Transmembrane proteins of tight junctions

The first transmembrane protein of TJs to be identified was occludin, following the generation of monoclonal antibodies against a liver junctional fraction, by the Tsukita laboratory in 1993 (Furuse et al., 1993). Occludin belongs to the TJ-associated MARVEL domain-containing protein family, which also includes tricellulin. Tricellulin is present at tricellular TJs (tTJs) (Ikenouchi et al., 2005), i. e. specialized structures occurring at the junction between three neighboring cells, but can localize along bicellular TJs when occludin is depleted (Ikenouchi et al., 2008). Spanning the plasma membrane four times, occludin harbors a cytoplasmic C-terminus that is rich in phosphorylatable residues (serine, threonine and tyrosine), and its phosphorylation has been linked to its accumulation at junctions and to the dynamic regulation of TJs (Wong, 1997; Sakakibara et al., 1997; Andreeva et al., 2001; Hirase et al., 2001). The C-terminal tail of occludin interacts directly with the actin cytoskeleton and also indirectly via adapters such as ZO proteins and cingulin (Furuse et al., 1994; Fanning et al., 1998; Itoh et al., 1999b; Haskins et al., 1998; Cordenonsi et al., 1999b).

It was recently shown that an actin-dependent stretching of ZO-1 allows the accumulation of occludin at TJs (Spadaro et al., 2017), possibly related to its partitioning into ZO-1 phase-separated condensates (Beutel et al., 2019). The role of occludin at TJs remains nevertheless elusive. Early studies highlighted its importance in TJ structure and function: occludin exogenously expressed in fibroblasts, which normally lack TJs, localized at cell-cell contacts, conferred them adhesiveness (Van Itallie and Anderson, 1997), and participated in the formation of TJ-like strands (Furuse et al., 1998), whereas the treatment of epithelial cells with synthetic occludin peptides disrupted TJ assembly and barrier function (Lacaz-Vieira et al., 1999; Chung et al., 2001; Nusrat et al., 2005). However, occludinknockdown (KD) or -knockout (KO) cells were able to form morphologically normal TJ strands and displayed

unaffected paracellular permeability (Yu et al., 2005; Saitou et al., 1998). In addition, TJs in occludin-KO mice were not disrupted and the intestinal epithelium showed a functional barrier despite the observation of histological abnormalities in the mutant animals (Saitou et al., 2000;

Schulzke et al., 2005). Therefore, although a substitutional redundancy exerted by tricellulin could obscure the roles of occludin (Furuse, 2010; Ikenouchi et al., 2005; Ikenouchi et al., 2008), additional transmembrane components of TJs must be involved in the formation of TJ strands and their barrier function.

Subsequent work from the Tsukita laboratory identified claudins as the main components of TJ strands (Furuse et al., 1998). Claudins are a protein family which encompasses 26 members in human and 27 in mouse, expressed tissue-specifically (Gunzel and Yu, 2013;

Anderson and Van Itallie, 2009; Furuse, 2010). They are the core structural components of TJ fibrils, and can generate TJ strands by themselves when overexpressed in fibroblasts lacking endogenous TJs (Furuse et al., 1998; Kubota et al., 1999). They span the membrane four times, and their C-terminal cytoplasmic tail can be modified by phosphorylation and palmitoylation, which regulate their accumulation at junctions (Van Itallie et al., 2005; D'Souza et al., 2005; Aono and Hirai, 2008; Ishizaki et al., 2003; Ikari et al., 2006). Furthermore, most of the claudins contain at their C-terminus a PsD-95/Disc-large/ZO-1 (PDZ)-binding motif, which allows them to bind to PDZ domain-containing proteins of the TJ cytoplasmic plaque such as ZO proteins (Itoh et al., 1999a). In addition to bridging claudins to the actin cytoskeleton, this interaction with the PDZ domains of ZO-1 and ZO-2 plays a fundamental role in the assembly of TJ fibrils (Umeda et al., 2006). The extracellular loops and transmembrane spans of claudins mediate their homo- and heterophilic interactions, both in cis, i. e. in the same plasma membrane, and in trans, i. e. with claudins from the neighboring cell (Furuse et al., 1999; Krause et al., 2008; Blasig et al., 2006; Rossa et al., 2014; Piontek et al., 2008; Zhao et al., 2018). These assemblies are critical for TJ formation and permeability barrier function. Indeed, by forming paracellular pores with their extracellular loops and controlling the passage of ions and small molecules, claudins are the major determinant of

