CLDN11在胰腺癌神经浸润动物模型中mRNA水平的表达

目的:神经浸润的发生预示胰腺癌预后不良,疼痛的发生与神经浸润密切相关,癌细胞和神经组织间相互作用、连接及粘附可能参与了神经浸润的发生,Claudins作为组成紧密连接的主要成份,在多种肿瘤中有所表达,本实验拟通过观察其成员CLDN11在体内、体外mRNA水平的表达,探讨CLDN11在胰腺癌神经浸润发病机制中的作用,为其诊断及治疗新方法的探索提供一定的实验依据。方法:通过裸鼠坐骨神经周围注射不同人胰腺癌细胞系的方法建立稳定的胰腺癌神经浸润动物模型,成瘤后检测肿瘤组织中CLDN11 mRNA表达水平的差异。同时检测不同人胰腺癌细胞株中CLDN11 mRNA的表达水平的差异。结果:CLDN11在神经侵犯发生率低的肿瘤中的表达高于神经侵犯发生率高的肿瘤,在正常胰腺组织中无表达。CLDN11的mRNA水平在panc-1细胞株中表达高于Capan-2组。结论:经本实验研究发现CLDN11在PNI发生率高的肿瘤组织及高神经浸润能力的细胞株中表达下调,而在PNI发生率低的肿瘤组织及神经浸润能力低的细胞株中高表达,可以得出在神经浸润发生中,CLDN11的表达受到抑制的结论,由此推断如果过表达CLDN11,有可能阻碍PNI的发生及发展;另外,CLDN11表达的下降也可能预示着PNI的发生及进展,因此CLDN11表达的下降可作为PNI发生的预警信号,也可作为胰腺癌基因治疗的靶点,为提高胰腺癌的早期诊断率、改善胰腺癌的预后提供初步的基础实验依据。     

Tight junction proteins ZO-1, occludin, and claudins in developing and adult human perineurium.

In peripheral nerves, groups of Schwann cell-axon units are isolated from the adjacent tissues by the perineurium, which creates a diffusion barrier responsible for the maintenance of endoneurial homeostasis. The perineurium is formed by concentric layers of overlapping, polygonal perineurial cells that form tight junctions at their interdigitating cell borders. In this study, employing indirect immunofluorescence and immunoelectron microscopy, we demonstrate that claudin-1 and -3, ZO-1, and occludin, but not claudin-2, -4, and -5, are expressed in the perineurium of adult human peripheral nerve. We also describe the expression of occludin, ZO-1, claudin-1, -3, and -5 in the developing human perineurium, showing that the expressions of claudin-1 and -3, ZO-1, and occludin follow similar spatial developmental expression patterns but follow different timetables in achieving their respective adult distributions. Specifically, claudin-1 is already largely restricted to perineurium-derived structures at 11 fetal weeks, whereas claudin-3 and occludin are weakly expressed in the perineurial structures at this age and acquire a well-defined perineurial distribution only between 22 and 35 fetal weeks. ZO-1 appears to acquire its mature profile even later during the third trimester. The results of the present and previous studies show that the perineurial diffusion barrier matures relatively late during human peripheral nerve development.     

Phosphorylation of claudin-4 by PKCepsilon regulates tight junction barrier function in ovarian cancer cells

Claudin proteins belong to a large family of transmembrane proteins essential to the formation and maintenance of tight junctions (TJs). In ovarian cancer, TJ protein claudin-4 is frequently overexpressed and may have roles in survival and invasion, but the molecular mechanisms underlying its regulation are poorly understood. In this report, we show that claudin-4 can be phosphorylated by protein kinase C (PKC) at Thr189 and Ser194 in ovarian cancer cells and overexpression of a claudin-4 mutant protein mimicking the phosphorylated state results in the disruption of the barrier function. Furthermore, upon phorbol ester-mediated PKC activation of OVCA433 cells, TJ strength is decreased and claudin-4 localization is altered. Analyses using PKC inhibitors and siRNA suggest that PKCepsilon, an isoform typically expressed in ovarian cancer cells, may be important in the TPA-mediated claudin-4 phosphorylation and weakening of the TJs. Furthermore, immunofluorescence studies showed that claudin-4 and PKCepsilon are co-localized at the TJs in these cells. The modulation of claudin-4 activity by PKCepsilon may not only provide a mechanism for disrupting TJ function in ovarian cancer, but may also be important in the regulation of TJ function in normal epithelial cells.     

