DRG1 Maintains Intestinal Epithelial Cell Junctions and Barrier Function by Regulating RAC1 Activity in Necrotizing Enterocolitis.

BACKGROUND: An immature intestine is a high-risk factor for necrotizing enterocolitis (NEC), which is a serious intestinal disease in newborns. The regulation of developmentally regulated GTP-binding protein 1 (DRG1) during organ development suggests a potential role of DRG1 in the maturation process of the intestine. AIM: To illustrate the function of DRG1 during the pathogenesis of NEC. METHODS: DRG1 expression in the intestine was measured using immunohistochemistry and q-PCR. Immunoprecipitation coupled with mass spectrometry was used to identify the interacting proteins of DRG1. The biological functions of the potential interactors were annotated with the Database for Annotation, Visualization and Integrated Discovery. Caco2 and FHs74Int cells with stable DRG1 silencing or overexpression were used to investigate the influence of DRG1 on cell junctions and intestinal barrier permeability and to elucidate the downstream mechanism. RESULTS: DRG1 was constitutively expressed during the intestinal maturation process but significantly decreased in the ileum in the context of NEC. Protein interaction analysis revealed that DRG1 was closely correlated with cell junctions. DRG1 deficiency destabilized the E-cadherin and occludin proteins near the cell membrane and increased the permeability of the epithelial cell monolayer, while DRG1 overexpression prevented lipopolysaccharide-induced disruption of E-cadherin and occludin expression and cell monolayer integrity. Further investigation suggested that DRG1 maintained cell junctions, especially adherens junctions, by regulating RAC1 activity, and RAC1 inhibition with NSC23766 attenuated intestinal injury and led to improved barrier integrity in experimental NEC. CONCLUSIONS: Our findings illustrate the mechanism underlying the effect of DRG1 deficiency on epithelial cell permeability regulation and provide evidence supporting the application of RAC1 inhibitors for protection against NEC.

Small GTPase Rap1A/B Is Required for Lymphatic Development and Adrenomedullin-Induced Stabilization of Lymphatic Endothelial Junctions.

Objective- Maintenance of lymphatic permeability is essential for normal lymphatic function during adulthood, but the precise signaling pathways that control lymphatic junctions during development are not fully elucidated. The Gs-coupled AM (adrenomedullin) signaling pathway is required for embryonic lymphangiogenesis and the maintenance of lymphatic junctions during adulthood. Thus, we sought to elucidate the downstream effectors mediating junctional stabilization in lymphatic endothelial cells. Approach and Results- We knocked-down both Rap1A and Rap1B isoforms in human neonatal dermal lymphatic cells (human lymphatic endothelial cells) and genetically deleted the mRap1 gene in lymphatic endothelial cells by producing 2 independent, conditional Rap1a/b knockout mouse lines. Rap1A/B knockdown caused disrupted junctional formation with hyperpermeability and impaired AM-induced lymphatic junctional tightening, as well as rescue of histamine-induced junctional disruption. Less than 60% of lymphatic- Rap1a/b knockout embryos survived to E13.5 exhibiting interstitial edema, blood-filled lymphatics, disrupted lymphovenous valves, and defective lymphangiogenesis. Consistently, inducible lymphatic- Rap1a/b deletion in adult animals prevented AM-rescue of histamine-induced lymphatic leakage and dilation. Conclusions- Rap1 (Ras-related protein) serves as the dominant effector downstream of AM to stabilize lymphatic junctions. Rap1 is required for maintaining lymphatic permeability and driving normal lymphatic development.

PAK6 targets to cell-cell adhesions through its N-terminus in a Cdc42-dependent manner to drive epithelial colony escape.

