Scribble and Discs-large direct initial assembly and positioning of adherens junctions during the establishment of apical-basal polarity.

Apical-basal polarity is a fundamental property of animal tissues. Drosophila embryos provide an outstanding model for defining mechanisms that initiate and maintain polarity. Polarity is initiated during cellularization, when cell-cell adherens junctions are positioned at the future boundary of apical and basolateral domains. Polarity maintenance then involves complementary and antagonistic interplay between apical and basal polarity complexes. The Scribble/Dlg module is well-known for promoting basolateral identity during polarity maintenance. Here, we report a surprising role for Scribble/Dlg in polarity initiation, placing it near the top of the network-positioning adherens junctions. Scribble and Dlg are enriched in nascent adherens junctions, are essential for adherens junction positioning and supermolecular assembly, and also play a role in basal junction assembly. We test the hypotheses for the underlying mechanisms, exploring potential effects on protein trafficking, cytoskeletal polarity or Par-1 localization/function. Our data suggest that the Scribble/Dlg module plays multiple roles in polarity initiation. Different domains of Scribble contribute to these distinct roles. Together, these data reveal novel roles for Scribble/Dlg as master scaffolds regulating assembly of distinct junctional complexes at different times and places.

Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis.

Epithelial barrier loss is a driver of intestinal and systemic diseases. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction and a potential therapeutic target, but enzymatic inhibition has unacceptable toxicity. Here, we show that a unique domain within the MLCK splice variant MLCK1 directs perijunctional actomyosin ring (PAMR) recruitment. Using the domain structure and multiple screens, we identify a domain-binding small molecule (divertin) that blocks MLCK1 recruitment without inhibiting enzymatic function. Divertin blocks acute, tumor necrosis factor (TNF)-induced MLCK1 recruitment as well as downstream myosin light chain (MLC) phosphorylation, barrier loss, and diarrhea in vitro and in vivo. Divertin corrects barrier dysfunction and prevents disease development and progression in experimental inflammatory bowel disease. Beyond applications of divertin in gastrointestinal disease, this general approach to enzymatic inhibition by preventing access to specific subcellular sites provides a new paradigm for safely and precisely targeting individual properties of enzymes with multiple functions.

Graft-versus-host disease propagation depends on increased intestinal epithelial tight junction permeability.

Graft-versus-host disease (GVHD) is a complication of hematopoietic stem cell transplantation (HSCT) that affects multiple organs. GVHD-associated intestinal damage can be separated into two distinct phases, initiation and propagation, which correspond to conditioning-induced damage and effector T cell activation and infiltration, respectively. Substantial evidence indicates that intestinal damage induced by pretransplant conditioning is a key driver of GVHD initiation. Here, we aimed to determine the impact of dysregulated intestinal permeability on the subsequent GVHD propagation phase. The initiation phase of GVHD was unchanged in mice lacking long MLCK (MLCK210), an established regulator of epithelial tight junction permeability. However, MLCK210-deficient mice were protected from sustained barrier loss and exhibited limited GVHD propagation, as indicated by reduced histopathology, fewer CD8+ effector T cells in the gut, and improved overall survival. Consistent with these findings, intestinal epithelial MLCK210 expression and enzymatic activity were similarly increased in human and mouse GVHD biopsies. Intestinal epithelial barrier loss mediated by MLCK210 is therefore a key driver of the GVHD propagation. These data suggest that inhibition of MLCK210-dependent barrier regulation may be an effective approach to limiting GVHD progression.

The scaffolding protein ZO-1 coordinates actomyosin and epithelial apical specializations in vitro and in vivo.

Polarized epithelia assemble into sheets that compartmentalize organs and generate tissue barriers by integrating apical surfaces into a single, unified structure. This tissue organization is shared across organs, species, and developmental stages. The processes that regulate development and maintenance of apical epithelial surfaces are, however, undefined. Here, using an intestinal epithelial-specific knockout (KO) mouse and cultured epithelial cells, we show that the tight junction scaffolding protein zonula occludens-1 (ZO-1) is essential for development of unified apical surfaces in vivo and in vitro We found that U5 and GuK domains of ZO-1 are necessary for proper apical surface assembly, including organization of microvilli and cortical F-actin; however, direct interactions with F-actin through the ZO-1 actin-binding region (ABR) are not required. ZO-1 lacking the PDZ1 domain, which binds claudins, rescued apical structure in ZO-1-deficient epithelia, but not in cells lacking both ZO-1 and ZO-2, suggesting that heterodimerization with ZO-2 restores PDZ1-dependent ZO-1 interactions that are vital to apical surface organization. Pharmacologic F-actin disruption, myosin II motor inhibition, or dynamin inactivation restored apical epithelial structure in vitro and in vivo, indicating that ZO-1 directs epithelial organization by regulating actomyosin contraction and membrane traffic. We conclude that multiple ZO-1-mediated interactions contribute to coordination of epithelial actomyosin function and genesis of unified apical surfaces.