Glial cells in peripheral nerves of the cockroach, Periplaneta americana

The ultrastructural organization and the junctional complexes of peripheral nerves have been investigated in the cockroach Periplaneta americana. Nerve 5 is surrounded by a layer of connective tissue, the neural lamella, beneath which is a layer of perineurial glial cells wrapping the axons. Adjacent perineurial cells are joined to one another by septate, gap and tight junctions. Septate and gap junctions were observed in freeze-fracture replicas of main trunk nerve 5. Septate junctions were found as rows of PF particles mainly in perineurial cell membranes. Gap junctions exhibited EF macular aggregates in perineurial and subperineurial glial cells. During incubations in vivo with extracellularly applied ionic lanthanum, the lanthanum did not penetrate beyond the perineurium. Where nerve 5 branches and contacts the muscle, lanthanum penetrated freely between the muscle fibres and the nerve branches. In small peripheral branches where the axons are surrounded by single a glial layer, lanthanum is unable to penetrate to the axolemma.     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions. I. Larval development

In the central nervous system (CNS) of full-grown larvae of the blowfly Calliphora erythrocephala, the glial-ensheathed nerve cells are completely surrounded by a layer of perineurial cells which form a “blood-brain barrier” between the circulating haemolymph and the CNS. A variety of intercellular junctions, including gap and tight junctions, are found between adjacent perineurial cells and some also between apposing glial cells; these have been characterized by freeze-fracturing as well as by tracer studies and analysis of thin sections. They are found not to be present between such cells in the undifferentiated CNS in the newly hatched larvae, nor are the nerve cells encompassed by glial cells; ionic lanthanum can penetrate to the axonal surfaces at this stage. However, over the 5 days of larval growth and development the glial cells produce attentuated cytoplasmic processes that ensheath the nerve cells, and the perineurium is formed; junctional complexes are assembled and a larval blood-brain barrier is produced which excludes tracers. Freeze-fracture preparations suggest that the inverted gap junctions which develop have done so by migration of individual intramembranous EF particles to form, at first, linear arrays and small clusters and, ultimately, macular aggregations in the perineurium; these lie between the undulating rows of PF particles forming the septate junctions. These septate junctions are formed by the organization of arrays of PF particles into multiple rows. Extensive PF particles fusing into ridges with EF grooves to form perineurial “tight” junctions are also observed, seemingly in the process of development; entry of exogenous lanthanum followed by its exclusion parallels the completion of ridge formation. These ridges are simple linear arrays of particles which may be discontinuous, lying in parallel with one another and the surface. Clustered particle arrays as well as scattered short ridges on the axonal PF, however, appear to be present unchanged throughout larval life; their role may therefore be associated with neural membrane function although there are suggestions that some may form axo-glial junctions. This is the first report on the lateral migration of intramembranous particles as the mode of formation of gap junctions in the nervous system of an invertebrate.     

Occludin-deficient Embryonic Stem Cells Can Differentiate into Polarized Epithelial Cells Bearing Tight Junctions

Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.     

The ultrastructural organization of peripheral nerves in two insect species (Periplaneta americana and Schistocerca gregaria)

The ultrastructural organization of various peripheral nerves, including the crural nerve, has been investigated in the locust and cockroach. In some cases the larger nerves are ensheathed by a fat body layer which is not always complete. However, like many nervous connectives, they do possess a continuous acellular neural lamella and a perineurial cell layer which surround the glial-axonal mass. Adjacent perineurial cells are associated with one another by septate desmosomes, gap junctions and tight junctions. These last may represent the morphological basis of the ‘blood-brain barrier’ observed electrophysiologically in these peripheral nerves in another report. Very small nerves of the cockroach, however, although lying embedded in a neural lamella, do not possess a specialized perineurial layer displaying junctional complexes, unless they contain one or more large axons. If they have only one or more small axons, these small nerves may either appear naked, or display a single glial cell process loosely enveloping them; in either case there is no structural basis for a ‘barrier’ system. Various comparisons have been made between locust crural nerve and the cockroach central nervous connectives in an attempt to correlate some aspects of their ultrastructural organization with relevant electrophysiological information.     

