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Domain organization of ZO proteins and the structure of the SH3-GUK domains of ZO-1. A, domain organization of the ZO MAGUKs. The canonical MAGUK protein binding motifs (PDZ, SH3, and GUK) are separated by " unique " (U) regions of high sequence diversity. TJ binding partners of ZO-1 are mapped above their known interaction domain with the transmembrane strand components labeled in red (reviewed in Ref. 14). Those interactions that are also conserved in ZO-2 and ZO-3 are indicated, and the section of ZO-1 whose structure is presented here is indicated by dashed square brackets. B, ribbon diagram of the ZO-1 SH3 (cyan) and GUK (green) domains. The fifth strand of the SH3 domain and the strand following the GUK domain form the interdomain linkage (pink). The ZO-1 construct used for the structure determination lacked the U5 region; arrow 1 points to the truncation point (orange highlight). The entire structure could be traced with confidence except for 3 residues located at the NMP-binding region at the GUK domain (arrow 2, orange highlight).
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Tight junctions are dynamic components of epithelial and endothelial cells that regulate the paracellular transport of ions, solutes, and immune cells. The assembly and permeability of these junctions is dependent on the zonula occludens (ZO) proteins, members of the membrane-associated guanylate kinase homolog (MAGUK) protein family, which are cha...
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MAGUK protein ZO-2 is present at tight junctions (TJs) and nuclei. In MDCK ZO-2 knockdown (KD) cells, nuclei exhibit an irregular shape with lobules and indentations. This condition correlates with an increase in DNA double strand breaks, however cells are not senescent and instead become resistant to UV-induced senescence. The irregular nuclear sh...
Citations
... ZO-1 consists of several domains, including three PDZ domains, a SH3 domain, a GUK domain, and actin binding region (Supplementary Fig. S4A) [39]. ZO-1 interacts with claudin via its PDZ domain [39]. ...
... ZO-1 consists of several domains, including three PDZ domains, a SH3 domain, a GUK domain, and actin binding region (Supplementary Fig. S4A) [39]. ZO-1 interacts with claudin via its PDZ domain [39]. Additionally, ZO-1 directly interacts with occludin or α-catenin through its region that includes SH3 domain and GUK domain [40]. ...
Abstracts
Mesenchymal stem cells are recruited from the bone marrow into breast tumors, contributing to the creation of a tumor microenvironment that fosters tropism for breast tumors. However, the intrinsic mechanisms underlying the recruitment of bone marrow-derived mesenchymal stem cells (MSCs) into the breast tumor microenvironment are still under investigation. Our discoveries identified zonula occludens-1 (ZO-1) as a specific intrinsic molecule that plays a vital role in mediating the collective migration of MSCs towards breast tumor cells and transforming growth factor beta (TGF-β), which is a crucial factor secreted by breast tumor cells. Upon migration in response to MDA-MB-231 cells and TGF-β, MSCs showed increased formation of adherens junction-like structures (AJs) expressing N-cadherin and α-catenin at their cell-cell contacts. ZO-1 was found to be recruited into the AJs at the cell-cell contacts between MSCs. Additionally, ZO-1 collaborated with α-catenin to regulate AJ formation, dependently on the SH3 and GUK domains of the ZO-1 protein. ZO-1 knockdown led to the impaired migration of MSCs in response to the stimuli and subsequent downregulation of AJs formation at the cell-cell contacts during MSCs migration. Overall, our study highlights the novel role of ZO-1 in guiding MSC migration towards breast tumor cells, suggesting its potential as a new strategy for controlling and re-engineering the breast tumor microenvironment.
... Of these, PDZ1 binds to claudins, while PDZ3 binds to JAMs [28][29][30] . Importantly, PDZ3 exists as part of structural module containing PDZ, Src homology 3 (SH3), and guanylate kinase (GUK) domains, termed the PSG module 29,31 . The ZO-1 PSG also forms a dense web of interactions with the cytoskeletal adaptor proteins afadin, αE-catenin, vinculin, and shroom2, signal transduction proteins such as ZONAB and Gα12, and the tight junction protein occludin 23,[31][32][33][34][35][36][37][38][39] . ...