The six serine/threonine kinases in the p21-activated kinase (PAK) family are important regulators of cell adhesion, motility and survival. PAK6, which is overexpressed in prostate cancer, was recently reported to localize to cell-cell adhesions and to drive epithelial cell colony escape. Here we report that PAK6 targeting to cell-cell adhesions occurs through its N-terminus, requiring both its Cdc42/Rac interactive binding (CRIB) domain and an adjacent polybasic region for maximal targeting efficiency. We find PAK6 localization to cell-cell adhesions is Cdc42-dependent, as Cdc42 knockdown inhibits PAK6 targeting to cell-cell adhesions. We further find the ability of PAK6 to drive epithelial cell colony escape requires kinase activity and is disrupted by mutations that perturb PAK6 cell-cell adhesion targeting. Finally, we demonstrate that all type II PAKs (PAK4, PAK5 and PAK6) target to cell-cell adhesions, albeit to differing extents, but PAK1 (a type I PAK) does not. Notably, the ability of a PAK isoform to drive epithelial colony escape correlates with its targeting to cell-cell adhesions. We conclude that PAKs have a broader role in the regulation of cell-cell adhesions than previously appreciated.

PAK5 mediates cell: cell adhesion integrity via interaction with E-cadherin in bladder cancer cells.

Urothelial bladder cancer is a major cause of morbidity and mortality worldwide, causing an estimated 150 000 deaths per year. Whilst non-muscle-invasive bladder tumours can be effectively treated, with high survival rates, many tumours recur, and some will progress to muscle-invasive disease with a much poorer long-term prognosis. Thus, there is a pressing need to understand the molecular transitions occurring within the progression of bladder cancer to an invasive disease. Tumour invasion is often associated with a down-regulation of E-cadherin expression concomitant with a suppression of cell:cell junctions, and decreased levels of E-cadherin expression have been reported in higher grade urothelial bladder tumours. We find that expression of E-cadherin in a panel of bladder cancer cell lines correlated with the presence of cell:cell junctions and the level of PAK5 expression. Interestingly, exogenous PAK5 has recently been described to be associated with cell:cell junctions and we now find that endogenous PAK5 is localised to cell junctions and interacts with an E-cadherin complex. Moreover, depletion of PAK5 expression significantly reduced junctional integrity. These data suggest a role for PAK5 in maintaining junctional stability and we find that, in both our own patient samples and a commercially available dataset, PAK5mRNA levels are reduced in human bladder cancer compared with normal controls. Taken together, the present study proposes that PAK5 expression levels could be used as a novel prognostic marker for bladder cancer progression.

Listeria monocytogenes antagonizes the human GTPase Cdc42 to promote bacterial spread.

The bacterial pathogen Listeria monocytogenes uses actin-based motility to spread from infected human cells to surrounding healthy cells. Cell-cell spread involves the formation of thin extensions of the host plasma membrane ('protrusions') containing motile bacteria. In cultured enterocytes, the Listeria protein InlC promotes protrusion formation by binding and antagonizing the human scaffolding protein Tuba. Tuba is a known activator of the GTPase Cdc42. In this work, we demonstrate an important role for Cdc42 in controlling Listeria spread. Infection of the enterocyte cell line Caco-2 BBE1 induced a decrease in the level of Cdc42-GTP, indicating that Listeria downregulates this GTPase. Genetic data involving RNA interference indicated that bacterial impairment of Cdc42 may involve inhibition of Tuba. Experiments with dominant negative and constitutively activated alleles of Cdc42 demonstrated that the ability to inactivate Cdc42 is required for efficient protrusion formation by Listeria. Taken together, these findings indicate a novel mechanism of bacterial spread involving pathogen-induced downregulation of host Cdc42.

Loss of glutathione S-transferase A4 accelerates obstruction-induced tubule damage and renal fibrosis.

