ZO-1 Stabilizes the Tight Junction Solute Barrier through Coupling to the Perijunctional Cytoskeleton

Itallie, Christina M. Van; Fanning, Alan S.; Bridges, Arlene S.; Anderson, James M.

doi:10.1091/mbc.e09-04-0320

Cited by 381 publications

(463 citation statements)

References 84 publications

(111 reference statements)

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“…Consistent with this, ZO-1 knockdown has been shown to increase large solute flux across the tight junction (26,27), and a recent report demonstrated enhanced sensitivity of ZO-1 knockdown monolayers to cytochalasin D or calcium chelation (27). In contrast, the TER increase induced by rho kinase inhibition in control monolayers did not occur in ZO-1-knockdown MDCK monolayers (27). Thus, previous data, as well as the data presented here, indicate that ZO-1 is essential for cytoskeletally-mediated barrier regulation.…”

Section: Discussionsupporting

confidence: 55%

“…Interestingly, ours is not the first study showing that loss of ZO-1 expression does not markedly affect TER (25)(26)(27). This suggests that other proteins, for example ZO-2, may compensate for ZO-1 loss (25).…”

Section: Discussionmentioning

confidence: 56%

“…Together with previous studies showing increased flux of large solutes following MLCK activation (19), our identification of a specific role for ZO-1 in MLCK-dependent barrier regulation suggests that ZO-1 interactions with the cytoskeleton regulate flux of large solutes via a tight junction leak pathway. Consistent with this, ZO-1 knockdown has been shown to increase large solute flux across the tight junction (26,27), and a recent report demonstrated enhanced sensitivity of ZO-1 knockdown monolayers to cytochalasin D or calcium chelation (27). In contrast, the TER increase induced by rho kinase inhibition in control monolayers did not occur in ZO-1-knockdown MDCK monolayers (27).…”

Section: Discussionmentioning

confidence: 63%

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MLCK-dependent exchange and actin binding region-dependent anchoring of ZO-1 regulate tight junction barrier function

Marchiando

Weber

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

224

221

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The perijunctional actomyosin ring contributes to myosin light chain kinase (MLCK)-dependent tight junction regulation. However, the specific protein interactions involved in this process are unknown. To test the hypothesis that molecular remodeling contributes to barrier regulation, tight junction protein dynamic behavior was assessed by fluorescence recovery after photobleaching (FRAP). MLCK inhibition increased barrier function and stabilized ZO-1 at the tight junction but did not affect claudin-1, occludin, or actin exchange in vitro. Pharmacologic MLCK inhibition also blocked in vivo ZO-1 exchange in wild-type, but not long MLCK −/− , mice. Conversely, ZO-1 exchange was accelerated in transgenic mice expressing constitutively active MLCK. In vitro, ZO-1 lacking the actin binding region (ABR) was not stabilized by MLCK inhibition, either in the presence or absence of endogenous ZO-1. Moreover, the free ABR interfered with full-length ZO-1 exchange and reduced basal barrier function. The free ABR also prevented increases in barrier function following MLCK inhibition in a manner that required endogenous ZO-1 expression. In silico modeling of the FRAP data suggests that tight junction-associated ZO-1 exists in three pools, two of which exchange with cytosolic ZO-1. Transport of the ABR-anchored exchangeable pool is regulated by MLCK. These data demonstrate a critical role for the ZO-1 ABR in barrier function and suggest that MLCK-dependent ZO-1 exchange is essential to this mechanism of barrier regulation.fluorescence recovery after photobleaching | mathematical models | myosin light chain kinase | paracellular permeability | intestinal epithelium G reat progress has been made toward identifying components of the tight junction. These include transmembrane, peripheral membrane, and regulatory proteins, many of which contain one or more domains that mediate interactions with other tight junction and cytoskeletal proteins (1). These and other observations drove development of models that depicted the tight junction as a static, heavily cross-linked protein complex (2). However, recent data showing rapid and continuous remodeling of the tight junction refuted the previous models and led to the hypothesis that modulation of protein remodeling behavior could be a mechanism of tight junction barrier regulation (3, 4).The perijunctional ring of F-actin and myosin II that supports the tight junction is essential to physiological and pathophysiological barrier regulation (5). For example, activation of perijunctional myosin light chain kinase (MLCK) is sufficient to enhance paracellular permeability (6, 7) and is required for tight junction barrier regulation in response to Na + -nutrient cotransport, inflammatory cytokines, or pathogenic bacteria (8). Thus, modulation of MLCK activity represents a point of convergence for multiple signaling pathways that regulate tight junction barrier function.To assess the role of molecular remodeling in barrier regulation, dynamic behaviors of tight junction proteins were assessed b...

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Section: Discussionsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 56%

Section: Discussionmentioning

confidence: 63%

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MLCK-dependent exchange and actin binding region-dependent anchoring of ZO-1 regulate tight junction barrier function

Marchiando

Weber

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

224

221

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“…All of these functions are orchestrated by complexes of transmembrane, cytoplasmic, and cytoskeletal proteins. Transmembrane proteins of TJ are anchored to the actin cytoskeleton by cytoplasmic scaffolding proteins, among which ZO-1 and ZO-2 play a critical role (11)(12)(13). The major transmembrane proteins of AJ are E-cadherin and nectin, which are anchored to the actin and microtubule cytoskeletons by distinct cytoplasmic protein complexes (9,10).…”

Section: Tight Junctions (Tj)mentioning

confidence: 99%

A Role for ZO-1 and PLEKHA7 in Recruiting Paracingulin to Tight and Adherens Junctions of Epithelial Cells

