Organization of multiprotein complexes at cell–cell junctions
The formation of stable cell–cell contacts is required for the generation of barrier-forming sheets of epithelial and endothelial cells. During various physiological processes like tissue development, wound healing or tumorigenesis, cellular junctions are reorganized to allow the release or the incorporation of individual cells. Cell–cell contact formation is regulated by multiprotein complexes which are localized at specific structures along the lateral cell junctions like the tight junctions and adherens junctions and which are targeted to these site through their association with cell adhesion molecules. Recent evidence indicates that several major protein complexes exist which have distinct functions during junction formation. However, this evidence also indicates that their composition is dynamic and subject to changes depending on the state of junction maturation. Thus, cell–cell contact formation and integrity is regulated by a complex network of protein complexes. Imbalancing this network by oncogenic proteins or pathogens results in barrier breakdown and eventually in cancer. Here, I will review the molecular organization of the major multiprotein complexes at junctions of epithelial cells and discuss their function in cell–cell contact formation and maintenance.
KeywordsAdherens junction Tight junction Cell polarity Cell–cell adhesion Protein complexes JAM PAR proteins
In multicellular organisms, cell–cell adhesion is involved in most developmental processes. It is necessary for example for the assembly of coherent sheets of barrier-forming epithelial and endothelial cells which line the inner and outer surfaces of the organism like those of the intestine, the skin or the blood vessels. However, also in adult tissues cell–cell contacts are far from being static structures which maintain the barriers by simply holding cells together. During the turnover of growing tissues such as the intestine or the skin, they are constantly remodeled to allow the extrusion of “old” cells and the incorporation of “young” cells derived from stem cells without a concomitant loss of the barrier function (Fuchs et al. 2004). Similarly, during leukocyte extravasation in secondary lymphoid organs or at sites of an ongoing immune response, the homotypic interactions between endothelial cells must be altered to allow the passage of the leukocytes without affecting the barrier properties of the endothelium (Ley et al. 2007). Finally, during wound healing and tissue repair, cells undergo a coordinated movement, proliferate and establish new cell–cell contacts once they encounter cells from the opposing site of the wound (Perez-Moreno and Fuchs 2006). These different demands in different cell types and different physiological situations require a sophisticated regulatory network which enables a partial dismantling and re-establishment of cell–cell contacts while simultaneously preventing the loss of an epithelial phenotype which in adult tissues frequently correlates with tumor progression and metastasis (Thiery 2002).
Not surprisingly, the organization of cell–cell contacts of epithelial has attracted a great deal of attention. Due to the easy accessibility of cultured epithelial cells many discoveries have been made in epithelial cells. Epithelial cell–cell contacts contain three major adhesive structures which can be identified at the ultrastructural level, the tight junctions (TJs), the adherens junctions (AJs), and the desmosomes. In polarized epithelial cells of certain tissues like the intestine, TJs and AJs are asymmetrically distributed at the apical region of the lateral cell contact forming the apical junctional complex (AJC) which encircles the apex of the cells and demarkates the border between the apical and the basolateral membrane domains (Nelson 2003). Common to all three types of structures is the presence of several adhesion molecules that link the neighbouring cells through homophilic and heterophilic adhesive interactions, and the presence of cytoplasmic scaffolding proteins that organize signaling complexes and which provide a mechanical link to the actin cytoskeleton (or intermediate filaments in the case of desmosomes). The scaffolding proteins might also link different protein complexes—at least temporarily—and thus organize supramolecular protein complexes. It should be noted that these protein complexes are dynamic, and that their composition is subject to regulation depending on junctional maturation and integrity. During the last few years, a rapid progress has been made in identifying new proteins at cell–cell contacts and in particular in deciphering the molecular composition of the TJs. Among the most exciting findings was probably the discovery of protein complexes at TJs which are highly conserved through evolution and which regulate cellular polarization in different organisms and various cell types. Here, I will review the major multiprotein complexes present at cell–cell contacts of vertebrate epithelial cells and highlight the most recent advances in the understanding of their role in the organization and functions of epithelial cell–cell contacts in vertebrates.