Glutathione transferase isozyme A4 (GSTA4) exhibits high catalytic efficiency to metabolize 4-hydroxynonenal (4-HNE), a highly reactive lipid peroxidation product that has been implicated in the pathogenesis of various chronic diseases. We investigated the role of 4-HNE in the mechanisms of unilateral ureteral obstruction (UUO)-induced fibrosis and its modulation by GSTA4-4 in a mouse model. Our data indicate that after UUO, accumulation of 4-HNE and its adducts were increased in renal tissues, with a concomitant decrease in the expression of GSTA4-4 in mice. As compared to wild-type (WT) mice, UUO caused an increased expression of fibroblast markers in the interstitium of GSTA4 KO mice. Additionally, increased autophagy and tubular cell damage were more severe in UUO-treated GSTA4 KO mice than in WT mice. Furthermore, GSK-3ß phosphorylation and expression of Snail, a regulator of E-cadherin and Occludin, was found to be significantly higher in UUO-inflicted GSTA4 KO mice. GSTA4 over-expression prevented 4-HNE-induced autophagy activation, tubular cell damage and Snail nuclear translocation in vitro. The effects of long-term expression of GSTA4 in restoration of UUO-induced damage in mice with the GSTA4 inducible transposon system indicated that release of obstruction after 3 days of UUO resulted in the attenuation of interstitial SMAα and collagen I expression. This transposon-delivered GSTA4 expression also suppressed UUO-induced loss of tubular cell junction markers and autophagy activation. Together, these results indicate that 4-HNE significantly contributes to the mechanisms of tubule injury and fibrosis and that these effects can be inhibited by the enhanced expression of GSTA4-4.

The small GTPase RhoA is required to maintain spinal cord neuroepithelium organization and the neural stem cell pool.

The regulation of adherens junctions (AJs) is critical for multiple events during CNS development, including the formation and maintenance of the neuroepithelium. We have addressed the role of the small GTPase RhoA in the developing mouse nervous system using tissue-specific conditional gene ablation. We show that, in the spinal cord neuroepithelium, RhoA is essential to localize N-cadherin and ß-catenin to AJs and maintain apical-basal polarity of neural progenitor cells. Ablation of RhoA caused the loss of AJs and severe abnormalities in the organization of cells within the neuroepithelium, including decreased neuroepithelial cell proliferation and premature cell-cycle exit, reduction of the neural stem cell pool size, and the infiltration of neuroepithelial cells into the lumen of the ventricle. We also show that, in the absence of RhoA, its effector, mammalian diaphanous-related formin1 (mDia1), does not localize to apical AJs in which it likely stabilizes intracellular adhesion by promoting local actin polymerization and microtubule organization. Furthermore, expressing a dominant-negative form of mDia1 in neural stem/progenitor cells results in a similar phenotype compared with that of the RhoA conditional knock-out, namely the loss of AJs and apical polarity. Together, our data show that RhoA signaling is necessary for AJ regulation and for the maintenance of mammalian neuroepithelium organization preventing precocious cell-cycle exit and differentiation.

Matrix-specific p21-activated kinase activation regulates vascular permeability in atherogenesis.

Elevated permeability of the endothelium is thought to be crucial in atherogenesis because it allows circulating lipoproteins to access subendothelial monocytes. Both local hemodynamics and cytokines may govern endothelial permeability in atherosclerotic plaque. We recently found that p21-activated kinase (PAK) regulates endothelial permeability. We now report that onset of fluid flow, atherogenic flow profiles, oxidized LDL, and proatherosclerotic cytokines all stimulate PAK phosphorylation and recruitment to cell-cell junctions. Activation of PAK is higher in cells plated on fibronectin (FN) compared to basement membrane proteins in all cases. In vivo, PAK is activated in atherosclerosis-prone regions of arteries and correlates with FN in the subendothelium. Inhibiting PAK in vivo reduces permeability in atherosclerosis-prone regions. Matrix-specific PAK activation therefore mediates elevated vascular permeability in atherogenesis.

Increased matriptase zymogen activation in inflammatory skin disorders.

