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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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

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.

Tight junctions form continuous intercellular contacts controlling solute movement through the paracellular pathway across epithelia. Paracellular barriers vary among epithelia in electrical resistance and behave as if they are lined with pores that have charge and size selectivity. Recent evidence shows that claudins, a large family (at least 24 members) of intercellular adhesion molecules, form the seal and its variable pore-like properties. This evidence comes from the study of claudins expressed in cultured epithelial cell models, genetically altered mice, and human mutants. We review information on the structure, function, and transcriptional and posttranslational regulation of the claudin family as well as of their evolutionarily distant relatives called the PMP22/EMP/MP20/claudin, or pfam00822, superfamily.

Tight junctions form selective barriers that regulate paracellular transport across epithelia. A large family of tetraspanning cell-cell adhesion proteins called claudins create the barrier and regulate electrical resistance, size, and ionic charge selectivity. Study of inherited human claudin diseases and the outcome of the genetic manupulation of claudins in mice, Drosophila, and Caenorhabditis elegans are furthering our understanding of paracellular physiology.

The tight junction seal formed between epithelial cells varies among tissues in both tightness and ionic charge selectivity. We recently demonstrated that the extracellular domains of the claudin family of proteins are determinants of both characteristics, but in that study other unidentified domains in the claudins clearly contributed to their physiological potency. To investigate the importance of the cytoplasmic carboxyl-terminal domains in determining the degree to which a claudin can influence barrier properties, we constructed chimeras by exchanging the tails of claudin-2 and -4 and expressing them in MDCK II cells. Although swapping these domains had little effect on claudin localization, we found that the tail of claudin-2 could stabilize claudin-4, with a concomitant increase in both protein level and physiologic influence. This difference in stability was not an artifact of their chimeric structure, since metabolic radio-labeling experiments revealed that the half-life of endogenous claudin-2 is more than three times longer than claudin-4 (>12 h and approximately 4 h respectively). Further, half-life was not affected by removing the carboxyl-terminal three amino acids, which form a PDZ-binding motif. The finding that cytoplasmic tails of claudins strongly influence stability reveals a potential mechanism by which cells can establish their tight junction protein composition and thus function.

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.

Tight junctions (TJ) regulate paracellular ionic charge selectivity and conductance across epithelial tissues and cell lines. These properties vary among epithelia, and recent evidence implicates the claudins, a family of TJ transmembrane proteins, as important determinants of both characteristics. To test the hypothesis that each claudin contributes a characteristic charge discrimination to the TJ, we expressed claudins-2, -4, -11, and -15 in both cation-selective Madin-Darby canine kidney (MDCK) II cells and in anion-selective LLC-PK1 cells and examined changes in transepithelial electrical resistance (TER) and paracellular charge selectivity. Regulated expression and localization were verified by immunoblot analysis and immunofluorescence microscopy, respectively. Expression of claudin-4 increased TER in both cell lines, whereas effects of the others on TER were variable. Claudin-4 and -11 decreased paracellular permeability for Na+ in MDCK II cells, whereas neither claudin-2 nor -15 had an effect. Conversely, in LLC-PK1 cells, claudin-2 and -15 increased the permeability for Na+, whereas claudin-4 and -11 were without effect. We conclude that the contribution of each claudin is most easily detectable when it reverses the direction of monolayer charge selectivity. These results are consistent with a model in which exogenous claudins add new charge-selective pores, leading to a physiological phenotype that combines endogenous and exogenous contributions. Additionally, it is possible to rationalize the direction of charge selectivity conferred by the individual claudins on the basis of electrostatic effects of the charged amino acids in their first extracellular loops.

Epithelia separate tissue spaces by regulating the passage of ions, solutes, and water through both the transcellular and paracellular pathways. Paracellular permeability is defined by intercellular tight junctions, which vary widely among tissues with respect to solute flux, electrical resistance, and ionic charge selectivity. To test the hypothesis that members of the claudin family of tight junction proteins create charge selectivity, we assessed the effect of reversing the charge of selected extracellular amino acids in two claudins using site-directed mutagenesis. Claudins were expressed in cultured Madin-Darby canine kidney cell monolayers under an inducible promoter, and clones were compared with and without induction for transmonolayer electrical resistance and dilution potentials. Expression and localization of claudins were determined by immunoblotting, immunofluorescence microscopy, and freeze-fracture electron microscopy. We observed that substituting a negative for a positive charge at position 65 in the first extracellular domain of claudin-4 increased paracellular Na+ permeability. Conversely, substituting positive for negative charges at three positions in the first extracellular domain of claudin-15, singly and in combination, reversed paracellular charge selectivity from a preference for Na+ to Cl-. These results support a model where claudins create charge-selective channels in the paracellular space.