The function of adherens junctions
A main function of AJs is to connect cells to regulate tissue formation and morphogenesis during development as well as the maintenance of solid tissues in the adult organism (Gumbiner 1996). The major cell adhesion molecules at AJs, the classical cadherins, connect adjacent cells through homophilic interactions and are linked to the cytoskeleton through proteins associated with their cytoplasmic tail, the catenins. This link generates a transcellular network of actin filaments running through the entire sheet of cells with the cadherin–catenin complexes serving as connectors of the actin filaments bundles at the intercellular space. During morphogenetic events, for example during neural tube formation, mechanical forces can thus be applied to the whole cellular sheet. In adult tissues, cadherin-mediated cell adhesion is absolutely required for cell–cell adhesion to be maintained as a loss of cadherin adhesion by Ca2+-depletion results in a loss of cell–cell interaction and rounding up of the cells despite a number of other adhesion molecules (which are not Ca2+-dependent) at cell–cell contacts (Takeichi 1977).
Cadherins and catenins
The association of E-cadherin with p120ctn is subject to a similar reciprocal regulation of cell–cell contact localization: The cadherin molecule is necessary to localize p120ctn at the cell contact, and p120ctn is required for the stable localization of the cadherin molecule at AJs (Reynolds and Roczniak-Ferguson 2004). In contrast to β-catenin, p120ctn stabilizes cadherin which is constitutively endocytosed (Bryant and Stow 2004) by regulating its turnover rate at the surface (Davis et al. 2003). Besides its additional role in regulating transcription by interacting with transcription factors (van Roy and McCrea 2005), p120ctn has a also a function in regulating the activity of Rho small GTPases. The formation of early cell–cell contacts correlates with the activation of Cdc42 and Rac1 and the inhibition of RhoA (Noren et al. 2001), and these changes in activities of the small GTPases are at least in part regulated by p120ctn which interacts with the Rac activator Vav2 (Noren et al. 2000) and the RhoA inhibitor p190RhoGAP (Wildenberg et al. 2006). The activation of Cdc42/Rac1 and the downregulation of RhoA activity serves to facilitate new cell adhesion by increasing the cell surface interacting with membranes of neighbouring cells through lamellipodia and filopodia formation and simultaneously inhibiting cell migration by inhibiting stress fiber formation, respectively (Perez-Moreno and Fuchs 2006).
The role of α-catenin is less clear. For a long time considered to bridge the AJs to the actin cytoskeleton through direct interactions with both β-catenin and F-actin, this role has been challenged by the observations that its binding to F-actin (occurs only as homodimer) and its heterodimeric association with β-catenin are mutually exclusive (Drees et al. 2005; Yamada et al. 2005). Therefore, the physical bridge between the cadherin–catenin complex and the actin cytoskeleton remains to be identified. It is possible that α-catenin present in the cadherin-associated heterodimeric β-catenin–α-catenin complex is able to bind to other proteins which associate with F-actin. The putative linker is required to fulfill two critera: to bind directly to α-catenin and simultaneously–either indirectly or directly—to F-actin. From several proteins which fulfill these requirements, vinculin and α-actinin turned out not to mediate actin binding to the cadherin–catenin complex excluding them from the list of possible candidates (Yamada et al. 2005). Other candidate proteins including AF-6/afadin (Pokutta et al. 2002), ZO-1 (Itoh et al. 1997), formin (Kobielak et al. 2004), spectrin (Pradhan et al. 2001) or the LIM protein ajuba (Marie et al. 2003) remain to be tested.