... Importantly, PDZ3 exists as part of structural module containing PDZ, Src homology 3 (SH3), and guanylate kinase (GUK) domains, termed the PSG module 29,31 . The ZO-1 PSG also forms a dense web of interactions with the cytoskeletal adaptor proteins afadin, αE-catenin, vinculin, and shroom2, signal transduction proteins such as ZONAB and Gα12, and the tight junction protein occludin 23,[31][32][33][34][35][36][37][38][39] . In addition to its central role in the construction of tight junctions, ZO-1 plays an integral role in the formation of new cell-cell contacts and is recruited to nascent adherens junctions through its interactions with afadin and αE-catenin [40][41][42][43][44][45] . ...
Intercellular adhesion complexes must withstand mechanical forces to maintain tissue cohesion, while also retaining the capacity for dynamic remodeling during tissue morphogenesis and repair. Most cell-cell adhesion complexes contain at least one PSD95/Dlg/ZO-1 (PDZ) domain situated between the adhesion molecule and the actin cytoskeleton. However, PDZ-mediated interactions are characteristically nonspecific, weak, and transient, with several binding partners per PDZ domain, micromolar dissociation constants, and bond lifetimes of seconds or less. Here, we demonstrate that the bonds between the PDZ domain of the cytoskeletal adaptor protein afadin and the intracellular domains of the adhesion molecules nectin-1 and JAM-A form molecular catch bonds that reinforce in response to mechanical load. In contrast, the bond between the PDZ3-SH3-GUK (PSG) domain of the cytoskeletal adaptor ZO-1 and the JAM-A intracellular domain becomes dramatically weaker in response to ~2 pN of load, the amount generated by single molecules of the cytoskeletal motor protein myosin II. These results suggest that PDZ domains can serve as force-responsive mechanical anchors at cell-cell adhesion complexes, and that mechanical load can enhance the selectivity of PDZ-peptide interactions. PDZ mechanosensitivity may thus help to generate the intricate molecular organization of cell-cell junctions and allow junctional complexes to dynamically remodel in response to mechanical load.
... Nevertheless, another study indicated that expect ZO-3, both full-length ZO-1 and ZO-2 could bind ZONAB in vitro [9]. The difference might be caused by the internal interaction of ZO proteins, which formed closed conformation to prevent the combination with other proteins [40,41] and specific protein modifications or something else are possibly needed to expose their crucial binding domains. Furthermore, the cell proliferation regulation function of endogenous ZO proteins is not obvious as expected in physiological condition. ...
Tight junction (TJ) is the barrier of epithelial and endothelial cells to maintain paracellular substrate transport and cell polarity. As one of the TJ cytoplasmic adaptor proteins adjacent to cell membrane, zonula occludens (ZO) proteins are responsible for connecting transmembrane TJ proteins and cytoplasmic cytoskeleton, providing a binding platform for transmembrane TJ proteins to maintain the barrier function. In addition to the basic structural function, ZO proteins play important roles in signal regulation such as cell proliferation and motility, the latter including cell migration, invasion and metastasis, to influence embryonic development, tissue homeostasis, damage repair, inflammation, tumorigenesis, and cancer progression. In this review, we will focus on the signal regulating function of ZO proteins in inflammation and tumorigenesis, and discuss the limitations of previous research and future challenges in ZO protein research.
... To form cell barriers, transmembrane proteins are necessary components, but they only accumulate in membrane and cannot form strands [37,38]. Structural analysis reveals that ZO proteins have a N-terminal fragment, containing three 3 PSD-95/discs-large/Zonula occludens-1 (PDZ) domains and ZO unique motifs (U5, U6 and GUK), which can interact with known TJ transmembrane proteins, such as PDZ domains bind CLDN and JAM, and unique motifs interact with OCLN, indicating that ZO protein links other proteins in TJs ( Figure 6) [38,39]. Umeda et al. identified that ZO-1 deficient cells could not detect assembled TJs. ...
... The mechanism of how PKCδ regulate ZO-1 is unknown, but based on current research, PKCδ may interact with mutltiple domain of ZO-1 to regulate translocation. ZO-1 contains a conserved region (Figure 6), SH3-GUK, which has various functions, such as contacting with transcription factors and TJ proteins [39]. Fanning et al. proved that U5 and GUK were critical in locating ZO-1 to TJs, but U6 inhibited the translocation process [41]. ...