Contributions of intestinal epithelial barriers to health and disease.

A core function of epithelia is to form barriers that separate tissue compartments within complex organisms. These barriers also separate the internal milieu from the external environment and are, therefore, an essential component of host defense. However, in many cases, a perfect barrier would be improbable with life itself. Examples include the air spaces within the lungs, the renal tubules, and the intestines. Here, we focus on the mechanisms by which barriers are assembled, maintained, and regulated in the context of health and disease. Because of its unique challenges and extensive study, we focus on the gastrointestinal tract as an organ-specific example of the essential contributions of the paracellular barrier to life.

ZO-1 interactions with F-actin and occludin direct epithelial polarization and single lumen specification in 3D culture.

Epithelia within tubular organs form and expand lumens. Failure of these processes can result in serious developmental anomalies. Although tight junction assembly is crucial to epithelial polarization, the contribution of specific tight junction proteins to lumenogenesis is undefined. Here, we show that ZO-1 (also known as TJP1) is necessary for the formation of single lumens. Epithelia lacking this tight junction scaffolding protein form cysts with multiple lumens and are defective in the earliest phases of polarization, both in two and three dimensions. Expression of ZO-1 domain-deletion mutants demonstrated that the actin-binding region and U5-GuK domain are crucial to single lumen development. For actin-binding region, but not U5-GuK domain, mutants, this could be overcome by strong polarization cues from the extracellular matrix. Analysis of the U5-GuK binding partners shroom2, α-catenin and occludin showed that only occludin deletion led to multi-lumen cysts. Like ZO-1-deficiency, occludin deletion led to mitotic spindle orientation defects. Single lumen formation required the occludin OCEL domain, which binds to ZO-1. We conclude that ZO-1-occludin interactions regulate multiple phases of epithelial polarization by providing cell-intrinsic signals that are required for single lumen formation.

Remodeling the zonula adherens in response to tension and the role of afadin in this response.

Morphogenesis requires dynamic coordination between cell-cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell-cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions.

Rap1 and Canoe/afadin are essential for establishment of apical-basal polarity in the Drosophila embryo.

The establishment and maintenance of apical-basal cell polarity is critical for assembling epithelia and maintaining organ architecture. Drosophila embryos provide a superb model. In the current view, apically positioned Bazooka/Par3 is the initial polarity cue as cells form during cellularization. Bazooka then helps to position both adherens junctions and atypical protein kinase C (aPKC). Although a polarized cytoskeleton is critical for Bazooka positioning, proteins mediating this remained unknown. We found that the small GTPase Rap1 and the actin-junctional linker Canoe/afadin are essential for polarity establishment, as both adherens junctions and Bazooka are mispositioned in their absence. Rap1 and Canoe do not simply organize the cytoskeleton, as actin and microtubules become properly polarized in their absence. Canoe can recruit Bazooka when ectopically expressed, but they do not obligatorily colocalize. Rap1 and Canoe play continuing roles in Bazooka localization during gastrulation, but other polarity cues partially restore apical Bazooka in the absence of Rap1 or Canoe. We next tested the current linear model for polarity establishment. Both Bazooka and aPKC regulate Canoe localization despite being "downstream" of Canoe. Further, Rap1, Bazooka, and aPKC, but not Canoe, regulate columnar cell shape. These data reshape our view, suggesting that polarity establishment is regulated by a protein network rather than a linear pathway.

A contractile actomyosin network linked to adherens junctions by Canoe/afadin helps drive convergent extension.

Integrating individual cell movements to create tissue-level shape change is essential to building an animal. We explored mechanisms of adherens junction (AJ):cytoskeleton linkage and roles of the linkage regulator Canoe/afadin during Drosophila germband extension (GBE), a convergent-extension process elongating the body axis. We found surprising parallels between GBE and a quite different morphogenetic movement, mesoderm apical constriction. Germband cells have an apical actomyosin network undergoing cyclical contractions. These coincide with a novel cell shape change--cell extension along the anterior-posterior (AP) axis. In Canoe's absence, GBE is disrupted. The apical actomyosin network detaches from AJs at AP cell borders, reducing coordination of actomyosin contractility and cell shape change. Normal GBE requires planar polarization of AJs and the cytoskeleton. Canoe loss subtly enhances AJ planar polarity and dramatically increases planar polarity of the apical polarity proteins Bazooka/Par3 and atypical protein kinase C. Changes in Bazooka localization parallel retraction of the actomyosin network. Globally reducing AJ function does not mimic Canoe loss, but many effects are replicated by global actin disruption. Strong dose-sensitive genetic interactions between canoe and bazooka are consistent with them affecting a common process. We propose a model in which an actomyosin network linked at AP AJs by Canoe and coupled to apical polarity proteins regulates convergent extension.