Nectins and afadin
The second major adhesive protein complex at AJs consists of members of the nectin family of adhesion molecules and a scaffolding protein that is directly associated with the cytoplasmic domain of nectins named AF-6/afadin (Takai and Nakanishi 2003). Nectins are immunoglobulin-like proteins and comprise a family consisting of four members (nectin-1 to -4), which are localized at AJs of epithelial cells (Reymond et al. 2001; Sakisaka and Takai 2004). Unlike classical cadherins which undergo only homophilic interactions in trans, nectins undergo both trans-homophilic and trans-heterophilic interactions. The major heterophilic binding partners are other members of the nectin family as well as members of nectin-related adhesion molecules Nectin-like (Necl)-1 to -5 (Sakisaka et al. 2007). Nectins are “true” adhesion molecules as they support cell aggregation when ectopically expressed in cells (Aoki et al. 1997; Lopez et al. 1998; Satoh-Horikawa et al. 2000; Takahashi et al. 1999). They also seem to influence the E-cadherin-mediated adhesion (Martinez-Rico et al. 2005; Sato et al. 2006) suggesting that they are contributing to the overall strength of cell–cell adhesion. Like E-cadherin, nectin-2 appears very early at cell–cell contacts during junction formation and is present at so-called primordial, spot-like AJs (pAJs) or puncta (Asakura et al. 1999), which are formed at the tips of protrusions of two contacting cells (Perez-Moreno and Fuchs 2006). An important function of nectins which is similar to that of cadherins is their ability to activate Cdc42 and Rac1 small GTPases. Trans-interaction of nectins results in the recruitment and activation of c-Src, followed by the activation of the two guanine-nucleotide exchange factors “FGD-1-related Cdc42 GEF” (FRG) and Vav2, which are specific for Cdc42 and Rac1, respectively (Fukuhara et al. 2004; Kawakatsu et al. 2002, 2005). As pointed out above, the activation of these small GTPases is probably required to facilitate junction formation suggesting that nectins cooperate with cadherins in the regulation of the actin cytoskeleton at sites of cell adhesion. However, in addition they might also help to regulate the formation of tight junctions and their physical separation from AJs during junctional maturation (see below).
All nectins directly associate with afadin (Reymond et al. 2001; Takahashi et al. 1999). L-Afadin, the longer version of two afadin isoforms with a F-actin-binding domain, is a scaffolding protein which interacts with both nectins and F-actin through independent domains suggesting that it directly links nectin-based adhesion sites to the actin cytoskeleton (Mandai et al. 1997) (Fig. 1). However, through additional protein interactions, it might also establish an indirect link to the actin cytoskeleton as well as to the cadherin–catenin complex by its interaction with “classical” AJ-associated proteins. Afadin directly associates with α-catenin (Pokutta et al. 2002; Tachibana et al. 2000), with vinculin through its association with ponsin/SH3P12 (Mandai et al. 1999), and with α-actinin through its association with “afadin Dil domain-interacting protein” (ADIP) and “Lim domain only 7” (LMO7) (Asada et al. 2003; Ooshio et al. 2004) (Fig. 1). It should be mentioned that some of these protein interactions might occur specifically in certain tissues, and that the molecular mechanisms underlying these interactions are not revealed in detail. Nevertheless, it is likely that through these multiple interactions the two major protein complexes at AJs are physically linked and that they influence each other in their localization and activity (Sakisaka et al. 2007).
The function of TJs
Integral membrane proteins at TJs
Multiprotein complexes at TJs
Since the discovery of ZO-1 as the first protein at TJs and its molecular cloning (Itoh et al. 1993; Stevenson et al. 1986), the number of proteins that are localized at TJs has steadily increased. These proteins comprise scaffolding and adapter proteins, regulatory proteins like small GTPases, G-proteins, kinases and phosphatases, as well as transcription factors or factors regulating RNA processing (Fig. 3). The large number and the functional diversity of these proteins suggest that TJs are a focus of incoming and outgoing signals and that their composition is dynamic. In accordance with this, many proteins identified at TJs are found at other compartments of the cell as well including the nucleus or the cytoskeleton and are actively shuttling between these compartments and the TJs (Matter and Balda 2007). The organization of these networks is regulated by proteins containing multiple protein–protein interaction domains such as PDZ domains, GuK domains, SH2 or SH3 domains (Pawson and Nash 2003). At the TJs three major protein complexes exist which involve one or several scaffolding proteins, the ZO protein complex, the Pals1–PATJ complex, and the PAR-3–aPKC–PAR-6 complex (Fig. 3).
