Histochem Cell Biol (2008) 130:55–70 57 Fig. 2 Freeze–fracture image of the rat intestinal epithelium. The freeze fracture electron micrograph showns the apical brush border, the intramembraneous particle strands of the TJs and the lateral cell sur- face. The replica shows a continous network of TJ strands. Adapted from (Achler et al. 1989) with permission from D. Drenckhahn, Uni- versität Würzburg. Bar 0.5 m coexpression of claudin-1 and claudin-2 (Furuse et al. 1998b, 1999). Balda et al. (1996) however reported that overexpression of both, wild type and COOH-terminally truncated occludin overexpression in cultured MDCK epi- thelial cells induces not only a modest increase in TER but also signiWcant increase in the Xux of the non-charged com- pound FITC-dextran 4 kDa. Using freeze–fracture electron microscopy and immunocytochemistry, they were able to attribute this increase in paracellular permeability to an altered distribution and membrane topology between neigh- bouring cells. The claudins Searching for the proteins forming the structural backbone of the TJ strands, the Tsukita group re-examined the junc- tional fraction prepared from chicken liver and discovered the Wrst members of the claudin family: two 22-kDa pro- teins, claudin-1 and claudin-2 (Furuse et al. 1998a). The claudins also have four transmembrane domains, but do not show any sequence similarity to occludin (Fig. 3b). So far, 24 members of the claudin family have been identiWed in diVerent species. Claudins appear to be expressed in a tissue-speciWc manner; some claudins are expressed only in speciWc cell types, e.g. claudin-5 is expressed primarily by vascular endothelial cells or clau- din-11 appears to be expressed solely in oligodendrocytes and Sertoli cells (Morita et al. 1999). Moreover, most cell types express more than two claudin isoforms conferring diVerent size and charge selectivity qualities by the amino acid sequence of their extracellular loops, and the combina- tion of those isoforms results in cell- or tissue-type speciWc barrier function by “heteropolymerisation” into the result- ing TJ strands (see below). As demonstrated by immuno- gold electron microscopy, there is accumulating evidence now that the claudins do constitute the TJ strands as observed by freeze–fracture electron microscopy (Tsukita et al. 2001). Exogenous expression of claudins in Wbro- blasts devoid of endogenous TJs resulted in the formation of TJ strands. Summarizing existing data, there is clear evidence that the claudins constitute the backbone of TJ strands, while occludin, which itself cannot reconstitute such TJ strands, seems to play a permeability regulating role by incorporating itself into the claudin-based strands, a process, the mecha- nism of which however is not yet understood. The func- tion(s) of occludin thus still remain to be elucidated. Junction-associated adhesion molecules (JAMs) A third class of integral membrane proteins comprises members of the immunoglobulin superfamily (Fig. 3c), and can be subdivided into a group consisting of JAM proteins and a group consisting of CAR, CLMP, ESAM and JAM-4 (Ebnet 2008; Ebnet et al. 2004). To date, at least three JAM isoforms have been described, which are expressed diVer- entially in epithelial and endothelial cells, but also in cells devoid of TJ strands like leukocytes (D’Atri and Citi 2002). Except for JAM-1, little is known about the role of these proteins in TJ assembly and function. JAM-1, formerly F11 receptor (Sobocka et al. 2000) (Fig. 3c), may be involved in immune cell transmigration or cell adhesion (Bazzoni et al. 2000) but has not been localized to the junction strands. A putative PDZ binding motif in the cytoplasmic domain of JAM-1 has been identiWed, which has been able to interact with PDZ domains of ZO-1, AF-6, PAR-3 and MUPP1 (D’Atri and Citi 2002; Itoh et al. 2001). Moreover, JAM-1 has been shown to interact with cingulin. Thus, even though JAM-1 is not part of the TJ strands, it may well be involved in the propagation of signal cascades resulting from homophilic and heterophilic adhesion. A role for JAM-1 in the complex process of adhesion and transmigra- tion of monocytes through endothelial cells has been dem- onstrated (Martin-Padura et al. 1998). As another function 123