Tight junction cytoplasmic proteins

Transmembrane components of TJs are directly linked to proteins underlying the membrane and forming the TJ cytoplasmic plaque (Figure 3). These proteins serve as interface between transmembrane components and the actin and MT cytoskeletons, and participate in TJ canonical fence and gate functions (Balda and Matter, 2008). They also confer additional roles by transmitting signals from the junction in order to regulate several cellular processes including cell migration, proliferation and differentiation, gene expression and cytoskeletal organization (Balda and Matter, 2008; Guillemot et al., 2008b; Schneeberger and Lynch, 2004). The TJ plaque is thus composed of a complex network of scaffolding, polarity, adaptor, cytoskeletal and signaling proteins (Guillemot et al., 2008b; Balda and Matter, 2008).

ZO proteins (Figure 4) comprise three structurally related proteins, ZO-1, -2 and -3, belong to the membrane-associated guanylate kinase (MAGUK) family and are at the core of the TJ

cytoplasmic plaque. ZO-1 was the first cytoplasmic TJ-associated component to be identified, following the generation of monoclonal antibodies against a TJ-enriched fraction from mouse liver (Stevenson et al., 1986). ZO-2 and ZO-3 were subsequently identified as proteins that co-immunoprecipitated with ZO-1 in cultured epithelial cell lysates (Gumbiner et al., 1991;

Haskins et al., 1998). ZO-1 and ZO-2 are essential, since mice KO for one or the other die at early embryonic stage (Katsuno et al., 2008; Xu et al., 2008), whereas ZO-3-KO mice are viable and do not show any TJ disruption phenotype (Xu et al., 2008; Adachi et al., 2006). ZO proteins contain three PDZ motifs and several additional structural domains, which mediate multiple interactions and allow them to be the pillars of TJs (Figure 4).

Figure 4. Domain organization and interactors of ZO proteins.

Domains are color-coded. Interacting partners are indicated with a line that delineates the approximate localization of the binding sites. SH: Src-homology; GUK: guanylate kinase. Adapted from Rouaud et al. (2020a).

ZO proteins associate with themselves by homo- and heterodimerization. The first PDZ domain of ZO-1 mediates its homodimerization, whereas the second is responsible for heterodimerization through binding to the second PDZ domain of either ZO-2 or ZO-3 (Utepbergenov et al., 2006). In addition, PDZ domains of ZO proteins directly bind the C-terminal cytosolic tail of the main TJ transmembrane components (claudins, occludin and JAMs) to promote their junctional recruitment and stabilization, and are indispensable for TJ formation, cell polarity and paracellular barrier functions (Itoh et al., 1999a; Furuse et al., 1994;

Fanning et al., 1998; Haskins et al., 1998; Ebnet et al., 2000; Umeda et al., 2006; Otani and

Furuse, 2020). It was recently shown that ZO proteins can phase separate and recruit into their condensates several TJ components, including occludin and claudins, driving the clustering of TJ-associated proteins required for TJ assembly (Beutel et al., 2019; Schwayer et al., 2019;

Otani and Furuse, 2020; Rouaud et al., 2020a). ZO proteins also ensure the crosstalk between TJs and the actomyosin cytoskeleton, directly by binding with actin and cortactin and indirectly via their interaction with other actin-binding proteins and regulators of Rho GTPases (Itoh et al., 1997; Fanning et al., 1998; Katsube et al., 1998; Rouaud et al., 2020a; Guillemot et al., 2008b; Citi et al., 2014; Itoh et al., 2012; Ikenouchi et al., 2007; Odenwald et al., 2018). Finally, ZO-1 and ZO-2 regulate gene expression by sequestering at junctions the transcription factor DbpA/ZONAB, mediating TJ mechanotransduction (Spadaro et al., 2014; Spadaro et al., 2017). Indeed, this ability to bind to DbpA, and also occludin, depends on the conformation of ZO-1 and ZO-2, related to their heterodimerization and the contractility of the actin cytoskeleton or the substrate stiffness (Spadaro et al., 2014; Spadaro et al., 2017).