The ZO protein complex
The CRB3–Pals1–PATJ complex
The CRB3–Pals1–PATJ complex has originally been described in Drosophila as a protein complex (the Crumbs–Stardust–Discs lost complex; this complex is now called Crumbs–Stardust (Sdt)–dPATJ complex (Pielage et al. 2003)) involved in the regulation of apico-basal polarization (Tepass et al. 2001). In Drosophila epithelial cells, this complex localizes to the apical region of the lateral membrane domain, called subapical region (SAR) or marginal zone (Knust and Bossinger 2002), that is positionally analogous to the TJs in vertebrate epithelial cells. The localization of the CRB3–Pals1–PATJ complex at the TJ is mediated through a direct and PDZ domain-dependent interaction of Pals1 with the C-terminal PDZ domain motif in CRB3, and a direct interaction between Pals1 and PATJ involving the L27N domain of Pals1 and the L27 domain present at the NH2-terminal region of PATJ (Fig. 3) (Roh et al. 2002b). RNA interference-mediated knockdown of Pals1 leads to defects in the formation of TJs as well as in the development of lumen-containing epithelial cysts (an assay system for apico-basal polarity development (O’Brien et al. 2002)), and is accompagnied by a loss of PATJ protein expression (Straight et al. 2004). Conversely, knockdown of PATJ impairs the barrier function of TJs and results in a loss of Pals1 at TJs, an internalization of CRB3, and a redistribution of other TJ components like occludin and ZO-3 (Michel et al. 2005; Shin et al. 2005). These observations strongly suggest that the CRB3–Pals1–PATJ complex is important for the development of functional TJs in vertebrate epithelial cells.
The PAR-3–aPKC–PAR-6 complex
In evolutionary terms, the PAR-3–aPKC–PAR-6 complex is the most ancient among the three major protein complexes at TJs. As opposed to the ZO protein complex and the CRB3–Pals1–PATJ complex, all three proteins of this complex exist in C.elegans where they cooperate to regulate the development of membrane asymmetry in the zygote (Kemphues 2000). The acronym Par stands for partitioning-defective and reflects the lack of partition of cytoplasmic P granules in C.elegans mutant embryos in response to sperm entry (Kemphues et al. 1988). The initial screen identified six par genes, and their molecular characterization revealed that they encoded proteins of different structures and functions which include scaffolding/adapter proteins with several protein–protein interaction domains (PAR-3, PAR-6), serine/threonine kinases (PAR-1, PAR-4), a protein containing a RING finger domain typical for E3 ubiquitin ligases (PAR-2) and a member of the 14-3-3 family of signaling proteins (PAR-5) (reviewed in (Goldstein and Macara 2007; Suzuki and Ohno 2006)). With the exception of PAR-2, all PAR proteins exist in Drosophila and vertebrates. Two PAR proteins, PAR-3 and PAR-6, form a functional unit with aPKC, the PAR-3–aPKC–PAR-6 complex (Ohno 2001). In this complex, PAR-3 and PAR-6 undergo direct interactions with aPKC (Fig. 3). The interaction of PAR-6 with aPKC is mediated by a heterotypic PB1–PB1 domain interaction, the interaction of PAR-3 with aPKC by the CR3 domain of PAR-3 and the kinase domain of aPKC (Ohno 2001). The interactions of the two scaffolding proteins PAR-3 and PAR-6 with aPKC are assumed to regulate the localization and the activity of aPKC, respectively. Among all integral membrane proteins tested, PAR-3 binds specifically to JAM-A, -B, and -C (Ebnet et al. 2001, 2003), and the interaction with JAM-A might serve to anchor the PAR–aPKC complex to TJs. The interaction with PAR-6 is assumed to regulate the activity of aPKC (Lin et al. 2000). In the absence of small GTPases like Cdc42 or Rac1 aPKC is inactive. The binding of active Cdc42 or Rac1 to the CRIB domain of PAR-6 activates aPKC, probably by inducing a conformational change of PAR-6 which allows aPKC to become active (Yamanaka et al. 2001).