The single Drosophila ZO-1 protein Polychaetoid regulates embryonic morphogenesis in coordination with Canoe/afadin and Enabled.

Adherens and tight junctions play key roles in assembling epithelia and maintaining barriers. In cell culture zonula occludens (ZO)-family proteins are important for assembly/maturation of both tight and adherens junctions (AJs). Genetic studies suggest that ZO proteins are important during normal development, but interpretation of mouse and fly studies is limited by genetic redundancy and/or a lack of null alleles. We generated null alleles of the single Drosophila ZO protein Polychaetoid (Pyd). Most embryos lacking Pyd die with striking defects in morphogenesis of embryonic epithelia including the epidermis, segmental grooves, and tracheal system. Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure. However, Pyd loss does affect several cell behaviors that drive dorsal closure. The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic null mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together. Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.

Protein expression in platelets from six species that differ in their open canalicular system.

Platelets contain an invaginated, tubular membranous structure called the surface-connected open canalicular system (SCCS or OCS), which is contiguous with the plasma membrane and serves as a site for granule fusion and as a reservoir of membrane for platelet spreading. According to ultrastructural studies, platelets from some species lack OCS. In an attempt to correlate biochemical and functional attributes with the presence of an OCS, platelets from human, mouse and dog (OCS(+)), and from cow, camel and horse (OCS(-)) were analysed for differential protein expression and aggregation in response to thrombin. Among the 18 different cytoskeletal and regulatory proteins examined, five (Rac1, RhoA, Ras, calmodulin and Src) were expressed at higher levels in OCS(+) platelets (p < 0.05). Given the role of Arf6 in the formation of tubular invaginations in nucleated cells, the levels of Arf6-GTP were analysed in OCS(+) and OCS(-) platelets. There was no significant correlation between the presence of OCS and total Arf6 or Arf6-GTP levels. Comparison of platelet aggregation between different species suggests that OCS(-) platelets have delayed responses. This comparison of platelets from six different species, which differ in their OCS, shows the differential expression of known signaling components and foreshadows future studies focusing on OCS formation and function.

Primary platelet signaling cascades and integrin-mediated signaling control ADP-ribosylation factor (Arf) 6-GTP levels during platelet activation and aggregation.

Previous studies showed that ADP-ribosylation factor 6 (Arf6) is important for platelet function; however, little is known about which signaling events regulate this small GTP-binding protein. Arf6-GTP was monitored in platelets stimulated with a number of agonists (TRAP, thrombin, convulxin, collagen, PMA, thapsigargin, or A23187) and all led to a time-dependent decrease in Arf6-GTP. ADP and U46619 were without effect. Using inhibitors, it was shown that the decrease of Arf6-GTP is a direct consequence of known signaling cascades. Upon stimulation via PAR receptors, Arf6-GTP loss could be blocked by treatment with U-73122, BAPTA/AM, Ro-31-8220, or Gö6976, indicating requirements for phospholipase C, calcium, and protein kinase C (PKC) alpha/beta, respectively. The Arf6-GTP decrease in convulxin-stimulated platelets showed similar requirements and was also sensitive to piceatannol, wortmannin, and LY294002, indicating additional requirements for Syk and phosphatidylinositol 3-kinase. The convulxin-induced decrease was sensitive to both PKCalpha/beta and delta inhibitors. Outside-in signaling, potentially via integrin engagement, caused a second wave of signaling that affected Arf6. Inclusion of RGDS peptides or EGTA, during activation, led to a biphasic response; Arf6-GTP levels partially recovered upon continued incubation. A similar response was seen in beta3 integrin-null platelets. These data show that Arf6-GTP decreases in response to known signaling pathways associated with PAR and GPVI. They further reveal a second, aggregation-dependent, process that dampens Arf6-GTP recovery. This study demonstrates that the nucleotide state of Arf6 in platelets is regulated during the initial phases of activation and during the later stages of aggregation.

Endobrevin/VAMP-8 is the primary v-SNARE for the platelet release reaction.

Platelet secretion is critical to hemostasis. Release of granular cargo is mediated by soluble NSF attachment protein receptors (SNAREs), but despite consensus on t-SNAREs usage, it is unclear which Vesicle Associated Membrane Protein (VAMPs: synaptobrevin/VAMP-2, cellubrevin/VAMP-3, TI-VAMP/VAMP-7, and endobrevin/VAMP-8) is required. We demonstrate that VAMP-8 is required for release from dense core granules, alpha granules, and lysosomes. Platelets from VAMP-8-/- mice have a significant defect in agonist-induced secretion, though signaling, morphology, and cargo levels appear normal. In contrast, VAMP-2+/-, VAMP-3-/-, and VAMP-2+/-/VAMP-3-/- platelets showed no defect. Consistently, tetanus toxin had no effect on secretion from permeabilized mouse VAMP-3-/- platelets or human platelets, despite cleavage of VAMP-2 and/or -3. Tetanus toxin does block the residual release from permeabilized VAMP-8-/- platelets, suggesting a secondary role for VAMP-2 and/or -3. These data imply a ranked redundancy of v-SNARE usage in platelets and suggest that VAMP-8-/- mice will be a useful in vivo model to study platelet exocytosis in hemostasis and vascular inflammation.