Peripheral nerve pericytes modify the blood–nerve barrier function and tight junctional molecules through the secretion of various soluble factors

The objectives of this study were to establish pure blood–nerve barrier (BNB) and blood–brain barrier (BBB)‐derived pericyte cell lines of human origin and to investigate their unique properties as barrier‐forming cells. Brain and peripheral nerve pericyte cell lines were established via transfection with retrovirus vectors incorporating human temperature‐sensitive SV40 T antigen (tsA58) and telomerase. These cell lines expressed several pericyte markers such as α‐smooth muscle actin, NG2, platelet‐derived growth factor receptor β, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, the inulin clearance was significantly lowered in peripheral nerve microvascular endothelial cells (PnMECs) through the up‐regulation of claudin‐5 by soluble factors released from brain or peripheral nerve pericytes. In particular, bFGF secreted from peripheral nerve pericytes strengthened the barrier function of the BNB by increasing the expression of claudin‐5. Peripheral nerve pericytes may regulate the barrier function of the BNB, because the BNB does not contain cells equivalent to astrocytes which regulate the BBB function. Furthermore, these cell lines expressed several neurotrophic factors such as NGF, BDNF, and GDNF. The secretion of these growth factors from peripheral nerve pericytes might facilitate axonal regeneration in peripheral neuropathy. Investigation of the characteristics of peripheral nerve pericytes may provide novel strategies for modifying BNB functions and promoting peripheral nerve regeneration. J. Cell. Physiol. 226: 255–266, 2010. © 2010 Wiley‐Liss, Inc.     

HGF/c-Met pathway facilitates the perineural invasion of pancreatic cancer by activating the mTOR/NGF axis

Perineural invasion (PNI) is a pathologic feature of pancreatic cancer and is associated with poor outcomes, metastasis, and recurrence in pancreatic cancer patients. However, the molecular mechanism of PNI remains unclear. The present study aimed to investigate the mechanism that HGF/c-Met pathway facilitates the PNI of pancreatic cancer. In this study, we confirmed that c-Met expression was correlated with PNI in pancreatic cancer tissues. Activating the HGF/c-Met signaling pathway potentiated the expression of nerve growth factor (NGF) to recruit nerves and promote the PNI. Activating the HGF/c-Met signaling pathway also enhanced the migration and invasion ability of cancer cells to facilitate cancer cells invading nerves. Mechanistically, HGF/c-Met signaling pathway can active the mTOR/NGF axis to promote the PNI of pancreatic cancer. Additionally, we found that knocking down c-Met expression inhibited cancer cell migration along the nerve, reduced the damage of the sciatic nerve caused by cancer cells and protected the function of the sciatic nerve in vivo. Taken together, our findings suggest a supportive mechanism of the HGF/c-Met signaling pathway in promoting PNI by activating the mTOR/NGF axis in pancreatic cancer. Blocking the HGF/c-Met signaling pathway may be an effective target for the treatment of PNI.Subject terms: Pancreatic cancer, Cancer microenvironment     

A blood-brain barrier without tight junctions in the fly central nervous system in the early postembryonic stage

Summary The anatomical basis of the vertebrate blood-brain barrier is a series of tight junctions between endothelial cells of capillaries in the central nervous system. Over two decades ago, tight junctions were also proposed as the basis of the blood-brain barrier in insects. Currently there is a growing understanding that septate junctions might possess barrier properties in various invertebrate epithelial cells. We now examine these two views by studying the blood-brain barrier properties of the early postembryonic larva of a dipteran fly (Delia platura) by transmission electron microscopy. Newly hatched larvae possess a functioning blood-brain barrier that excludes the extracellular tracer, ionic lanthanum. This barrier is intact throughout the second instar stage as well. The ultrastructural correlate of this barrier is a series of extensive septate junctions that pervade the intercellular space between adjacent perineurial cells. No tight junctions were located in either nerve, glial or perineurial cell layers. We suggest that the overall barrier might involve septate junctions within extensive, meandering intercellular clefts.     

