MARCKS-related protein regulates cytoskeletal organization at cell-cell and cell-substrate contacts in epithelial cells.

Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines. However, deletion of MRP did not abrogate the cytokine responses, suggesting that MRP is not required in the occludin-dependent IFN/TNF response. Instead, our results reveal a key role for MRP in epithelial cells in control of multiple actin-based structures, likely by regulation of integrin signaling. Changes in focal adhesion organization and basal actin stress fibers in MRP-knockout (KO) cells were reminiscent of those seen in FAK-KO cells. In addition, we found alterations in cell-cell interactions in MRP-KO cells associated with increased junctional tension, suggesting that MRP may play a role in focal adhesion-adherens junction cross talk. Together, our results are consistent with a key role for MRP in cytoskeletal organization of cell contacts in epithelial cells.

Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo.

UNLABELLED: Liver kinase B1 (LKB1) and its downstream effector AMP-activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich-cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver-specific (albumin-Cre) LKB1 knockout mice (LKB1(-/-) ) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1(-/-) livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6-carboxyfluorescein diacetate (6-CFDA), which is processed in hepatocytes into its fluorescent derivative 6-carboxyfluorescein (6-CF) and secreted into the canaliculi. In LKB1(-/-) mice, 6-CF remained largely in hepatocytes, canalicular secretion was delayed, and 6-CF appeared in the blood. To test whether 6-CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6-CFDA. In wild-type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1(-/-) mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. CONCLUSION: LKB1 plays a critical role in regulating the maintenance of TJ and paracellular permeability, which may explain how various drugs, chemicals, and metabolic states that inhibit the LKB1/AMPK pathway result in cholestasis. (Hepatology 2016;64:1317-1329).

Architecture of tight junctions and principles of molecular composition.

The tight junction creates an intercellular barrier limiting paracellular movement of solutes and material across epithelia. Currently many proteins have been identified as components of the tight junction and understanding their architectural organization and interactions is critical to understanding the biology of the barrier. In general the architecture can be conceptualized into compartments with the transmembrane barrier proteins (claudins, occludin, JAM-A, etc.), linked to peripheral scaffolding proteins (such as ZO-1, afadin, MAGI1, etc.) which are in turned linked to actin and microtubules through numerous linkers (cingulin, myosins, protein 4.1, etc.). Within this complex network are associated many signaling proteins that affect the barrier and broader cell functions. The PDZ domain is a commonly used motif to specifically link individual junction protein pairs. Here we review some of the key proteins defining the tight junction and general themes of their organization with the perspective that much will be learned about function by characterizing the detailed architecture and subcompartments within the junction.

Structural Basis of a Key Factor Regulating the Affinity between the Zonula Occludens First PDZ Domain and Claudins.

The molecular seal between epithelial cells, called the tight junction (TJ), is built by several membrane proteins, with claudins playing the most prominent role. The scaffold proteins of the zonula occludens family are required for the correct localization of claudins and hence formation of the TJ. The intracellular C terminus of claudins binds to the N-terminal PDZ domain of zonula occludens proteins (PDZ1). Of the 23 identified human claudin proteins, nine possess a tyrosine at the -6 position. Here we show that the claudin affinity for PDZ1 is dependent on the presence or absence of this tyrosine and that the affinity is reduced if the tyrosine is modified by phosphorylation. The PDZ1 ß2-ß3 loop undergoes a significant conformational change to accommodate this tyrosine. Cell culture experiments support a regulatory role for this tyrosine. Plasticity has been recognized as a critical property of TJs that allow cell remodeling and migration. Our work provides a molecular framework for how TJ plasticity may be regulated.

Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cell-cell and cell-substrate adhesion.

