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First published online November 27, 2006
doi: 10.1242/10.1242/jcs.03233

Journal of Cell Science 119, 4819-4827 (2006)
Published by The Company of Biologists 2006
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Distinct subdomain organization and molecular composition of a tight junction with adherens junction features

Fabio D. Nunes1,*, Lanier N. Lopez1,*, Harrison W. Lin1,*, Caroline Davies1, Ricardo B. Azevedo1, Alexander Gow2,{ddagger} and Bechara Kachar1,{ddagger}

1 Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892, USA
2 Center for Molecular Medicine and Genetics, Carman and Ann Adams Dept of Pediatrics, Dept of Neurology, Wayne State University School of Medicine, Detroit, MI 48201, USA


Figure 1
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  		Fig. 1. Morphological features of the novel DC-OHC heterotypic junction. (a) Organ of Corti schematic. Pillar Cells (stippled) separate IHC (hatching) from OHC (black asterisks). Vertical black arrows, reticular lamina. (b) DIC micrograph of the reticular lamina apical surface. Green, DC-OHC junctions; red, DC-DC junctions. P, pillar cells. (c) SEM of the apical surface and vertical cut away of the reticular lamina. (d) TEM of two DC phalanges (left and center) and an OHC (right) reveals the morphologies of DC-DC and DC-OHC junctions. Square brackets, TJ in the DC-DC AJC. (e) Freeze-etching of a DC-DC AJ (left rectangle in d) reveals fibrils across the intercellular space (white arrowheads) likely corresponding to E-cadherin cross-bridges. (f) Freeze-etching of a DC-OHC junction (right rectangle in d) reveals fibrils in a DC-OHC junction (black arrowheads) similar to those in conventional freeze-fracture replicas (Fig. 5). (g) TEM of an OHC flanked by DCs shows DC-OHC junction morphology. (h) Freeze-etching reveals a DC-OHC junction (rectangle in g). The plasma membranes (brackets) are flanked by a dense cytoplasmic protein coat (green dots) and a large actin cytoskeleton. Tissue samples from: guinea pig, (b,e-h); rat (d) and mouse (c). Black stars, DC phalanges; white arrows, DC-OHC interface; white asterisks, apices of OHCs; MT, microtubule bundle. Bar in h, 7.6 µm (b); 6.4 µm (c); 0.6 µm (d); 0.1 µm (e,f); 0.85 µm (g); 0.3 µm (h).

 

Figure 2
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  		Fig. 2. Immunolocalization of TJ proteins in the reticular lamina. (a-d) Immunofluorescence labeling of the reticular lamina for ZO-1 (a), claudin-9 (b), claudin-6 (c) and claudin-14, with the antibody PB196 (d) at DC-OHC (white arrowheads) and DC-DC junctions (black arrows). (e-n) Immunogold electron microscopy gold labeling of DC-OHC junctions showing ZO-1 (e), occludin (f), claudin-9 (g,h), claudin-6 (i,j) and claudin-14, with the antibody PB196 (k-n). OHCs are to the right. Black arrowheads, apices of junctions. (o) Single confocal optical section near the apical surface of the reticular lamina showing immunofluorescence for claudin 14 (with the antibody PB321) in DC-OHC junctions (green), ZO-1 in DC-DC and DC-OHC junctions (blue) and actin (labeled with rhodamine phalloidin) in DC-DC and DC-OHC junctions and stereocilia. (p) Immunogold electron microscopy of claudin 14 labeling (with the antibody PB321) of DC-OHC junctions (arrowheads). The absence of background away from the junctions attests to the specificity of antibody labeling. Tissue samples from: guinea pig, (a-f,o,p); rat (g,i,k,l) or mouse (h,j,m,n). Bar in d, 10 µm (a-d); 0.4 µm (e-n); 10 µm (o); 0.3 µm (p).