Enhanced tight junction function in human breast cancer cells by antioxidant, selenium and polyunsaturated lipid

Paracellular permeability (PCP) is governed by tight junctions (TJs) in epithelial cells, acting as cell-cell adhesion structures, the aberration of which is known to be linked to the dissociation and metastasis of breast cancer cells. This study hypothesized that the function of TJs in human breast cancer cells can be augmented by gamma linolenic acid (GLA), selenium (Se), and iodine (I) in the presence of 17-beta-estradiol, as these molecules are known to increase TJ functions in endothelial cells, using assays of trans-epithelial resistance (TER), PCP, immunofluorescence, and in vitro invasion and motility models. GLA, I, and Se individually increased TER of MDA-MB-231 and MCF-7 human breast cancer cells. The combination of all three agents also had a significant increase in TER. Addition of GLA/Se/I reduced PCP of both breast cancer cell lines. GLA/Se/I reversed the effect of 17-beta-estradiol (reduced TER, increased PCP). Immunofluorescence revealed that after treatment with Se/I/GLA over 24 h, there was increasing relocation to breast cancer cell-cell junctions of occludin and ZO-1 in MCF-7 cells. Moreover, treatment with GLA/Se/I, alone or in combination, significantly reduced in vitro invasion of MDA-MB-231 cells through an endothelial cell barrier (P < 0.0001) and reduced 17-beta-estradiol induced breast cancer cell motility (P < 0.0001). Our previous work has demonstrated that GLA, I, and Se alone, or in combination are able to strengthen the function of TJs in human endothelial cells; this has now proved to be true of human breast cancer cells. This combination also completely reversed the effect of 17-beta-estradiol in these cells.     

Perineurium in the Drosophila (Diptera : Drosophilidae) embryo and its role in the blood-brain/nerve barrier

A monolayer of perineurial cells overlies glia and neurons, and this stratum of the central nervous system is the principal site of the Drosophila (Diptera : Drosophilidae) blood-brain barrier. Perineurial cells are bonded together by pleated-sheet septate junctions that are the anatomical correlate of the vertebrate tight junction. The blood-brain barrier maintains the ionic homeostasis necessary for proper nerve function. It was known that a functioning blood-brain barrier is present in mature (Stage 17) Drosophila embryos, but the genesis of this barrier was not known. We surveyed the central nervous system of late stage embryos (15 through 17) to determine when perineurial cells could first be detected. These cells take their place in (on) the central nervous system and are joined together by pleated-sheet septate junctions, during Stage 17. Those septate junctions are quickly occlusive to lanthanum tracer. This development step occurs during the same time as when chemical synapses first become functional. Such concurrent maturation is far from coincidental, because partitioning nerves and their synapses from hemolymph (with its variable ionic constitution) are essential for normal electrophysiology. We discuss details of the germ line derivation of perineurial cells, their first detection in the embryonic central nervous system, their functional properties, and the polygonal cell-packing pattern seen in the larval central nervous system.     

