A feedback circuitry involving γ-actin, β-actin and nonmuscle myosin-2 A controls tight junction and apical cortex mechanics.

Cytoplasmic β- and γ-actin isoforms, along with non-muscle myosin 2 isoforms, are tightly regulated in epithelial cells and compose the actomyosin cytoskeleton at the apical junctional complex. However, their specific role in regulating the mechanics of the membrane cortex and the organization of junctions, and which biomechanical circuitries modulate their expression remain poorly understood. Here, we show that γ-actin depletion in MDCK and other epithelial cells results in increased expression and junctional accumulation of β-actin and increased tight junction membrane tortuosity, both dependent on nonmuscle myosin-2A upregulation.

Cingulin binds to the ZU5 domain of scaffolding protein ZO-1 to promote its extended conformation, stabilization, and tight junction accumulation.

Zonula occludens-1 (ZO-1), the major scaffolding protein of tight junctions (TJs), recruits the cytoskeleton-associated proteins cingulin (CGN) and paracingulin (CGNL1) to TJs by binding to their N-terminal ZO-1 interaction motif. The conformation of ZO-1 can be either folded or extended, depending on cytoskeletal tension and intramolecular and intermolecular interactions, and only ZO-1 in the extended conformation recruits the transcription factor DbpA to TJs. However, the sequences of ZO-1 that interact with CGN and CGNL1 and the role of TJ proteins in ZO-1 TJ assembly are not known.

PLEKHA5, PLEKHA6, and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis.

Copper homeostasis is crucial for cellular physiology and development, and its dysregulation leads to disease. The Menkes ATPase ATP7A plays a key role in copper efflux, by trafficking from the Golgi to the plasma membrane upon cell exposure to elevated copper, but the mechanisms that target ATP7A to the cell periphery are poorly understood. PDZD11 interacts with the C-terminus of ATP7A, which contains sequences involved in ATP7A trafficking, but the role of PDZD11 in ATP7A localization is unknown.

WW, PH and C-Terminal Domains Cooperate to Direct the Subcellular Localizations of PLEKHA5, PLEKHA6 and PLEKHA7.

PLEKHA5, PLEKHA6, and PLEKHA7 (WW-PLEKHAs) are members of the PLEKHA family of proteins that interact with PDZD11 through their tandem WW domains. WW-PLEKHAs contribute to the trafficking and retention of transmembrane proteins, including nectins, Tspan33, and the copper pump ATP7A, at cell-cell junctions and lateral membranes. However, the structural basis for the distinct subcellular localizations of PLEKHA5, PLEKHA6, and PLEKHA7 is not clear.

Tension-Dependent Stretching Activates ZO-1 to Control the Junctional Localization of Its Interactors.

Tensile forces regulate epithelial homeostasis, but the molecular mechanisms behind this regulation are poorly understood. Using structured illumination microscopy and proximity ligation assays, we show that the tight junction protein ZO-1 exists in stretched and folded conformations within epithelial cells, depending on actomyosin-generated force. We also show that ZO-1 and ZO-2 regulate the localization of the transcription factor DbpA and the tight junction membrane protein occludin in a manner that depends on the organization of the actin cytoskeleton, myosin-II activity, and substrate stiffness, resulting in modulation of gene expression, cell proliferation, barrier function, and cyst morphogenesis.

PLEKHA7 Recruits PDZD11 to Adherens Junctions to Stabilize Nectins.

PLEKHA7 is a junctional protein implicated in stabilization of the cadherin protein complex, hypertension, cardiac contractility, glaucoma, microRNA processing, and susceptibility to bacterial toxins. To gain insight into the molecular basis for the functions of PLEKHA7, we looked for new PLEKHA7 interactors. Here, we report the identification of PDZ domain-containing protein 11 (PDZD11) as a new interactor of PLEKHA7 by yeast two-hybrid screening and by mass spectrometry analysis of PLEKHA7 immunoprecipitates.

ZO proteins redundantly regulate the transcription factor DbpA/ZONAB.