Known proteins associated with the cell-adhesion protein E-cadherin include catenins and proteins involved in signaling, trafficking and actin organization. However, the list of identified adherens junction proteins is likely to be incomplete, limiting investigation into this essential cell structure. To expand the inventory of potentially relevant proteins, we expressed E-cadherin fused to biotin ligase in MDCK epithelial cells, and identified by mass spectrometry neighboring proteins that were biotinylated. The most abundant of the 303 proteins identified were catenins and nearly 40 others that had been previously reported to influence cadherin function. Many others could be rationalized as novel candidates for regulating the adherens junction, cytoskeleton, trafficking or signaling. We further characterized lipoma preferred partner (LPP), which is present at both cell contacts and focal adhesions. Knockdown of LPP demonstrated its requirement for E-cadherin-dependent adhesion and suggested that it plays a role in coordination of the cell-cell and cell-substrate cytoskeletal interactions. The analysis of LPP function demonstrates proof of principle that the proteomic analysis of E-cadherin proximal proteins expands the inventory of components and tools for understanding the function of E-cadherin.

The N and C termini of ZO-1 are surrounded by distinct proteins and functional protein networks.

BACKGROUND: Biotin ligase tagging with ZO-1 was applied to identify a more complete tight junction proteome. RESULTS: Identical but also different proteins and functional networks were identified near the N and C ends of ZO-1. CONCLUSION: The ends of ZO-1 are embedded in different functional subcompartments of the tight junction. SIGNIFICANCE: Biotin tagging with ZO-1 expands the tight junction proteome and defines subcompartments of the junction. The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier-forming proteins like occludin and the claudin family; scaffolding proteins like ZO-1; and some cytoskeletal, signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we identified the proteins that are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N or C terminus, expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Of a predicted proteome of ∼9000, we identified more than 400 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C-terminal ends. Those proximal to the N terminus were enriched in transmembrane tight junction proteins, and those proximal to the C terminus were enriched in cytoskeletal proteins. We also identified many unexpected but easily rationalized proteins and verified partial colocalization of three of these proteins with ZO-1 as examples. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly, the technique demonstrates high spatial resolution, which could be generally applied to defining other subcellular protein compartmentalization.

Phosphorylation of claudin-2 on serine 208 promotes membrane retention and reduces trafficking to lysosomes.

Claudins are critical components of epithelial and endothelial tight junction seals, but their post-transcriptional regulation remains poorly understood. Several studies have implicated phosphorylation in control of claudin localisation and/or function, but these have focused on single sites or pathways with differing results, so that it has been difficult to draw general functional conclusions. In this study, we used mass spectrometry (MS) analysis of purified claudin-2 from MDCK II cells and found that the cytoplasmic tail is multiply phosphorylated on serines, a threonine and tyrosines. Phos-tag SDS PAGE revealed that one site, S208, is heavily constitutively phosphorylated in MDCK II cells and in mouse kidney; this site was targeted for further study. Mutational analysis revealed that the phosphomimetic mutant of claudin-2, S208E, was preferentially localised to the plasma membrane while claudin-2 S208A, which could not be phosphorylated at this site, both immunolocalized and co-fractionated with lysosomal markers. Mutations at sites that were previously reported to interfere with plasma membrane targeting of claudin-2 reduced phosphorylation at S208, suggesting that membrane localisation is required for phosphorylation; however phosphorylation at S208 did not affect binding to ZO-1 or ZO-2 Administration of forskolin or PGE2 resulted in dephosphorylation at S208 and transient small increases in transepithelial electrical resistance (TER). Together these data are consistent with phosphorylation at S208 playing a major role in the retention of claudin-2 at the plasma membrane.

Claudin-2 forms homodimers and is a component of a high molecular weight protein complex.

Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.

Occludin is required for cytokine-induced regulation of tight junction barriers.

The function of occludin remains elusive. Proposed roles include maintenance of tight junction barriers, signaling and junction remodeling. To investigate a potential role in mediating cytokine-induced changes in barrier properties, we measured barrier responses to interferon-gamma plus TNFalpha in control, occludin-overexpressing and occludin knockdown MDCK II monolayers. MDCK cells show a complex response to cytokines characterized by a simultaneous increase in the transepithelial electrical resistance and a decrease in the barrier for large solutes. We observed that overexpression of occludin increased and occludin knockdown decreased sensitivity to cytokines as assessed by both these parameters. It is known that caveolin-1 interacts with occludin and is implicated in several models of cytokine-dependent barrier disruption; we found that occludin knockdown altered the subcellular distribution of caveolin-1 and that partitioning of caveolin into detergent-insoluble lipid rafts was influenced by changing occludin levels. Knockdown of caveolin decreased the cytokine-induced flux increase, whereas the increase in the electrical barrier was unaltered; the effect of double knockdown of occludin and caveolin was similar to that of occludin single knockdown, consistent with the possibility that they function in the same pathway. These results demonstrate that occludin is required for cells to transduce cytokine-mediated signals that either increase the electrical barrier or decrease the large solute barrier, possibly by coordinating the functions of caveolin-1.