Fine structure of a lepidopteran nervous system and its accessibility to peroxidase and lanthanum

Summary The avascular ventral nerve cord of the moth, Manduca sexta, possesses an extensive dorsal mass of connective tissue in which lie fibroblasts that produce a collagen-like protein. The lateral and ventral surfaces of the nerve cord are ensheathed by an acellular neural lamella. Beneath this lies a layer of microtubule-laden perineurial cells which are separated from one another at their peripheral borders by lacunae containing electron-opaque material to which the cells are attached by hemi-desmosomes. Beyond these spaces, narrow intercellular clefts occur between the interdigitating perineurial plasma membranes; these are then connected by both gap and tight junctions. The axons beneath are surrounded by glia which also contain many microtubules and which are linked to one another by desmosomes and tight junctions.When intact nerve cords are incubated in horseradish peroxidase, reaction product is subsequently found within the neural lamella as well as in the lacunae and clefts between perineurial cells, but not beyond this level. Desheathed preparations, however, contain peroxidase within the cytoplasm of the exposed glial cells. Lanthanum penetrates the neural lamella and the lacunae, clefts and gap junctions between adjacent perineurial cells, but no further. It therefore appears that the tight junctions in the perineurium may be the site of restriction to the entry of ions and molecules, the existence of which has been suggested previously by electrophysiological investigations.I am grateful to Miss Yvonne R. Carter for her invaluable technical assistance and to Dr. J.E. Treherne and Dr. D.B. Sattelle for helpful discussions.     

Endothelial tight junctions and their regulatory signaling pathways in vascular homeostasis and disease

Endothelial tight junctions (TJs) regulate the transport of water, ions, and molecules through the paracellular pathway, serving as an important barrier in blood vessels and maintaining vascular homeostasis. In endothelial cells (ECs), TJs are highly dynamic structures that respond to multiple external stimuli and pathological conditions. Alterations in the expression, distribution, and structure of endothelial TJs may lead to many related vascular diseases and pathologies. In this review, we provide an overview of the assessment methods used to evaluate endothelial TJ barrier function both in vitro and in vivo and describe the composition of endothelial TJs in diverse vascular systems and ECs. More importantly, the direct phosphorylation and dephosphorylation of TJ proteins by intracellular kinases and phosphatases, as well as the signaling pathways involved in the regulation of TJs, including and the protein kinase C (PKC), PKA, PKG, Ras homolog gene family member A (RhoA), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Akt, and Wnt/β-catenin pathways, are discussed. With great advances in this area, targeting endothelial TJs may provide novel treatment for TJ-related vascular pathologies.     

Claudin-11 Tight Junctions in Myelin Are a Barrier to Diffusion and Lack Strong Adhesive Properties

The radial component is a network of interlamellar tight junctions (TJs) unique to central nervous system myelin. Ablation of claudin-11, a TJ protein, results in the absence of the radial component and compromises the passive electrical properties of myelin. Although TJs are known to regulate paracellular diffusion, this barrier function has not been directly demonstrated for the radial component, and some evidence suggests that the radial component may also mediate adhesion between myelin membranes. To investigate the physical properties of claudin-11 TJs, we compared fresh, unfixed Claudin 11-null and control nerves using x-ray and neutron diffraction. In Claudin 11-null tissue, we detected no changes in myelin structure, stability, or membrane interactions, which argues against the notion that myelin TJs exhibit significant adhesive properties. Moreover, our osmotic stressing and D₂O-H₂O exchange experiments demonstrate that myelin lacking claudin-11 is more permeable to water and small osmolytes. Thus, our data indicate that the radial component serves primarily as a diffusion barrier and elucidate the mechanism by which TJs govern myelin function.     

Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins

Tight junctions (TJs) are dynamic, multiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiological development and pathological mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a biosensor of TJ remodeling, we observed increased claudin 4 fluorescence recovery after photobleaching (FRAP) dynamics in response to inflammatory cytokines. Interferon γ and tumor necrosis factor α increased the proportion of mobile claudin 4 in the TJ. Up-regulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin-binding incompatibility.     