What is the function of the PAR–aPKC complex in TJ physiology? A large body of evidence indicates a critical role of the PAR complex in TJ formation rather than in TJ maintenance (Chen and Macara 2005, 2006; Gao et al. 2002; Hirose et al. 2002; Joberty et al. 2000; Mizuno et al. 2003; Nagai-Tamai et al. 2002; Ooshio et al. 2007; Suzuki et al. 2001, 2002; Yamanaka et al. 2001). Many of these studies applied dominant-negative mutants of either PAR-3 or PAR-6 or aPKC. The negative effects on TJ formation were only observed when these mutants were expressed during the process of cell–cell contact formation but not when expressed in fully polarized epithelial cells where TJ formation had already been completed (Gao et al. 2002; Nagai-Tamai et al. 2002; Suzuki et al. 2001, 2002; Yamanaka et al. 2001). This strongly suggests that the PAR–aPKC complex develops its polarizing activity at an early stage of cell–cell contact formation and that it is critical for the formation of TJs rather than for their maintenance.
Regulation of membrane asymmetry and TJ formation by the PAR-3–aPKC–PAR-6 complex
The pAJs are positive for typical AJ proteins like E-cadherin, α-catenin, β-catenin, nectin-2, AF-6/afadin and ponsin but also for typical TJ proteins like ZO-1 and JAM-A (Adams et al. 1996; Asakura et al. 1999; Ebnet et al. 2001; Suzuki et al. 2002; Yonemura et al. 1995). During maturation, occludin is recruited to these sites, and during further maturation, claudin-1, PAR-3 and aPKC appear (Suzuki et al. 2002) (Fig. 5). Although direct comparison has not been performed yet, it is likely that aPKC together with PAR-6 appear slightly later than PAR-3 (Suzuki et al. 2002). The formation of cadherin-based pAJs marks the early sites of cell–cell adhesion and probably serves as a “landmark” or “positional cues” for membrane growth and for the recruitment of other integral and peripheral membrane proteins (Yeaman et al. 1999). After the localization of the first set of proteins at pAJs other proteins can be recruited through direct physical interactions with those already present. For example, α-catenin-associated ZO-1 could serve to recruit occludin and claudins, afadin could serve to recruit JAM-A and nectin-2 (or vice versa), and JAM-A or nectin-2 could recruit PAR-3 which serves as scaffold to assemble the PAR-3–aPKC–PAR-6 complex.
Once the PAR-3–aPKC–PAR-6 complex has been recruited to nascent cell–cell contacts aPKC has to be activated as suggested by the observation that ectopic expression of a kinase-dead, dominant-negative mutant of aPKC prevents the maturation of pAJs into belt-like AJs and TJs (Suzuki et al. 2001, 2002). The activation occurs most likely by the Rho GTPases Cdc42 and Rac1 which bind to the Crib domain of PAR-6 thereby inducing a conformational change which leads to the activation of PAR-6-associated aPKC (Garrard et al. 2003; Ohno 2001; Peterson et al. 2004; Yamanaka et al. 2001). Both E-cadherin and nectin-2 could be responsible for the activation of Cdc42 and Rac1. Whereas E-cadherin seems to activate Rac1 but not Cdc42 (Betson et al. 2002; Kovacs et al. 2002; Nakagawa et al. 2001; Noren et al. 2001; Yamada and Nelson 2007), nectin-2 induces the activation of both Cdc42 and Rac1 after ectopic expression in cultured epithelial cells (Fukuhara et al. 2003, 2004; Fukuyama et al. 2005; Kawakatsu et al. 2002, 2005). The association of the Rac1 GEF Tiam1 with PAR-3 (Chen and Macara 2005; Mertens et al. 2005) could regulate a locally restricted activation of Rac1 specifically at those sites where cell–cell adhesion has occured and where the activity of aPKC is required to promote the maturation of cell–cell contacts and the development of TJs from pAJs.