Net intestinal transport of oxalate reflects passive absorption and SLC26A6-mediated secretion.

Mice lacking the oxalate transporter SLC26A6 develop hyperoxalemia, hyperoxaluria, and calcium-oxalate stones as a result of a defect in intestinal oxalate secretion, but what accounts for the absorptive oxalate flux remains unknown. We measured transepithelial absorption of [(14)C]oxalate simultaneously with the flux of [(3)H]mannitol, a marker of the paracellular pathway, across intestine from wild-type and Slc26a6-null mice. We used the anion transport inhibitor DIDS to investigate other members of the SLC26 family that may mediate transcellular oxalate absorption. Absorptive flux of oxalate in duodenum was similar to mannitol, insensitive to DIDS, and nonsaturable, indicating that it is predominantly passive and paracellular. In contrast, in wild-type mice, secretory flux of oxalate in duodenum exceeded that of mannitol, was sensitive to DIDS, and saturable, indicating transcellular secretion of oxalate. In Slc26a6-null mice, secretory flux of oxalate was similar to mannitol, and no net flux of oxalate occurred. Absorptive fluxes of both oxalate and mannitol varied in parallel in different segments of small and large intestine. In epithelial cell lines, modulation of the charge selectivity of the claudin-based pore pathway did not affect oxalate permeability, but knockdown of the tight-junction protein ZO-1 enhanced permeability to oxalate and mannitol in parallel. Moreover, formation of soluble complexes with cations did not affect oxalate absorption. In conclusion, absorptive oxalate flux occurs through the paracellular "leak" pathway, and net absorption of dietary oxalate depends on the relative balance between absorption and SLC26A6-dependent transcellular secretion.

Setting up a selective barrier at the apical junction complex.

Across the animal kingdom the apical junction complex of epithelial cells creates both a permeability barrier and cell polarity. Although based on overlapping and evolutionarily conserved proteins, the cell-cell contacts of nematodes, flies and mammals appear to differ in morphology and functional organization. Emerging evidence shows that the selective pore-like properties of vertebrate and invertebrate barriers are created by the claudin family. Similarly, assembly of the barriers requires a conserved set of polarity-generating protein complexes, particularly the PAR protein complexes.

Newly synthesized claudins but not occludin are added to the basal side of the tight junction.

A network of claudin strands creates continuous cell-cell contacts to form the intercellular tight junction barrier; a second protein, occludin, is associated along these strands. The physiological barrier remains stable despite protein turnover, which involves removal and replacement of claudins both in the steady state and during junction remodeling. Here we use a pulse-block-pulse labeling protocol with fluorescent ligands to label SNAP/CLIP-tags fused to claudins and occludin to identify their spatial trafficking pathways and kinetics in Madin-Darby canine kidney monolayers. We find that claudins are first delivered to the lateral membrane and, over time, enter the junction strand network from the basal side; this is followed by slow replacement of older claudins in the strands. In contrast, even at early times, newly synthesized occludin is found throughout the network. Taking the results together with our previous documentation of the mechanism for claudin strand assembly in a fibroblast model, we speculate that newly synthesized claudins are added at strand breaks and free ends; these are most common in the basalmost edge of the junction. In contrast, occludin can be added directly within the strand network. We further demonstrate that claudin trafficking and half-life depend on carboxy-terminal sequences and that different claudins compete for tight junction localization.

Phosphorylation of tight junction transmembrane proteins: Many sites, much to do.

Phosphorylation is a dynamic post-translational modification that can alter protein structure, localization, protein-protein interactions and stability. All of the identified tight junction transmembrane proteins can be multiply phosphorylated, but only in a few cases are the consequences of phosphorylation at specific sites well characterized. The goal of this review is to highlight some of the best understood examples of phosphorylation changes in the integral membrane tight junction proteins in the context of more general overview of the effects of phosphorylation throughout the proteome. We expect as that structural information for the tight junction proteins becomes more widely available and the molecular modeling algorithms improve, so will our understanding of the relevance of phosphorylation changes at single and multiple sites in tight junction proteins.

