56 Histochem Cell Biol (2008) 130:55–70 Fig. 1 Molecular composition of tight junctions. The transmembrane proteins occludin, the claudin(s) and junctional adhesion molecule-1 (JAM-1) constitute the barrier formed by TJs sealing the paracellular space. They appear to be interacting in a homophilic manner, and occludin seems to co-polymerase into claudin-based TJ strands. Clau- dins adhere with each other in a homotypic as well as a heterotypic manner. ZO-1, -2, and -3 bind the cytoplasmic tail of occludin and link the TJ to the actin cytoskeleton. Proteins of the ZO family can shuttle to the nucleus to inXuence transcriptional processes in cellular proliferation and diVerentiation. The ZO-proteins have also been shown to interact with claudins and provide molecular scaVolds for TJ assembly. Cingulin is a 140 kDa TJ plaque protein which assoicates with the actomyosin cytoskeleton. Its putative function is transduction yielding diVerences in permeability barrier function between diVerent tissues (Staehelin 1973). However, since a direct linear relationship between the complexity of the TJ strand network and the measured transepithelial electri- cal resistance (TER) could not unambiguousy be estab- lished for all cell types analysed, it was predicted that the strands might contain pores that Xuctuate between open and closed conformations, suggesting that the TJ strands appear to be remarkably dynamic (Claude 1978). Occludin Recent identiWcation of the TJ-speciWc integral membrane proteins forwarded our understanding of TJ molecular com- position in mammals: occludin (ca. 60 kDa) was identiWed as the Wrst integral membrane protein localised at TJs in chicken (Furuse et al. 1993) and then also in mammals 123 of the mechanical force generated by the actomyosin cytoskeleton important for cellular diVerentiation. The Ras target AF-6 interacts with ZO-1 and serves as a. peripheral component of tight junctions in epithelial cells. Symplekin is a 126 kDa protein that occurs and probably functions in the nucleus as well as in the TJ plaques. Tyrosine phosphorylated Par3 regulates tight junction assembly and promotes cellular polarity by intracellular signalling. Localization of 7H6 TJ-associated antigen along the cell border of vascular endothelial cells has been shown to correlate with paracellular barrier function. Additional proteins have been localised to the TJs but a function has presently not been assigned. Moreover diverse signaling proteins are detected at the apical junctional complex but they are not uniquely conWned to the TJ (Ando-Akatsuka et al. 1996). The occludin transmembrane domain spans the membrane four times with a short cyto- plasmic N-terminus and an especially long corboxy-termi- nal cytoplasmic domain (Fig. 3a). Occludin localizes to the TJs, and its overexpression increases TER in mammalian epithelial cells (Balda et al. 1996). However, transfection of insect cells devoid of endogenous TJs with occludin cDNA surprisingly demonstrated that occludin is not by itself suYcient to form TJ strands but its expression merely pro- duced focal homophilic adhesion sites (Furuse et al. 1996). Moreover, disruption of both occludin alleles in embryonic stem cells did not prevent the formation of an eVective diVusion barrier and the polarisation of epithelial cells, as shown by freeze–fracture analysis and TER assessment (Saitou et al. 1998). It was thus concluded that occludin is not required for the formation of TJ strands. However, occludin appears to interact, directly or indirectly, with claudins and is recruited into the long strands formed by