Arf6 plays an early role in platelet activation by collagen and convulxin.

Small GTPases play critical roles in hemostasis, though the roster of such molecules in platelets is not complete. In this study, we report the presence of Ras-related GTPases of the ADP-ribosylation factor (Arf) family. Platelets contain Arf1 or 3 and Arf6, with the latter being predominantly membrane associated. Using effector domain pull-down assays, we show, counter to other GTPases, that Arf6-GTP is present in resting platelets and decreases rapidly upon activation with collagen or convulxin. This decrease does not completely rely on secondary agonists (ADP and thromboxane A2) or require integrin signaling. The decrease in free Arf6-GTP temporally precedes activation of Rho family GTPases (RhoA, Cdc42, and Rac1). Using a membrane-permeant, myristoylated peptide, which mimics the N-terminus of Arf6, we show that the Arf6-GTP decrease is essential for collagen- and convulxin-induced aggregation, platelet adherence, and spreading on collagen-coated glass. Treatment with this peptide also affects the activation of Rho family GTPases, but has little effect on RalA and Rap1 or on agonist-induced calcium mobilization. These data show that Arf6 is a key element in activation through GPVI, and is required for activation of the Rho family GTPases and the subsequent cytoskeletal rearrangements needed for full platelet function.

Platelets from Munc18c heterozygous mice exhibit normal stimulus-induced release.

A critical aspect of hemostasis is the release of clot-forming components from the three intra-platelet stores: dense core granules, alpha-granules and lysosomes. Exocytosis from these granules is mediated by soluble (SNAPs and NSF) and integralmembrane proteins (v- and t-SNAREs). Three SM (Sec1/Munc18) proteins are present in mouse platelets (Munc18a, 18b and 18c) and each potentially regulates exocytosis via modulation of their cognate syntaxin binding partner. To define the molecular machinery required for platelet exocytosis, we analyzed platelets from Munc18c heterozygous knockout mice. These platelets show a decrease in Munc18c but no apparent reduction in other secretory machinery components. No differences in the rates of aggregation or of secretion of [(3)H]-5HT (dense core granules), platelet factor 4 (alpha-granules), or hexosaminidase (lysosomes) were detected between platelets from Munc18c heterozygous knockout or wild-type mice. The platelets also show normal morphology. Contrary to a predicted requirement for Munc18c in platelet secretion, data reported here show that reducing Munc18c levels does not substantially alter platelet function. These data show that despite Munc18c's role in platelet secretion, the lack of a secretion defect may be attributed to compensation by other Munc18 isoforms or that one allele is sufficient to maintain secretion under standard conditions.

PCB 104-induced proinflammatory reactions in human vascular endothelial cells: relationship to cancer metastasis and atherogenesis.

Polychlorinated biphenyls (PCBs) are widespread environmental contaminants that are known to induce carcinogenic and possibly atherogenic events. Recent evidence suggests that selected PCBs may be potent developmental agents of vascular inflammatory responses by inducing cellular oxidative stress and activating redox-responsive transcription factors. Therefore, the aim of this paper is to investigate PCB-induced proinflammatory reactions in human vascular endothelial cells. To determine the proinflammatory effects, cellular oxidative stress and expression of genes encoding for monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules, such as E-selectin and intercellular adhesion molecule-1 (ICAM-1), were assessed in human umbilical vein endothelial cells (HUVEC) exposed to 2,2',4,6,6'-pentachlorobiphenyl (PCB 104), a representative of ortho-substituted, non-coplanar PCB congeners. PCB 104 increased the oxidative stress in endothelial cells, as determined by the increased 2',7'-dichlorofluorescein (DCF) and rhodamine 123 fluorescence. In addition, PCB 104 markedly upregulated the expression of MCP-1, E-selectin, and ICAM-1 at both the mRNA and protein levels. These effects were time- and concentration-dependent. The maximum expression of inflammatory genes was observed in endothelial cells exposed to 20 microM of PCB 104 for 1 or 2 h, depending on the specific gene. In addition, PCB 104 elevated the adhesion of THP-1 cells (a human acute monocytic leukemia cell line) to endothelial cell monolayers. These results indicate that PCB 104 is a potent stimulant of inflammatory mediators in human vascular endothelial cells. We hypothesize that these proinflammatory processes may contribute to the development of cancer metastasis and/or atherogenesis in patients exposed to PCBs.