Pulimeno

Paschoud

Citi

2011

Journal of Biological Chemistry

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Paracingulin is a 160-kDa protein localized in the cytoplasmic region of epithelial tight and adherens junctions, where it regulates RhoA and Rac1 activities by interacting with guanine nucleotide exchange factors. Here, we investigate the molecular mechanisms that control the recruitment of paracingulin to cell-cell junctions. We show that paracingulin forms a complex with the tight junction protein ZO-1, and the globular head domain of paracingulin interacts directly with ZO-1 through an N-terminal region containing a conserved ZIM (ZO-1-Interaction-Motif) sequence. Recruitment of paracingulin to cadherinbased cell-cell junctions in Rat1 fibroblasts requires the ZIMcontaining region, whereas in epithelial cells removal of this region decreases the junctional localization of paracingulin at tight junctions but not at adherens junctions. Depletion of ZO-1, but not ZO-2, reduces paracingulin accumulation at tight junctions. A yeast two-hybrid screen identifies both ZO-1 and the adherens junction protein PLEKHA7 as paracingulin-binding proteins. Paracingulin forms a complex with PLEKHA7 and its interacting partner p120ctn, and the globular head domain of paracingulin interacts directly with a central region of PLEKHA7. Depletion of PLEKHA7 from Madin-Darby canine kidney cells results in the loss of junctional localization of paracingulin and a decrease in its expression. In summary, we characterize ZO-1 and PLEKHA7 as paracingulin-interacting proteins that are involved in its recruitment to epithelial tight and adherens junctions, respectively. Tight junctions (TJ)2 and adherens junctions (AJ) are key elements of the apical junctional complex of vertebrate epithelial cells and are of fundamental importance in the morphogenesis and homeostasis of epithelial tissues. TJ and AJ carry out several functions: canonical roles in cell-cell adhesion and tissue sorting (AJ), cell polarization and permeability barrier functions (TJ), and signaling functions through pathways that control the organization of the cytoskeleton and the activity of transcription factors (for review, see Refs.

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“…The C-terminal domains mediate interactions with the cortical actin cytoskeleton, whereas the N-terminal MAGUK domains bind to the transmembrane components of tight junction strands and other scaffolding or signaling molecules that regulate TJ assembly. The three ZO MAGUKs clearly share some functional roles, and recent genetic studies indicate that there is a certain amount of functional redundancy between ZO-1 and ZO-2 within the TJ (13,15,16). Either ZO-1 or ZO-2 appears to be sufficient for formation of a functional barrier in cultured cells, but the deletion of both ZO-1 and ZO-2 in epithelial cells disrupts the TJ barrier and prevents the assembly of TJ transmembrane proteins described above into strands (13).…”

mentioning

confidence: 99%

Insights into Regulated Ligand Binding Sites from the Structure of ZO-1 Src Homology 3-Guanylate Kinase Module

Lye

Fanning

et al. 2010

Journal of Biological Chemistry

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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 characterized by a core Src homology 3 (SH3)-GUK module that coordinates multiple protein-protein interactions. The structure of the ZO-1 SH3-GUK domain confirms that the interdependent folding of the SH3 and GUK domains is a conserved feature of MAGUKs, but differences in the orientation of the GUK domains in three different MAGUKs reveal interdomain flexibility of the core unit. Using pull-down assays, we show that an effector loop, the U6 region in ZO-1, forms a novel intramolecular interaction with the core module. This interaction is divalent cation-dependent and overlaps with the binding site for the regulatory molecule calmodulin on the GUK domain. These findings provide insight into the previously observed ability of the U6 region to regulate TJ assembly in vivo and the structural basis for the complex protein interactions of the MAGUK family.The assembly and functional organization of plasma membrane domains, such as synapses and cell adhesions, depends on the temporally and spatially regulated assembly of multiprotein complexes. In one common paradigm, the trafficking and assembly of the transmembrane receptors and adhesion molecules that form these complexes is organized by a series of cytosolic scaffolding proteins. One of the most common and ubiquitous families of scaffolding molecules is the membraneassociated guanylate kinase homologs (MAGUKs).2 Founding members of this family include the discs large tumor suppressor in Drosophila, Lin2, which is required for RT kinase signaling in Caenorhabditis and PSD-95 and its related synaptic scaffolding proteins, which also control channel activity. This diverse family of proteins is characterized by a core motif of protein-binding domains, including a PSD-95/DLG/ZO-1 (PDZ) domain, an Src homology 3 (SH3) domain, and a region of homology to guanylate kinase referred to as the GUK domain ( Fig. 1A) (reviewed in Ref. 1). Most also include additional PDZ domains or other conserved protein-binding domains. These domains are separated by unique regions (U regions), which can also have protein-binding and regulatory roles (1, 2). However, it is the core SH3-GUK module and its adjacent unique domains that often have a dominant role in the regulation of transmembrane ligand binding, protein cross-linking, and localization to the appropriate subcellular domain.The structural basis for the regulatory properties of the various MAGUKs is poorly understood. To date, the only core motif that has been resolved at an atomic level belongs to PSD-95 (postsynaptic density protein 95) (3, 4), a neuronal protein involved in the regulation of postsynaptic gated ion channel. The structure reveals that the SH3 and GUK domains are not simply...

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ZO-1 Stabilizes the Tight Junction Solute Barrier through Coupling to the Perijunctional Cytoskeleton

Cited by 381 publications

References 84 publications

MLCK-dependent exchange and actin binding region-dependent anchoring of ZO-1 regulate tight junction barrier function

MLCK-dependent exchange and actin binding region-dependent anchoring of ZO-1 regulate tight junction barrier function

A Role for ZO-1 and PLEKHA7 in Recruiting Paracingulin to Tight and Adherens Junctions of Epithelial Cells

Insights into Regulated Ligand Binding Sites from the Structure of ZO-1 Src Homology 3-Guanylate Kinase Module

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