Matriptase, a type 2 transmembrane serine protease, and its inhibitor hepatocyte growth factor activator inhibitor (HAI)-1 are required for normal epidermal barrier function, and matriptase activity is tightly regulated during this process. We therefore hypothesized that this protease system might be deregulated in skin disease. To test this, we examined the level and activation state of matriptase in examples of 23 human skin disorders. We first examined matriptase and HAI-1 protein distribution in normal epidermis. Matriptase was detected at high levels at cell-cell junctions in the basal layer and spinous layers but was present at minimal levels in the granular layer. HAI-1 was distributed in a similar pattern, except that high-level expression was retained in the granular layer. This pattern of expression was retained in most skin disorders. We next examined the distribution of activated matriptase. Although activated matriptase is not detected in normal epidermis, a dramatic increase is seen in keratinocytes at the site of inflammation in 16 different skin diseases. To gain further evidence that activation is associated with inflammatory stimuli, we challenged HaCaT cells with acidic pH or H(2)O(2) and observed matriptase activation. These findings suggest that inflammation-associated reactive oxygen species and tissue acidity may enhance matriptase activation in some skin diseases.

Phospholipid scramblase 1 is secreted by a lipid raft-dependent pathway and interacts with the extracellular matrix protein 1 in the dermal epidermal junction zone of human skin.

We examined the interaction of ECM1 (extracellular matrix protein 1) using yeast two-hybrid screening and identified the type II transmembrane protein, PLSCR1 (phospholipid scramblase 1), as a binding partner. This interaction was then confirmed by in vitro and in vivo co-immunoprecipitation experiments, and additional pull-down experiments with GST-tagged ECM1a fragments localized this interaction to occur within the tandem repeat region of ECM1a. Furthermore, immunohistochemical staining revealed a partial overlap of ECM1 and PLSCR1 in human skin at the basal epidermal cell layer. Moreover, in human skin equivalents, both proteins are expressed at the basal membrane in a dermal fibroblast-dependent manner. Next, immunogold electron microscopy of ultrathin human skin sections showed that ECM1 and PLSCR1 co-localize in the extracellular matrix, and using antibodies against ECM1 or PLSCR1 cross-linked to magnetic immunobeads, we were able to demonstrate PLSCR1-ECM1 interaction in human skin extracts. Furthermore, whereas ECM1 is secreted by the endoplasmic/Golgi-dependent pathway, PLSCR1 release from HaCaT keratinocytes occurs via a lipid raft-dependent mechanism, and is deposited in the extracellular matrix. In summary, we here demonstrate that PLSCR1 interacts with the tandem repeat region of ECM1a in the dermal epidermal junction zone of human skin and provide for the first time experimental evidence that PLSCR1 is secreted by an unconventional secretion pathway. These data suggest that PLSCR1 is a multifunctional protein that can function both inside and outside of the cell and together with ECM1 may play a regulatory role in human skin.

Biochemical characterization of the cellular glycosylphosphatidylinositol-linked membrane type-6 matrix metalloproteinase.

Ubiquitously expressed membrane type-1 matrix metalloproteinase (MT1-MMP), an archetype member of the MMP family, binds tissue inhibitor of metalloproteinases-2 (TIMP-2), activates matrix metalloproteinase-2 (MMP-2), and stimulates cell migration in various cell types. In contrast with MT1-MMP, the structurally similar MT6-MMP associates with the lipid raft compartment of the plasma membrane using a GPI anchor. As a result, MT6-MMP is functionally distinct from MT1-MMP. MT6-MMP is insufficiently characterized as yet. In addition, a number of its biochemical features are both conflicting and controversial. To reassess the biochemical features of MT6-MMP, we have expressed the MT6-MMP construct tagged with a FLAG tag in breast carcinoma MCF-7 and fibrosarcoma HT1080 cells. We then used phosphatidylinositol-specific phospholipase C to release MT6-MMP from the cell surface and characterized the solubilized MT6-MMP fractions. We now are confident that cellular MT6-MMP partially exists in its complex with TIMP-2. Both TIMP-1 and TIMP-2 are capable of inhibiting the proteolytic activity of MT6-MMP. MT6-MMP does not stimulate cell migration. MT6-MMP, however, generates a significant level of gelatinolysis of the fluorescein isothiocyanate-labeled gelatin and exhibits an intrinsic, albeit low, ability to activate MMP-2. As a result, it is exceedingly difficult to record the activation of MMP-2 by cellular MT6-MMP. Because of its lipid raft localization, cellular MT6-MMP is inefficiently internalized. MT6-MMP is predominantly localized in the cell-to-cell junctions. Because MT6-MMP has been suggested to play a role in disease, including cancer and autoimmune multiple sclerosis, the identity of its physiologically relevant cleavage targets remains to be determined.