The exact mechanism how the maturation of cell–cell contacts is regulated by aPKC is not clear. One could imagine that aPKC phosphorylates components of TJs and thereby regulates their specific localization or their specific functions at the TJs. Phosphorylation of occludin, claudin-1 and ZO-1 by aPKCζ has been found in vitro (Nunbhakdi-Craig et al. 2002). However, a physiological relevance of these phosphorylations has not been demonstrated, yet. Alternatively, aPKC could regulate TJ formation indirectly by regulating the development of membrane asymmetry along the lateral cell–cell contacts. The following example might serve to illustrate an example for this activity. In polarized epithelial cells, aPKC and PAR-1, another Ser/Thr kinase, are separately localized along the apico-basal axis: aPKC localizes to TJs whereas PAR-1 localizes to the basolateral membrane domain (Bohm et al. 1997). PAR-1 is a substrate for aPKC, and aPKC-mediated phosphorylation of PAR-1 leads to its dissociation from the membrane into the cytoplasm (Hurov et al. 2004; Suzuki et al. 2004). As a result, PAR-1 is absent from aPKC-containing membrane domains. A reciprocal mechanism has been described in Drosphila epithelial cells (Benton and St Johnston 2003). Drosphila PAR-1 phosphorylates PAR-3/Bazooka thereby inhibiting its dimerization and blocking its ability to assemble a functional PAR-3–aPKC–PAR-6 complex. As a result, the PAR-3–aPKC–PAR-6 complex is absent from PAR-1-containing membrane domains. Through these reciprocal inhibitory interactions two distinct membrane domains are generated characterized by the mutual exclusion of aPKC and PAR-1. Thus, by regulating the formation of a specific membrane domain from which certain proteins are actively excluded, aPKC could indirectly promote the formation of TJs. It is not clear, yet, if the abilities of the PAR-3–aPKC–PAR-6 complex to regulate TJ formation and to regulate membrane asymmetry are mechanistically linked.
Regulation of TJ maintenance by the Rich1–Amot complex
Besides Amot, PATJ and Pals1, the Rich1 immunoprecipitates contain PAR-3 and aPKC (Wells et al. 2006). Surprisingly, the Rich1 immunoprecipitates do not contain PAR-6, and they contain the 100 kDa isoform of PAR-3 which lacks the aPKC-interacting domain and thus cannot directly associate with aPKC (Lin et al. 2000). This suggests that PAR-3 and aPKC can undergo interactions with the Rich1–Amot complex which are independent of the PAR-3–aPKC–PAR-6 interaction with the Pals1–PATJ complex (Hurd et al. 2003) (Fig. 6). The functional relevance of this interaction is not clear. Two Amot-like (Amotl) proteins—Amotl1/JEAP and Amotl2/MASCOT—which have been described earlier as TJ components (Nishimura et al. 2002; Patrie 2005) are present in Amot but not Rich1-containing protein complexes. All three Amot proteins directly interact with the scaffolding protein MUPP1 and its paralogue PATJ (Sugihara-Mizuno et al. 2007) (Fig. 6). These findings suggest that additional Amot protein-containing complexes exist with functions different from regulating Cdc42 activity.
Signaling from TJs
In addition to the relatively stable protein complexes described so far (Fig. 4), many protein complexes at TJs assemble only transiently. In addition, some proteins are not exclusively associated with TJ but shuttle between the TJ and other compartments in the cell. The identification of such proteins has revealed that TJ proteins are engaged in receiving signals but also in delivering signals to the cell interiour and thereby regulate epithelial proliferation and differentiation (Matter and Balda 2003). The mechanism by which TJ proteins influence for example gene expression is most likely indirect through binding and sequestration of regulatory molecules at the TJs as exemplified by ZO-1.
ZO-1 associates with ZONAB/DbpA, a transcription factor which promotes proliferation of epithelial cells, in part by interacting with the cell division kinase CDK4 and also by regulating the expression of genes involved in proliferation like cyclin D1 and PCNA (Balda et al. 2003; Balda and Matter 2000; Sourisseau et al. 2006). In proliferating cells, which have little ZO-1, ZONAB/DbpA expression is high and ZONAB/DbpA protein is localized in the nucleus. When cells reach confluence and develop intercellular junctions, ZO-1 is accumulating at cell–cell contacts and recruits ZONAB/DbpA to the junctions thus sequestering it away from the nucleus (Balda and Matter 2000). Interestingly, during cellular stress ZONAB/DbpA associated with ZO-1 can be re-activated. The heat shock protein Apg-2 that is distributed in the cytoplasm under normal conditions is recruited to cell–cell contacts in response to heat shock where it binds directly to ZO-1 using the same binding interface like ZONAB/DbpA, i.e. the SH3 domain of ZO-1 (Tsapara et al. 2006). This leads to a loss of ZONAB/DbpA from cell junctions and in activation of the transcriptional activity of ZONAB/DbpA (Tsapara et al. 2006). Thus, ZO-1 influences gene expression and cell cycle progression in a cell density-dependent manner, and this function can be regulated during cellular stress.
