexin IV (Nrx IV), a SJ-specific protein, coincides with the location of SJs in the cone and pigment cells. Somatic mosaic analysis of nrx IV null mutants shows that loss of Nrx IV leads to defects in ommatidial morphology and integrity. nrx IV hypomorphic allelic combinations generated viable adults with defective SJs and displayed a compromised blood-eye barrier (BEB) function. These findings establish that SJs are essential for ommatidial integrity and in creating a BEB around the ion and light sensitive photoreceptors. Our studies may provide clues towards understanding the vertebrate BEB formation and function.     

A Laminin G-EGF-Laminin G module in Neurexin IV is essential for the apico-lateral localization of Contactin and organization of septate junctions

Septate junctions (SJs) display a unique ultrastructural morphology with ladder-like electron densities that are conserved through evolution. Genetic and molecular analyses have identified a highly conserved core complex of SJ proteins consisting of three cell adhesion molecules Neurexin IV, Contactin, and Neuroglian, which interact with the cytoskeletal FERM domain protein Coracle. How these individual proteins interact to form the septal arrays that create the paracellular barrier is poorly understood. Here, we show that point mutations that map to specific domains of neurexin IV lead to formation of fewer septae and disorganization of SJs. Consistent with these observations, our in vivo domain deletion analyses identified the first Laminin G-EGF-Laminin G module in the extracellular region of Neurexin IV as necessary for the localization of and association with Contactin. Neurexin IV protein that is devoid of its cytoplasmic region is able to create septae, but fails to form a full complement of SJs. These data provide the first in vivo evidence that specific domains in Neurexin IV are required for protein-protein interactions and organization of SJs. Given the molecular conservation of SJ proteins across species, our studies may provide insights into how vertebrate axo-glial SJs are organized in myelinated axons.     

The claudins

The claudin multigene family encodes tetraspan membrane proteins that are crucial structural and functional components of tight junctions, which have important roles in regulating paracellular permeability and maintaining cell polarity in epithelial and endothelial cell sheets. In mammals, the claudin family consists of 24 members, which exhibit complex tissue-specific patterns of expression. The extracellular loops of claudins from adjacent cells interact with each other to seal the cellular sheet and regulate paracellular transport between the luminal and basolateral spaces. The claudins interact with multiple proteins and are intimately involved in signal transduction to and from the tight junction. Several claudin mouse knockout models have been generated and the diversity of phenotypes observed clearly demonstrates their important roles in the maintenance of tissue integrity in various organs. In addition, mutation of some claudin genes has been causatively associated with human diseases and claudin genes have been found to be deregulated in various cancers. The mechanisms of claudin regulation and their exact roles in normal physiology and disease are being elucidated, but much work remains to be done. The next several years are likely to witness an explosion in our understanding of these proteins, which may, in turn, provide new approaches for the targeted therapy of various diseases.     

Calmodulin-Dependent Protein Phosphorylation in Synaptic Junctions

Synaptic junctions (SJs) from rat forebrain were examined for Ca2+/calmodulin (CaM)-dependent kinase activity and compared to synaptic plasma membrane (SPM) and postsynaptic density (PSD) fractions. The kinase activity in synaptic fractions was examined for its capacity to phosphorylate endogenous proteins or exogenous synapsin I, in the presence or absence of Ca2+ plus CaM. When assayed for endogenous protein phosphorylation, SJs contained approximately 25-fold greater amounts of Ca2+/CAM-dependent kinase activity than SPMs, and fivefold more activity than PSDs. When kinase activities were measured by phosphorylation of exogenous synapsin I, SJs contained fourfold more activity than SPMs, and 10-fold more than PSDs. The phosphorylation of SJ proteins of 60- and 50-kilodalton (major PSD protein) polypeptides were greatly stimulated by Ca2+/CaM; levels of phosphorylation for these proteins were 23- and 17-fold greater than basal levels, respectively. Six additional proteins whose phosphorylation was stimulated 6-15-fold by Ca2+/CAM were identified in SJs. These proteins include synapsin I, and proteins of 240, 207, 170, 140, and 54 kilodaltons. The 54-kilodalton protein is a highly phosphorylated form of the major PSD protein and the 170-kilodalton component is a cell-surface glycoprotein of the postsynaptic membrane that binds concanavalin A. The CaM-dependent kinase in SJ fractions phosphorylated endogenous phosphoproteins at serine and/or threonine residues. Ca2+-dependent phosphorylation in SJ fractions was strictly dependent on exogenous CaM, even though SJs contained substantial amounts of endogenous CaM (15 micrograms CaM/mg SJ protein). Exogenous CaM, after being functionally incorporated into SJs, was rapidly removed by sequential washings. These observations suggest that the SJ-associated CaM involved in regulating Ca2+-dependent protein phosphorylation may be in dynamic equilibrium with the cytoplasm. These findings indicate that a brain CaM-dependent kinase(s) and substrate proteins are concentrated at SJs and that CaM-dependent protein phosphorylation may play an important role in mechanisms that underlie synaptic communication.     

Developmental regulation of claudin localization by fetal alveolar epithelial cells

Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.     

Double gene deletion reveals lack of cooperation between claudin 11 and claudin 14 tight junction proteins

Members of the claudin family of proteins are the main components of tight junctions (TJs), the major selective barrier of the paracellular pathway between epithelial cells. The selectivity and specificity of TJ strands are determined by the type of claudins present. An understanding of the cooperation between different claudins in various tissues is thus important. To study the possible cooperation between claudin 11 and claudin 14, we have generated claudin 11/claudin 14 double-deficient mice, which exhibit a combination of the phenotypes found in each of the singly deficient mutants, including deafness, neurological deficits, and male sterility. These two claudins have distinct and partially overlapping expression patterns in the kidney. Claudin 11 is located in both the proximal and distal convoluted tubules, whereas claudin 14 occurs in both the thin descending and thick ascending limbs of the loop of Henle and in the proximal convoluted tubules. Although daily urinary excretion of Mg(++), and to a lesser extent of Ca(++), tends to be higher in claudin 11/claudin 14 double mutants, these changes do not reach statistical significance compared with wild-type animals. Thus, under normal conditions, co-deletion of claudin 11 and claudin 14 does not affect kidney function or ion balance. Our data demonstrate that, despite the importance of each of these claudins, there is probably no functional cooperation between them. Generation of additional mouse models in which different claudins are abolished should provide further insight into the complex interactions between claudin proteins in various physiological systems.     

Ochratoxin A displaces claudins from detergent resistant membrane microdomains

Ochratoxin A (OchA) is a food-borne mycotoxin with multiple effects in vivo. Previously, we have demonstrated that the toxin can significantly impair the barrier function of the gut epithelial cell line, Caco-2. Barrier disruption involved loss of claudins 3 and 4, but not claudin 1 from the tight junction complex. In this study, we demonstrate for the first time, that OchA is able to remove claudins 3 and 4 from the detergent insoluble membrane microdomains associated with the tight junctions. However, cholesterol distribution within the microdomain was unaffected by the toxin. In addition, the thiol antioxidant, N-acetyl cysteine, preserved the microdomain localisation of claudins and also the barrier function of Caco-2 cells. This work suggests that OchA-mediated barrier toxicity is due to removal of claudins from detergent insoluble membrane microdomains. Moreover, loss of microdomain association may be due to oxidative events.