... Structure of ZO proteins[39]. ZO proteins share similar N-terminal domain, including PDZ, SH3, unique (U5,GUK, U6) motif. First and third PDZ motifs bind to claudins and JAM, and the second PDZ link ZO proteins together. ...
PKC isozymes are involved in the modulation of cellular pathways related with tumor progression, acting as a suppressor or promoter. In cancer cells, PKCs are mutated, and most common type is loss of function. This paper focuses on the effect of PKCδ mutation in gastric cancer. LOF mutation occurs throughout catalytic and kinase domains of PKCδ, disrupting activation and function of kinase. In catalytic domain, there are various potential mutation targets, such as binding groove and zinc finger. Mutation residues detected in the kinase domain, such as DFG and APE motifs, can alter catalytic function, causing interruption of activation. Also, a critical region, called hinge region, modulates caspase-3 dependent cleavage, and such tyrosine mutation in this region reduces cleavage activity, inhibiting fully activation of kinase. Importantly, LOF mutation affects cellular activity of downstream protein, p53, through inhibiting transcription, localization, and phosphorylation. For instance, C1 domain mutant suppresses binding capacity with p53, reducing transcription of p53. Disruption of cellular component, tight junction, assembling related to PKC mutation. As identified, PKCδ correlates with ZO-1, and LOF mutation prevent translocation of ZO-1 to TJ area, leading to errors in TJ assembling, promoting tumor invasion.
... These junctional complexes mostly interact with ZO1 within its NT half-comprising in turn its PDZ3, SH3, U5 and GUK domains. For example, many members of the tight junction protein family of claudins bind to the first PDZ (PDZ1) domain [70], whereas connexins mostly interact at the second PDZ domain (PDZ2) [14,28] The focal adhesion protein vinculin binds to the third PDZ domain (PDZ3) [71] and occludin [72] and AJ molecules [69,73,74] interact with the GUK domain ( Figure 2). Interestingly, ZO1 is force sensitive and is considered to be a "tension transducer", exhibiting tension-dependent intramolecular interactions between its NT and CT portions [75]. ...
Barrier function is a vital homeostatic mechanism employed by epithelial and endothelial tissue. Diseases across a wide range of tissue types involve dynamic changes in transcellular junctional complexes and the actin cytoskeleton in the regulation of substance exchange across tissue compartments. In this review, we focus on the contribution of the gap junction protein, Cx43, to the biophysical and biochemical regulation of barrier function. First, we introduce the structure and canonical channel-dependent functions of Cx43. Second, we define barrier function and examine the key molecular structures fundamental to its regulation. Third, we survey the literature on the channel-dependent roles of connexins in barrier function, with an emphasis on the role of Cx43 and the actin cytoskeleton. Lastly, we discuss findings on the channel-independent roles of Cx43 in its associations with the actin cytoskeleton and focal adhesion structures highlighted by PI3K signaling, in the potential modulation of cellular barriers. Mounting evidence of crosstalk between connexins, the cytoskeleton, focal adhesion complexes, and junctional structures has led to a growing appreciation of how barrier-modulating mechanisms may work together to effect solute and cellular flux across tissue boundaries. This new understanding could translate into improved therapeutic outcomes in the treatment of barrier-associated diseases.
... It is important to note that there are multiple organizational forms of actin that are regulated by actin-binding proteins which may affect dynamics of TJ proteins and barrier integrity (Tornavaca et al., 2015). GuK are kinases that normally catalyze ATP-dependent transformation of GMP into GDP; however, the GuK domain on the ZO subfamily of MAGUKs is likely enzymatically inactive due to a missing GMP binding-site and steric hindrance at the ATP-binding site (Lye et al., 2010;Zhu et al., 2012). Instead, the GuK domain of ZO proteins has been shown to bind occludin and PDZ motifs of the claudins (Fanning et al., 1998;Chattopadhyay et al., 2014). ...