paracellular permeability (Gunzel and Yu, 2013). Since TJ strands are a “mosaic” of multiple claudin types, it allows the modulation of the permeability function (water, size and charge selectivity), depending on the specialization of the tissue, by combining different claudin types (Gunzel and Yu, 2013; Furuse et al., 1999). Physiologically, two pathways of paracellular permeability have been observed, one that forms charge-selective small-pores (estimated diameter of ~4Å), and another that is size-selective and allows the permeation of molecules up to ~60Å (Otani and Furuse, 2020; Zihni et al., 2016; Shen et al., 2011; Watson et al., 2005).

It was reported that claudin-5-KO mouse endothelial cells and claudin quintuple-KO MDCK cells have an altered permeability barrier to ions and small molecules but retain their barrier to macromolecules intact (Nitta et al., 2003; Otani et al., 2019), suggesting the involvement of additional proteins in the function of gate to larger molecules. The role of claudins in the fence function of TJs is also not clear: epithelial polarity and lipid apicobasal distribution were not disorganized in claudin quintuple-KO MDCK cells (Otani et al., 2019), suggesting that TJ strands per se are not essential for membrane fence formation.

In addition to tetraspan proteins, other transmembrane components of TJs include single-span transmembrane immunoglobin-like adhesion molecules, such as JAMs (Junctional Adhesion Molecules) (Martin-Padura et al., 1998; Luissint et al., 2014), CAR (Cocksackie and Adenovirus Receptor) (Cohen et al., 2001b), ESAM (Endothelial cell Selective Adhesion Molecule) (Nasdala et al., 2002; Hirata et al., 2001) and the tTJs-specific angulins (Masuda et al., 2011). ESAM is specifically present in endothelial cells, whereas CAR is detected in epithelial cells (Bazzoni, 2003). JAM-A and -C have a wide distribution, whereas JAM-B is restricted to the endothelial cells of specific blood vessels (Bazzoni, 2003). JAMs bind in cis and trans in an homo- and heterophilic manner, but not in all combinations (Bazzoni, 2003). They do not constitute TJ strands, but are involved in the membrane apposition observed at TJs since claudin quintuple-KO MDCK cells lack TJ strands but keep the close juxtaposition of adjacent membranes, and a widening of the intercellular space is observed when JAM-A is additionally removed (Otani et al., 2019). JAM-A appears to stabilize the

paracellular barrier (Bazzoni, 2003), since the intestinal epithelium of JAM-A-KO mice is easily disrupted when facing inflammation (Laukoetter et al., 2007; Vetrano et al., 2008). In support of this idea, JAM-A-KD cultured epithelial monolayers are leakier to large molecules (Liu et al., 2000). Furthermore, claudin quintuple-KO MDCK monolayers have a disrupted permeability to small molecules and ions but keep their barrier role to macromolecules until JAM-A is also removed, indicating the critical role of JAMs in macromolecule paracellular permeability control in coordination with claudins (Ebnet et al., 2000; Laukoetter et al., 2007; Otani et al., 2019).

The cytoplasmic C-terminal tail of JAMs interact with several proteins of the cytoplasmic plaque of junctions, including ZO-1 via its PDZ domain (Ebnet et al., 2000; Bazzoni et al., 2000) and CGN (Bazzoni et al., 2000). These bindings connect JAMs to the cytoskeletons and support its role in TJ assembly (Bazzoni et al., 2000; Bazzoni and Dejana, 2001; Martinez-Estrada et al., 2001; Bazzoni, 2003). JAMs also bind and recruit polarity complex proteins (Par3, Par6 and aPKC) and have thus been implicated in the fence function of TJs and the establishment of epithelial polarity (Ebnet et al., 2001; Itoh et al., 2001; Rehder et al., 2006; Tuncay et al., 2015; Otani et al., 2019; Otani and Furuse, 2020).