Differential role of Rho GTPases in intestinal epithelial barrier regulation in vitro.

Maintenance of intestinal epithelial barrier functions is crucial to prevent systemic contamination by microbes that penetrate from the gut lumen. GTPases of the Rho-family such as RhoA, Rac1, and Cdc42 are known to be critically involved in the regulation of intestinal epithelial barrier functions. However, it is still unclear whether inactivation or activation of these GTPases exerts barrier protection or not. We tested the effects of Rho GTPase activities on intestinal epithelial barrier functions by using the bacterial toxins cytotoxic necrotizing factor 1 (CNF-1), toxin B, C3 transferase (C3 TF), and lethal toxin (LT) in an in vitro model of the intestinal epithelial barrier. Incubation of cell monolayers with CNF-1 for 3 h induced exclusive activation of RhoA whereas Rac1 and Cdc42 activities were unchanged. As revealed by FITC-dextran flux and measurements of transepithelial electrical resistance (TER) intestinal epithelial permeability was significantly increased under these conditions. Inhibition of Rho kinase via Y27632 blocked barrier destabilization of CNF-1 after 3 h. In contrast, after 24 h of incubation with CNF-1 only Rac1 and Cdc42 but not RhoA were activated which resulted in intestinal epithelial barrier stabilization. Toxin B to inactivate RhoA, Rac1, and Cdc42 as well as Rac1 inhibitor LT increased intestinal epithelial permeability. Similar effects were observed after inhibition of RhoA/Rho kinase signaling by C3 TF or Y27632. Taken together, these data demonstrate that both activation and inactivation of RhoA signaling increased paracellular permeability whereas activation of Rac1 and Cdc42 correlated with stabilized barrier functions.

MAGIs regulate aPKC to enable balanced distribution of intercellular tension for epithelial sheet homeostasis.

Constriction of the apical plasma membrane is a hallmark of epithelial cells that underlies cell shape changes in tissue morphogenesis and maintenance of tissue integrity in homeostasis. Contractile force is exerted by a cortical actomyosin network that is anchored to the plasma membrane by the apical junctional complexes (AJC). In this study, we present evidence that MAGI proteins, structural components of AJC whose function remained unclear, regulate apical constriction of epithelial cells through the Par polarity proteins. We reveal that MAGIs are required to uniformly distribute Partitioning defective-3 (Par-3) at AJC of cells throughout the epithelial monolayer. MAGIs recruit ankyrin-repeat-, SH3-domain- and proline-rich-region-containing protein 2 (ASPP2) to AJC, which modulates Par-3-aPKC to antagonize ROCK-driven contractility. By coupling the adhesion machinery to the polarity proteins to regulate cellular contractility, we propose that MAGIs play essential and central roles in maintaining steady state intercellular tension throughout the epithelial cell sheet.

Antiangiogenic and antimitotic effects of aspirin in hypoxia­reoxygenation modulation of the LOX-1-NADPH oxidase axis as a potential mechanism.