A vertebrate-like blood--brain barrier, with intraganglionic blood channels and occluding junctions, in the scorpion

India ink and ionic lanthanum injections have revealed that the central nervous system (CNS) of the scorpion possesses a highly vascularized cephalothoracic ganglionic mass. It, together with other abdominal ganglia which form a ventral nerve cord, are all ensheathed by an outer layer of modified glial, or perineurial, cells. These cells resemble those which line the blood channels permeating the CNS, in exhibiting both inverted gap and tight junctions. Although the latter show close or fused membrane appositions, lanthanum appears to penetrate past a number, but not all, of them. Freeze-fracturing reveals that these junctions are composed of E-face particles aligned into a network of rows, or ridges, which are frequently discontinuous, especially near the periphery of the perineurium. This produces a somewhat 'leaky' system but occlusion to tracers occurs ultimately, for in the CNS none can be found beyond the perineurium. The existence of this perineurial blood-brain barrier is also demonstrable electrophysiologically where cations such as Mg2+ are unable to penetrate beyond the perineurial layer although they can, it seems, leak in via the blood vascular system. Relative differences in tightness between the perineurium and the cells lining the blood channels may be attributed to differences in the relative number of discontinuous ridges. This is borne out by the observation that the peripheral nervous system has a highly attenuated perineurium with many fewer junctions, and some of these nerves tend to be leaky with respect to tracer penetration. In fixed material the junctional ridges may fracture on to the E-face or partly on both the EF and PF, while in unfixed tissue they are usually found on the PF. In both cases they exhibit complementary grooves that are coincident with the ridges across membrane transitions; in such cases the cell membranes are fused with concomitant obliteration of the intercellular space. These tight junctions, often closely associated with EF gap junctional particle aggregates which may be very loosely clustered, appear to form the basis of the observed blood-brain barrier in the scorpion CNS.     

Molecular composition of tight and adherens junctions in the rat olfactory epithelium and fila

Tight and adherens junctions (TJs, AJs) between neurons, epithelial and glial cells provide barrier and adhesion properties in the olfactory epithelium (OE), and subserve functions such as compartmentalization and axon growth in the fila olfactoria (FO). Immunofluorescence and immunoelectronmicroscopy were combined in sections of rat OE and FO to document the cellular and subcellular localization of TJ proteins occludin(Occl), claudins(Cl) 1-5 and zonula occludens(ZO) proteins 1-3, and of AJ proteins N-cadherin(cad), E-cad, and alpha-, beta- and p120-catenin(cat). With the exception of Cl2, all TJ proteins were colocalized in OE junctions. Differences in relative immunolabeling intensities were noted between neuronal and epithelial TJs. In the FO, Cl5-reactivity was localized in olfactory ensheathing cell (OEC) junctions, Cl1-reactivity in the FO periphery, with differential colocalization with ZOs. Supporting cells formed N-cad-immunoreactive (ir) AJs with olfactory sensory neurons, E-cad-ir junctions with microvillar and gland duct cells, and both N-cad and E-cad-ir junctions in homotypic contacts. Alpha, beta- and p120-cat were localized in all AJs of the OE. AJs were scarce in the globose basal cell layer. Immature and mature neurons formed numerous contacts. In the FO, AJs were documented between OECs, between OECs and axons, and between axons. Most AJs colocalized N-cad with catenins, occasionally E-cad-ir AJs were found in the FO periphery. Characteristics of molecular composition suggest differential properties of TJs formed by neuronal, epithelial and glial cells in the OE and FO. The presence and molecular composition of AJs are consistent with a role of AJ proteins in neuroplastic processes in the peripheral olfactory pathway.     

RalA and RalB differentially regulate development of epithelial tight junctions

Tight junctions (TJs) are structures indispensable to epithelial cells and are responsible for regulation of paracellular diffusion and maintenance of cellular polarity. Although many interactions between TJ constituents have been identified, questions remain concerning how specific functions of TJs are established and regulated. Here we investigated the roles of Ral GTPases and their common effector exocyst complex in the formation of nascent TJs. Unexpectedly, RNA interference-mediated suppression of RalA or RalB caused opposing changes in TJ development. RalA reduction increased paracellular permeability and decreased incorporation of components into TJs, whereas RalB reduction decreased paracellular permeability and increased incorporation of components into TJs. Activities of both Ral GTPases were mediated through the exocyst. Finally, we show that TJ-mediated separation of apical-basal membrane domains is established prior to equilibration of barrier function and that it is unaffected by Ral knockdown or specific composition of TJs.