Multiple claudin-claudin cis interfaces are required for tight junction strand formation and inherent flexibility.

Tight junctions consist of a network of sealing strands that create selective ion permeability barriers between adjoining epithelial or endothelial cells. The current model for tight junction strands consists of paired rows of claudins (Cldn) coupled by a cis interface (X-1) derived from crystalline Cldn15. Here we show that tight junction strands exhibit a broad range of lateral bending, indicating diversity in cis interactions. By combining protein-protein docking, coevolutionary analysis, molecular dynamics, and a mutagenesis screen, we identify a new Cldn-Cldn cis interface (Cis-1) that shares interacting residues with X-1 but has an ~ 17° lateral rotation between monomers. In addition, we found that a missense mutation in a Cldn14 that causes deafness and contributes stronger to Cis-1 than to X-1 prevents strand formation in cultured cells. Our results suggest that Cis-1 contributes to the inherent structural flexibility of tight junction strands and is required for maintaining permeability barrier function and hearing.

Visualizing the dynamic coupling of claudin strands to the actin cytoskeleton through ZO-1.

The organization and integrity of epithelial tight junctions depend on interactions between claudins, ZO scaffolding proteins, and the cytoskeleton. However, although binding between claudins and ZO-1/2/3 and between ZO-1/2/3 and numerous cytoskeletal proteins has been demonstrated in vitro, fluorescence recovery after photobleaching analysis suggests interactions in vivo are likely highly dynamic. Here we use superresolution live-cell imaging in a model fibroblast system to examine relationships between claudins, ZO-1, occludin, and actin. We find that GFP claudins make easily visualized dynamic strand patches between two fibroblasts; strand dynamics is constrained by ZO-1 binding. Claudin association with actin is also dependent on ZO-1, but colocalization demonstrates intermittent rather than continuous association between claudin, ZO-1, and actin. Independent of interaction with ZO-1 or actin, claudin strands break and reanneal; pulse-chase-pulse analysis using SNAP-tagged claudins showed preferential incorporation of newly synthesized claudins into break sites. Although claudin strand behavior in fibroblasts may not fully recapitulate that of epithelial tight junction strands, this is the first direct demonstration of the ability of ZO-1 to stabilize claudin strands. We speculate that intermittent tethering of claudins to actin may allow for accommodation of the paracellular seal to physiological or pathological alterations in cell shape or movement.

Sorbin and SH3 domain-containing protein 2 (SORBS2) is a component of the acto-myosin ring at the apical junctional complex in epithelial cells.

SORBS2 is a scaffolding protein associated with Abl/Arg non-receptor tyrosine kinase pathways and is known to interact with actin and several other cytoskeletal proteins in various cell types. Previous BioID proximity labeling of tight and adherens junction proteins suggested that SORBS2 is a component of the apical junction complex of epithelial cells. We asked whether SORBS2 plays a previously unappreciated role in controlling perijunctional actin and tight junction barrier function. Using super resolution imaging we confirmed that SORBS2 is localized at the apical junction complex but farther from the membrane than ZO-1 and located partially overlapping both the tight- and adherens junctions with a periodic concentration that alternates with myosin IIB in polarized epithelial cells. Overexpression of GFP-SORBS2 recruited alpha-actinin, vinculin and N-WASP, and possibly CIP4 to cellular junctions. However, CRISPR-Cas9 knock-out of SORBS2 did not alter the localization- or immunofluorescent staining intensity of these or several other junctional- and cytoskeletal proteins. SORBS2 knock-out also did not affect the barrier function as measured by TER and dextran flux; nor did it change actin-dependent junction re-assembly as measured by Ca2+-switch and Latrunculin-B wash-out assays. The kinetics of HGF-induced cell scattering and wound healing, and dextran flux increase induced by PDGF also were unaffected by SORBS2 knock-out. SORBS2 concentrates with apical junctional actin that accumulates in response to knock-down of ZO-1 and ZO-2. In spite of our finding that SORBS2 is clearly a component of the apical junction complex, it does not appear to be required for either normal tight- or adherens junction assembly, structure or function or for growth factor-mediated changes in tight junction dynamics.