The blood-brain barrier (BBB) allows the brain to selectively import nutrients and energy critical to neuronal function while simultaneously excluding neurotoxic substances from the peripheral circulation. In contrast to the highly permeable vasculature present in most organs that reside outside of the central nervous system (CNS), the BBB exhibits a high transendothelial electrical resistance (TEER) along with a low rate of transcytosis and greatly restricted paracellular permeability. The property of low paracellular permeability is controlled by tight junction (TJ) protein complexes that seal the paracellular route between apposing brain microvascular endothelial cells. Although tight junction protein complexes are principal contributors to physical barrier properties, they are not static in nature. Rather, tight junction protein complexes are highly dynamic structures, where expression and/or localization of individual constituent proteins can be modified in response to pathophysiological stressors. These stressors induce modifications to tight junction protein complexes that involve de novo synthesis of new protein or discrete trafficking mechanisms. Such responsiveness of BBB tight junctions to diseases indicates that these protein complexes are critical for maintenance of CNS homeostasis. In fulfillment of this vital role, BBB tight junctions are also a major obstacle to therapeutic drug delivery to the brain. There is an opportunity to overcome this substantial obstacle and optimize neuropharmacology via acquisition of a detailed understanding of BBB tight junction structure, function, and regulation. In this review, we discuss physiological characteristics of tight junction protein complexes and how these properties regulate delivery of therapeutics to the CNS for treatment of neurological diseases. Specifically, we will discuss modulation of tight junction structure, function, and regulation both in the context of disease states and in the setting of pharmacotherapy. In particular, we will highlight how these properties can be potentially manipulated at the molecular level to increase CNS drug levels via paracellular transport to the brain.
... However, this latter region is also crucial for the indirect interaction of ZO proteins with the actomyosin cytoskeleton: the SG module binds to -catenin [18,77,104], vinculin [105], afadin [20,106], the spectrin-binding protein protein 4.1R [107], and the myosinVII-and actin-binding protein shroom2 [108] (Figure 1). Furthermore, intramolecular interactions occur between the SH3-GUK, U5 and U6 domains [109], which control ZO protein targeting to junctions and coordinate its activities [16,32,75,[110][111][112]. Importantly, the PDZ3-SH3-GUK supramodule of ZO-1 (ZPSG) also undergoes a mechano-sensitive intramolecular interaction with the C-terminal ZU5 domain (Figure 1), which controls the scaffolding of its interactors occludin and DbpA/ZONAB [21], this latter a transcription factor that binds to ZO-1, as described below. In summary, the PDZ and ZPSG regions are critically involved in scaffolding functions of ZO proteins, through both intra-and intermolecular interactions, and these interactions are subjected to regulation by actomyosin-dependent force [21]. ...
Tight and adherens junctions are specialized sites of cell-cell interaction in epithelia and endothelia, and are involved in barrier, adhesion, and signaling functions. These functions are orchestrated by a highly organized meshwork of macromolecules in the membrane and cytoplasmic compartments. In this review, we discuss the structural organization and functions of the major cytoplasmic scaffolding and adaptor proteins of vertebrate apical junctions (ZO proteins, afadin, PLEKHA7, cingulin, paracingulin, polarity complex proteins, and a few others), focusing on their interactions with cytoskeletal and signaling proteins. Furthermore, we discuss recent results highlighting how mechanical tension, protein-protein interactions and post-translational modifications regulate the conformation and function of scaffolding proteins, and how spontaneous phase separation into biomolecular condensates contributes to apical junction assembly. Using a sequence-based algorithm, a large fraction of cytoplasmic proteins of apical junctions are predicted to be phase separating proteins (PSPs), suggesting that formation of biomolecular condensates is a general mechanism to organize cell-cell contacts by clustering proteins.
... Following the three N-terminal PDZ domains, ZO proteins contain a SH3-GUK module. Crystal structure analysis of the ZO-1 SH3-GUK tandem domain confirmed independent folding of the SH3 and GUK domains, and pulldown assays identified the downstream U6 loop as an intramolecular ligand of the SH3-GUK core with a potential role in regulating TJ assembly in vivo [102]. Crystal structures of the complete MAGUK core module of ZO-1 comprising the PDZ3-SH3-GUK region and its complex with the cytoplasmic tail of adhesion molecule JAM-A revealed that residues from the adjacent SH3 domain are involved in ligand binding to the ZO-1 PDZ3 [103]. ...