Hypoxia­reoxygenation (HR) is a primary driver of angiogenesis in both atherogenesis and tumorigenesis. The main target of hypoxia-driven proangiogenic signaling is adherens junctions responsible for contact inhibition of endothelial cells. We analyzed the effects of hypoxia (8­12 hours) followed by a brief period of reoxygenation (2 hours) (HR) on angiogenesis and integrity of adherens junction in cultured human umbilical vein endothelial cells as well as the effects of aspirin on modulation of human umbilical vein endothelial cells' response to HR. Cells exposed to HR displayed considerable enhancement of tube formation (angiogenesis) on matrigel. Immunocytostaining of near-confluent cells revealed that HR caused disruption of adherens junctions and internalization of their components VE-cadherin, p120 catenin, and b-catenin. Additionally, HR resulted in the appearance of binucleated cells, and VE-cadherin in colocalization with b-catenin was found to be positioned between the separating nuclei. Presence of aspirin (acetylsalicylic acid, 1 mM) resulted in preservation of adherens junctions on the cellular membrane and prevented angiogenesis as well as mitosis. HR caused upregulation LOX-1, the p47(phox) subunit of NADPH, while reducing transcription of endothelial nitric oxide synthase. Aspirin had no effect on endothelial nitric oxide synthase and canceled the transcriptional activation of the LOX-1 and p47(phox) subunit of NADPH oxidase. Based on these data, we hypothesize that aspirin preserves the integrity of adherens junctions and thus blunts angiogenic response to HR through downregulation of LOX-1 and the LOX-1-mediated p47(phox) component of NADPH oxidase transcription, thus preventing NADPH oxidase assembly and function.

Rho/Rho-associated kinase-II signaling mediates disassembly of epithelial apical junctions.

Apical junctional complex (AJC) plays a vital role in regulation of epithelial barrier function. Disassembly of the AJC is observed in diverse physiological and pathological states; however, mechanisms governing this process are not well understood. We previously reported that the AJC disassembly is driven by the formation of apical contractile acto-myosin rings. In the present study, we analyzed the signaling pathways regulating acto-myosin-dependent disruption of AJC by using a model of extracellular calcium depletion. Pharmacological inhibition analysis revealed a critical role of Rho-associated kinase (ROCK) in AJC disassembly in calcium-depleted epithelial cells. Furthermore, small interfering RNA (siRNA)-mediated knockdown of ROCK-II, but not ROCK-I, attenuated the disruption of the AJC. Interestingly, AJC disassembly was not dependent on myosin light chain kinase and myosin phosphatase. Calcium depletion resulted in activation of Rho GTPase and transient colocalization of Rho with internalized AJC proteins. Pharmacological inhibition of Rho prevented AJC disassembly. Additionally, Rho guanine nucleotide exchange factor (GEF)-H1 translocated to contractile F-actin rings after calcium depletion, and siRNA-mediated depletion of GEF-H1 inhibited AJC disassembly. Thus, our findings demonstrate a central role of the GEF-H1/Rho/ROCK-II signaling pathway in the disassembly of AJC in epithelial cells.

Septate-junction-dependent luminal deposition of chitin deacetylases restricts tube elongation in the Drosophila trachea.

The function of tubular epithelial organs like the kidney and lung is critically dependent on the length and diameter of their constituting branches. Genetic analysis of tube size control during Drosophila tracheal development has revealed that epithelial septate junction (SJ) components and the dynamic chitinous luminal matrix coordinate tube growth. However, the underlying molecular mechanisms controlling tube expansion so far remained elusive. Here, we present the analysis of two luminal chitin binding proteins with predicted polysaccharide deacetylase activities (ChLDs). ChLDs are required to assemble the cable-like extracellular matrix (ECM) and restrict tracheal tube elongation. Overexpression of native, but not of mutated, ChLD versions also interferes with the structural integrity of the intraluminal ECM and causes aberrant tube elongation. Whereas ChLD mutants have normal SJ structure and function, the luminal deposition of the ChLD requires intact cellular SJs. This identifies a new molecular function for SJs in the apical secretion of ChLD and positions ChLD downstream of the SJs in tube length control. The deposition of the chitin luminal matrix first promotes and coordinates radial tube expansion. We propose that the subsequent structural modification of chitin by chitin binding deacetylases selectively instructs the termination of tube elongation to the underlying epithelium.

Calmodulin-dependent protein phosphatase: immunocytochemical localization in chick retina.