The localization and activities of DbpA/ZONAB and YAP transcription factors are in part regulated by the density-dependent assembly of epithelial junctions. DbpA activity and cell proliferation are inhibited by exogenous overexpression of the tight junction (TJ) protein ZO-1, leading to a model whereby ZO-1 acts by sequestering DbpA at the TJ. However, mammary epithelial cells and mouse tissues knock-out for ZO-1 do not show increased proliferation, as predicted by this model.

PLEKHA7 modulates epithelial tight junction barrier function.

PLEKHA7 is a recently identified protein of the epithelial zonula adhaerens (ZA), and is part of a protein complex that stabilizes the ZA, by linking it to microtubules. Since the ZA is important in the assembly and disassembly of tight junctions (TJ), we asked whether PLEKHA7 is involved in modulating epithelial TJ barrier function. We generated clonal MDCK cell lines in which one of four different constructs of PLEKHA7 was inducibly expressed.

MgcRacGAP interacts with cingulin and paracingulin to regulate Rac1 activation and development of the tight junction barrier during epithelial junction assembly.

The regulation of Rho-family GTPases is crucial to direct the formation of cell-cell junctions and tissue barriers. Cingulin (CGN) and paracingulin (CGNL1) control RhoA activation in epithelial cells by interacting with RhoA guanidine exchange factors. CGNL1 depletion also inhibits Rac1 activation during junction assembly.

Cingulin is dispensable for epithelial barrier function and tight junction structure, and plays a role in the control of claudin-2 expression and response to duodenal mucosa injury.

Cingulin (CGN) is a 140 kDa protein, which is localized to the cytoplasmic region of vertebrate tight junctions (TJ), and regulates gene expression and RhoA signaling in cultured cells. To investigate the function of CGN at the organism level, we generated CGN knockout (CGN(-/-)) mice by homologous recombination. CGN(-/-) mice are viable and fertile, and are born at the expected mendelian ratios.

Cingulin and paracingulin show similar dynamic behaviour, but are recruited independently to junctions.

Cingulin (CGN) and paracingulin (CGNL1) are structurally related proteins that regulate Rho family GTPases by recruiting guanine nucleotide exchange factors to epithelial junctions. Although the subcellular localization of cingulin and paracingulin is likely to be essential for their role as adaptor proteins, nothing is known on their in vivo localization, and their dynamics of exchange with the junctional membrane. To address these questions, we generated stable clones of MDCK cells expressing fluorescently tagged cingulin and paracingulin.

The tight junction protein cingulin regulates gene expression and RhoA signaling.

Tight junctions (TJ) regulate the passage of solutes across epithelial sheets, contribute to the establishment and maintenance of epithelial apico-basal polarity and are involved in the regulation of gene expression and cell proliferation. Cingulin, a Mr 140 kDa protein localized in the cytoplasmic region of TJ, is not directly required for TJ formation and epithelial polarity but regulates RhoA signaling, through its interaction with the RhoA activator GEF-H1, and gene expression. Here we describe in more detail the effect of cingulin mutation in embryoid bodies (EB) on gene expression, by identifying the genes that show the highest degree of up- or downregulation, and the putative canonical pathways that might be affected by cingulin.

Paracingulin regulates the activity of Rac1 and RhoA GTPases by recruiting Tiam1 and GEF-H1 to epithelial junctions.

Small GTPases control key cellular events, including formation of cell-cell junctions and gene expression, and are regulated by activating and inhibiting factors. Here, we characterize the junctional protein paracingulin as a novel regulator of the activity of two small GTPases, Rac1 and RhoA, through the functional interaction with their respective activators, Tiam1 and GEF-H1. In confluent epithelial monolayers, paracingulin depletion leads to increased RhoA activity and increased expression of mRNA for the tight junction protein claudin-2.

Disruption of the cingulin gene does not prevent tight junction formation but alters gene expression.

Cingulin, a component of vertebrate tight junctions, contains a head domain that controls its junctional recruitment and protein interactions. To determine whether lack of junctional cingulin affects tight-junction organization and function, we examined the phenotype of embryoid bodies derived from embryonic stem cells carrying one or two alleles of cingulin with a targeted deletion of the exon coding for most of the predicted head domain. In homozygous (-/-) embryoid bodies, no full-length cingulin was detected by immunoblotting and no junctional labeling was detected by immunofluorescence.