Tight junctions are complex supramolecular entities composed of integral membrane proteins, membrane-associated and soluble cytoplasmic proteins engaging in an intricate and dynamic system of protein–protein interactions. Three-dimensional structures of several tight-junction proteins or their isolated domains have been determined by X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy. These structures provide direct insight into molecular interactions that contribute to the formation, integrity, or function of tight junctions. In addition, the known experimental structures have allowed the modeling of ligand-binding events involving tight-junction proteins. Here, we review the published structures of tight-junction proteins. We show that these proteins are composed of a limited set of structural motifs and highlight common types of interactions between tight-junction proteins and their ligands involving these motifs.
... Linear sequence of ZO1, ZO2, and ZO3 and their N-terminal, PSG, and C-terminal regions are depicted as two circles to indicate proteinprotein interactions between the same homolog (red), between different homologs (green), and for intra-molecular interactions (blue). References to previous interaction studies are indicated by numbers: *1 (Utepbergenov et al., 2006), *2 (Fanning et al., 1998), *3 (Wu et al., 2007), and *4,5 (Lye et al., 2010;Spadaro et al., 2017). separation of ZO protein is sufficient to enrich and compartmentalize junctional proteins, we expressed and purified a number of known ZO interaction proteins (clients; Figure S4C) and measured their partitioning into the condensed phase of ZO proteins in vitro ( Figure 5B). ...
... In particular, we focused on the U6 domain, which has been shown to regulate tight-junction formation but is not known to bind other proteins. It was previously shown that deletion of the U6 domain causes assembly of ectopic tight-junction strands in the lateral membrane domain in MDCK-II cells (Fanning et al., 2007;Lye et al., 2010;Rodgers et al., 2013). ...
... Phase separation was significantly enhanced in comparison with that of ZO1-FL. The U6 has previously been shown to bind to the GuK domain via electrostatic interactions (Lye et al., 2010). Our data now suggest that U6 back-binding prevents the PSG module from oligomerization. ...
Tight junctions are cell-adhesion complexes that seal tissues and are involved in cell polarity and signaling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands are dependent on the membrane-associated scaffolding proteins ZO1 and ZO2. To understand how zona occludens (ZO) proteins organize junction assembly, we performed quantitative cell biology and in vitro reconstitution experiments. We discovered that ZO proteins self-organize membrane-attached compartments via phase separation. We identified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight-junction proteins, including adhesion receptors, cytoskeletal adapters, and transcription factors. Our results suggest that an active-phase transition of ZO proteins into a condensed membrane-bound compartment drives claudin polymerization and coalescence of a continuous tight-junction belt.
... Indeed, ZO-1 can undergo multivalent interactions with other ZO proteins such as ZO-2 and ZO-3 as well as other TJ scaffolding proteins, such as Cingulins (Fanning et al., 1998(Fanning et al., , 2007Utepbergenov et al., 2006). There is also evidence for intra-molecular interaction sites for ZO-1 and other members of the MAGUK (membrane-associated guanylate kinases) protein family (Ye et al., 2018); (Lye et al., 2010;Spadaro et al., 2017) (Ye et al., 2018). This suggests that ZO-1b might be capable of undergoing phase separation, and that this property might contribute to its previously demonstrated scaffolding function in recruiting other proteins to TJ (Bauer et al., 2010;Fanning and Anderson, 2009;Matter and Balda, 2003). ...
Cell-cell junctions respond to mechanical forces by changing their organization and function. To gain insight into the mechanochemical basis underlying junction mechanosensitivity, we analyzed tight junction (TJ) formation between the enveloping cell layer (EVL) and the yolk syncytial layer (YSL) in the gastrulating zebrafish embryo. We found that the accumulation of Zonula Occludens-1 (ZO-1) at TJs closely scales with tension of the adjacent actomyosin network, revealing that these junctions are mechanosensitive. Actomyosin tension triggers ZO-1 junctional accumulation by driving retrograde actomyosin flow within the YSL, which transports non-junctional ZO-1 clusters toward the TJ. Non-junctional ZO-1 clusters form by phase separation, and direct actin binding of ZO-1 is required for stable incorporation of retrogradely flowing ZO-1 clusters into TJs. If the formation and/or junctional incorporation of ZO-1 clusters is impaired, then TJs lose their mechanosensitivity, and consequently, EVL-YSL movement is delayed. Thus, phase separation and flow of non-junctional ZO-1 confer mechanosensitivity to TJs.