ZO-2 is another scaffolding proteins at TJs which is involved in signaling. In contrast to ZO-1, however, ZO-2 seems to actively shuttle between TJs and the nucleus. ZO-2 contains functional nuclear localization and nuclear export signals (Gonzalez-Mariscal et al. 2006; Jaramillo et al. 2004) and interacts with various proteins that have nuclear functions including the transcription factors AP-1 and C/EBP (Betanzos et al. 2004), the DNA-binding protein SAF-B (Traweger et al. 2003), and the p120ctn family member ARVCF (Kausalya et al. 2004). ZO-2 probably inhibits the activity of the transcription factors AP-1 and C/EBP by regulating their export from the nucleus which is consistent with the predominant nuclear localization of ZO-2 in sparse cells and the localization of ZO-2 as well as AP-1 and C/EBP at TJs in confluent cells (Betanzos et al. 2004). In the case of ARVCF, ZO-2 regulates its nuclear import (Kausalya et al. 2004) where it might regulate transcription similar to other p120ctn family members (Hatzfeld 2005). In summary, the identification of protein complexes formed by typical TJ proteins and typical nuclear proteins involved in the regulation of transcription indicates that the TJs participate in the regulation of proliferation and differentiation.
A protein complex at the lateral membrane which regulates TJ formation: the Scribble–Discs Large–Lethal Giant Larvae complex
Protein complexes at cell junctions and cancer
Protein complexes as targets for pathogens
Many pathogens need to overcome epithelial and endothelial barriers to invade the host and establish infection. For this purpose, various strategies have evolved to disrupt the barrier and allow the pathogen the passage into tissues. These strategies include the release of proteolytic enzymes that cleave adhesion molecules like occludin, E-cadherin or desmoglein (Hanakawa et al. 2004; Pentecost et al. 2006; Wu et al. 1998, 2000), or the release of toxins that act via cell surface receptors to induce intracellular changes (e.g. of the actin cytoskeleton) which eventually lead to alterations of the barrier (Hopkins et al. 2003; Nusrat et al. 2001). More “advanced” strategies involve the delivery of pathogen-derived proteins via secretion systems into the host cell cytoplasm where these proteins directly associate with host cell proteins to influence their function. One example is the Helicobacter pylori (H.pylori) effector protein “Cytotoxin-associated gene A antigen” (CagA). H.pylori induces morphological changes of epithelial cells, alterations of the composition of the apical junctional complex as well as a breakdown of the epithelial barrier function (Amieva et al. 2003; Bagnoli et al. 2005), and H.pylori infections can result in mucosal damage (ulceration), inflammation (gastritis) and cancer (gastric carcinoma) (Peek and Blaser 2002). The CagA protein is involved in many of these processes through multiple interactions with a number of host proteins. After the translocation into the host cell, CagA is phosphorylated by src-family kinases and recruits the phosphotyrosine phosphatase SHP-2 (Higashi et al. 2002). In addition, CagA associates with several proteins involved in the regulation of TJs and in the formation of apico-basal polarity. First, it recruits and thereby mislocalizes the TJ proteins ZO-1 and JAM-A to the site of bacterial attachment (Amieva et al. 2003). Second, it directly interacts with the serine/threonine kinase PAR-1 (Saadat et al. 2007). Under normal conditions, PAR-1 cooperates with the PAR-3–aPKC–PAR-6 complex through reciprocal phosphorylations to regulate the formation of distinct membrane domains (Hurov et al. 2004; Suzuki et al. 2004) (see also above). The binding of CagA to PAR-1 blocks the kinase activity of PAR-1 thus preventing the phosphorylation of PAR-3; at the same time, it prevents the phosphorylation of itself by aPKC. As a result, the integrity of cell–cell contacts is disturbed and cells are extruded from the monolayer (Saadat et al. 2007). Thus, through its multiple interactions with signaling molecules, scaffolding proteins and cell polarity proteins, CagA disregulates critical cellular functions to enter the sub-epithelial tissues which also leads to inflammation and eventually to cancer (Hatakeyama 2004).