A canonical function of TJs is to mediate the establishment and maintenance of apicobasal cell polarity. The coordinated activity of the apical polarity complexes Par3/Par6/aPKC and Crumbs/PALS1/PATJ/MUPP1, which are localized at TJs, is fundamental for this process (Rodriguez-Boulan and Macara, 2014; Assemat et al., 2008; Suzuki and Ohno, 2006; Riga et al., 2020). Partitioning defective 3 (Par3) interacts with the transmembrane proteins JAMs through its first (out of three) PDZ domain (Itoh et al., 2001). It subsequently recruits the other members of the complex, i. e. partitioning defective 6 (Par6) and the catalytical partner atypical protein kinase C (aPKC) activated by the Rho GTPases Cdc42 or Rac1 (Lin et al., 2000;

Peterson et al., 2004; Johansson et al., 2000). The second polarity complex is composed of the transmembrane component Crumbs bound to the PDZ protein PALS1 that is in turn linked to the multi-PDZ proteins PATJ and MUPP1 (Tepass et al., 1990; Roh et al., 2002b; Roh et al., 2002a; Assemat et al., 2013; Bulgakova and Knust, 2009). PATJ also directly interacts with the transmembrane TJ components claudins, and MUPP1 with claudins, JAMs and CAR, serving thus as scaffolds for both cytoplasmic and transmembrane proteins (Roh et al., 2002a;

Hamazaki et al., 2002; Jeansonne et al., 2003; Coyne et al., 2004; Rouaud et al., 2020a). All these interactions bring together several proteins and their downstream effectors at the specific localization of TJs and thus drive junction assembly and polarity formation (Assemat et al., 2008; Suzuki and Ohno, 2006; Riga et al., 2020).

Two related proteins, cingulin (CGN) and paracingulin (CGNL1, also known as JACOP), are crucial scaffolds for the crosstalk between TJs and actomyosin and MT cytoskeletons (Rouaud et al., 2020a). CGN was discovered as a protein that co-purified with non-muscle myosin II from chicken intestinal epithelial cells (Citi et al., 1988; Citi et al., 1989), and CGNL1 was detected in a junction-enriched fraction isolated from mouse liver (Ohnishi et al., 2004). Both form parallel homodimers and contain an N-terminal globular head domain, a long α-helical rod domain and a small C-terminal globular tail (Cordenonsi et al., 1999a; Ohnishi et al., 2004;

Citi et al., 2000). They are localized in a distal layer of the TJs, further away from the membrane than ZO proteins (Citi et al., 1988; Citi et al., 1989; Stevenson et al., 1989; Van Itallie and Anderson, 2014; Rouaud et al., 2020a). Depending on the tissue, CGNL1 is also detected at the ZA in addition to TJs (Ohnishi et al., 2004). The junctional recruitment of CGN depends on its interaction with ZO-1 through its N-terminal ZO-1-interaction motif (ZIM) (D'Atri et al., 2002), whereas CGNL1 is brought to junctions either by ZO-1 via the same conserved ZIM domain, or by PLEKHA7, or both, depending on the cell type (Tornavaca et al., 2015; Ohnishi et al., 2004; Pulimeno et al., 2011). In addition to its interaction with ZO-1, CGN binds actin and myosin, and co-pellets and bundles actin filaments in vitro, thus directly tethering the actomyosin cytoskeleton to ZO proteins and TJs (D'Atri and Citi, 2001; Cordenonsi et al., 1999a; Yano et al., 2018; Rouaud et al., 2020a). Moreover, CGN and CGNL1 are adaptors of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), thus fine-tuning the activity of Rho small GTPases and therefore regulating indirectly cytoskeletal organization (Citi et al., 2014). CGN and CNGL1 are also linked to MTs, since CGN binds to MTs in a phosphorylation-dependent manner via its head domain (Yano et al., 2013; Mangan et al., 2016; Yano et al., 2018) and CGNL1 co-pellets with MTs in vitro and is less recruited to

junctions when MTs are depolymerized (Vasileva and Citi, 2018; Paschoud et al., 2011).

Depletion of either CGN or CGNL1 in cellular models does not result in striking changes of the TJ morphology and functions, neither in cytoskeleton architecture (Guillemot et al., 2008a;

Guillemot and Citi, 2006; Guillemot et al., 2004; Guillemot et al., 2012). CGN-KO mice are viable but have an impaired mucosal repair (Guillemot et al., 2012), and CGN modulates the endothelial barrier both in human cultured cells and in mouse brains (Schossleitner et al., 2016). Angiogenesis in CGNL1-KO endothelial cells is disturbed (Chrifi et al., 2017), and CGNL1-KO and CGN-KO cells exhibit an altered dynamic of junction formation and gene expression due to the impacts on RhoA and Rac1 activation (Aijaz et al., 2005; Guillemot et al., 2008a; Guillemot and Citi, 2006; Guillemot et al., 2004; Guillemot et al., 2013).

Therefore, proteins of the TJ cytoplasmic plaque exert crucial roles in junction assembly and stabilization, and represent a signaling platform regulating several cellular processes such as gene expression, mechanotransduction, cytoskeletal organization, cell migration, proliferation and differentiation. They are thus fundamental players of the canonical and non-canonical functions of TJs.