 

Figure 3
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  		Fig. 3. Ultrastructure of the DC-OHC junction. (a) Freeze fracture shows the organization of intramembranous fibrils in two subdomains (brackets) of a DC-OHC junction at a tricellular DC-OHC-DC interface. (b) Electron micrograph from a 30 nm thin section shows the actin cytoskeleton (green dots) at a DC-OHC junction. Two subdomains (brackets) are apparent. Bar, 0.34 µm.

 

Figure 4
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  		Fig. 4. Immunolocalization of AJ proteins in the reticular lamina. (a,c,e) Whole-mount preparations show ß-catenin (a), p120ctn (c) and ß-actin (e) localized at DC-DC junctions (asterisks and arrows) and DC-OHC junctions (arrowheads). P in (e), pillar cells. (b,d,f) ImmunoEM shows ß-catenin (b), p120ctn (d) and ß-actin (f) at DC-OHC junctions. OHCs are to the right. Black arrowheads, claudin-14-rich apical subdomain. (g) Whole-mount preparation shows E-cadherin (green) at DC-DC junctions (arrows and asterisks) and ZO-1 (red) at DC-DC (arrows) and DC-OHC junctions (arrowheads). Tissue samples are from guinea pig. Bar in g, 10 µm (a,c,e,g); 0.7 µm (b,d,f).

 

Figure 5
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  		Fig. 5. Ultrastructure of the DC-OHC and DC-DC junctions. (a) Wide field freeze-fracture view of DC-OHC and DC-DC junctions reveals the overall organization of tight junction strands and the distinct morphologies between the DC-DC and DC-OHC junctions. (b) Anti-ß-actin immunoEM of DC-OHC and DC-DC junctions showing the dense labeling of the cytoplasmic plaque. Importantly, the plane of section passes through the plaque (electron opaque band) revealing it to be a continuous band across the DC phalanges between DC-DC and DC-OHC junctions. Gold particles also label other components of the apical region of the OHC, which correspond to the actin-rich cuticular plate of these cells. Bar, 0.2 µm.

 

Figure 6
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  		Fig. 6. Heterologous expression of claudins in transfected COS-7 cells. (a-c) Freeze fracture of unfixed transfected COS-7 cells shows fibrils (black arrowheads) for GFP-claudin-9 (a), -6 (b) and -14 (c) on the P- and E-fracture faces. (d-f) Confocal colocalization (white arrowheads) of endogenous ß-catenin (red) with GFP-claudin-9 (d) and -6 (e) but not -14 (f) (green) at transfected cell-cell contacts. Arrows in (d), GFP-claudin-9 in filopodia of a transfected cell. (g-i) Confocal colocalization (white arrowheads) of endogenous p120ctn (red) and GFP-claudin-9 (d) and -6 (e) but not -14 (f) (green). Bar in i, 0.15 µm (a-c); 3.4 µm (d-i).

 

Figure 7
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  		Fig. 7. Morphology, subdomain architecture and molecular components of AJCs and TAJs. (a) Diagram of a canonical AJC depicting a TJ and an AJ. The TJ comprises claudin-based anastomosing contacts between adjacent cell membranes. A ZO-1 scaffold assembles the underlying dense plaque and recruits a discrete actin cytoskeletal network. The AJ comprises a belt of cadherin-based transmembrane bridges and a catenin molecular complex assembles the underlying dense cytoplasmic plaque and recruits an extensive actin cytoskeletal network. (b) Diagram of a unitary, hybrid tight-adherens junction (TAJ) at the DC-OHC interface. The apical-most region of the TAJ is a claudin-14-rich subdomain of parallel TJ strands and the underlying ZO-1 scaffold assembles a small actin cytoskeletal network. A second subdomain enriched in claudin-9/6 forms an extensive belt of anastomosing membrane contacts. The underlying ZO-1 scaffold and the catenin molecular complex assemble a dense cytoplasmic plaque and recruit an extensive actin cytoskeletal network. The dense cytoplasmic plaque is localized to TAJs at a location that is analogous to their classical location in the canonical AJC.

 

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© The Company of Biologists Ltd 2006