Conclusions and perspectives
The last decade has witnessed a steady increase in the number of new proteins localized at cell–cell contacts of epithelial cells. The identification of claudins at TJs has strongly increased the understanding of the molecular basis of TJ function. The identification of cell polarity protein complexes like the PAR-3–aPKC–PAR-6 complex and the CRB3–Pals1–PATJ complex at TJs has added new aspects on the mechanisms underlying the development of TJs. The identification of the nectin–afadin system provided evidence for a second major adhesive system besides the cadherin–catenin system at AJs. It also became evident that TJs and AJs are signaling centers which are actively engaged in regulating proliferation and differentiation through feed-back mechanisms with the cytoskeleton and the nucleus.
It is not clear if these proteins form the same complexes like in epithelial cells, and it is likely that differences exist in the composition of protein complexes to regulate the specific requirements in the context of the given cell or tissue. As one example, JAM-C deficiency in mice leads to a mislocalization of PAR-6, aPKC and PATJ bot not PAR-3 in spermatids (Gliki et al. 2004) suggesting that PAR-3 is not part of the JAM-C-associated polarity complex in spermatids despite its ability to interact with PAR-6, aPKC and also directly with JAM-C (Ebnet et al. 2003; Suzuki and Ohno 2006). As another example, in endothelial cells two PAR protein complexes have been identified, a “conventional” PAR-3–aPKC–PAR-6 complex and a second PAR complex in which PAR-3 and PAR-6 are independently associated with VE-cadherin and which lacks aPKC (Iden et al. 2006). Nevertheless, the use of a set of conserved proteins by morphologically diverse cell types to regulate cell–cell contact formation highlights both the importance of the proteins for cellular function as well as their versatility that allows the regulation of similar aspects in different cell types.
Many open questions remain. For example, what is the molecular nature of the intramembrane diffusion barrier (fence function) of the TJs? The absence of TJ strands in cells lacking all three ZO proteins results in a complete loss of the barrier function of the epithelial sheet but, unexpectedly, the fence function which has been attributed to the presence of TJ strands is retained (Umeda et al. 2006). Membrane diffusion barriers exist also in other cells even in the absence of a physical cell–cell contact, for example at the axonal hillock of neurons to separate somatodendritic and axonal membrane domains (Winckler et al. 1999). It has been suggested that in these cells the accumulation of integral membrane proteins that are anchored to the submembranous cytoskeleton function as rows of pickets which prevent the free diffusion of even small molecules (Nakada et al. 2003). A second unresolved issue is if the activity of the PAR-3–aPKC–PAR-6 complex to regulate membrane asymmetry is mechanistically linked to its role in TJ formation. Finally, the functional interrelationship of the three major polarity complexes at TJs (depicted in Figs 3, 4) has not been resolved, yet. Members of individual complexes interact with each other. For example, CRB3 can also interact with PAR-6 (Lemmers et al. 2004), PAR-6 can also interact with Pals1 (Hurd et al. 2003), and ZO-3 can interact with PATJ (Roh et al. 2002a). Genetic evidence in Drosophila suggests a functional hierarchy among the protein complexes (Johnson and Wodarz 2003) but it is unclear if a similar hierarchy exists in vertebrate epithelial cells and which aspects of cell–cell contact formation are regulated by these interactions. Most likely, many of these interactions are dynamically regulated and occur in a temporally and spatially restricted manner. The large number of scaffolding and signaling molecules identified at cell–cell contacts and the multitude of physical interactions described so far among these proteins indicates that cell–cell contact formation, the development of TJs from pAJs and the aquisition of membrane polarity is a highly dynamic process the complexity of which is still far from being completely understood.
I would like to thank Volker Gerke for discussions and continuous support. I would also like to thank Karl Matter for helpful discussions and Atsushi Suzuki for critically reading the manuscript and for ongoing collaborations. I apologize to authors of work in the field that could not be cited due to space limitations. Work from my group is supported by grants from the German Research Foundation (DFG) and from the Medical Faculty of the University Münster.
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