Calmodulin-dependent protein phosphatase, previously called CaM-BP80 or calcineurin, is present in high concentrations in the central nervous system. The level of the phosphatase has been shown by radioimmunoassay to increase during development in the retinas of embryonic and hatching chicks (Tallant, E.A., and W.Y. Cheung, 1983, Biochemistry, 22:3630-3635). The aims of this study are to immunocytochemically localize the phosphatase in developing and mature retinas and to determine if the phosphatase is present in fractions of retinal synaptic membranes and synaptic junctions. Vibratome slices of fixed chick retina and Western blots of detergent-solubilized retinal fractions are both treated sequentially with rabbit primary antisera and goat anti-rabbit Fab fragments conjugated to peroxidase, and then reacted with hydrogen peroxide and diaminobenzidine. The tissue slices are further processed for electron microscopy. This paper demonstrates the presence of peroxidase reaction product in the retina just before synapse formation. In the outer plexiform layer the product is confined to photoreceptor synaptic terminals, whereas in the inner plexiform layer it is present in synaptic terminals of bipolar cells and in dendrites of ganglion cells. In this latter site the product is present postsynaptically at bipolar and amacrine synapses. The phosphatase is detected in Western blots of both synaptic plasma membrane and synaptic junction fractions.

Isolation of synaptic junctional complexes of high structural integrity from rat brain.

A new method has been developed for isolating synaptic junctional complexes (SJC) of high structural integrity. The major step in the isolation involves homogenization of a synaptosomal membrane (SM) fraction in a biphasic system consisting of Freon 113 and an aqueous phase containing 0.2% Triton X-100. Well-preserved SJCs, along with membrane vesicles, were recovered in the aqueous phase after low-speed centrifugation of the homogenate. The membranes were subsequently separated from the SJCs by centrifugation on a discontinuous sucrose density gradient. The purity and identity of subcellular fractions were monitored by thin sectioning electron microscopy, using specific and nonspecific staining methods. From the electron microscope studies we conclude that SJCs and their components occupy about 65% of the area covered by structures in this fraction. The assay of enzyme activities indicates that homogenization in Triton-Freon and subsequent steps of the isolation procedure affect the activities of Na, K-ATPase, cytochrome oxidase, and acid phosphatase to different extents, but do not cause total inactivation. Electrophoresis of the SJC-enriched fraction on sodium dodecyl sulfate-polyacrylamide gels has demonstrated that a polypeptide which co-migrates with tubulin is the major component in this fraction, and that a polypeptide co-migrating with actin is also present.

Cellular redistribution of protein tyrosine phosphatases LAR and PTPsigma by inducible proteolytic processing.

Most receptor-like protein tyrosine phosphatases (PTPases) display a high degree of homology with cell adhesion molecules in their extracellular domains. We studied the functional significance of processing for the receptor-like PTPases LAR and PTPsigma. PTPsigma biosynthesis and intracellular processing resembled that of the related PTPase LAR and was expressed on the cell surface as a two-subunit complex. Both LAR and PTPsigma underwent further proteolytical processing upon treatment of cells with either calcium ionophore A23187 or phorbol ester TPA. Induction of LAR processing by TPA in 293 cells did require overexpression of PKCalpha. Induced proteolysis resulted in shedding of the extracellular domains of both PTPases. This was in agreement with the identification of a specific PTPsigma cleavage site between amino acids Pro821 and Ile822. Confocal microscopy studies identified adherens junctions and desmosomes as the preferential subcellular localization for both PTPases matching that of plakoglobin. Consistent with this observation, we found direct association of plakoglobin and beta-catenin with the intracellular domain of LAR in vitro. Taken together, these data suggested an involvement of LAR and PTPsigma in the regulation of cell contacts in concert with cell adhesion molecules of the cadherin/catenin family. After processing and shedding of the extracellular domain, the catalytically active intracellular portions of both PTPases were internalized and redistributed away from the sites of cell-cell contact, suggesting a mechanism that regulates the activity and target specificity of these PTPases. Calcium withdrawal, which led to cell contact disruption, also resulted in internalization but was not associated with prior proteolytic cleavage and shedding of the extracellular domain. We conclude that the subcellular localization of LAR and PTPsigma is regulated by at least two independent mechanisms, one of which requires the presence of their extracellular domains and one of which involves the presence of intact cell-cell contacts.

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.