Apical surface supracellular mechanical properties in polarized epithelium using noninvasive acoustic force spectroscopy.

Maintenance of epithelial tissue integrity requires coordination between cell-cell adherens junctions, tight junctions (TJ), and the perijunctional actomyosin cytoskeleton. Here we addressed the hypothesis that alterations in TJ structure and remodeling of the actomyosin cytoskeleton modify epithelial mechanics. Current methods to measure supracellular mechanical properties disrupt intact monolayers, therefore, we developed a novel method using noncontact acoustic frequency-modulation atomic force microscopy (FM-AFM) and tested it on MDCK polarized monolayers. Our results show that double knockdown (dKD) of ZO-1/ZO-2 elevates the apical epithelial tension and effective viscosity. Interestingly, epithelial tension is more sensitive to inhibition of myosin II ATPase activity than to inhibition of ROCK activity, but viscosity is highly sensitive to both. Additionally, we showed epithelial intercellular pulling forces at tricellular junctions and adhesion forces in dKD cells are elevated with an increase in contractility. In conclusion, FM-AFM enables the physiological and quantitative investigation of mechanics in intact epithelium.

Claudin profiling in the mouse during postnatal intestinal development and along the gastrointestinal tract reveals complex expression patterns.

Members of the claudin protein family are key regulators of tight junction selectivity and are implicated in influencing development and cellular differentiation in the intestine and other tissues. The goal of the present study was to profile claudin gene expression and protein location during postnatal development of the mouse jejunum and in the adult mouse gut from duodenum to distal colon as a first step in understanding both normal claudin function and the pathologic implications of altered expression patterns. The relative expression of claudins 1-19 and other tight and adherens junction genes was determined by quantitative RT-PCR from six regions of normal mouse intestine and colon. Immunofluorescent localization was performed for claudins 1-5, 7, 8, 10, 12, 15, and 18. Transcripts for claudins 1-5, 7-13, 17, and 18 were all detected in adult intestine, although their relative abundance differed up to 1000-fold within individual segments. In contrast to the unchanging expression and localization of ZO-1, occludin, and JAM, most claudins were expressed in decreasing or increasing gradients or in more complex patterns along the longitudinal axis of the intestine and the crypt to villus/surface differentiation axis. During neonatal development at days 1, 14, 28, and 90 several claudins showed striking increases or decreases in transcript expression as well as changes in tissue localization along the crypt-villus axis. Claudin-19 was only detected at days 1 and 14. This database provides a resource for investigating regional and developmental differences in permselectivity, crypt to villus/surface differentiation and neoplastic changes along the gut and during postnatal development.

A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction.

Assembly and sealing of the tight junction barrier are critically dependent on the perijunctional actin cytoskeleton, yet little is known about physical and functional links between barrier-forming proteins and actin. Here we identify a novel functional complex of the junction scaffolding protein ZO-1 and the F-BAR-domain protein TOCA-1. Using MDCK epithelial cells, we show that an alternative splice of TOCA-1 adds a PDZ-binding motif, which binds ZO-1, targeting TOCA-1 to barrier contacts. This isoform of TOCA-1 recruits the actin nucleation-promoting factor N-WASP to tight junctions. CRISPR-Cas9-mediated knockout of TOCA-1 results in increased paracellular flux and delayed recovery in a calcium switch assay. Knockout of TOCA-1 does not alter FRAP kinetics of GFP ZO-1 or occludin, but longer term (12 h) time-lapse microscopy reveals strikingly decreased tight junction membrane contact dynamics in knockout cells compared with controls. Reexpression of TOCA-1 with, but not without, the PDZ-binding motif rescues both altered flux and membrane contact dynamics. Ultrastructural analysis shows actin accumulation at the adherens junction in TOCA-1-knockout cells but unaltered freeze-fracture fibril morphology. Identification of the ZO-1/TOCA-1 complex provides novel insights into the underappreciated dependence of the barrier on the dynamic nature of cell-to-cell contacts and perijunctional actin.