Tight junctions (TJ) consist of networks of linear sealing strands between adjoining cells, forming cell-cell contacts in epithelial cell sheets that regulate the ion permeation through the intercellular space. Claudins, a large family of transmembrane proteins, are the main components of the TJ. Crystal structure of claudin-15 monomer has been reported recently. However, it has remained largely unknown what are the crucial domains involved in the polymerization toward highly-ordered strands, what is the architecture of claudin strands and paracellular pores in atomic level resolution, and how claudins interact with other TJ proteins to achieve barrier and pore functions. Multiple claudins are expressed in the inner ear and are required for normal hearing. We have previously shown that in outer hair cells (OHCs) claudin-14 and claudin-9/6 segregate into morphologically distinguishable TJ subdomains. This contrasts with the common view that most claudins co-assemble into heteromeric strands. Our long term goal is to attain a molecular-level description of TJ assembly and a complete ultrastructural model of the TJ strand backbone in the inner ear. These findings will collectively provide the basis to understand how various claudins, and TJ accessory proteins expressed in hair cells and supporting cells, assemble to form the homotypic and heterotypic TJs of the inner ear. To identify and characterize interacting claudin interfaces we use a combination of computational methods, including evolutionary coupling, residue conservation mapping, and solvent accessible surface area evaluation. To validate our in silico data we perform mutagenesis analysis, cultured cell transfections, live-cell imaging, and freeze fracture EM. Based on the validated in silico and in vitro data, we use experimental data driven molecular docking and molecular dynamics (MD) to generate claudin strand models and to study the paracellular ion conductivity mechanisms. We have just completed the screening and evaluation of 1200 intermolecular claudin-claudin interfaces by in silico methods. Among them, four interfaces, including three lateral (cis-interaction) and one head-to-head (trans-interaction) interfaces, are consistent with the mutagenesis experimental data. Our interface analysis indicates that hydrophobic residues on extracellular loop1 (i.e. Ile39 and Ile44) and loop2 (i.e. Phe146) are essential for the trans interactions. Mutations on these residues either eliminated or greatly reduced the TJ strand formation. We further uncovered that intermolecular hydrogen bonds between Ser67 and Glu157 are important for lateral (-cis) interactions. Based on these interfaces we generated an octameric model of the claudin strand. In our model, we observe a 10A extracellular pore formed by two strongly associated claudin tetramers similar to claudin that is consistent with the selectivity, and pore architecture data, of claudins reported elsewhere. Our model provides a mechanistic insight of TJ polymerization, paracellular molecular flow through TJ and potential targets for TJ modulation. A complete molecular level model of the TJ strands backbone will provide the basis to understand how claudin isoforms and TJ accessory proteins express and assemble in the inner ear epithelium to form the various homotypic and heterotypic TJs essential for hearing and vestibular function. Tight junction fibrils or strands have been extensively studied by freeze fracture electron microscopy. Freeze fracture as well as freeze etching methods, rely on creating a metal cast of the surface of the freeze-fractured samples which is then imaged by TEM. The platinum used to create the contrast in replicas has a tendency to crystallize during deposition creating a coarse representation of the original sample, and limiting the resolution of the method. In an attempt to extend the resolution of the method and better analyze the substructure of the TJ strands we have been exploring the use of new metal alloys called glass metals where a combination of metals with atoms with different sizes creates a mismatch and prevents the growth of regular crystals. We have tested several non-crystal forming alloys of either zirconium (LM-105), hafnium (LM-X6), or platinum (Pt-850). The replicas generated had significantly smaller grain size and showed electron diffraction patterns characteristic of non-crystal structures. Glass-metal replicas of cells expressing claudin fibrils confirmed the predicted anti-parallel double row arrangement of a claudin strand. We further explored the use of phase plate TEM with amorphous and carbon-only replicas, resulting in increased ability to resolve the substructure and periodicity of the TJ strands. Our new amorphous replica method extends the resolution of freeze-fracture and freeze-etching microscopy and shows great promise for studying structures that would be difficult to study by cryo-EM. 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. We helped Dr. James M. Andersons laboratory (NHLBI) identify and characterize a novel functional complex of the junction scaffolding protein ZO-1 and the F-BAR-domain protein TOCA-1. Ultrastructural analysis shows actin accumulation at the adherens junction in TOCA-1-knockout cells but the morphology of the TJ strand as seen in freeze-fracture replicas was unaltered. 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. We had previously reported that the apical junction in the cochlear epithelium is organized in a fashion similar to muscle sarcomeres, in that NMII, constituting the hair cell's equivalent of the M-line, alternates with actin and alpha-actinin, which are localized at the hair cell's equivalent of the Z-disk. In collaboration with the laboratory of Dr. Jung-Bum Shin we identified a novel component of the apical junctional sarcomeric belt. This collaborative work showed that multiple isoforms of Xin-Actin Binding Repeat Containing 2 (XIRP2) protein are expressed in hair cells, co-localizing with actin-rich structures in bundles, the underlying cuticular plate, as well as the apical junctional circumferential actin belt. In cardiomyocytes, XIRP2 localizes to the intercalated disk, which is a site of mechanical and electrical coupling between neighboring cardiomyocytes and thus can be considered analogous to the hair cell/supporting cell junction. Since XIRP2 binds to alpha-actinin, we hypothesized that XIRP2 also localizes to adherens junctions of hair cells. Triple-labeling experiments with antibodies specific for XIRP2, alpha--actinin, and non-muscle myosin IIC (NMIIC) showed that all three proteins localized at periodic loci along the apical hair cell junction. Interestingly, XIRP2-immunoreactive foci at the apical junction localize closer to NMII than alpha--actinin. The co-localization data also suggests that the C-terminus of the long XIRP2 isoform binds to alpha--actinin embedded in the Z-disk, with the Xin repeats coextending with F-actin; in contrast, the N-terminus, may be positioned closer to the M-line, which explains the colocalization with NMII (rather than alpha--actinin) in triple-labeling experiments. Such a model is reminiscent of the role of nebulin, which is believed to act as a regulator of thin filament length and Z-disk structure. A concerted role of XIRP2 and nebulin is supported by the fact that these two proteins interact with each other, and contain repeat domains (Xin repeats in XIRP2 and nebulin-like repeats in nebulin) that bind F-actin in a similar manner.
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